Chemistries for DNA Nanotechnology - Chemical Reviews (ACS

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Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

Chemistries for DNA Nanotechnology Mikael Madsen and Kurt V. Gothelf*

Chem. Rev. Downloaded from pubs.acs.org by MIAMI UNIV on 02/04/19. For personal use only.

Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark ABSTRACT: The predictable nature of DNA interactions enables the programmable assembly of highly advanced 2D and 3D DNA structures of nanoscale dimensions. The access to ever larger and more complex structures has been achieved through decades of work on developing structural design principles. Concurrently, an increased focus has emerged on the applications of DNA nanostructures. In its nature, DNA is chemically inert and nanostructures based on unmodified DNA mostly lack function. However, functionality can be obtained through chemical modification of DNA nanostructures and the opportunities are endless. In this review, we discuss methodology for chemical functionalization of DNA nanostructures and provide examples of how this is being used to create functional nanodevices and make DNA nanostructures more applicable. We aim to encourage researchers to adopt chemical modifications as part of their work in DNA nanotechnology and inspire chemists to address current challenges and opportunities within the field.

CONTENTS 1. Introduction 2. Synthesis of Modified Oligonucleotides 2.1. Phosphoramidite-Based Oligonucleotide Synthesis 2.2. In-Synthesis Modification of DNA 2.3. Handles for Postsynthesis Modification of DNA 2.3.1. Amino Handles 2.3.2. Thiol Handles 2.3.3. Alkyne Handles 2.3.4. Electrophilic Handles 2.3.5. Azide Handles 2.3.6. Modifications for Postsynthesis Diels− Alder Reactions 2.3.7. Hydrazide and Aminooxy Modifications 2.3.8. Modifications for Palladium-Catalyzed Couplings 2.4. Introduction of Modifications in DNA Nanostructures 2.4.1. Fluorescent Dyes and Quenchers 2.4.2. Azobenzenes and Other Photoreactive Compounds 2.4.3. Hydrophobic Modifications 2.4.4. Biotinylation of DNA 2.4.5. Redox-Active Modifications 2.4.6. Other Less Common DNA Modifications 2.5. Modification of DNA Using Terminal Deoxynucleotidyl Transferase 3. Nucleic Acid Analogues 3.1. Peptide Nucleic Acid (PNA) 3.2. Locked Nucleic Acid (LNA) 3.3. Phosphorothioate-Modified DNA (psDNA) 4. Reactions on DNA Nanostructures © XXXX American Chemical Society

4.1. DNA-Templated Organic Synthesis 4.1.1. DNA-Templated Synthesis for Preparation of Sequence-Specific Oligomers 4.1.2. DNA-Templated Synthesis for Formation of Nanowires 4.2. Organic Chemistry on DNA Nanostructures 4.2.1. Single Molecule Reactions on DNA Origami 4.2.2. Chemical Cross-Linking of DNA Nanostructures 4.2.3. Polymerization on DNA Nanostructures 4.3. Metallization of DNA Nanostructures 5. Chemical Methods for Immobilization of Nanomaterials in DNA Nanostructures 5.1. Carbon Nanotubes 5.2. Conjugated Polymers and Oligomers 5.3. Other Polymers 5.4. Inorganic Nanomaterials 5.4.1. Gold Nanoparticles 5.4.2. Quantum Dots 6. Proteins in DNA Nanotechnology 6.1. Covalently Linked Protein−DNA Conjugates 6.1.1. Conjugation Using Heterobifunctional Linkers 6.1.2. Site-Specific Attachment of DNA to Proteins 6.1.3. Peptide Tags for Formation of Protein− DNA Conjugates 6.1.4. Self-Labeling Polypeptides for Formation of Protein−DNA Conjugates

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Special Issue: Nucleic Acid Nanotechnology Received: September 17, 2018

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DOI: 10.1021/acs.chemrev.8b00570 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews 6.1.5. Affinity-Guided Attachment of DNA to Proteins 6.2. Noncovalent Binding for Immobilization of Proteins in DNA Nanostructures 6.2.1. DNA-Binding Proteins 6.2.2. Small Molecule−Protein Interactions 6.2.3. Electrostatic and Hydrophobic Interactions 7. Surface-Immobilization of DNA Nanostructures 7.1. Electrostatic Interactions 7.2. Covalent Interactions 7.3. Biotin−Avidin Interactions 7.4. Hydrophobic Interactions 7.5. Other Methods for Surface-Immobilization 8. Conclusion and Outlook Author Information Corresponding Author ORCID Notes Biographies Acknowledgments List of Abbreviations References

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that researchers in DNA nanotechnology have backgrounds in a variety of fields such as physics, molecular biology, computer science, mathematics, and chemistry. For those not familiar with chemical functionalization of DNA, it is logical to avoid excessive chemical manipulations and settle for commercially available standard modifications of DNA. Over the past decade, more and more attention has, however, been focused on functional DNA structures and their potential applications in for instance biophysics, nano-optics and -electronics, and medicine.5 Fortunately, oligonucleotides with a wide variety of modifications including dyes, biotin, and chemical handles are commercially available, and Section 2 will provide an overview of the most common modifications and a discussion of their applications in DNA nanotechnology. While the area of nucleic acid chemistry is closely related to DNA nanotechnology, there has been surprisingly little overlap between the fields. Currently, this seems to be changing as exemplified by the application of nucleic acid analogues in DNA nanotechnology described in Section 3. Once integrated in DNA sequences and DNA nanostructures, chemical functionalities can control chemical reactions ranging from individual covalent reactions, oligomerizations, and polymerizations, to cross-linking and metallization of DNA nanostructures, all of which are topics addressed in Section 4. In Section 5 macromolecular entities and inorganic nanomaterials are described, both in terms of how they are prepared and functionalized for use in DNA nanotechnology and their actual integration in DNA nanostructures. In extension of this, Section 6 is devoted to proteins in DNA nanostructures since the formation of protein−DNA conjugates and the integration of proteins in DNA nanostructures often come with particular challenges. The seventh and final section provides a brief overview of the chemistry underlying surface-immobilization of DNA nanostructures which is a highly important, but often overlooked, process in terms of investigating DNA nanostructures. In the seven sections, we have aimed at including a selection of important contributions and covering the field broadly; however, the review is not exhaustive, since there are too many cases where similar or related chemistries have been applied to include them all. Altogether, we hope to show chemists the opportunities residing in the application of different types of chemistries to DNA nanotechnology and indeed also to show researchers from other fields the palette of opportunities for functionalization that various chemistries provide DNA nanotechnology. With this dual purpose, we have attempted to show both the detailed chemistry applied to DNA nanostructures and also to include illustrated examples of the functionalized nanostructures at work in a wide range of applications.

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1. INTRODUCTION Ned Seeman’s visions and pioneering work on immobile DNA junctions and folding of DNA into artificial structures in the early 1980s founded the field of DNA nanotechnology.1,2 With further developments such as DNA tiles, AFM imaging techniques, and DNA strand displacement reactions, the field gained more attention from the late 1990s.3,4 In particular, since the invention of DNA origami by Poul Rothemund in 2006, DNA nanotechnology has expanded with an impressive pace.5,6 Several reviews have covered the development of DNA nanotechnology5,7−15 and its application in biomedicine,16−19 photonics,20−24 optics,25−27 biotechnology,28−33 and nanofabrication.34−37 The main reasons for the success of DNA as the molecule of choice for programmed self-assembly are (i) the reliable digital encodability of Watson−Crick base pairing, (ii) the high predictability of the structure of the DNA helix with its hydrophobic interior and negatively charged exterior, which, unlike proteins, leaves little space for secondary interactions, (iii) the dynamic nature of DNA hybridization, (iv) the availability of oligonucleotides of almost any sequence through automated synthesis, and (v) the chemical stability and inertness of DNA. Overall, these properties make DNA a unique molecule for programmed self-assembly of nanostructures with welldefined geometry, which can even be expanded with dynamic properties through various hybridization exchange reactions. However, unlike proteins, the chemical inertness of DNA renders such structures limited in chemical functionality and therefore they mainly serve as structurally well-defined scaffolds. Already in his first papers on DNA nanotechnology, Seeman envisioned integration of other molecules and materials in DNA nanostructures such as proteins and electronically active molecules to obtain functional structures.1,38 In spite of this, the main focus of the field was for many years devoted to the design and folding of DNA-only structures. This is no surprise since structure design has been the key driver of the field and a territory with many new ways to pave and hallmarks to achieve. Yet another reason behind the focus on DNA-only structures is

2. SYNTHESIS OF MODIFIED OLIGONUCLEOTIDES This section describes the origin of the building blocks for creating chemically modified DNA nanostructures. The automated synthetis of DNA strands is the technical foundation of DNA nanotechnology, and therefore the chemistry of DNA synthesis will briefly be described. In DNA nanostructures, the individual DNA strands are the building blocks and their nature eventually determines the structure and properties of the desired design. Tailoring the chemical functionality of the DNA building blocks is the first step toward obtaining nanostructures with desired function. This section, therefore, subsequently provides a discussion of methods for functionalizing DNA strands along B


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Figure 1. Phosphoramidite-based oligonucleotide synthesis cycle. CPG = controlled pore glass, Activator = 5-(ethylthio)-1H-tetrazole, CE = Cyanoethyl, DMT = 4,4′-Dimethoxytrityl. The bottom panel shows a typical base-modified CPG solid support for oligonucleotide synthesis and the standard protection groups used for the respective nucleobases. While isobutyryl protection is traditionally used for guanine, the DMF-protecting group is also widely used. Acetyl protection is occasionally used for dC.

thesis from stable phosphoramidite nucleosides using highly optimized synthesis cycles provides coupling yields of more than 99% per cycle, resulting in good yields of oligonucleotides as long as 200mers.43 Chemical DNA synthesis using phosporamidite chemistry is commonly performed from the 3′ to the 5′ end, opposite the direction of DNA synthesis by polymerases. The building blocks contain a phosphoramidite group on the 3′hydroxyl group of the deoxyribose sugar and an acid labile tritylbased (typically dimethoxytrityl, DMT) protecting group on the 5′-hydroxyl group (Figure 1). The starting point of oligonucleotide synthesis is typically a nucleoside-modified solid support or a universal solid support such as UnyLinker supports.44,45 For

with some of the most common DNA modifications and their applications in DNA nanotechnology. 2.1. Phosphoramidite-Based Oligonucleotide Synthesis

With the discovery of DNA’s structure and biological importance, the bottom-up synthesis of DNA became an obvious and appealing challenge to the organic synthesis community.39 The initial progress in oligonucleotide synthesis started in the 1950s and is well described elsewhere.40 Eventually, with the invention of phosphoramidite chemistry and the adaption of solid-phase synthesis, oligonucleotide synthesis became widely applicable.41,42 Oligonucleotide synC


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Figure 2. In-synthesis modifications of DNA. (A) Scheme showing the general strategy for 5′-modification of DNA using a modified phosphoramidite without hydroxyl-handle for further chain elongation. (B) 3′-modification of DNA using a modified solid support. (C) Internal modification of DNA using a non-nucleobase phosphoramidite containing the modification. (D) Internal modification of DNA using a modified nucleobase in order to maintain Watson−Crick base pairing in the oligonucleotide product. In this case modified dT is shown, but all four bases can in principle be employed, although dT and dA remain the most popular sites for modification. Note that the oligonucleotide products on the right-hand side are rotated 180° to display the sequences in the 5′ to 3′ direction. (E) Branching modifications can also be employed in DNA synthesis. The example shown her is based on work by von Kiedrowski and co-workers who used the combination of reversed DNA synthesis and a trisubstituted benzene branching modifier to obtain branched structures with all 5′-extensions.49

nucleoside-modified supports, the first 3′-nucleoside NS1 is defined (Figure 1), whereas any phosphoramidite can be incorporated as a 3′-terminus when using universal solid supports. To begin the oligonucleotide synthesis cycle, the hydroxyl group is deprotected by detritylation on the solid support (Figure 1). A phosphoramidite reagent is mixed with an activator, typically a tetrazole derivative, providing a reactive species that adds to the solid support hydroxyl groups within a

minute. Excess reagents are washed off, and if any unreacted hydroxyl groups remain, they are acetylated in a subsequent capping step. Upon capping, phosphorus is oxidized from P(III) to P(V) (phosphite triester to phosphate triester) by treatment with I2-based oxidizing mixtures. This sets the stage for the next synthesis cycle starting with detritylation of the nucleotide that was just incorporated. During synthesis, all exocyclic amines of the nucleobases as well as the phosphodiester backbone are protected. Various protecting group schemes are available for D


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able, they are difficult to make, or the modification is not compatible with DNA synthesis. In many cases, a chemical handle is therefore introduced to the DNA strand in-synthesis and the desired modification is subsequently coupled to the DNA strand in a postsynthesis conjugation reaction between the chemical handle on the DNA and a complementary chemical functionality on the modification. One of the most pronounced differences between the methods is that the postsynthesis modification occurs on the unprotected and charged DNA strand in aqueous or water-containing solvents, limiting the chemistries that can be applied. The most common oligonucleotide modifications are amino groups, thiols, and alkynes (Figure 3). Oligonucleotides containing numerous variations of these

the exocyclic amines, and standard protection groups are shown at the bottom of Figure 1. The phosphodiester is typically protected by the cyanoethyl group. Synthesis cycles are repeated until the desired sequence is obtained. At this point, the oligonucleotide can be deprotected and cleaved from the solid support by treatment with base. DNA sequences of up to 150− 200 nts are synthesized using this stategy, but yields decrease significantly when increasing the length of the oligonucleotides. After synthesis, the crude oligonucelotide can be purified using various methods. An elegant and efficient purification method, that is widely applied, relies on leaving the final DMT group on the oligonucleotide during cleavage and deprotection. The hydrophobic nature of the DMT group allows separation from truncated oligonucelotides that have been capped during the synthesis. This can for instance be achieved using small, commercially available reversed phase purification cartridges. In the same step, the liberated protecting group residues are also removed from the oligonucleotide. Other options for purification are HPLC, electrophoresis, and thin-layer chromatography.46,47 2.2. In-Synthesis Modification of DNA

The development of a streamlined process for oligonucleotide synthesis revolutionized biotechnology by providing rapid access to any short DNA strand of choice for primers. Importantly, the chemical synthesis of oligonucleotides is not limited to incorporation of naturally occurring nucleotides. Basically, any chemical functionality compatible with the synthesis conditions and the structure of the protected oligonucleotide strand can be incorporated, as long as it is equipped with a phosphoramidite group. For the creation of chemically modified DNA-based nanostructures, this provides access to a wide range of building blocks in the form of chemically modified oligonucleotides. Whereas many modifications can be directly incorporated in oligonucleotides during solid-phase synthesis, others require postsynthesis conjugation. To enable postsynthesis conjugation, a number of phosphoramidite reagents containing functional handles have been developed. While Goodchild’s excellent review on oligonucleotide modifications dates back to 1990, it still provides a good overview of the most common modifications incorporated using phosphoramidite chemistry.48 In general, phosphoramidites are used for 5′- and internal modifications (Figures 2A and C, respectively) and modified solid supports for 3′-modifications (Figure 2B). Modified nucleobases are mostly used for internal modifications to maintain Watson−Crick base pairing (Figure 2D). Due to their wide availability, C8-modified dA and C5modified dU, also known as modified dT, are commonly used. Modifications can also be installed on dG and dC, but nucleoside phosphoramidites of these species are less widespread. Modifications can also enable branching of oligonucleotides, and the use of orthogonal protecting groups facilitates absolute control over all sequences extruding from the branching point. We used Fmoc- and DMT-protecting groups to obtain tripoidal DNA structures, whereas von Kiedrowski and co-workers combined the use of orthogonal allyloxy carbonyl and DMT protecting groups with reverse-direction oligonucleotide synthesis to form tripoidal structures with all strands extruding from a benzene core in the 3′ to 5′ direction (Figure 2E).49,50

Figure 3. Common chemical handles for postsynthesis DNA modifications and their reaction partners (from top to bottom): Amines and N-hydroxysuccinimide esters, thiols and maleimides, alkynes and azides.

reactive handles with different linkers and protecting groups are commercially available. Both 3′-, 5′-, and internally modified nucleotides can be obtained, and these modified oligoes are used routinely for postsynthesis conjugation to DNA as described in the following sections. Besides this, numerous other reactive handles can be incorporated in DNA using in-synthesis modification. These are also described below, and an overview of the functional handles is provided in Table 1. 2.3.1. Amino Handles. Amino modifications allow selective reaction with activated carboxylates in amide bond forming reactions. Due to the wide range of commercially available probes containing activated carboxylates, such as N-hydroxysuccinimide (NHS) esters, amino-modified oligonucleotides provide access to a broad range of functionality. During DNA synthesis, the amino group has to be protected. For 5′modification, trityl-based (monomethoxytrityl (MMT)) protection can be used, facilitating purification on commercially available reversed phase purification cartridges. For internal and 3′-modifications, base-labile groups such as trifluoroacetyl (TFA) and fluorenylmethyloxycarbonyl (Fmoc) are generally used. To avoid interfering with the base pairing properties of DNA, internal modifications are generally attached to a nucleoside, most often dT (Figure 2D).51,52 While the coupling of NHS esters with amino-modified oligonucleotides remains highly popular, this method suffers some important drawbacks. Amines are protonated at low to neutral pH and therefore hardly react with NHS esters under these conditions. At high pH, NHS esters undergo hydrolysis, which also complicates the conjugation. To obtain high yields from conjugation of NHS ester to amino-modified DNA, appropriately buffered solutions

2.3. Handles for Postsynthesis Modification of DNA

For a large share of reported DNA modifications, the corresponding phorphoramidites are not commercially availE


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also very selective even in the presence of reactive primary amines. A drawback of the thiol-maleimide conjugation is the risk of thiol-exchange when present in complex medium, but significant stabilization can be obtained by hydrolytic ringopening of the thioether product.58 Linkage using disulfide formation is also an attractive feature of thiol-modified DNA usually achieved by activation of one thiol with 2-mercaptopyridine.59 Thiol modifiers are commonly protected as disulfides, and reduction of the resulting disulfide modified oligonucleotides with reagents such as dithiothreitol or tris(2-carboxyethyl)phosphine is required before further conjugation. Alternatively, DNA modified with free thiols can be obtained using for instance benzoyl or MMT protection, although the obtained thiols are prone to intermolecular disulfide formation and reduction may still be required prior to further reactions.55,60 Other common, thiol-based modifications include lipoic acidbased cyclic disulfides. Such modifications generally provide stronger binding to gold surfaces, which will be discussed further in section 5.4.1.2.61,62 2.3.3. Alkyne Handles. Terminal alkynes can also be readily incorporated in oligonucleotides during solid phase synthesis both internally as well as in the 5′ or 3′ end, and they are important species for conjugation with azides in copper(I)catalyzed alkyne−azide cycloaddition (CuAAC).63−65,106 The reliable nature of the copper-catalyzed “click” reaction and the large number of available azide-probes make alkyne-modified oligonucleotides popular in bioconjugation. Generally, terminal alkynes do not require protection during oligonucleotide synthesis. However, the introduction of orthogonally removable protecting groups provides the possibility to selectively react at several alkyne groups in sequence.66 The invention of strainpromoted alkyne−azide cycloaddition (SPAAC) provides another route to efficient reactions with azide probes without the need for copper(I), which tends to complicate certain applications of oligonucleotides due to its cytotoxicity and tendency to generate reactive oxygen species that may degrade DNA.67 Dibenzocyclooctynes can be directly incorporated during oligonucleotide synthesis to allow subsequent copperfree “click” reaction which for instance enables conjugation of DNA to conjugated polymers,68 quantum dots, gold nanoparticles, etc.69,70 SPAAC is popular for its ease of use and the mild reaction conditions required, but this comes at the cost of a significantly lower reaction rate compared to CuAAC. 2.3.4. Electrophilic Handles. Amines, alkynes, and thiols are very common oligonucleotide modifications, but sometimes the complementary reactivity is required to enable reactions with other amine-, alkyne-, or thiol-species. Aldehydes are readily incorporated using for instance 5-formylindole phosphoramidites.71 The aldehyde functionality enables reductive amination reactions with amines as well as conjugation to aminooxy groups, hydrazides, 1,2-amino thiols, etc.72,73 Carboxylates can also be incorporated for reactions with amines in amide-bond forming reactions. NHS esters incorporated at the 5′-end of oligonucleotides allow direct conjugation of the protected oligonucleotide to amines on the solid support. If appropriate oxidizing conditions are used during the synthesis cycle, NHS esters may be incorporated internally in oligonucleotides without significant hydrolysis during the synthesis.74 Alternatively, trityl-based protection of carboxylic acids enables direct formation of carboxylate-modified oligos to be used in solution phase amide bond forming reactions.75,76 For reactions with thiols, maleimides are attractive functional groups although they are not compatible with general cleavage and

Table 1. Overview of the Most Important Functional DNA Handles and Their Reactions

and high concentrations of reactants are required. In some cases, it is desirable to replace the amino group by hydrazides, which will be discussed in an upcoming section. Besides conjugations with activated carboxylates, amino modified oligonucleotides are also commonly used for conjugation to isothiocyanates and aldehydes.53 2.3.2. Thiol Handles. Similar to the amino-modification, thiol-modified DNA also provides access to functionalization with a wide range of probes due to their efficient reaction with maleimides, α-halocarbonyls, and vinylsulfones.54−57 Especially the reaction of thiols with maleimides is popular due to its high reaction rate, and when carried out at pH 6.5−7.5, the reaction is F


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Figure 4. Integration of modified DNA oligonucleotides in DNA nanostructures. DNA origami is assembled through the annealing of several short synthetic staple strands with a long scaffold strand. (A) Modified staple strands are added prior to annealing to directly incorporate modifications in the DNA origami. (B) Alternatively, modified oligonucleotides are hybridized to extended staple strands to allow modification of many sites using a single modified sequence.

amidite chemistry.93 Furthermore, 1,2,4,5-tetrazines have also been used for reaction with DNA containing trans-cyclooctene in a reaction that was fully orthogonal to CuAAC.94 2.3.7. Hydrazide and Aminooxy Modifications. As discussed above, amino-modifications are very useful for functionalization with activated esters. However, at low to neutral pH, the reactions are slow due to protonation of the amines and at higher pH, the reactions are fast but degradation of the activated ester tends to cause problems. An alternative functionality allowing fast reaction with activated esters at lower pH is therefore often desired. Hydrazides can be readily incorporated in DNA using phosphoramidite chemistry, and with a pKa of 4−5 they remain neutral at low to neutral pH, thus providing an excellent alternative to amines for reaction of oligonucleotides with electrophiles.95,96 Furthermore, the hydrazide functionality reacts rapidly with the dialdehyde formed upon periodate-cleavage of the terminal, vicinal diol of RNA in a reaction leading to the formation of a 6-membered ring. Aminooxy groups are also attractive alternatives to amino groups. The oximes formed upon reaction of aminooxy groups with aldehydes are significantly more stable than imines, and aminooxy groups can readily be incorporated in DNA using phosphoramidite chemistry.97,98 For instance, oxime formation using aminooxy functionalized DNA enables formation of peptide−DNA conjugates and incorporation of proteins in DNA nanostructures.80,99 Hydrazones and oximes are both significantly more stable than imines, which is important for biological applications.100 2.3.8. Modifications for Palladium-Catalyzed Couplings. The era of transition metal-catalyzed cross-couplings has also made its mark on oligonucleotide functionalization. Incorporation of 8-bromoguanine or 5-iodoracil provides valuable substrates for palladium-catalyzed cross couplings. Although many of these are incompatible with the aqueous environment required for working with DNA, the Suzuki− Miyaura couplings are compatible with water and have been used for postsynthetic functionalization of 8-bromoguanine as well as 5-iodoracil.101,102 Alternatively, oligonucleotide crosscouplings performed on the solid support are possible to circumvent solvent restrictions and significantly broaden the scope of reactions applicable for DNA functionalization.103,104

deprotection schemes. Therefore, incorporation of maleimides during synthesis requires protection. Maleimido-2,5-dimethyl furan cyclo-adducts are stable during cleavage and deprotection and can be transformed to the corresponding maleimide by thermally induced retro-Diels−Alder reaction, providing access to maleimide−oligonucleotide conjugates.77 Since the introduction of electrophilic groups often requires specialized reagents and optimized synthesis conditions, handles such as maleimides, NHS esters, and aldehydes are commonly incorporated by postsynthesis conjugation of amino- or thiolmodified DNA with homo- or heterobifunctional linkers (Section 6.1.1).78−82 2.3.5. Azide Handles. Reaction of oligonucleotides with alkynes in CuAAC or SPAAC requires incorporation of azides although they are considered incompatible with phosphoramidite formation due to Staudinger reduction of the azide in the presence of phosphoramidite reagents.83 Thus, azides are generally introduced in a separate step subsequent to oligonucleotide synthesis either by reaction of sodium azide with haloalkyl modifications on the solid support, diazo transfer reactions to alkyl amines, or reaction of amines with bifunctional NHS-azide molecules etc.84−87 Other studies suggest that azide incorporation using phosphoramidite chemistry is indeed possible when using appropriate building blocks.88,89 Oligonucleotide azides are also useful for Staudinger coupling to probes, which further emphasizes the value of methods to readily incorporate azide groups in DNA.90 2.3.6. Modifications for Postsynthesis Diels−Alder Reactions. Incorporation of several other reactive handles by phosphoramidite chemistry has also been described, some of which will be included here. Diels−Alder (DA) reactions have been investigated for use in conjugation to oligonucleotides. Through incorporation of a 1,3-hexadiene moiety to the 5′-end of oligonucleotides using a simple phosphoramidite reagent, efficient DA reaction with maleimide-functionalized molecules can be achieved.91 Likewise, introduction of furan-based functionalities enables subsequent DA reactions with maleimides.92 Furthermore, handles for inverse electron demand DA reactions, which are extremely fast conjugation reactions, have been described. As demonstrated by Schoch et al., efficient coupling to 1,2,4,5-tetrazines was enabled by the incorporation of norbonene dienophiles in DNA strands using phosphorG


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Figure 5. Applications of Förster resonance energy transfer in DNA nanostructures. (A) Illustration of the setup used for monitoring the dynamic B−Z transformation in a DNA nanostructure. Adapted with permission from ref 121. Copyright 1999 Springer Nature. (B) Schematic representation of the DNA DX tile used to monitor the development of FRET between two fluorophores throughout switching between 11 different states. Adapted with permission from ref 122. Copyright 2011 Angewandte Chemie International Edition. (C) DNA Origami positioner for tuning the average distance between two molecules with atomic-scale precision. Adapted with permission from ref 123. Copyright 2016 Springer Nature. (D) Illustration of a FRET cascade on DNA origami gated by a “jumper dye” (green). Adapted with permission from ref 126. Copyright 2011 American Chemical Society. (E) Schematic rerpresentation of a three-station light harvesting device. Adapted from ref 127. Copyright 2011 American Chemical Society.

place at room temperature. Since the extension of the staple strands can be identical, only one modified DNA strand is required to introduce modifications at several positions. However, depending on the position of the modification in the oligonucleotide, this method may offer less positional control because the staple strand extends from the surface of the origami. Furthermore, hybridization to staple strand extensions at room temperature may not be as efficient as the integration of modified staple strands. 2.4.1. Fluorescent Dyes and Quenchers. The most common modification in DNA nanotechnology is chromophores. In particular, fluorophores are widely used as markers in gels and microscopy, for sensing devices, to monitor dynamics and distances or kinetics, for light harvesting and energy transfer, and for single molecule microscopy.108−110 A plethora of fluorophores with different optical properties are commercially available for preparation of fluorophore−DNA conjugates using either in-synthesis or postsynthesis modification. Likewise, several fluorescence quenchers are available for conjugation to DNA strands. Some fluorophores may be damaged during DNA synthesis due to the alkaline conditions used for cleavage and deprotection. For instance, the widely used Cy5 fluorophore is susceptible to degradation under standard deprotection conditions. This can be overcome by using milder deprotection conditions although it requires protecting groups of DNA bases compatible with Ultra-Mild deprotection. Therefore, oligonucleotide−dye conjugates are often prepared using postsynthesis conjugation to avoid degradation of fluorophores that are suceptible to degradation. A table showing the compatibility of various dyes and quenchers with different deprotection conditions is provided by Glen Research.111 Chromophores commonly introduced to DNA using phosphoramidite chemistry include flat aromatic molecules such as pyrenes, perylenes, porphyrenes, and binaphthyls as well

Palladium-mediated functionalization of nucleic acids has recently been reviewed by Defrancq and Messaoudi.105 The above section provides an overview of some of the most important functional handles that can be incorporated in DNA during phosphoramidite oligonucleotide synthesis. There are many other reactive handles, and the overview provided here is by no means exhaustive, as the chemistry compatible with DNA functionalization is extremely broad. 2.4. Introduction of Modifications in DNA Nanostructures

In this section, the most common small molecule functionalities introduced in DNA nanostructures will be reviewed. Figure 4 illustrates the two basic methods to introduce modified oligonucleotides in DNA nanostructures, exemplified for DNA origami: The modified DNA strand is either (A) directly integrated in the origami structure as a staple strand that hybridizes to the long scaffold strand and contributes to the integrity of the structure or (B) introduced after folding of the DNA origami structure by hybridization to staple strand extensions on the origami. The first method (Figure 4A) is simpler and more direct and requires only one hybridization step. Furthermore, this approach offers higher positional control as the modification is closely integrated within the origami structure rather than extending from it. One of the drawbacks to this procedure is the high temperature employed in the denaturation step prior to annealing of the DNA origami. This tends to cause degradation of proteins in protein−DNA conjugates, and it may also be harmful to other materials such as inorganic nanoparticles etc. Furthermore, if several copies of the same modification are to be integrated at different positions in the same origami structure, a unique DNA strand is required for each conjugate. Both of these problems are circumvented in the second method (Figure 4B) where hybridization to the staple strand extensions and the modified DNA strands takes H


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fluorescent energy transfer.26,125,126 Tinnefeld and collaborators placed 5 dyes on a line in DNA origami with a distance between each dye of approximately 4.5 nm (Figure 5D). DNA strands with 4 different dyes were prepared using postsynthesis conjugation and supplied commercially. The central ATTO 488 dye served as donor, ATTO 565 served as the middle station or so-called “jumper-dye”, and the two terminal ATTO 647N and Alexa 750 served as distinguishable acceptors. They demonstrated that the absence or presence of one of the jumper dyes served as a gate to control the direction of the cascade.126 In related studies, Yan and collaborators created a light harvesting, three-stage fluorescent energy transfer device based on a DNA 7helix bundle (Figure 5E).127 The first donor (Pyrene) and the second donor (Cy3) were placed in a ring with up to six copies around the bundle, and finally the excitation energy was transferred to a single acceptor (Alexa Fluor 647) at the extended middle helix. The excitation energy was efficiently funneled to the acceptor in this design. In most of the FRET examples described above, only the averaged ensemble information was obtained. Single molecule fluorescence techniques, on the other hand, can provide dynamic and spatial characterization of individual DNA nanostructures.128 In one example, Nir and collaborators characterized the dynamics of a single molecule DNA walker by single molecule FRET (Figure 6A).129 Amino-modified DNA strands were modified postsynthetically through reaction with the NHS ester-functionalized dyes ATTO 550 and ATTO 647N. In a recent study, rotational movement of a DNA robotic arm was characterized using single molecule FRET (Figure 6B).130 By applying an external electrical field to the device, the position of the arm could be controlled. In recent years, the super-resolution microscopy technique, “DNA points accumulation for imaging in nanoscale topography” (DNA-PAINT) has emerged as a powerful method to image DNA nanostructures and fixed cells, occasionally with a resolution of just a few nanometers.27,109 The technique employs short dye-labeled ssDNA strands that bind transiently to stretches of 8−10 nt ssDNA strands on the imaging target, e.g. staple strands extending from DNA origami or DNA strands on an antibody binding to a cellular target. This was beautifully demonstrated by Jungmann et al., who imaged digits on DNA origami using consecutive multiplexed imaging by PAINT (Figure 6C).108 2.4.2. Azobenzenes and Other Photoreactive Compounds. Chromophores may also be used to induce structural changes in DNA-nanostructures. In one approach, a so-called photocaged DNA sequence is employed where the DNA strand contains photocleavable protecting groups at the nucleobases which impedes base pairing. Recently, Stephanopoulos and collaborators made use of photocaged DNA in a tweezer-like structure with a ssDNA hairpin connecting the two halves as illustrated in Figure 7A.131 In the relaxed state it is closed, but the structure is opened in the presence of a trigger DNA strand that binds to the ssDNA hairpin connector. Seven thymidines in the integrated trigger strand are protected by 6-nitropiperonyloxymethyl groups that are released upon photoirradiation at 365 nm. The main advantage of a phototriggered reaction is its fast kinetics compared to strand displacement reactions. In the actual case, the phototriggered reaction is 60-fold faster. One disadvantage of the approach is its irreversibility. This may not be an issue in for instance drug-delivery, but it constitutes a major barrier for constructing nanoactuators capable of undergoing reversible mechanical transformations.

as several cyanine and coumarin dyes. Incorporation of these species in DNA strands has been reviewed more thoroughly elsewhere.112 These chromophores replace nucleobases and participate directly in the double helix base stacking. Alternatively, they can be attached to nucleobases via linkers serving to conserve the natural base pairing pattern. Incorporation of large aromatic chromophores in DNA will inevitably affect the physical properties of the DNA including the melting temperature of duplexes.113 The use of fluorophore phosphoramidites enables incorporation of multiple fluorophores in the same DNA strand in a straightforward manner and saves time by avoiding postsynthesis conjugation. DNA labeled with multiple chromophores has been subject to intense research and is for instance used to develop sensors,114 photonic wires,115 light-harvesting systems,116 etc. The preparation and applications of multichromophore DNA systems have been reviewed in detail by Kool et al. and Wagenknecht et al., respectively.117,118 One of the most common applications of fluorophores in DNA nanotechnology is Förster Resonance Energy Transfer (FRET) studies. In brief, FRET is the process of nonradiative energy transfer between two fluorophores, a donor and an acceptor. Typically, the donor is excited at a lower wavelength and the transfer of energy to the acceptor results in emission of light at a higher wavelength from the acceptor. The energy transfer is highly dependent on the distance between the dyes; thus, it is very useful for monitoring dynamics and distancedependent processes in DNA-nanostructures. A similar process takes place between fluorophore and quencher, where the excited quencher decays nonradiatively and thereby turns off fluorescence when it is in proximity of a fluorophore. FRET processes are highly compatible with the sizes of DNA nanostructures because typical distances for these processes are up to 5−10 nm depending on the dyes and their orientation in space. A more detailed description of FRET is found elsewhere.119 The first common application of FRET and fluorophorequencher pairs in nucleic acids was molecular beacons, in which DNA hairpin hybridization probes are used to optically detect specific nucleic acid sequences in biological samples.120 FRET was soon adopted by Ned Seeman for the first DNA based mechanical device constructed by two double crossover tiles connected by a single helix region (Figure 5A).121 Driven by the B-Z conversion of the single helix domain, the device underwent a 180° rotation controlled by the concentration of ions and monitored by the FRET between fluorescein and Cy3 dyes conjugated to the device. In the first example of a device driven by strand displacement, FRET was also applied to monitor the opening and closing of the device.4 Since then, FRET has become one of the most common methods for monitoring dynamic events in DNA nanotechnology. Our laboratory used it for monitoring the opening of a box with a dynamic lid.110 We also designed a dynamic DNA tile actuator that can obtain 11 different positions, and in this case FRET was used to precisely monitor the position of the dyes (Figure 5B).122 Funke and Dietz designed a 3D origami positioning device, illustrated in Figure 5C, where the distance between dyes was monitored with Ångstrom precision using FRET.123 In another application, FRET, and in particular fluorophore−quencher pairs, was used for studying kinetics of logic circuits based on DNA stranddisplacement reactions as shown by Qian and Winfree.124 DNA nanostructures also constitute excellent platforms for organizing more than two dyes to investigate cascades of I


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destabilization and denaturation of the duplex due to the controlled mechanical switching from the planar trans-structure to the nonplanar cis-structure.132 This makes azobenzenemodified DNA a highly useful tool for introducing dynamics in DNA nanotechnology. Kuzyk, Liu, and collaborators applied azobenzene-modified DNA for photochemical switching of a plasmonic device built from gold nanorods (AuNRs) integrated in a switchable DNA structure (Figure 7C).134 In another example, azobenzenes controlled the reversible assembly and disassembly of hexagon-shaped 2D origami diffusing on a phospholipid surface as imaged by high speed AFM (Figure 7D).135 Famulok and coworkers used dimethyl azobenzene modifications to control the shuttling of dsDNA rotaxanes upon lightinduced conformational switching which eventually enabled logic gating.136 Finally, Yan, Yang, and collaborators used azobenzenes to control the efficiency of an enzymatic cascade mediated by the two proteins, glucose-6-phosphate dehydrogenase (G6pDH) and lactate dehydrogenase (LDH) (Figure 7E).137 The cascade was electronically coupled by the cofactor nicotinamide adenine dinucleotide (NAD+), which was conjugated to a DNA strand as an integral part of the origami structure. The state of the photoregulated azobenzene duplex determined whether the NAD+ cofactor diffused freely between the enzymes or stayed fixed at a position away from the enzymes. 2.4.3. Hydrophobic Modifications. Various hydrophobic groups can be easily incorporated in DNA using commercially available phosphoramidites. In the 3′-end, cholesterol modifications are incorporated through modified solid supports, while internal and 5′-modification is achieved using cholesteryl phosphoramidites.138−140 Several other lipophilic moieties including tocoperol,141 stearyl,142 diacyl lipids,143 multi chain lipids,144 etc. have also been incorporated in oligonucleotides during phosphoramidite synthesis.145 Postsynthesis coupling of these hydrophobic moieties is possible but the combination of their inherent hydrophobicity and the hydrophilicity of DNA often makes it difficult to find an appropriate solvent mixture for the conjugation. Therefore, incorporation during synthesis or subsequent coupling while the oligonucleotide is still immobilized on the solid support are preferred strategies for incorporation of hydrophobic moieties in DNA. Patwa et al. have thoroughly reviewed the preparation and applications of lipid−DNA conjugates.146 Cholesterol, in particular, and other hydrophobic groups have been conjugated to DNA strands to enhance interaction with phospholipid membranes. Their hydrophobic groups insert into lipid bilayers and thus facilitate manipulation of cellular systems or micelles by DNA nanostructures.147 In 2004, Höök and collaborators applied cholesterol−DNA conjugates to anchor DNA in vesicles and solid supported membranes, which allowed immobilization of vesicles on membranes via DNA hybridization.148 Several groups later used similar set-ups to control the assembly and fusion of lipid vesicles (Figure 8A).149−152 Albinsson and collaborators synthesized porphyrin modified nucleosides and incorporated them in DNA strands for fluorescent energy and electron transfer studies (Figure 8B).153,154 A dsDNA containing two of these moieties on one of the strands was incorporated in liposomes prepared from 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC). The complementary sequence was modified with flourescein, and energy transfer from the excited dye to the membrane-embedded zincporphyrene followed by electron transfer to benzoquinone moieties in the membrane was beautifully demonstrated.

Figure 6. Single molecule imaging of DNA nanostructures. (A) Illustration of a DNA walker that can be followed by FRET measurements. Adapted with permission from ref 129. Copyright 2013 American Chemical Society. (B) Illustration (top) and single molecule fluorescence time trace (bottom) of a DNA origami robotic arm that can be controlled by external electric fields. Adapted with permission from ref 130. Copyright 2018 American Association for the Advancement of Sciences. (C) Multiplexing, super-resolution single molecule imaging using DNA-PAINT. Transient binding of dye labeled DNA strands to different staple strands extending from the origami results in blinking that allows super resolution imaging. Adapted with permission from ref 108. Copyright 2014 Springer Nature.

For this purpose, the photoswitchable azobenzenes come in handy. Their use in DNA chemistry was pioneered by Asanuma, and today azobenzenes can readily be incorporated in DNA using commercially available phosphoramidites (Figure 7B).132,133 When exposed to UV-light, azobenzenes undergo a transition from the trans to the cis conformation. It will return thermally to the trans form after milliseconds to hours depending of the structure of the azobenzene substituents, but it can also be switched photochemically from cis to trans upon higher wavelength excitation. Incorporation of approximately 6 azobenzenes in a DNA double helix facilitates controlled J


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Figure 7. Azobenzene and other compounds as photochemical regulators in DNA structures. (A) DNA tweezer controlled by a photocaged trigger strand. Adapted with permission from ref 131. Copyright 2018 John Wiley & Sons, Inc. (B) Typical azobenzene-phosphoamidite structure and function in DNA strands. (C) Switching between a fixed and a relaxed organization of two gold nanorods by the azobenzene mechanism. (B) and (C) are adapted from ref 134. Copyright 2016 Springer Nature. Licensed under the creative commons agreement (https://creativecommons.org/licenses/ by/4.0/). (D) Illustration of the azobenzene-controlled assembly and disassembly of DNA structures anchored in a lipid membrane. Adapted from ref 135. Copyright 2014 American Chemical Society. (E) Illustration of the azobenzene-regulated enzymatic cascade between glucose-6-phosphate dehydrogenase (G6pDH) and lactate dehydrogenase (LDH). Adapted from ref 137. Copyright 2018 American Chemical Society.

via cholesterol-modified oligonucleotides assembled in different ordered 1D and 2D assemblies depending on the connector strands added to the monomers. Smaller DNA structures have also been organized on membranes. Recently, long-range ordered assembly of 3 point-star tiles anchored in a supported lipid bilayer by a TEG-cholesterol moiety was shown, where the assembly into hexagonal lattices was steered by blunt end stacking.157 Surface anchored DNA nanostructures have been applied for a range of different biophysical investigations. A system to study substrate-enhanced diffusion of enzymes in 2D was designed by Li, Fan, and co-workers (Figure 9A).158 In this work, the enzymatic cascade between glucose oxidase and catalase was imaged by total internal reflection fluorescence (TIRF) microscopy. The oxidase was immobilized on DNA origami anchored to a supported membrane bilayer through 22

Since the emergence of DNA origami, anchoring of such structures in lipid membranes through hydrophobic modification has been studied by several groups.155 In work by Endo, Sugiyama, and collaborators, mentioned earlier (Figure 7D), a hexagon-shaped DNA origami containing six triethylene glycol (TEG)-tethered cholesterol groups for anchoring of the structure in a lipid bilayer was designed.135 In addition to serving as a model for interactions of DNA nanostructures with cell membranes, the immobilization of DNA origami in lipid bilayers also offered the opportunity for AFM imaging of dynamic events. The study revealed that origami monomers anchored in lipid bilayers with six cholesterols are able to diffuse, whereas dimers and higher order assemblies are more immobile. Membrane-assisted assembly of large arrays of cholesterol functionalized DNA nanostructures has been studied by Liedl and collaborators.156 In their work, multilayer origami anchored K


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Figure 9. Interactions of DNA-origami with lipid-bilayers via hydrophobic anchors. (A) Illustration of an enzymatic cascade reaction studied by TIRF microscopy, which revealed higher 2D diffusion of catalase in the vicinity of glucose oxidase. Adapted from ref 158. Copyright 2017 American Chemical Society. (B) Structures of concave, convex, and flat DNA origami structures (top) used for sculpting DOPC membranes. The bottom panel shows fluorescence confocal microscopy images of membranes treated with the cholesterol-modified DNA structures corresponding to the illustrations shown above. Scale bars = 5 μm. Adapted from ref 161. Copyright 2018 Springer Nature. Licensed under a creative commons agreement (https://creativecommons.org/ licenses/by/4.0/).

Figure 8. Early examples on interactions between DNA modified with hydrophobic groups and lipid membranes. (A) Illustration of the assembly and fusion of vesicles containing cholesterol-modified DNA sequences. Adapted from ref 152. Copyright 2007 American Chemical Society. (B) Depiction of a DNA helix anchored in a lipid membrane via a zinc-porphyrine unit. The system was designed to study energy transfer from flourescein (yellow) to the zinc-porphyrine (red) followed by electron transfer from the zinc-porphyrene to 2,6-di-tertbutyl-p-benzoquinone (blue) dissolved in the membrane. Adapted from ref 153. Copyright 2009 American Chemical Society.

Many groups have investigated the interaction between DNA nanostructures and lipids and their ability to serve as a scaffold for the controlled formation of vesicles. Perrault and Shih designed a wireframe DNA−origami octahedron to serve as an endoskeleton for the size-controlled formation of small unilamellar vesicles (Figure 10A).162 The structures were designed with 48 extended staple strand handles for hybridization to the same number of DNA strands conjugated to 1,2O-dioctadecyl-glycerol via phosphoamidite chemistry. The structure was encapsulated by directing a lipid bilayer to assemble around the structure through recruitment of phospholipids at the surface of the DNA octahedron. In contrast to this, Liu and his lab used gold nanoparticles as the endoskeleton for templating the formation of uniform vesicles using a DNA-conjugate of hydrophobic dendrons.163 To prepare the vesicles, 13 nm AuNPs were first incubated with thiolated ssDNA followed by hybridization to the DNA− dendron sequences and the remaining space between the dendrons was filled by addition of a second type of DNA− dendron conjugates. The formed vesicles exhibited narrow size dispersity and the size could be tuned by altering the size of the AuNP or the length of the DNA strands. In continuation of this work, Liu, Yan, and co-workers used a cuboidal DNA origami scaffold to form cuboidal vesicles using the same dendrons.164 In yet another study, the dendrons were applied to form free floating sheets of the amphiphilic molecules on 2D origami sheets.165 Vesicles can also be shaped using DNA nanostructures as exoskeletons which was demonstrated by Rothmann, Shih, Lin, and collaborators (Figure 10B), and the chemistry used in this

cholesterol-DNA conjugates, and very slow diffusion of the origami was observed due to the high number of anchoring points. In contrast, a catalase−DNA conjugate was anchored to the bilayer via a single cholesterol molecule and thus diffused with high mobility. The glucose oxidase modified origami and the catalase were functionalized with different fluorescent dyes, and TIRF microscopy imaging revealed higher 2D diffusion of catalase in the vicinity of glucose oxidase where the H2O2 substrate was available. This indicates that the substrate conversion enhances the mobility of the enzyme. Simmel’s lab investigated the behavior of a 2D rectangular origami structure covered with up to 35 cholesterol moieties and investigated its tendency to collapse into an envelope type structure, depending on the amount of cholesterols and detergents.159 The closed envelope structures opened in the presence of small unilamellar vesicles (SUVs) made from palmitoyl-oleoyl-phosphatidylcholine (POPC) through interactions of the cholesterol groups with the lipid layer. The interaction between multilayer origami structures functionalized with cholesterol at one face and giant unilamellar vesicles has been investigated by Schwille and co-workers.160,161 Here, the authors observed how the curvature of the origami structures could induce vesicle deformation (Figure 9B). Interaction with concave origami surfaces triggered outward membrane tubules, while the convex structure triggered evagination/invagination-type deformation. The flat origami had no visible effect on the vesicle surface. L


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Figure 10. Shaping vesicles using DNA-templates. (A) Illustration and TEM images (bottom left) of a DNA origami octahedron functionalized with lipids at the exterior to function as an endoskeleton during the formation of vesicles. Scale bars = 50 nm. Adapted from ref 162. Copyright 2014 American Chemical Society. (B) Schematic showing the use of a DNA origami nanoring with lipids on the interior as exoskeleton for the formation of uniform, small vesicles that can be released from the DNA ring. Adapted with permission from ref 166. Copyright 2016 Springer Nature.

Figure 11. Artificial DNA membrane channels. (A) Illustration showing the first example of a DNA-based lipid membrane channel formed by DNA origami and functionalized with cholesterol for insertion in a membrane. The TEM image (right panel) shows several channels inserted into a small unilamellar vesicle. Adapted with permission from ref 170. Copyright 2012 American Association for the Advancement of Sciences. (B) Depiction of a six-helix bundle membrane channel functionalized with two porphyrins. Adapted with permission from ref 171. Copyright 2013 John Wiley & Sons, Inc. (C) A six-helix bundle containing 72 phosphorothioates in a band around the structure is reacted with ethyl iodide to make the belt hydrophobic for subsequent insertion into a lipid membrane. Adapted from ref 174. Copyright 2013 American Chemical Society.

approach is very similar to the work by Perrault and Shih described above.166 Here, a ring-shaped DNA origami was functionalized with up to 16 DNA strands modified with 1,2dioleoyl-glycerol derived lipids by hybridization to staple strand extensions on the inside of the ring. After mixing with extra lipid and detergent, subsequent dialysis led to formation of highly monodisperse vesicles which could even be isolated from the

DNA templates. In continuation of this, stacking of more origami ring templates on top of each other facilitates fusion and reshaping of the vesicles.167 Vesicles in DNA nanorings can also serve as a platform to organize SNARE proteins for membrane fusion.168 SNARE proteins were conjugated to DNA, and the products were hybridized to extended staples in the interior of the DNA-rings. In addition, the incorporation of lipid−DNA M


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Figure 12. Monodisperse polymer nanoparticles using DNA nanocages. (A) Illustration of the strategy used for formation of entangled hydrophobic chains in the interior of a cubic DNA cage. Cross-linking with a bis-NHS reagent afforded monodisperse hydrophobic polymers with DNA anchors. The polymer particles could be removed from the DNA cage by denaturation of the DNA template. (B) Chemical structures of the modified DNA probes and the bis-NHS cross-linker used in the study. Adapted with permission from ref 182. Copyright 2017 Springer Nature.

conjugates to the ring interior allowed capturing of small unilamellar vesicles and subsequent insertion of SNARE− proteins into the vesicles. In this way, the number of SNARE proteins per vesicle could be controlled, and details of SNARE− protein mediated membrane fusion were studied. Proteins and peptides that form channels through the membrane of living cells are crucial to the interaction of cells with the surrounding environment and for the transmembrane transport of water, ions, and other molecules. The first example of an artificial DNA origami channel in the form of a solid state pore was reported by Liedl, Keyser, and co-workers early in 2012.169 Later that year, Dietz and Simmel described an artificial DNA-origami channel spanning a lipid bilayer membrane.170 This was the first example of the interaction between a DNA origami structure and membranes through functionalization with hydrophobic molecules. The structure contained a barrelshaped body with a central six-helix bundle stem that extended from the barrel and penetrated the membrane (Figure 11A). Membrane adhesion was mediated by 26 cholesterol moieties extending from the face of the barrel. One of the major challenges of handling origami structures modified with several hydrophobic moieties is to avoid aggregation caused by hydrophobic interactions. This can either be achieved by postassembly introduction of the cholesterol− DNA conjugates, by prior insertion of the conjugates into the membrane followed by hybridization to the origami, or through the use of detergents. Alternatively, appropriate structural design of the employed nanostructures can serve to overcome aggregation. In Dietz’s and Simmel’s work, the membranespanning origami channel was cleverly designed with no large cholesterol-modified surfaces being able to overlap. As a consequence, no severe aggregation was observed, and TEM images showed that the channels inserted in unilamellar vesicles in the desired orientation (Figure 11A, right). Electro-

physiological measurements showed similarities with the responses of natural membranes, and programmable gating of the current was obtained by blocking and deblocking the channel with DNA hairpins. Several other groups have reported on membrane-spanning DNA nanostructures, and in some cases, other chemistries have been applied. Howorka and his lab designed a six-helix bundle functionalized with two alkyne-tethered porphyrins (Figure 11B),171 and this relatively simple design inserted into the membrane as a channel which was verified by electrophysiology measurements.172 Later, Keyser and collaborators showed that even a single DNA duplex functionalized with 3 porphyrins would insert into a lipid bilayer and function as an ion channel.173 In another study, a DNA six-helix bundle containing 72 phosphorothioate groups in a band around the structure was reacted with ethyl iodide. This served to remove the charge from the phosphorothioates and provided a hydrophobic surface to guide the insertion of the structure into a lipid membrane (Figure 11C).174 DNA nanotechnology has also made use of DNA−lipid conjugates for other applications than membrane insertion. Sleiman’s lab has thoroughly investigated the use of lipids as well as PEG chains conjugated to DNA strands for creating small functional DNA devices. In one example, Edwardson et al. synthesized dendritic DNA amphiphiles based on DNA with a branched hydrophobic moiety attached to it. The molecules were attached to the edges of DNA nanocages facilitating programmed assembly of multiple cages via hydrophobic interactions. Moreover, hydrophobic guests were hosted in the interior of the cages when recognized by the amphiphiles.175 The same lab has made use of alkyl linkers in the form of dodecyl groups for incorporation in DNA. Using a dodecanediol phosphoramidite, the hydrophobic modification was readily incorporated during oligonucleotide synthesis and when N


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cially available, and in addition, a range of probes can be obtained for postsynthesis incorporation of biotin. Several examples of the applications of the biotin−Streptavidin interaction in DNA nanotechnology will be given later in this review. 2.4.5. Redox-Active Modifications. Incorporation of electroactive groups is an important method to functionalize DNA nanostructures. Electron transfer provides a sensitive output for detection using cyclic voltammetry, and common modifications such as methylene blue191,192 and ferrocene193194 are readily incorporated using phosphoramidite chemistry. Especially ferrocene has been intensely studied, and phosphoramidites are available for modifications of DNA both internally and in the termini. Porphyrin and pyrene chromophores have also been incorporated in DNA for their electrochemical behavior.195 Metal-coordinating groups are also of interest due to their electronical properties. Duprey et al. showed that incorporation of a cyclidene group coordinating either copper or nickel ions could be used for electrochemical detection of the nucleobase positioned opposite to the tag in DNA duplexes.196 Ruthenium has also been incorporated through metal coordination and used for electrochemical detection.197,198 It is highly challenging to estimate the distance from the surface to the electro active group when studying charge transfer from electroactive groups in dsDNA attached to electrodes. This challenge arises from the fact that dsDNA anchored on for instance a gold electrode via a thiol group may be tilted. Furthermore, it has been difficult to distinguish between charge transport mediated by the dsDNA helix and charge transport through space. To avoid tilting, Fan and co-workers elegantly used a DNA tetrahedral structure anchored to a gold surface via thiols in 3 of its 4 corners.199 The redox active group can then be placed anywhere in the triangle with very precise control over the distance to the electrode surface (Figure 13). Using this setup, intercalating redox groups such as methylene blue were shown to transfer charge through the DNA with little distance dependence. For ferrocene redox groups, that do not intercalate,

protruding from the edges of DNA nanocages, this also enabled assembly of multicage structures via hydrophobic interactions.176 The incorporation of hydrophobic alkyl linkers was also combined with incorporation of hydrophilic ethylene glycol based linkers to enable tuning of hydrophobic interaction and modulate the means of structure assembly.176,177 The structures obtained using these approaches have shown promise in drug delivery applications,178,179 mediation of reactions between DNA and hydrophobic probes,180 improvement of stability toward nucleases,181 etc. In another application, monodisperse polymer nanoparticles were formed by attachment of hydrophobic modifications at each corner of a cubic DNA cage (Figure 12A).182 The hydrophobic modifications were based on the dodecanediol phosphoramidite, and 6 consequtive hydrophobic modifications were attached to the cube through the 5′-end of ssDNA (Figure 12B). Some of the hydrophobic tags were equipped with amino groups using standard phosphoramidite based amino-modifiers to allow bis-NHS mediated cross-linking of the hydrophobic groups in the interior of the cubic cage upon assembly. As such, monodisperse hydrophobic polymer particles with DNA handles could be formed using the cubic DNA cage as a template. Both Häner’s group183,184 and Sleiman’s group185 have investigated the formation of nanoscopic fibers formed from single DNA strands conjugated to hydrophobic molecules. In the first case, pyrene moieties containing short linkers were incorporated in DNA during phosphoramidite oligonucleotide synthesis. The pyrene units stacked on each other forming a hydrophobic interior, while the DNA protruded into the aqueous environment. Under the right conditions, this led to the formation of long (up to 500 nm) supramolecular polymers with helical ribbon-like structure.183 In Sleiman’s work, 12 hexaethylene monomers followed by one or two Cy3 dyes were added to the 5′-end of a DNA strand during solid phase synthesis and proved essential for the formation of micrometer long fibers that were connectable to DNA origami and gold nanoparticles through hybridization.185 2.4.4. Biotinylation of DNA. Although simple in its nature, the small biotin molecule (Vitamin B7) has been of significant importance in DNA nanotechnology. Biotin makes a very strong noncovalent interaction with tetravalent avidin proteins. The biotin−avidin interaction thus provides a reliable method for specific attachment of a protein to DNA. Streptavidin, which is commonly employed for biotin binding, is a 53 kDa protein that can be readily observed in AFM imaging to spatially locate individual biotinylated DNA strands in DNA nanostructures. As discussed in more detail later, we have used this setup for probing chemical reactions at the single molecule level.186 The multivalence of avidin proteins also enables immobilization of DNA nanostructures on biotinylated surfaces for single molecule spectroscopy studies. Biotin phosphoramidites are available for 5′- and internal modifications, while biotinmodified CPG is available for formation of 3′-modified DNA.187,188 Internal and 3′-modifications require a protecting group such as 4-tert-butyl benzoyl to avoid branching of the biotin urea moiety during synthesis. Postsynthesis conjugation or enzymatic couplings of biotin to DNA is possible, and biotinylated DNA is also commercially available. In some cases, it is desirable to remove avidin proteins from the DNA. For this, cleavable linkers containing disulfides or photocleavable groups are introduced between the DNA and biotin during oligonucleotide synthesis.189,190 Biotinylated DNA is commer-

Figure 13. Precise positioning of ferrocene relative to a gold electrode surface. A triangular face of a DNA tetrahedron is anchored to the surface via three thiol groups. Ferrocene (orange dot) is incorporated either in the bottom, in the middle, or in the top of the structure. The charge transfer is monitored by alternating current voltammetry (bottom right) where the curve shows the amplitude of the current in response to an oscillating potential as the potential is increased. Adapted from ref 199. Copyright 2012 American Chemical Society. O


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including dideoxyribonucleotide triphosphates (ddNTPs) (Figure 14).209 The ddNTPs have the advantage that only one

charge transfer was shown to proceed in a highly distance dependent manner, most likely by tunneling through space. 2.4.6. Other Less Common DNA Modifications. In addition to the most common DNA modifications in DNA nanotechnology described above, a few other less common modifications will briefly be mentioned here. For instance, functionalization of DNA with polymers has gained significant interest due to the increased stability in plasma and resistance to nucleases observed for the conjugates. Kwak and Herrmann have reviewed the formation of various polymer−DNA conjugates, and some examples showing the applicability of polymers in DNA nanostructures will be discussed in a subsequent section.200 Carbohydrate−DNA conjugates and peptide−DNA conjugates are biologically important in relation to cellular internalization, and numerous strategies, including ones based on phosporamidite chemistry, enabled the formation of these conjugates. For more insight into this topic, the reader is directed to Lö nnberg’s review201 and to the review by Venkatesan and Kim.202 Photo-cross-linking agents such as psoralenes, which intercalate into dsDNA and induce covalent cross-linking between strands upon exposure to UV-light, are also popular. Psoralenes are readily introduced into DNA using phosphoramidite chemistry.203 Both psoralene and 3-cyanovinylcarbazole have been introduced to DNA nanostructures to increase the thermal stability through cross-linking (see section 4.2.2 for further information).204,205 Without much difficulty, small molecule protein ligands are incorporated into DNA strands.206 Most often this is achieved using postsynthesis conjugation although phosphoramidite chemistry often provides a practical alternative. For instance, using phosphoamidite chemistry, the receptor molecule folic acid, which binds to the folate receptor involved in cellular internalization, has been incorporated into DNA. Evidently, phosphoramidite-based solid phase DNA synthesis provides access to a wide range of modified DNA for use in DNA nanotechnology. However, not all laboratories working in DNA nanotechnology have access to automated DNA synthesizers, and therefore postsynthesis modification of DNA continues to be an extremely valuable tool for DNA nanotechnology.

Figure 14. Nucleoside triphosphates for incorporation in the 3′-end of DNA strands by terminal deoxynucleotidyl transferase according to the scheme shown (TdT). The enzyme accepts a variety of modifications R such as small molecules, macromolecules, and proteins.

nucleotide is incorporated since the 3′-OH is required for further coupling steps. TdT has been used for incorporation of various small molecules in the 3′-end of DNA including halogenated and thiolated nucleobases,211,212 nitrophenyl modified nucleobases,213 fluorophores,214 and various handles for click chemistry.215 TdT can even be used for incorporation of very large substrates such as a macrocyclic peptide, a PEG chain, a dendrimer, and even Streptavidin to the 3′-end of DNA strands.216 It was further demonstrated that TdT can be used for functional patterning of DNA nanostructures.217 For functionalization of DNA nanostructures, TdT provides the attractive opportunity to label multiple staple strands in parallel in the same vial. The synthesis and purification of the dNTP substrates are sometimes challenging, but the promiscuous nature of TdT combined with its straightforward use makes it a strong tool for incorporation of complex molecules into DNA nanostructures.

3. NUCLEIC ACID ANALOGUES This review mainly focuses on DNA and DNA modifications. Nevertheless, other nucleic acid analogues need mentioning for their importance in nucleic acid nanotechnology. RNA nanotechnology is increasingly evolving as a field of its own, and it has recently been reviewed by Jasinsky et al. 218 Chemical modification of RNA in nucleic acid nanotechnology is less developed than that of DNA. However, many of the methods for introduction of modifications in-synthesis or postsynthetically are the same, although the 2′-OH and the lower stability of RNA may pose challenges. One of the unique opportunities for chemical modifications in RNA is periodate cleavage of the viccinal diol of the 3′-terminal ribonucleoside followed by reaction with an amine in a reductive reaction which allows selective, chemical modification at the RNA 3′-end.219 Several artificial nucleic acid analogues (XNAs) including phosphorothioate-modified DNA (psDNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA) have been developed. They show high stability toward nucleases and are thus of significant importance for biological and clinical applications. When hybridized to ssDNA, several XNAs including LNA, PNA, and 2′-fluoro RNA (2′-F RNA) have increased helix stability compared to dsDNA which may be of importance for structural

2.5. Modification of DNA Using Terminal Deoxynucleotidyl Transferase

Besides in-synthesis modification and postsynthesis chemical functionalization, enzymatic methods are also important alternatives for preparation of modified DNA. Several methods have been developed for installing modified nucleobases using DNA polymerases.207 Many polymerases have been used for templated incorporation of base-modified deoxyribonucleoside triphosphates (dNTPs) into DNA, and an overview of the scope can be found in the review by Pinheiro and Holliger.208 For DNA nanotechnology, however, it is often desirable to be able to attach modifications to shorter DNA strands in a nontemplated fashion. The template-independent terminal deoxynucleotidyl transferase (TdT) is an efficient tool for this and will therefore be the focus of this section.209 TdT is a commercially available template-independent polymerase that attaches nucleotides to the 3′-end of singlestranded DNA.210 Notably, TdT is not restricted to adding one nucleotide to the DNA chain, and often multiple nucleotides will be attached to the 3′-end of the oligonucleotide substrate. The nucleoside triphosphate substrates are almost solely dNTPs, but TdT also accepts a range of other nucleotide analogues P


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Figure 15. PNA for nucleic acid nanotechnology. (A) Structure of PNA repeat unit (top) and a building block (bottom) for incorporating cytosine in PNA during solid phase peptide synthesis. The Bhoc protecting group for exocyclic amines is highlighted by the blue circle. (B) Illustration of the setup used for incorporating peptide segments in a DNA nanocage using readily prepared PNA−peptide conjugates. (B) is reprinted from ref 234. Copyright 2013 American Chemical Society.

dryloxycarbonyl (Bhoc) protecting groups for the exocyclic amines of the nucleobase (Figure 15A). Therefore, PNA− peptide conjugates can easily be prepared and incorporated into DNA nanostructures. Flory et al. used peptide−PNA conjugates for efficient incorporation of peptides into DNA nanocages at room temperature (Figure 15B).234 While PNA provides many opportunities for DNA nanotechnology, its use is in some cases challenged by insufficient solubility in aqueous environment, especially for longer PNA strands. Addition of charged groups can help overcome this, and although this requires some chemical synthesis efforts, PNA has the potential to play a significant role in the future development of DNA nanotechnology.

DNA nanotechnology. As opposed to DNA, the backbone of XNAs such as phosphorodiamidate morpholino oligomers (PMOs) and PNA is not negatively charged. This provides an important means for changing the electrostatics of nanostructures and optimizing them for cell internalization. The following sections will contain brief descriptions of PNA, LNA, and psDNA and their role in DNA nanotechnology. Several other XNAs have been reported, but detailed descriptions of these are beyond the scope of this review. For more insight to this, the review by Pinheiro and Holliger is recommended.208 3.1. Peptide Nucleic Acid (PNA)

PNA is a polymer based on an N-(2-aminoethyl)-glycine backbone with nucleobases attached to each repeating unit. It was first described by Nielsen et al. in 1991,220,221 and since then it has been widely recognized as an important research tool with great potential for use in therapeutics as well as in bionanotechnology. The development of systems for DNAtemplated polymerization of PNA further highlights its potential.222−224 The importance of PNA is partly due to its very strong binding to both DNA and RNA. DNA and PNA mixtures can form DNA−PNA duplexes, PNA2−DNA triplexes (for PNA with high pyrimidine content), and PNA2−DNA2 quadruplexes that are all much more stable than the corresponding pure DNA counterparts.225−228 Due to the charge-neutral backbone, hybridization of PNA does not require stabilization by cations.229 The high stability of PNA−DNA complexes was shown to significantly improve incorporation of PNA compared to DNA into the center of rectangular DNA origami.230 Besides improving incorporation and adding stability to structures, the stronger binding of PNA to DNA also allows for invasion of DNA−DNA duplexes without the need for toeholds.220,231,232 This is mainly possible at low cation concentrations where the DNA−DNA duplex is less stabilized as demonstrated by a DNA−DNA duplex invasion using a DNA origami structure that switched from a closed to an open conformation upon sequence-specific PNA invasion.233 The importance of PNA in DNA nanotechnology is exemplified by the straightforward method it provides for incorporating peptides in DNA nanostructures. PNA is prepared using Fmoc-based solid-phase peptide synthesis using benzhy-

3.2. Locked Nucleic Acid (LNA)

LNA was first reported in 1997−1998 by the groups of Imanishi and Wengel, respectively.235−237 It interacts very strongly and sequence-specifically with DNA and RNA and shows significantly increased stability toward nucleases.238,239 Although some of the properties of LNA are similar to those of PNA, the structure of LNA resembles to a much larger extent naturally occurring nucleic acids. Like DNA and RNA, LNA is based on a negatively charged phosphodiester backbone (Figure 16A). The bicyclic nature of the sugar moiety, where the 2′-O and 4′-C are linked via a methylene bridge, provides higher stability of LNA− DNA and LNA−RNA duplexes compared to DNA−DNA and DNA−RNA duplexes. Structurally, LNA is a constricted or “locked” version of RNA and its preorganization favors formation of an A-helix with both DNA and RNA, whereas dsDNA under normal conditions exists in a B-helix conformation. The two isomers of LNA monomers, α-L-LNA and β-D-LNA, are defined by the orientation of the methylene bridge. In this review, unless otherwise stated, the term LNA refers to oligomers made up from β-D-LNA monomers. Due to its phosphodiester backbone, LNA chains can readily be synthesized using phosphoramidite-based oligonucleotide synthesis. It can easily be incorporated into DNA where each added LNA nucleotide adds several degrees to the melting temperature of the duplex formed with DNA or RNA.240 This is a major advantage of LNA and the reason for intense investigations toward making LNA-based therapeutics, such as the mixmer Q


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nanotechnology. Despite this, the unique properties of LNA make it an attractive tool for DNA nanotechnology. 3.3. Phosphorothioate-Modified DNA (psDNA)

Numerous modified nucleotides have been developed in recent years, and some of the first backbone modifications investigated in biotechnology were psDNA and 2′F RNA. PsDNA is closely related to DNA with only a single oxygen atom exchanged for sulfur. This exchange however implies that phosphorus of psDNA, unlike DNA, is chiral. This subtle backbone modification has a great impact on the properties of the resulting nucleic acid. Most importantly, psDNA is much more stable toward nucleases than DNA, and therefore, psDNA has played an important role in the development of antisense platforms.250 The chiral nature of the thiophosphates in psDNA implies that the phosphorothioate antisense oligonucleotides (AONs) are generally mixtures of 1000s of different isomers. Efforts have been made to develop a solid-phase synthesis protocol providing control over stereochemistry of phosphorus since the early 90s, and several approaches have been suggested.251−255 The synthesis of phosphorothioate backbones deviates from standard phosphoramidite oligonucleotide synthesis in that the oxidation step is exchanged for a sulfurization step where Beaucage’s reagent (Figure 17A) remains popular,

Figure 16. LNA for nucleic acid nanotechnology. (A) Structure of LNA repeating unit (top) and an LNA thymidine phosphoramidite for LNA synthesis (bottom). (B) Two set-ups (i and ii) for time-gated energy transfer cascade initiated by terbium (light-blue sphere) emission. Purple letters denote LNA nucleotides required for formation of stable duplexes at elevated temperature. (C) Illustration of 45-nucleotide energy transfer cascade (left) and data demonstrating significant A647 emission and hence efficient energy transfer in the cascade (right). (B) and (C) are adapted from ref 243. Copyright 2015 American Chemical Society.

drug “Miravirsen” against hepatitis C, which is currently in phase 2 clinical trials.241,242 LNA is a highly attractive and readily applicable tool for DNA nanotechnology because of its ability to significantly increase double helix stability through straightforward incorporation. For instance, LNA was used for increasing the double helix melting temperature to investigate a time-gated cascade energy transfer setup based on four different fluorophores hybridized to an oligonucleotide scaffold (Figure 16B−C).243 The high affinity of LNA for DNA has also proved useful in toeholds for strand-displacement where strong binding to the toehold increases the displacement kinetics and minimizes leakage in the system.244,245 In this way, LNA-based toeholds provide a strong tool for dynamic DNA nanotechnology as demonstrated by Maune et al., who used an LNA toehold for removal of a protecting strand when immobilizing carbon nanotubes on DNA origami.246 Moreover, it has been demonstrated that incorporation of α-L-LNA into DNAzymes can markedly enhanced DNAzyme activity, and recently, LNA proved efficient for detection of single-nucleotide polymorphisms using nanopore technology.247,248 Likewise, the use of LNA nucleotides proved crucial to Mirkin and co-workers for the creation of superlattices from oligonucleotide-modified gold nanoparticles (AuNPs) of various well-defined shapes.249 Incorporation of LNA into DNA nanostructures provides a straightforward route to fine-tuning and optimizing nanostructure function. Although DNA−LNA mixmers are commercially available, their cost significantly exceeds that of DNA oligonucleotides. For laboratories with limited access to inhouse oligonucleotide synthesis, this might prevent the adoption of LNA and hence its more widespread use in DNA

Figure 17. In-synthesis formation of phosphorothioates. (A) Sulfurization of phosphorus using Beaucage’s reagent. (B) Structure of chiral phosphoramidite used by Iwamoto et al. for stereoselective synthesis of oligonucleotides with phosphorothioate backbones.256 (C) Baran’s P(V) reagent for synthesis of homochiral phosphorothioates.257

although several other reagents exist. Because it is possible to switch between sulfurization and oxidation between each synthesis cycle, mixtures of phosphorothioate and phosphate groups can readily be incorporated in the nucleic acid backbone. Stereochemistry at phosphorus is defined during the coupling step, and specialized phosphoramidites are required for stereoselective synthesis of psDNA.256 Only recently, a scalable synthesis of stereochemically pure psDNA suitable for AON synthesis has been described, and the pharmacological properties of single isomers deviate from those of mixed isomer samples R


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Figure 18. DNA-templated synthesis. (A) Illustration of the DNA-templated synthesis concept. ssDNA is conjugated to reactive handles, and subsequent DNA hybridization increases the local concentration of reactive handles, thereby enabling reaction. Due to the low overall concentration, background reaction in a hybridization-independent fashion is insignificant.262 Adapted with permission from ref 262. Copyright 2004 John Wiley and Sons, Inc. (B) Gel analysis of matched (M) vs mis-matched (X) templated reaction of thiol (S) and amino (N) nucleophiles with various electrophiles. H and E denote hairpin and end-of-helix arrangement, respectively. Adapted from ref 263. Copyright 2001 American Chemical Society.

(Figure 17B).256 In very recent work from the Baran group, a new P(V) reagent was developed for stereospecific synthesis of chiral phosphorothioates in DNA with high selectivity (Figure 17C).257 Although the stabilizing effect of psDNA toward nucleases still has not found extensive use in larger DNA nanostructures, their chemical advantages have been exploited for different purposes. For instance, charge-neutral hydrophobic backbones used for DNA nanopores in lipid bilayers can be synthesized through alkylation of the phosphorotioate group using alkylhalides (Figure 11C).174,258 Alkylation of phosphorothioate has also been used for functionalization of DNA nanostructures with proteins and metal nanoparticles at specific positions along double helices.259,260 It should, however, be noted that alkylation makes psDNA more prone to hydrolysis, especially at elevated pH. In another application of psDNA in DNA nanotechnology described by Liedl and co-workers, DNA nanotubes were coated with CpG oligodeoxynucleotides that contained phosphorothioate linkages, and the resulting structures were investigated for immunostimulation.261 Incorporation of phosphorothioate-modified backbones in larger DNA nanostructures should provide a straightforward method for increasing stability of the structures toward nucleases, but further investigations are required to shed light on the feasibility of this method for in vivo stabilization of DNA nanostructures.

of the reacting orbitals in space during collision. Because collisions are more frequent when the reaction partners are present in high concentrations, reaction rates of bimolecular reactions are highly concentration dependent. The concept of DNA-templated synthesis is to increase the local concentration of the two reactants by conjugating them to DNA strands that can be brought into close proximity by DNA hybridization (Figure 18A). The two reactants are then present in high local concentrations, thereby facilitating the reaction although their overall concentrations in solution are low, i.e. in the low micromolar range. The Liu group has pioneered DNA-templated synthesis, and in their proof of concept study from 2001, they investigated the use of amines and thiols as the nucleophilic groups, with iodoacetamides, bromoacetamides, vinylsulfones, and maleimides as electrophiles in bimolecular reactions (Figure 18B).262,263 With reaction partners attached to ssDNA, the authors consistently observed product formation when the ssDNA strands were complementary, but no reaction took place when the sequences were mismatched. Insertion of DNA spacers between the reacting groups did not obstruct the reactions, pointing to the fact that the increased local concentration, rather than hybridization-induced alignment of the reaction partners, facilitated the DNA-templated reactions. The chemical functionalities compatible with DNA-templated synthesis were significantly expanded by Liu’s group, who further made use of DNA-templated synthesis to introduce orthogonality to otherwise nonorthogonal reactive groups.264 This enabled programming of the synthesis output into the DNA sequences attached to the reacting groups.265 Since its discovery, several research groups have adapted the method for a variety of applications including DNA nanotechnology as reviewed elsewhere by Gorska and Winssinger.266 The initial work on DNA-templated synthesis also showed potential for reaction discovery such as the finding of a new palladiumcatalyzed alkene-alkyne coupling.267 Creation of DNA encoded libraries using DNA-templated synthesis is of great interest in the pharmaceutical industry. This

4. REACTIONS ON DNA NANOSTRUCTURES In this section, focus is directed toward the use of DNA, and in particular DNA nanostructures, for controlling the formation and breaking of covalent bonds. The section will be introduced with a brief description of DNA-templated chemistry which prepares the ground for a more in-depth discussion of organic reactions taking place on DNA nanostructures. The remaining part of the section is devoted to inorganic reactions at DNA nanostructures with a main focus on DNA-metallization. 4.1. DNA-Templated Organic Synthesis

The reaction rate of bimolecular reactions depends both on the probability of the two reactants colliding and on the orientation S


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Figure 19. Autonomous synthesis of an oligomer in a single solution with a DNA walker. The DNA walker binds to a DNA overhang in station 0 (step 1). Subsequently, another region of the walker hybridizes to the next station (Step 2) where the amino group of the walker reacts with an incorporated NHS ester (Step 3). At the same time, the DNAzyme (purple region) of the walker cleaves a ribonucleotide (green dot) and the station’s building block is transferred to the walker (Step 4). A new cycle is initiated by hybridization to the next station (Step 5) and eventually a 3mer product attached to the walker is obtained (Step 6). Adapted with permission from ref 273. Copyright 2010 Springer Nature.

Figure 20. DNA-templated synthesis of oligomers linked through double bonds. (A) The templated Wittig reaction where the growing polymer is transferred from one DNA strand to another.274 (B) The strand exchange mechanism of oligomer synthesis, where waste strands are removed by strand displacement and new building blocks are brought in.278 (C) Example of a 10-mer product obtained using the strategy depicted in (A). Reprinted with permission from refs 274 and 278. Copyright 2012 Royal Society of Chemistry and 2017 American Chemical Society.

of a DNA 3-way or 4-way junction where they react and form a library of products that upon primer ligation are located to a linear DNA duplex.270 The Yoctoreactor enabled the preparation of compound libraries from which new drug leads such as kinase inhibitors were selected.271 Although fascinating in their nature, a further discussion of DNA encoded libraries is beyond the scope of this review, and the reader is directed to the review by Goodnow Jr. et al. for further insight into this topic.272

technology has been pioneered by the Liu lab, who initially described the preparation of a small 65-membered library of macrocyclic compounds that was screened for binding to carbonic anhydrase.268 Recently, the Liu lab created a 256.000membered library of macrocyclic compounds using DNAtemplated synthesis, thereby highlighting the method’s relevance in medicinal chemistry.269 The so-called Yoctoreactor is a platform for DNA-programmed synthesis of small molecule drug libraries. Here, the building blocks are placed in the middle T


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4.1.1. DNA-Templated Synthesis for Preparation of Sequence-Specific Oligomers. In Nature, the central roles of DNA are to store information and template the formation of sequence specific-polymers in replication, transcription, and translation. Therefore, artificial systems for DNA-templated synthesis of sequence-specific artificial polymers are an attractive research goal. Through modification of Nature’s own machinery, polymerases that accept some semiartificial nucleoside triphosphates and translational machinery that allow incorporation of artificial amino acids have been engineered. However, it is a major challenge to design nonenzymatic chemical systems for DNA-templated synthesis of sequence-specific polymers. In some of the examples described above, oligomers of up to 4 building blocks were created although the stepwise synthesis and its efficiency were far from that of Nature’s own machinery. One of the few examples of autonomous oligomer synthesis was presented by He and Liu in 2010 (Figure 19).273 The system consists of a DNA walker migrating along a DNA template (step 1). A DNAzyme in the walker comes into close proximity of a ribonucleotide in the station strands (step 2) before its amino group reacts with an NHS ester at the first station strand S1 (step 3). Amide bond formation and DNAzyme-mediated ribonucleotide cleavage leads to transfer of a building block to the walker (step 4). Separated from the NHS ester by a rigid backbone, the building block contains another amine that can react with the NHS at the next station following another step of the walker (step 5). Autonomous 3mer synthesis was achieved in this manner (step 6). All amines were protected with photocleavable protecting groups until the initiation of the autonomous synthesis to avoid premature reaction with the highly reactive NHS ester. The Turberfield and O’Reilly laboratories explored DNAdirected synthesis of oligomers in a series of papers starting in 2010.274−276 Using the Wittig reaction, they coupled building blocks in a DNA-controlled manner with an efficiency of approximately 85% in each step (Figure 20A). Because the building blocks (apart from the first and the last) contained both the phosphorus ylide and the aldehyde reactive handles, they had to be separated by the building blocks’ rigid backbones to avoid intramolecular reactions. The synthesis progressed by consecutive strand displacement reactions where the growing oligomer was transferred from strand to strand after each step (Figure 20B). Thus, the reactions were not autonomous, but impressively, sequence-controlled oligomers of up to 10 building blocks were obtained as illustrated in the product exemplified in Figure 20C. Later, they also developed an autonomous version of the reactions. Using a cascade of hybridization reactions between DNA hairpins, they synthesized oligomers composed of up to 4 building blocks that did not require any interference with the system that utilized both Wittig chemistry and amide couplings.277 4.1.2. DNA-Templated Synthesis for Formation of Nanowires. One of the promises of DNA nanotechnology is its potential to provide control over molecular wires in a bottom-up fashion, thereby providing a viable alternative to lithographybased top-down preparation of wires and semiconductors. Czlapinski and Sheppard demonstrated the formation of metalsalen bridges using DNA-templated synthesis.279 In our group, we have used this chemistry for modular assembly of linear and tripoidal nanoarchitectures. As illustrated in Figure 21, the structures were based on conjugated phenylene−ethynylene moieties linked together by metal-salen bridges using DNAtemplated synthesis.50 Linear building blocks containing

Figure 21. DNA-templated covalent coupling of multiple molecular wire fragments. (A) Scheme showing the DNA-templated formation of metal-salen linkages between the building blocks. (B) Structures of the linear (lef t) and tripoidal (right) phosphoramidite building blocks for formation of nanowires using DNA-templated synthesis. (C) Pictographic representation of the linear and tripoidal structures after DNA synthesis and their DNA-programmed coupling into linear and branched oligomers. Black spheres denote the metal−salen linkages.

salicylaldehyde moieties in each end were linked to a phosphoramidite in one end and a DMT-protected hydroxyl group in the other end (Figure 21B, left). The tripoidal building blocks were based on a central benzene ring connected to three salicylaldehyde moieties linked to an Fmoc-protected hydroxyl group, a DMT-protected hydroxyl group, and a phosphoramidite, respectively (Figure 21B, right). These building blocks were functionalized with DNA using solid phase phosphoramidite synthesis where orthogonal sequences were synthesized at each handle. Subsequently, DNA-templated synthesis enabled the formation of architectures composed of multiple covalently linked building blocks such as linear structures up to tetramers (Figure 21C). Through the introduction of elongated linear building blocks and cleavable linkers between the building blocks and the DNA, the modular assembly of conjugated building blocks was further developed.280−282 Eventually, a setup providing fully conjugated molecular wires of up to 4 building blocks was developed using DNA-templated Glaser−Eglinton couplings for oligomer synthesis.283 Sleiman’s group also synthesized conjugated molecular wires using nucleobase-templating.284 First, they prepared nonconjugated polymers with nucleobase-terminated linkers attached to them. Importantly, the nonconjugated polymers U


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Figure 22. Single molecule chemical reactions monitored on DNA origami. (A) Model of the DNA origami scaffold with incorporated biotin reference and three functional groups (an alkyne, an amine, and an azide). The diagram schematically illustrates their reactions with the complementary functionalities (Streptavidin is represented by a yellow dumbbell structure). (B) Chemical structures of the biotin-tethered functional groups. (C−E) Conditions and expected outcome of the reactions of the different functional groups. (F−H) AFM images of the respective reaction products depicted directly above in (C−E). (I−J) Model and AFM imaging of the expected products after three successive reactions. Adapted with permission from ref 186. Copyright 2010 Springer Nature.

linking of DNA nanostructures and formation of well-defined polymers. These topics will be covered in the next sections. 4.2.1. Single Molecule Reactions on DNA Origami. DNA origami immobilized on a solid surface may serve as a discrete solid support for studying single molecule chemical reactions. Because various functional groups can be placed at different geometrically predetermined positions, it may be possible to distinguish various chemical reactions at different locations at the origami surface in, for instance, a standard rectangular Rothemund origami. Although, the making and breaking of bonds in small molecules cannot be distinguished by AFM in this setting, the appearance and disappearance of these groups can be visualized with good resolution if the small molecule is tethered to a larger moiety such as a protein. To accomplish this, our group positioned three different functional groups, an azide, an amine, and an alkyne, at the origami surface, with a tethered biotin as a reference point (Figure 22A).186 Addition of the chemically complementary reagents to the reactions (azide, NHS ester, and alkyne, respectively) all tethered to biotin (Figure 22B) allowed imaging of the outcome of the reactions by AFM after addition of Streptavidin (Figures 22C−H). Streptavidin, which binds very strongly to biotin, provides an excellent contrast for AFM imaging because it is a relatively rigid protein with a diameter of approximately 5 nm. Only the expected reactions took place, which was verified by comparison with the reference position, and yields were

were prepared using living polymerization, which provided access to polymers of very narrow size distributions. Subsequently, phenylene-based monomers functionalized with alkyne and iodide groups para to each other were prepared, also with nuclebase-terminated linkers. Base pairing between the nucleobase-modified monomers and nonconjugated polymers facilitated the assembly of monomers on the nonconjugated polymer template. Lastly, Sonogashira polymerization provided conjugated phenylene-ethynylene polymers with lengths and dispersities resembling those of the nonconjugated polymer. Canary and Seeman used another strategy to synthesize socalled DNA nylon.285 In this approach, 2′-deoxy-2′-alkylthiouridine phosphoramidites containing either a protected diamine or a dicarboxylic acid appending from the 2′-thiol were used in automated oligonucleotide synthesis. The alternating dicarboxylic acids and diamines were then coupled after activation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form short nylon oligomers linked to the oligonucleotide sequence in a parallel manner. 4.2. Organic Chemistry on DNA Nanostructures

DNA nanostructures provide a highly attractive platform for investigation of chemical transformation with an amazing degree of control. Individual reactant molecules can be positioned with nanometer control, and to some extent, their orientation may also be steered. This enables studies of reactions at the single molecule level, which can be further used for controlled crossV


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resulting structures was investigated and compared to noncross-linked reference samples, revealing significantly increased stabilities (Figure 24A).204 In a study by Rusling et al., psoralen-

determined by AFM to be 84−90%. If performed sequentially, the three reactions gave a total yield of 69%. It is noteworthy that immobilization on mica renders the origami tolerant to a variety of buffers containing organic solvents. While the above description concerns bond-making, single molecule cleavage reactions were also imaged using the same concepts. As shown in Figure 23, this setup was also applied to monitor singlet oxygen diffusion to cleavable groups positioned at each

Figure 23. Setup for AFM monitoring of singlet oxygen diffusion from an indium pyropheophorbide singlet oxygen sensitizer (Green) to cleavable 1,9-di(alkoxy)anthracene moieties (Red). Blue triangles represent biotin. After irradiation, singlet oxygen generation, and cleavage, Streptavidin is added and the degree of cleavage relative to the noncleavable reference (outer left) is measured. Adapted from ref 286. Copyright 2010 American Chemical Society.

side of an indium pyropheophorbide singlet oxygen sensitizer conjugated to DNA and placed in the middle of the origami.286 Two pairs of biotins (blue triangles) were placed 18 or 36 nm from the sensitizer and linked to the origami via a DNA tether containing a 1,9-di(alkoxy)anthracene moiety that is specifically cleaved by singlet oxygen while a biotin containing a noncleavable linker was placed in the corner for reference. Upon irradiation, singlet oxygen generated through the catalytic action of the sensitizer diffused and reacted with the cleavable linkers, among other things. After irradiation and addition of Streptavidin, partial cleavage of the linkers was observed. The linkers closest to the sensitizer were significantly more prone to cleavage. Interestingly, if Streptavidin was added to the origami before irradiation, no cleavage was observed upon irradiation, probably because of Streptavidin reacting with singlet oxygen and depleting the surrounding few nanometers. In another example, disulfide-reduction driven by photoexcitation of pyrene was visualized at the single molecule level on DNA origami.287 Disulfide-reduction enabled a pyrene-containing DNA walker strand to take a step and reach another disulfide-containing extended staple strand. The process could be repeated, allowing the walker to take several steps as visualized by high-speed AFM imaging. 4.2.2. Chemical Cross-Linking of DNA Nanostructures. The stability of DNA nanostructures in buffers with different salt concentrations, at elevated temperatures, and in vivo is of great significance to many applications. While structural design provides some access to tuning nanostructure stability, chemical cross-linking is another important strategy to accomplish this. Psoralen is known for its ability to photo cross-link DNA. This has also been used for cross-linking DNA origami structures, thereby increasing their thermal stability. Sugiyama and collaborators used 8-methoxypsoralen to cross-link DNA origami and upon cross-linking, the thermal stability of the

Figure 24. Chemical cross-linking of DNA nanostructures. (A) Structure of 8-methoxypsoralene (top) used to cross-link and hence stabilize DNA nanostructures. The bottom panel shows 8-methoxypsoralene cross-linked to two thymine bases. Bottom panel is adapted from ref 204. Copyright 2011 American Chemical Society. (B) Setup for reversible cross-linking of DNA nanostructures using disulfide reduction and oxidation. The bottom panel shows the DNA routing of the structure in more detail. Adapted with permission from ref 290. Copyright 2016 John Wiley & Sons, Inc. (C) Illustration of the CuAAC approach for cross-linking DNA nanostructures (top).291 Orange trapezia and blue triangles denote alkyne and azide groups, respectively. Green pentagons denote click triazole products. The bottom panel shows an agarose gel of an unmodified 6 helix tube (6 HT), a 6 helix tube with handles for 24 click reactions without CuAAC ligation (24 ÷ click) and with CuAAC ligation (24 + click). The different structures were left untreated (Lane C), heated to 65 °C (Lane 1), and heated to 65 °C followed by exposure to an exonuclease (Lane 2). Adapted with permission from ref 291. Copyright 2015 John Wiley & Sons, Inc.

modified DNA was introduced into DNA nanostructures via triplex formation. In this way, site-selective photo-cross-linking of the DNA nanostructure was achieved.288 Tagawa et al. used photoligation of DNA nicks to stabilize DNA nanostructures. They functionalized DNA tiles with 5-carboxyvinyl-2′-deoxyuridine, which upon UV-exposure formed cross-links with cytosine bases.289 Later, the same group introduced 3-cyanovinylcarbazol into DNA arrays using phosphoramidite chemistry, and when W


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exposed to UV-irradiation, efficient cross-linking to neighboring pyrimidines on the complementary strand was observed.205 To stabilize double crossover tiles, our group utilized disulfide formation for reversible, covalent cross-linking where interlocked strands rendered the structures thermally more stable (Figure 24B).290 Another strategy for cross-linking DNA nanostructures is the addition of bis-functional agents. As an example of this, Endo et al. used a bis-maleimido reagent to cross-link thiol-modified DNA into micrometer long DNA rods.292 Recently, Manetto and collaborators introduced alkynes and azides into single-stranded tile-based nanostructures designed to position the reaction handles in close proximity.291 CuAAC was used to triazole-link the DNA strands within the structure before gel electrophoresis verified formation of catenated strands. When the number of alkyne and azide modifications was increased, the DNA structures showed improved stability toward heating as well as exonucleases (Figure 24C). Using a similar approach, the same group used 6helix bundle DNA nanostructures functionalized with azides and alkynes to direct the synthesis of a 762 nt gene from 14 oligonucleotides.293 Synthesis of a smaller 365 nt gene from splint oligonucleotides by templated CuAAC had been demonstrated earlier by Kukwikila et al.294 Very recently, Dietz’s lab described a platform for crosslinking of DNA origami by insertion of an additional pair of thymines at specific positions in DNA staple strands, thus enabling thymine dimerization upon exposure to 310 nm light (Figure 25).295 These cross-linked structures remained stable in buffers containing 5 mM Mg2+ at a wide range of temperatures with only minor degradation observed even at 90 °C, and the cross-linked structures also showed increased stability toward certain nucleases compared to the native structures. Importantly, this method could also cross-link higher-order assemblies which then remained mainly intact in 5 mM Mg2+-buffers opposed to non cross-linked assemblies. 4.2.3. Polymerization on DNA Nanostructures. Another attractive feature of DNA nanostructures is their potential use for creating polymer-based nanomaterials, and electrostatic interactions of dsDNA with cationic monomers, oligomers, and polymers have been used to template the formation of conjugated polymer (CP) nanowires. Nagarajan et al. first demonstrated that polyaniline (PANI) could be formed on a DNA template by oxidative polymerization catalyzed by the enzyme Horseradish peroxidase (HRP).296 Later, Nickels et al. demonstrated that PANI could be synthesized on DNA templates using three different oxidative polymerizations.297 Aniline’s pKaH is 4.6, and hence, the molecule is positively charged at lower pH. The positively charged monomers and oligomers interact with the negatively charged DNA template during polymerization. Eventually, the whole DNA template becomes imbedded in polyaniline, thus forming a polyaniline− DNA nanowire. Ma et al. used the same approach to synthesize conducting polyaniline nanowires on silicon surfaces.298 The mechanism for CP nanowire growth on DNA templates was described by Watson et al.299 Briefly, polymers nucleate in spherical particles that are dispersed over the length of the DNA template. With time, the particles grow and more nucleation sites appear until eventually, the size and number of particles has increased sufficiently to recombine and form the nanowire (Figures 26A and B). Using this strategy, nanowires based on several different conducting polymers have been formed, including polypyrrole (PPy),300,301 poly-2,5-bis(2-thienyl)-

Figure 25. Stabilization of 3D DNA origami by sequence-programmable thymine dimerization. Insertion of thymine pairs in DNA staple strands facilitates photoinduced thymine dimerization leading to intraor interhelical covalent bonds depending on the thymine positioning. Reprinted with permission from ref 295. Copyright 2018 American Association for the Advancement of Science under the creative commons agreement (https://creativecommons.org/licenses/by-nc/ 4.0/legalcode).

pyrrole (PTPT),302 and poly(3,4-ethylenedixoxythiophene) (PEDOT) (Figure 26C).303,304 Based on the same mechanisms, Ding’s group took advantage of this concept to form well-defined PANI nanostructures on DNA nanoarchitectures (Figure 27A−B).305,306 The DNA nanostructures were modified with guanosine-quadruplex based DNAzymes that show HRP-activity in the presence of the small molecule hemin. Addition of aniline to the structures caused PANI particles to selectively form at the DNAzymemodified positions as demonstrated on simple Y-shaped structures as well as on more complex triangular DNA origami structures.305,306 In a related approach, Weil’s group used rectangular DNA origami for templated formation of differently shaped polydopamine nanoparticles dependent on the position of protruding DNAzymes.307 Moreover, the same group investigated DNA origami as a template for atom-transfer radical polymerization (ATRP).308 SsDNA modified with an ATRP initiator was hybridized to extended staple strands protruding from rectangular origami. Addition of a PEGylated acrylate monomer along with sacrificial initiator, tris(2pyridylmethyl)amine, CuBr2, and ascorbic acid initiated the polymerization, and the formed polymer patterns on the origami template could be visualized by AFM topography (Figure 27C). Recently, Weil’s group combined the two concepts and formed DNA tubes with interior polydopamine growth and exterior ATRP polymerization.309 The authors also removed the DNA template to see if polydopamine or PEG acrylate polymer particles of defined shapes were released. On-surface treatment X


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Figure 27. Polymerization on specific domains of DNA nanostructures. (A) Illustration of the setup used to form PANI on triangular DNA origami using guanine-quadruplex DNAzymes with HRP activity. (B) Illustration of two different patterns of PANI formed on triangular DNA origami along with corresponding AFM topography images (scale bar = 200 nm) of the structures and height profiles of selected structures. (A) and (B) are adapted from ref 306. Copyright 2014 American Chemical Society. (C) Illustration of the strategy for performing ATRP on DNA origami. First, DNA initiator is patterned on DNA origami before the polymerization is initiated by addition of the required reagents. Use of a cross-linker allowed heat-stable polymer nanoparticles to form and DNA templates to be subsequently removed by heat denaturation. The AFM image (scale bar = 50 nm) shows an expected shape-defined polymer particle observed after removal of the DNA template. Adapted with permission from ref 308. Copyright 2016 John Wiley & Sons, Inc.

Figure 26. Templating polymerization on dsDNA. (A) Four-step illustration of the mechanism behind DNA-templated growth of conjugated nanowires starting with a few polymer nucleation sites emerging on the DNA template. (1) This is followed by formation of new nucleation sites and growth of the polymer particles (2, 3) until the particles merge on the template to form the DNA-CP nanowire (4). (B) Tapping mode AFM images showing the steps described above. Scale bars = 500 nm. (C) Structures of CPs commonly investigated in DNAtemplated nanowire formation. (A) and (B) are reprinted from ref 299. Copyright 2014 American Chemical Society.

with aqueous hydrochloric acid showed some promise for isolation of polydopamine particles, and there were indications of shape-defined polymer particles being left on the surface after DNA degradation. Cross-linking of the PEG acrylate polymer particles followed by origami template heat denaturation allowed isolation of a few shape-defined polymer particles (Figure 27C). In another approach to grow polymers from DNA origami, Okholm et al. used enzymatic polymerization by TdT (vide supra) to grow thymine polymers from DNA origami with the assistance of bovine serum albumin (BSA).310

terials for numerous applications including photonics, therapeutics, electronics, and sensing. The progress of DNAtemplated metallization including its preparation and applications was recently reviewed thoroughly by Chen et al.312 Shen et al.’s review focuses more specifically on the role of DNA nanotechnology in DNA metallization.36 As mentioned above, the formation of silver nanowires described by Braun et al. relied on chemical reduction of silverions. Introduction of reducing aldehyde groups by treatment with glutaraldehyde therefore provided a method for obtaining more efficient metallization, and importantly, metallization could be directed by precise positioning of reducing groups on the DNA.313 Carell’s group showed that efficient direction of DNA metallization was obtained through one of two strategies: Either aldehyde groups were introduced into DNA using click chemistry or dialdehydes were formed by periodate cleavage of cis-3,4-dihydroxypyrrolidine incorporated during oligonucleotide synthesis.314,315 In another approach, Ma et al. showed how anchoring of gold seeds directed metallization from the seeds along the dsDNA. 316 While initial research on DNA metallization utilized dsDNA, the use of more elaborate DNA nanostructures was eventually also employed. Yan, Reif, LaBean, and others showed that DNA nanoribbons assembled from

4.3. Metallization of DNA Nanostructures

In addition to organic transformations at DNA nanostructures, significant efforts have also been devoted to forming inorganic structures in DNA nanotechnology. In 1998, Braun et al. showed that DNA could be used as a template for formation of metal nanowires (Figure 28).311 They used λ-DNA with 12-mer sticky ends that hybridized to electrodes functionalized with complementary DNA. As such, the λ-DNA was stretched between the electrodes and then sodium cations were exchanged for Ag+-ions, to allow silver cluster formation upon treatment with hydroquinone as reducing agent. Following assembly of the silver-clusters on the DNA template, further chemical reduction in the presence of excess AgNO3 at acidic pH led to the formation of silver nanowires on the DNA template. Since this discovery, researchers have focused on using DNA nanostructures to template the formation of well-defined metal-nanomaY


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Figure 28. Metallization of dsDNA. (A) General scheme for silver seeding followed by metal plating in metallization of a dsDNA template. For seeding, reductive aldehyde groups are introduced by treatment with glutaraldehyde. Seeding can be performed with various metal ions, but silver remains widely used. (B) AFM topography image from seminal work on dsDNA metallization showing the granular silver nanowire formed between electrodes on a λ-DNA template.311 Reprinted with permission from ref 311. Copyright 1998 Springer Nature. Figure 29. Metallization of DNA nanostructures. (A) Diagram of tilebased formation of DNA nanoribbons from 4 × 4 DNA tiles (top) and AFM images of the formed nanoribbons (middle). The bottom panel shows SEM images of the bare nanoribbons and a silver nanowire formed upon metallization of the ribbon. Scale bars = 500 nm. Reprinted with permission from ref 317. Copyright 2003 American Association for the Advancement of Science. (B) Schematic representation of the metallization of branched DNA origami by psoralene cross-linking and glutaraldehyde treatment followed by silver seeding and gold plating (top). AFM topography images of metallization on a mica surface without any immobilized DNA (bottom lef t) and with metallized, branched DNA origami (bottom right). Scale bars = 500 nm. Adapted from ref 322. Copyright 2011 American Chemical Society.

DNA tiles could be metallized to yield very well-defined, highly conducting silver nanowires (Figure 29A).317 The same group broadened the concept and demonstrated that DNA nanotubes as well as DNA lattices and filaments were also suitable for metallization and, hence, silver nanowire formation.318,319 Around the same time, Zinchenko et al. used compact DNA toroids prepared by mixing T4 DNA with spermidine. The resulting DNA toroids were metallized, and very well-defined silver nanorings were obtained.320 Instead of incorporating reducing aldehyde groups in the DNA, Wilner et al. investigated the use of enzymatic reduction. AuNP-functionalized glucose oxidase (GOx)-DNA conjugates were prepared and annealed to DNA obtained by rolling circle amplification. In the presence of oxygen, glucose, and silver ions, the AuNPs grew, and metallic nanowires were formed.321 The invention of DNA origami brought new opportunities for preparation of metal nanoarchitectures by metallization of DNA nanostructures. Liu et al. used DNA origami cross-linked with psoralen to create branched silver nanowires.322 Besides crosslinking, an increase of the overall staple-strand concentrations also proved necessary to obtain sufficient stability of the structures. Upon introduction of aldehydes using the glutaraldehyde approach, silver seeding was performed before gold-plating successfully produced branched metal nanowires (Figure 29B). In a follow-up study, the yield was improved using palladium-seeding.323

Schreiber et al. used seeding with 1.4 nm gold nanoclusters followed by Au3+-ions plating to form AuNPs of various shapes such as nanorings, rectangles, and kites defined by the origami structure templates.324 Uprety et al. immobilized gold nanorods (AuNRs) on rectangular DNA origami followed by anisotropic gold plating to create small-diameter nanowires.325 In another important advancement, Finkelstein, LaBean, and co-workers immobilized AuNPs in various patterns on rectangular DNA origami. Subsequently, they enlarged the particles by reducing Ag+-ions from solution, eventually leading to recombination of the AuNPs and hence the formation of joined metallic nanostructures.326 Taking this concept a step further, Liedl’s group formed helical AuNP patterns around a DNA origami Z


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Figure 30. 3D DNA nanostructures as molds for preparation of shape-defined MNPs. (A) Illustration of the concept used for casting 3D gold nanoparticles using DNA origami molds. A small AuNP-seed is positioned in the interior of the 3D DNA origami. The structure is capped with lids, and MNPs are grown from the seed using either silver or gold ions. (B) Illustrations and TEM images of the shape-defined 3D MNPs formed. Scale bars = 20 nm. (A) and (B) are adapted with permission from ref 329. Copyright 2014 American Association for the Advancement of Science. (C) Illustration and TEM images of structures formed in the initial work from the Seidel lab. Scale bar = 40 nm. Reprinted from ref 328. Copyright 2014 American Chemical Society. (D) Illustration and TEM image of long gold nanowire formed in multiblock hollow DNA tube. Scale bar = 50 nm. Reprinted from ref 330. Copyright 2018 American Chemical Society.

rod.327 Both right- and left-handed helices were formed from AuNP-DNA conjugates hybridized to the template DNA rod. The AuNPs were further joined using Ag+ enlargement, and the resulting helical metal nanoparticles (MNPs) showed extremely high molar circular dichroism values. With this, the control over MNP shape moved into 3D. The formation of defined 3D metallic nanostructures from DNA templates was further investigated by the groups of Seidel and Yin.328,329 Yin’s lab prepared AuNPs and silver nanoparticles (AgNPs) of various prescribed 3D shapes by metal casting in stiff 3D DNA origami molds. A single or a few MNPs were used as seeds in the interior of the structure. After seeding, the 3D structures were capped with lids and the MNPs were grown (Figure 30A−B). In the work by Seidel and co-workers, stiff and hollow 3D DNA tubes served as molds for casting MNPs (Figure 30C). Because their structures were not capped, the MNPs could grow past the ends of the DNA tube. In a very recent study, this ability enabled growth of long homogeneous gold nanowires in the DNA tube molds (Figure 30D).330 With the recent achievement of 3D DNA bricks assembled into extremely large DNA nanostructures with interior cavities, it seems logical that future research will involve the formation of larger and more complex MNP structures from DNA templates.331 Also, there should be ample opportunity for organic chemists to help improve the quality of the MNPs obtained from DNA metallization. It is possible that functionalization of DNA molds with metal-coordination groups could help define the resulting MNPs more strictly. The 3D MNPs discussed here were prepared using metal-ion reduction mediated by externally added ascorbic acid or hydroxylamine. Alternatively, aldehyde functionalization of the

interior walls of the DNA structures may enable the formation of a very well-defined metal layer along the inner boundaries potentially initiating the formation of 3D MNPs with sharper edges and more well-defined boundaries.

5. CHEMICAL METHODS FOR IMMOBILIZATION OF NANOMATERIALS IN DNA NANOSTRUCTURES Structural DNA nanotechnology has provided an exceptional tool for controlling matter with high precision at the nanoscale, and various molecules and nanomaterials have been successfully arranged on DNA nanostructures. This chapter will discuss the chemistry used for positioning of different classes of nanomaterials such as carbon nanotubes (CNTs), organic polymers, metal nanoparticles (MNPs), and quantum dots (QDs) in DNA nanostructures. The immobilization of proteins in DNA nanostructures will be treated in section 6. 5.1. Carbon Nanotubes

Intense research within DNA nanotechnology has originated from the vision of using DNA nanostructures as templates for electronics. Ever since Iijima’s discovery of CNTs, their exceptional physical properties have given rise to very intense research efforts.332 CNTs exhibit extremely high mechanical strength and thermal stability, the electronic properties of CNTs vary from semiconducting to metallically conducting, and importantly, state of the art purification methods allow isolation of CNTs based on their electronic properties.333−335 The discovery of CNTs also spurred massive efforts toward chemical functionalization. Although CNTs are chemically rather resistant, a large number of methods were developed for CNT derivatization. Carboxylation, fluorination, and reductive AA


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(SWCNTs), double-walled CNTs (DWCNTs), and multiwalled CNTs (MWCNTs) are not discussed here. For more information on this, the reader is directed to Popov’s comprehensive review.338 This section will mainly focus on SWCNTs and their use in DNA nanotechnology. An important discovery regarding the properties of CNTs was made by Zheng et al. in 2003.339 They showed that ssDNA stacks on CNTs and efficiently helps disperse the nanotubes (Figure 32A). By screening a large number of DNA strands of varying sequences, certain sequences were found to, at least to some degree, enable separation of CNTs based on their structural and electronical properties.335 The physical characteristics of CNTs make them attractive candidates for assembly of electronics using DNA nanotechnology. In early studies toward this, DNA was attached to CNTs using amide-bond formation between amino-modified DNA and carboxyl defects of the CNTs.340 These CNT-DNA conjugates were used for multi-CNT assembly and for assembling gold nanoparticles on CNTs.341 Also using amide bond formation, Dekker and colleagues demonstrated the functionalization of CNTs with PNA to increase compatibility with organic solvents and hence improve the processing opportunities. The PNA was mainly attached to the ends of the CNTs and could be used for attaching long dsDNA with 12mer sticky ends to the CNTs.342 While the initial attempts to address CNTs using DNA interactions relied on covalently coupling nucleic acids or their analogues to CNTs, focus later turned to noncovalent interactions. This was based on the fact that strong π−π stacking interactions exist between CNTs and other aromatic molecules and hence enable manipulation of CNTs without covalently modifying their structure with the risk of impairing

couplings using diazonium salts are just a few of the many chemical methods for CNT functionalization that allow further chemical modification (Figure 31). Several reviews on the

Figure 31. Examples of methods for chemical modification of carbon nanotubes. Carboxylation allows derivatization by amide-bond formation and esterification, whereas fluorination provides handles for nucleophilic aromatic substitution. Reductive coupling with diazonium salts enables functionalization with substituted phenyl rings.

chemistry for CNT modification are available, and the ones by Tasis et al. and Balasubramanian et al. are recommended for indepth discussions.336,337 The many different configurations of CNTs along with the differences between single-walled CNTs

Figure 32. (A) Side view model (top) of ssDNA wrapping around SWCNT due to stacking interactions between the nucleobases and the CNT surface. Front view of the model is shown in the bottom left inset. The bottom right inset shows an AFM image of CNTs dispersed by DNA and isolated by ionexchange chromatography. Scale bar = 500 nm. Adapted with permission from ref 339. Copyright 2003 Springer Nature. (B) Scheme for the synthesis of a polyfluorene-block-DNA polymer using phosphoramidite based oligonucleotide synthesis (top). Lower left inset shows a front view model of the hydrophobic polyfluorene block (cyan) stacking on the CNT with the ssDNA still accessible for further interactions. Lower right inset shows a TEM micrograph of AuNPs immobilized on a CNT by interaction with the DNA part of polyfluorene-block-DNA wrapped around the CNT. Scale bar = 50 nm. Lower panels in (B) are adapted with permission from ref 343. Copyright 2011 John Wiley & Sons, Inc. AB


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the electronic properties. Conjugated polymers for instance showed promise for manipulation of CNTs. Kwak et al. synthesized a poly(9-alkylfluorene) end-functionalized with ssDNA where the hydrophobic polymer wrapped around CNTs while the DNA remained accessible. The structure was used for purification of CNTs based on their conductivity and also for immobilization of AuNPs on CNTs (Figure 32B).343 The direct stacking of DNA on CNTs also provided an important method for manipulating CNTs, which has become very important within DNA nanotechnology. After the invention of DNA origami, the use of flat rectangular origami plates as breadboards for nano electronics became an appealing research area. In the landmark study from 2009, Maune et al. set out to investigate how the tendency of ssDNA to wrap around CNTs could be exploited for immobilization of CNTs on DNA origami. They designed specific nucleotide sequences containing a dispersal domain of 40 thymines (40T), a 15mer double-stranded region (to avoid stacking of this region on the CNT), and a 5mer LNA toehold region (Figure 33A).246 Rectangular DNA origami structures were functionalized with extended staple strands positioned in tracks for binding to the CNTs, and the CNTs were successfully aligned on the origami structures, although in modest yield (Figure 33B). The authors successfully created a CNT-based field-effect transistor templated on DNA origami by positioning of two CNTs orthogonally to each other on the origami template. The following e-beam lithography deposition of electrodes onto the ends of the two CNTs enabled their use as conduction and gate channels. In another approach to immobilize CNTs on DNA origami, Eskelinen et al. exploited the strength of the biotin−Streptavidin interaction.344 Biotinylated staple strands were used for making CNT-binding tracks before Streptavidin was added to create a Streptavidin bridge (Figure 34A). Biotinylated ssDNA was wrapped around CNTs, and due to the tetravalent biotinbinding of Streptavidin, the CNTs were immobilized through the Streptavidin bridge although the yields were modest. Norton and co-workers also immobilized CNTs on DNA origami simply by dispersing CNTs using 40T DNA.345 The DNA origami structures were prepared without the edge staple strands to provide single-stranded rich regions, which then wrapped around the CNTs and caused immobilization. Similar to the other approaches, the yield of the desired structures was low. The poor immobilization yields for CNTs on DNA nanostructures have been ascribed to the variation in lengths and quality of the CNTs and the unreliable nature of the noncovalent ssDNA wrapping. Recently, Atsumi and Belcher demonstrated the use of DNA origami functionalized with G-quadruplex DNAzymes for creating CNTs with narrow length dispersity (Figure 34B− C).346 The CNTs were bound to the origami templates using the biotin−Streptavidin approach (Figure 34A), and CNTs were with some success cleaved at the DNAzyme restriction sites. In DNA nanotechnology, it has been appealing to use noncovalent ssDNA wrapping for binding to CNTs, and so far, the studies focusing on CNT immobilization on DNA nanostructures made use of this benign approach that avoids rendering the CNT backbone structure. However, the binding is reversible, the DNA coverage is limited, and the immobilization yields are typically poor. Future studies should focus on forming novel types of CNT-DNA conjugates by covalent coupling chemistries. Perhaps, DWCNTs could be used to protect the inner tube from being harmed during treatment with harsh chemicals. CNTs functionalized with a higher density of DNA

Figure 33. DNA-directed immobilization of carbon nanotubes in origami. (A) Illustration of the concept for immobilization of SWCNTs on DNA origami. A DNA construct consisting of a 40T dispersal domain, a 15mer binding strand with a complementary 15mer protecting strand, and a 5mer LNA toehold sequence is used to disperse CNTs. Extended staple strands bind the LNA toehold and displace the 15mer protecting strand, thereby immobilizing the CNT. The upper right panel shows an example of a well-aligned CNT on DNA origami. The bottom panel depicts how the use of two different binding sequences allows immobilization of two CNTs on the same DNA origami structure in an orthogonal alignment. (B) AFM image (top lef t, scale bar = 100 nm) and illustration (bottom left) of CNT cross-junction on DNA origami before electrode deposition. The green tail denotes a DNA ribbon grown from the rectangular origami for ease of determining the orientation of the origami. The right panel shows an AFM image of a field-effect transistor device templated on DNA origami. S and D denote source and drain electrodes, respectively, whereas G and g denote the gate electrodes. Scale bar = 100 nm. All figures are adapted with permission from ref 246. Copyright 2010, Springer Nature.

should be easier to work with in aqueous environment and potentially provide significantly improved immobilization yields. 5.2. Conjugated Polymers and Oligomers

Conjugated polymers and oligomers have been used intensively in bulk for organic electronics such as field-effect transistors, light-emitting diodes, and photovoltaics.347,348 They have also been studied for their single-molecule properties using singlemolecule spectroscopy, scanning tunneling microscopy (STM), and mechanically controlled break junctions, but the utilization of these molecules at the single-molecule level has remained largely unexplored.349−352 DNA nanotechnology provides a potential tool for controlling and using individual conjugated molecules, but to take full advantage of this, novel chemical methods are required as reviewed in this section. In 2015, our AC


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Figure 34. Directed immobilization of carbon nanotubes in origami using biotin−Streptavidin interactions. (A) Illustration of Streptavidin immobilization on tracks of biotinylated extended staple strands. CNTs, with biotinylated ssDNA wrapped around them, bind to the Streptavidin tracks thereby immobilizing the CNTs. Adapted with permission from ref 344. Copyright 2011 John Wiley & Sons, Inc. (B) Illustration of the concept (left) used for cutting CNTs in specific lengths determined by DNA origami. In the presence of hemin and hydrogen peroxide, the G-quadruplex DNAzymes facilitate radical formation that induces local CNT breaks. The right panel of B shows DNA origami architectures used for cutting CNTs in different lengths. Roman numerals in parentheses correspond to the designations in C. (C) Length distributions of CNTs after exposure to different origami architectures (iii−vi) as well as pristine DNA-dispersed CNTs (i) and randomly cut CNTs (ii). The lengths were determined from AFM imaging. B and C are adapted from ref 346. Copyright 2018 American Chemical Society.

group described the synthesis of a PPV-based bottle-brush type polymer.353 We attached DNA to the PPV backbone via triethylene glycol linkers in a grafting-from approach using phosphoramidite based oligonucleotide synthesis (Figure 35A). To enable this, we first prepared a PPV-type polymer containing one triethylene glycol linker per monomer unit. The linkers were terminated with TBDPS protecting groups, and after removal of 20−40% of the protecting groups, hydroxyl groups were liberated to use for immobilization of the polymer on a CPG solid support. By removing the remaining protecting groups, oligonucleotide synthesis could be performed directly onto the polymer’s triethylene glycol linkers. Upon cleavage, deprotection, and purification, a fully water-soluble material (poly(APPV-DNA)) was obtained that could be positioned with high efficiency along tracks of complementary extended staple strands on 2D and 3D DNA origami. In extended studies, methods for controlling the dynamics of single polymer molecules on DNA origami were developed.354 By toeholdmediated strand displacement, the position of individual polymer molecules was switched between two different tracks on flat rectangular DNA origami (Figure 35B). The position of the polymer could be determined by either AFM or by measuring Förster Resonance Energy Transfer (FRET) between

Figure 35. Immobilization of conjugated polymers on DNA origami. (A) Scheme depicting the synthesis of poly(APPV-DNA) (top). Bottom panel shows AFM topography images of poly(APPV-DNA) immobilized in designed patterns on flat rectangular DNA origami. Scale bars = 200 nm. AFM images with illustration insets are reprinted from ref 353. Copyright 2015 Springer Nature. (B) Illustration (top) and data (bottom) for switching the conformation of single polymer molecules on DNA origami by toehold-mediated strand displacement reactions. One end of the polymer is directed to any of two tracks by the help of linker strands. Linker strands can be removed by toeholdmediated strand displacement with remover strands allowing switching to the other track. The other part of the polymer remains bound to the so-called hinge region track. The position of the polymer is determined by fluorescence from two different acceptor fluorophores positioned in each of the two reversibly binding tracks upon FRET from the polymer to the acceptor. (B) and the synthesis overview in (A) are reprinted from ref 354. Copyright 2016 American Chemical Society.

the fluorescent polymer and two different dyes positioned along the respective tracks on the origami. More recently, we investigated the development of polymer− DNA conjugates based on different conjugated polymer AD


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Figure 36. Immobilization of polyfluorene and polythiophene on DNA origami. (A) Synthesis scheme devised for the preparation of poly(F-DNA) from the THP-protected polyfluorene precursor. Partial THP-deprotection allows phosphoramidite formation on exposed hydroxyl groups and subsequent immobilization on 3000 Å CPG using phosphoramidite coupling. THP-deprotection followed by DNA synthesis, cleavage, deprotection, and purification provides poly(F-DNA). (B) Illustration and AFM topography image of poly(F-DNA) and poly(APPV-DNA) immobilized in an orthogonal alignment on opposite surfaces of the same flat rectangular DNA origami structure. Scale bar = 300 nm. Adapted with permission from ref 355. Copyright 2017 John Wiley and Sons, Inc. (C) Overview of the synthesis of a DNA-block-polythiophene copolymer. An aniline-containing endgroup is incorporated during Kumada catalyst transfer polycondensation by the use of an ex-situ initiator. The block-copolymer is prepared by installation of an azide followed by strain-promoted azide−alkyne cycloaddition with DBCO-functionalized DNA. (D) AFM topography images of the block-copolymers immobilized in different patterns on DNA origami and illustration of the respective origami setups (bottom). Scale bar for type I image = 100 nm. Scale bar for type II and III images = 50 nm. Adapted from ref 68. Copyright 2017 American Chemical Society.

architectures.355 Using a polyfluorene backbone, we obtained a polymer−DNA conjugate (poly(F-DNA)) also capable of undergoing controlled routing on tracks of extended staple strands of DNA origami (Figure 36A). Moreover, the polymer was investigated in combination with poly(APPV-DNA). The two polymers could be positioned together on the same origami structure with orthogonal as well as parallel orientations relative to each other (Figure 36B), pointing toward future applications based on the interactions between different conjugated systems at the single molecule level. Mertig’s group also studied conjugated polymers using the DNA origami platform.68 They synthesized end-functionalized polythiophene polymers containing hydrophilic triethylene glycol side chains (Figure 36C). An aniline-based end-group was installed using an ex-situ initiator for the Kumada catalysttransfer polycondensation, and the obtained polymer was

functionalized with terminal azide groups using amide bond formation. Finally, a single DNA strand was installed at the end of individual polymer chains using DBCO-functionalized DNA to provide a block-type polymer, which theoretically allowed immobilization of one polythiophene per extended staple strand on DNA origami. Using this strategy, patterns composed of multiple thiophene polymers were constructed on flat rectangular DNA origami (Figure 36D). The interactions between the polymer chains were affected by introduction of detergent, which also significantly affected the fluorescence of the immobilized polymers. Recently, the Seeman and Canary laboratories have applied monodisperse conjugated oligomers within DNA nanotechnology. In their work from 2017, they described the synthesis of aniline octamers by consecutive Buchwald−Hartwig aminations using a protocol adapted from the Buchwald group.356,357 The AE


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Figure 37. Immobilization of octaniline in DNA crystals. (A) Overview of the incorporation of oligoaniline in DNA during oligonucleotide synthesis. The azide in one end of the oligoaniline allows coupling to an alkyne of the protected DNA grown on CPG. The other end contains a DMT-protected hydroxyl group used as starting point for further oligonucleotide synthesis upon deprotection. (B) Images of DNA crystals with incorporated oligoaniline. Changing the charge and oxidation state of oligoaniline leads to color changes of the crystals directly correlated to the oligoaniline form present under the specific conditions. Reprinted with permission from ref 357. Copyright 2017 John Wiley & Sons, Inc.

terminal amino groups were transformed into azides by treatment with tert-butyl nitrite followed by TMSN3. One end of the octamer was then functionalized with a DMT-protected hydroxyl group, while the other end was attached to alkynefunctionalized DNA still attached to CPG for solid phase synthesis. Removal of the DMT group allowed subsequent DNA synthesis, and aniline octamers with ssDNA at each end could be obtained (Figure 37A). The DNA oligoaniline conjugates were successfully incorporated into 3D DNA crystals. Interestingly, the visual appearance of the crystals directly revealed the oxidation state of the oligoaniline, and chemical treatment allowed switching between different oxidation states (Figure 37B). This opens an appealing opportunity for controlling the conductivity of DNA-based materials in a reversible fashion, although conductivity of the structures was not investigated in this study.357 Very recently, the same groups reported on a new method for attaching DNA to the ends of conjugated oligomers. In this study, oligomers containing azide groups at both ends were reacted in CuAAC reactions with alkyne-modified DNA attached to CPG to afford oligomers with identical DNA sequences attached to each end of the chain (Figure 38A).358 DNA conjugates were successfully obtained for both oligoanilines and oligo(phenylene vinylene)s using this strategy, and they were subsequently incorporated into flat rectangular DNA origami structures with central cavities. The authors succeeded in positioning the different oligomers in the cavity to create cross-type structures as visualized by AFM (Figure 38B). Furthermore, the application of such structures as electrooptical modulators was discussed, although this feature was only studied briefly. These examples highlight current activities in utilizing conjugated polymers and oligomers at the single molecule level within the field of DNA nanotechnology. While some efficient chemical tools have been developed to handle these semiconducting molecules using DNA interactions, efforts are

still required to shed light on the applications of the materials in single molecule devices. 5.3. Other Polymers

Cationic polymers are another fascinating type of materials that have gained interest for immobilization on DNA nanostructures through strong electrostatic interactions. The Shih lab used cationic oligolysines to coat DNA nanostructures and showed effective protection of the structures from denaturation under low salt conditions. Furthermore, attachment of 10 kDa polyethylene glycol chains to the oligolysines provided very efficient protection against serum nucleases.359 Kostiainen’s group investigated cationic block-copolymers for coating DNA nanostructures and found that the polymer coating could be used for tuning the catalytic activity of enzymes attached to the nanostructures.360 Research from the same group also showed that the positively charged N-terminal region of virus capsid proteins can coat DNA origami and mediate efficient uptake in HEK293 cells (see Figure 70A, section 6.2.3).361 The strategy hence provided an appealing method for creating DNA origami drug delivery systems. Polymers can also mediate actuation of DNA nanostructures. Recently, Keyser, Baumberg, and collaborators demonstrated that thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) could be used for actuation of a DNA origami flexor.362 pNIPAM was attached to DNA using strain-promoted click chemistry on both sides of the flexor hinge. Upon heating, the two polymer particles associated, thereby bringing the flexor into its closed conformation whereas cooling induced the reversible reaction, with reopening of the flexor upon polymer dissociation. The actuation was visualized by AFM as well as by energy transfer between a 16 nm AuNP and a Cy5 fluorophore positioned on opposite legs of the flexor (Figure 39). As a final example of immobilization of carbon-based macromolecular materials in DNA nanostructures, we will highlight a study on nanodiamonds from Liedl, Weil, Högele, and co-workers.363 Nanodiamonds have interesting fluoresAF


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Figure 39. Illustration of pNIPAM-mediated actuation of a DNA flexor. At elevated temperatures, the pNIPAM particles associate, while they dissociate upon cooling. Inset shows normalized fluorescence intensities demonstrating that surface enhanced fluorescence is observed when the AuNP approaches the Cy5 dye at elevated temperatures. Adapted with permission from ref 362. Copyright 2018 John Wiley & Sons, Inc.

materials in DNA nanostructures is discussed and accompanied by a selection of examples from the vast number of applications demonstrated for AuNPs and QDs in DNA nanotechnology. Numerous other inorganic nanomaterials have been investigated within DNA nanotechnology such as silver nanoparticles and complex silica composite materials.364−367 A recent review by Samanta and Medintz provides a broader discussion of DNAbased manipulation of inorganic nanoparticles.368 5.4.1. Gold Nanoparticles. Gold nanoparticles (AuNPs) are by far the most studied type of inorganic nanomaterials within the field of DNA nanotechnology. Unlike many DNA nanostructures, AuNPs are readily observed in electron microscopy and hence provide a great tool for probing DNA nanostructures and they have unique optical properties in terms of their plasmonic activities enabling creation of plasmonic hotspots, plasmonic waveguides, and plasmonic chiroptics.368 These applications rely on controlled nanoscale positioning of single particles for which DNA nanostructures are excellent tools. Furthermore, AuNPs allow detection at extremely low concentration,369 and they are also highly useful as templates for creating metal nanowires. 5.4.1.1. Synthesis of AuNPs. AuNPs have been known for centuries, and the first synthesis of stable gold colloids was already described by Faraday in 1857.370 Because the properties of AuNPs depend heavily on their size and shape, methods for controlling AuNP synthesis have been intensively studied. Today, high-quality AuNPs prepared using patented technologies are commercially available in various sizes and shapes. Most spherical AuNPs used in DNA nanotechnology are obtained as citrate-capped particles, generally prepared using variations of Turkevich’s original simplistic method based on citratemediated reduction of Au3+-salts.371 In brief, trisodium citrate is added to a heated, aqueous solution of HAuCl4 under vigorous stirring and spherical, citrate-capped AuNPs are formed when a color-change is observed (Figure 40, top). The synthesis conditions have been fine-tuned in numerous studies focusing on shape, size, stability, and other physical parameters. The size of the particles is of particular interest, and it can be adjusted

Figure 38. Immobilization of two intersecting conjugated oligomers in DNA origami. (A) Overview of the synthesis for preparation of oligoaniline and oligo(phenylene vinylene) with DNA in both termini using a double CuAAC reaction with DNA alkynes on the CPG solid support. (B) Illustration of DNA origami scaffold for assembling the two conjugated oligomers in a cross-junction (left). AFM image of the two oligomers crossing each other upon immobilization in the DNA origami structure. Scale bar = 100 nm. Adapted from ref 358. Copyright 2018 American Chemical Society.

cence properties and single nitrogen-vacancy color centers that provide unique opportunities for spin−spin studies. In this study, nanodiamonds were functionalized with denatured bovine serum albumin (dBSA) through electrostatic and hydrophobic interactions. The dBSA was linked by thiolmaleimide chemistry to biotins via polyethylene glycol tethers thus facilitating immobilization at specific positions on the origami structures using Streptavidin. Importantly, the surface chemistry involved in functionalization and immobilization did not alter the optical properties of the nitrogen vacant nanodiamond. 5.4. Inorganic Nanomaterials

In DNA nanotechnology, a large number of different inorganic nanomaterials have been investigated although the majority of studies focus on a narrow set of nanoparticles. This section will review gold nanoparticles (AuNPs) and fluorescent quantum dots (QDs), which have both attracted significant interest. The chemistry employed for positioning and immobilizing these AG


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toluene, where Au3+-reduction is mediated by sodium borohydride in the presence of alkane thiols. A phase transfer agent such as tetra-N-octylbromide is required to transfer goldions from the aqueous to the organic phase (Figure 40, bottom). Due to the formation of an alkanethiol monolayer shell on the particle, the resulting AuNPs are extremely stable particles tolerating both drying and redissolving in organic solvents without degradation or aggregation.374 Strategies based on the Turkevich and Brust−Shiffrin methods are still widely used for AuNP synthesis. However, several new methods including bioinspired synthesis and nonchemical approaches have been developed and a detailed discussion of these can be found in a review by Sengani et al.375 5.4.1.2. DNA-Functionalization of AuNPs. The first examples of DNA-functionalization of AuNPs were described in back-to-back articles by Mirkin et al.376 and Alivisatos et al.377 In Alivisatos’ work, AuNPs with a single or a few DNA strands were targeted and conjugates of different DNA-valence were separated by gel electrophoresis. Mirkin’s work focused on obtaining a higher density of DNA-functionalization using a method referred to as salt-aging. Thiol-modified DNA is employed to obtain strong gold−thiol bonds between the nanoparticle and the DNA. However, the citrate-capped AuNPs and DNA are both highly negatively charged species, and cations are required to screen negative charges and facilitate interaction between DNA and AuNP. Directly adding a high concentration of NaCl to AuNPs, however, disturbs the stability of the colloid and causes AuNP aggregation. In the original salt-aging method, the nanoparticles and thiol-modified DNA were initially mixed in solution without addition of salt to conjugate only a few DNA strands to each particle, thus increasing colloid stability in saltcontaining buffers (Figure 41). Subsequently, mixtures of thiolmodified DNA and nanoparticles are further incubated in NaCl

Figure 40. Synthesis of spherical AuNPs. Typically, the Turkevich method based on citrate reduction of HAuCl4 is employed providing citrate-capped AuNPs. In the Brust−Shiffrin method, NaBH4 is introduced as a reducing agent along with alkane thiols that cap the AuNPs and provide highly stable AuNPs.

simply by altering the pH of the synthesis solution. Changes in reducing or stabilizing agents, reactant concentrations, and pH all affect the nature of the obtained particles.372 The review by Jimenez-Ruiz et al. further discusses some of the refinements to Turkevich’s method.373 The Brust-Schiffrin method is an alternative strategy for the synthesis of spherical AuNPs that has also gained widespread use.374 It employs a two-phase system composed of water and

Figure 41. DNA-functionalization of citrate-capped AuNPs using the traditional salt-aging conditions,379 the low pH method,385 BSPP-stabilization, which is used extensively within DNA nanotechnology,384 and surfactant stabilization. The different thiol modifications illustrated below the diagram are arranged with increasing binding strength toward AuNP from top to bottom. The trithiol is derived from commercially available Trebler phosphoramidite and thiol modifier phosphoramidite. AH


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Figure 42. Site-selective DNA functionalization of AuNPs templated by DNA nanostructures. (A) Illustration of the DNA cage and bis-disulfide DNA probes for patterning DNA site-selectively on the surface of spherical AuNPs. Adapted with permission from ref 397. Copyright 2016 Springer Nature. (B) Illustration of the approach utilizing flat rectangular DNA origami for site-selective transfer of DNA strands to spherical AuNPs by means of hybridization to coating strands followed by toehold-mediated displacement from the origami. Reprinted with permission from ref 398. Copyright 2016 John Wiley & Sons, Inc. (C) Illustration showing the use of a clamp-like DNA origami structure for encapsulation of a DNA-functionalized AuNR. This allows further derivatization in a site-selective manner by hybridization of probes to the exterior of the DNA origami clamp. Adapted from ref 399. Copyright 2016 American Chemical Society.

otherwise possible.380 The use of a nonionic fluorosurfactant allowed DNA-functionalization of AuNPs in 2 h at 1 M NaCl.381 Sonication of the AuNP−DNA mixtures has also been shown to be effective for decreasing the conjugation time.380 Bis(psulfonatophenyl)phenylphosphine (BSPP) is another popular agent used for stabilizing AuNPs under higher salt concentrations.327,382,383 Citrate ligands can readily be exchanged for BSPP by repeated cycles of BSPP addition, centrifugation, and supernatant disposal. This enables loading with a large amount of DNA through salt-aging with final Na+-concentration of up to 700 mM.384 Another important advancement for the functionalization of AuNPs with DNA comes from adjusting the pH. Zhang et al. demonstrated that AuNPs could be functionalized with a high

containing solutions (originally 0.1 M) to significantly increase the DNA loading of the AuNPs.378,379 In further refinement of the salt-aging concept, the Na+concentration is gradually increased by addition of small volumes of high concentration NaCl solutions. Higher final Na+-concentrations afford higher DNA-loading, though at the cost of increased risk of aggregation during the process. Although the original protocols for the salt-aging process were time-consuming, it has been the preferred method for preparation of AuNP−DNA conjugates. Important modifications to the process have enabled much more rapid DNAfunctionalization of AuNPs. For instance, the use of detergents effectively stabilizes AuNPs under much higher salt concentrations and allows addition of salt at a much higher pace than AI


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Figure 43. Immobilization of AuNP−DNA conjugates in 2D DNA nanostructures. (A) Illustration of the specific hybridization of spherical AuNP− DNA conjugates on 2D DNA tiles by hybridization to the template (top). Bottom panels show AFM topography images and height profiles of 2D DNA tiles with (right) and without (left) AuNPs attached to the tile. Scale bars = 64 nm. Adapted from ref 401. Copyright 2004 American Chemical Society. (B) Illustration (left) and SEM image (right) of spherical AuNPs of three different sizes positioned on the same triangular DNA origami structure. Scale bar = 200 nm. Inset shows a close-up of one origami-AuNP construct. Scale bar = 20 nm. Adapted from ref 383. Copyright 2010 American Chemical Society. (C) Illustration (left) depicting the formation of hydrophilic patches matching the size and geometry of triangular DNA origami. DNA origami structures with AuNPs attached to the corners self-assemble in the hydrophilic patches and form long-range ordered AuNP assemblies. The right inset shows an AFM topography image of long-range ordered AuNPs on triangular DNA origami assembled in hydrophilic patches. Scale bar = 500 nm. Adapted with permission from ref 406. Copyright 2010 Springer Nature.

formed in a few hours without the need for any ligand exchange.393 An important challenge for the preparation of AuNP−DNA conjugates is to reliably control the valence and pattern of DNA strands on the particles. Suzuki et al. managed to control the number of DNA strands on spherical AuNPs as well as the spacing between them by introducing a 1D DNA scaffold to template the reactive thiol-modified oligonucleotides.394 The Mirkin group, on the other hand, used DNA functionalized magnetic nanoparticles to template the transfer of DNA siteselectively to AuNPs using a DNA ligase.395 In another approach, Huey Tan et al. used competition between hydrophilic and hydrophobic ligands to create amphiphilic AuNPs where AuNPs were partly covered in a hydrophobic polymer shell and partly exposed for reaction with thiolated DNA.396 In an elegant approach exploiting DNA nanostructures’ ability to form discrete patterns on AuNPs, Sleiman’s group used DNA nanostructures to template DNA functionalization of AuNPs.397 DNA nanocages of different shapes were modified with extended strands containing cyclic disulfides that reacted with AuNPs, and upon conjugation, the nanostructures were denatured. In this way, the DNA extensions were transferred to the AuNPs in a pattern defined by the nanostructure (Figure 42A). In a similar approach, the Fan lab used flat rectangular

density of DNA in as little as three minutes by lowering the pH to 3.0 during the conjugation step.385 In contrast to standard thiol modifications, DNA modified with lipoic acid62 or trithiol groups386 often conjugates more efficiently to AuNPs due to the stronger binding caused by the presence of several, rather than just one, gold−thiol bond per DNA strand. Furthermore, higher robustness and yields of the immobilization in DNA origami were observed in these experiments.62 Various other shapes of gold nanomaterials such as gold nanorods (AuNRs) are also important for DNA nanotechnology due to their unique plasmonic properties. The synthesis of these types of materials has been pioneered by the Mirkin and Murphy groups and reviewed thoroughly by Li et al. and Vigderman et al.387−390 However, the DNA-conjugation principles are largely similar to those discussed above. An important exception to this is that nonsperical AuNPs are typically obtained with alternative capping ligands such as cationic cetyl trimethylammonium bromide (CTAB). Before conjugation with DNA, CTAB has been commonly removed by for instance dual phase transfer steps followed by exchange for mercaptocarboxylic acids.391 Modern methods, however, enable the formation of AuNR− DNA conjugates by more direct salt-aging processes.392 Using the low pH approach, AuNR−DNA conjugates can even be AJ


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Figure 44. Examples of chiral plasmonics created using DNA nanostructures and AuNPs. (A) Positioning of spherical AuNPs in right- and left-handed helical shapes around a 24-helix bundle DNA origami structure as illustrated in the upper left inset and as visualized using TEM (top right). Scale bar = 100 nm. The bottom insets show CD spectra of the assemblies prior to (left) and after (right) plating of the AuNPs. Note the dramatic increase in CD signal observed upon plating. Adapted with permission from ref 327. Copyright 2012 Springer Nature. (B) Dynamic chiral plasmonic structure enabling chirality-switching in response to toehold-mediated strand displacement. Bottom inset shows the distinct switching events as observed from time-resolved CD spectroscopy. Adapted with permission from ref 417. Copyright 2014 Springer Nature. (C) Chiral plasmonic structure based on a 3D DNA tripod. The angles of the structure could be modified by toehold-mediated strand displacement, which enabled tuning of the chiral plasmonics. Adapted from ref 419. Copyright 2017 American Chemical Society. (D) A plasmonic walker composed of AuNRs positioned orthogonal to each other on opposite sides of a DNA origami structure. One of the AuNRs walks between different stations in response to addition of blocking and removal strands. Each station gives rise to discrete signals in CD spectroscopy. Adapted from ref 420. Copyright 2012 Springer Nature under a creative commons agreement (https://creativecommons.org/licenses/by/4.0/). (E) Reconfigurable plasmonic nanostructure based on dimers of triangular DNA origami with AuNRs positioned in orthogonal alignments. The structures, and hence the plasmonic signal observed in CD spectroscopy, can be reconfigured in response to GSH-mediated disulfide reduction, pH-controlled structural change of iMotif DNA sequences, or photoinduced conformational switching of a G-quadruplex with intercalated azobenzene probes. The bottom inset shows the mobility shift in gel electrophoresis and the signal decrease in CD-spectroscopy observed upon GSH-mediated disulfide reduction. Adapted from ref 422. Copyright 2017 American Chemical Society.

specific DNA anchoring to the encapsulated AuNR (Figure 42C).399 Conjugation of DNA to AuNPs remains an active field of research, and although this section should provide the reader with an overall understanding of the methodology for AuNP− DNA conjugation, more elaborate reviews such as the recent one by Liu et al. provide additional information.400 5.4.1.3. AuNPs in DNA Nanotechnology. AuNPs have been extensively studied in DNA nanotechnology, and an exhaustive exposition of this work is impossible within the scope of this

DNA origami to pattern DNA strands onto AuNPs by hybridization to coating ssDNA preattached to the AuNPs (Figure 42B). The coating DNA tags could be released from the origami template by toehold-mediated strand displacement (Figure 42B).398 Along the same lines, Shen et al. encapsulated a DNA functionalized AuNR in a DNA origami clamp by hybridization to capture strands extending in the interior of the origami. Extended staple strands protruding from the exterior of the DNA origami capsule could then be used for siteAK


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ments matched theoretically predicted ones (Figure 44A). When growing the metal nanoparticles, significantly increased CD signals (400-fold increase) were observed, also in good agreement with the theoretical models. These studies demonstrated the promise of DNA nanotechnology for templating chiral plasmonics, which has attracted significant interest since then and was recently reviewed by the Liu and Willner groups, respectively.23,414 While the initial studies within chiral plasmonics used spherical AuNPs, focus quickly turned to AuNRs which feature much higher extinction coefficients and provide significantly stronger plasmonic couplings. Lan et al. used bifacial assembly of DNA−AuNR conjugates in orthogonal patterns on flat rectangular DNA origami to generate architectures with tunable, chiral plasmonic activities.415 Building on this work, helical superstructures were created from multiple flat DNA rectangles assembled on top of each other through annealing to AuNRs. The size of the helical superstructures was adjustable by changing the origami/AuNR ratio, and larger structures gave rise to larger signals when investigated by CD spectroscopy.416 Recent focus within AuNR-based plasmonics has evolved around the introduction of dynamics to chiral plasmonic systems. Kuzyk, Liu, and collaborators linked two 14-helix bundles together by assembling them from the same scaffold strand.417 Connector strands added to the bundles allowed connection in two different locked conformations through DNA hybridization with a reversible locking mechanism enabled by introduction of a toehold to the linker strands. AuNRs were immobilized on the free faces of the bundles, and the two locked conformations formed left-handed and right-handed chiral plasmonic assemblies, respectively. Using toehold-mediated strand-displacement, the chiral plasmonic assembly could now be switched between right-handed, left-handed, and relaxed states, which was observed readily from CD spectroscopy (Figure 44B). In a refined version of this concept, introduction of azobenzene-modifications allowed photoswitching between locked and relaxed conformations.134 Switching in response to changes in pH was later realized through the incorporation of pH sensitive DNA motifs to the system.418 Reconfigurable plasmonic assemblies have also been obtained by immobilizing AuNRs on a DNA origami tripod. The structure was designed such that the angles of the tripod could be adjusted through toehold-mediated strand displacement reactions (Figure 44C). Using dark-field microscopy, the change in plasmonic coupling was demonstrated upon changing the tripod angles.419 In another elegant approach to achieve dynamic, plasmonic assemblies based on AuNRs on DNA nanostructures, a AuNR-walker was developed.420,421 A static AuNR (stator) was positioned on one face of the DNA structure by hybridization to a track of extended staple strands while, on the other face, a AuNR walker was introduced. The walking was mediated by selective binding to certain extended staple strands upon removal of blocking strands by toehold-mediated strand displacement. In this way, the AuNR walker could be directed back and forth between several stations on the origami structure (Figure 44D). All individual AuNR positions were distinguishable by CD spectroscopy due to the change in plasmonic coupling between the walker and the stator. Recently, Jiang et al. used triangular DNA origami dimers to realize plasmonic reconfiguration in response to changes in pH, chemical reduction, or by light-stimulus (Figure 44E).422 Another important application of AuNPs in DNA nanotechnology is the formation of crystalline 3D lattices. Such

review. Therefore, a few highlights will be used to exemplify how AuNPs are employed in DNA nanotechnology. Spatial organization of two or more gold nanoparticles at the nanoscale theoretically gives rise to plasmonic effects not obtained in bulk gold. DNA-based assembly provides a unique tool to realize such experiments, and DNA-assembled plasmonic architectures were recently reviewed by Liu and Liedl.24 Immobilization of AuNPs on DNA nanostructures can be achieved using AuNP−DNA conjugates hybridizing to complementary DNA protruding from DNA nanostructures. Kiehl and co-workers investigated the positioning of AuNPs on 2D DNA nanoarrays using AuNPs modified with multiple thiol-modified oligonucleotides (Figure 43A),401 and later they moved on to position particles of two different sizes along separate tracks on 2D DNA nanoarrays.402 At around the same time, Yan’s and LaBean’s laboratories demonstrated the positioning of Streptavidin-conjugated AuNPs on biotinylated DNA nanostructures to demonstrate the formation of a linear chain of 6 AuNPs on a DNA template.403 Ding et al. used AuNPs loaded with a high density of DNA for positioning AuNPs on triangular DNA origami.383 AuNPs of three different sizes (15, 10, and 5 nm) were used, and three extended staple strands specifically bound each of the particles. Using this approach, they prepared selfsimilar linear chains from particles of decreasing size which gave rise to plasmonic interactions between the particles (Figure 43B). Bringing plasmonic phenomena into macroscopic applications requires positioning of AuNPs with nm precision over macroscopic length-scales.404 To enable this, Wallraff, Rothemund, and co-workers developed a method for positioning DNA origami structures in lithographically defined patterns over macroscopic length scales.35,405 Cha and co-workers combined this strategy with AuNP immobilization and demonstrated how nanometer precise positioning of AuNPs on DNA origami could be applied for macroscopic patterning (Figure 43C).406 In a different approach to immobilize AuNPs in DNA nanostructures independent of hybridization, Sleiman’s lab prepared DNA nanotubes with capsules of different sizes along the tube.407 Each capsule was designed to match AuNPs of a certain size, and interestingly, citrate-capped AuNPs assembled in the dedicated capsules to form pea-pod-like structures. Several recent studies have focused on achieving high-yielding self-assembly of ordered 1D and 2D arrays of AuNPs using DNA nanostructures.384,408−410 AuNPs were also encapsulated in 3D DNA origami nanocages, where they hybridized to extended staple strands protruding into the cavity of the structure.411 Based on this setup, later studies used DNA origami as molds for creating shape-defined metal nanostructures as discussed in section 4.3 about DNA metallization.328−330,412 Immobilization of AuNPs in 3D DNA nanostructures has been widely investigated. In an attempt to form three-dimensional helices from AuNPs, Liu, Ding, and co-workers used a flat rectangular DNA origami that transformed into a DNA nanotube upon addition of folding strands.413 AuNPs were positioned on the flat origami sheet to form a helix around the tube upon folding as visualized in TEM and confirmed by CD spectroscopy of the chiral plasmonic assembly, which indicated helix formation. At around the same time, Kuzyk, Liedl, and collaborators published an elegant study demonstrating the formation of helical plasmonic nanostructures based on AuNPs positioned in a helix around 24-helix bundle DNA origami structures.327 Intriguingly, structures of opposite helicity could be formed and CD spectral measureAL


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nanopillar equipped with large gold nanoparticles to form plasmonic hotspots and eventually provide more than 100-fold signal enhancements for a dye positioned in the hotspot (Figure 45B).429 More recently, the same group achieved more than 5000-fold fluorescence enhancement using a modified version of the previously published setup.430 Importantly, NiCl2 was included to diminish the intrinsic quantum yield of the fluorophore, which was important for achieving a higher signal enhancement factor. AuNPs precisely positioned on DNA nanostructures have also been used for achieving surface-enhanced Raman scattering and recently for assessing the structure of DNA origami sheets in solution by SAXS.431−433 AuNPs continue to be of tremendous importance within DNA nanotechnology, and efforts seeking to harness the intriguing nanoscale properties of AuNPs through immobilization on DNA templates continue. 5.4.2. Quantum Dots. Quantum dots (QDs) are small inorganic nanocrystals. The term QD refers to systems where the quantum confinement effect is at play because the particle size is comparable to the exciton Bohr radius of the material. In this review, semiconducting QDs are most relevant and QD will therefore refer to semiconducting nanocrystals of sizes between 1 and 10 nm. These QDs are most commonly based on CdS, CdSe, CdTe, ZnSe, or CdZnSe alloys, and they are of tremendous importance due to their optical properties caused by the quantum confinement. Typically, QDs feature very narrow fluorescence emission bands while they have continuous absorbance bands at shorter wavelengths. At the same time, they typically have high quantum yields and stabilities toward photobleaching. Importantly, the optical properties of QDs can be tuned by controlling the size of the particles and through manipulation of their chemical composition. QDs should therefore be optimal fluorescent reporters for use within DNA nanotechnology, but so far, the challenges associated with handling and preparation of QD−DNA conjugates compared to small molecule dyes have prevented the widespread use of QDs in DNA nanotechnology. However, recent developments have eased the handling of QDs, and their use in DNA nanotechnology is likely to gather speed in the near future. 5.4.2.1. Synthesis of QDs. QDs are complex nanomaterials, and their synthesis composes a broad research field on its own; therefore, this discussion will focus on a few aspects relevant for the use of QDs in DNA nanotechnology. Several reviews cover the synthesis of QDs in detail, and a few of them are cited here.434,435 QDs are generally synthesized in nonpolar organic medium using either the hot-injection (HI) method or the heat up (HU) method. Both of these methods enable nucleation and growth in separate steps, which is crucial for obtaining monodisperse QDs. In short, the HI method relies on rapid injection of organometallic reagents (such as CdMe2, TMS2S, TMS2Se, TMS2Te, TOPSe, and TOPTe) into hot solvent containing ligands serving to prevent agglomeration. Nucleation occurs immediately but is then abolished due to the sharp temperature decrease caused by the injection. Instead, the nuclei enter the growth phase at lower temperatures, and highly monodisperse QDs are obtained. In the HU method, organometallic reagents and ligands are mixed and gradually heated. When suitable precursors are used, they will only nucleate within a narrow temperature range, thus providing separate nucleation and growth phases. Precursors featuring these optimal qualities are not always available, and this may cause formation of polydisperse QDs. QDs obtained from both of these processes are coated in hydrophobic molecules such as trioctyl phosphine/

lattices were formed directly from AuNP−DNA conjugates in simultaneous studies by the Mirkin and Gang laboratories.423,424 In another approach to arrange AuNPs in 3D crystal lattices, the Gang lab used 3D DNA nanostructures based on 6-helix bundles, which eventually assembled into ordered 3D lattices such as a diamond-like 3D lattice (Figure 45A).425−427 Very

Figure 45. Crystalline lattices and plasmonic hotspots based on AuNPs. (A) Illustration of the setup used for assembling FCC and diamond AuNP-lattices from tetrahedral DNA origami. The origami structure has sticky ends at its corners to enable binding of AuNPs modified with complementary DNA sequences. Further binding to other tetrahedrons leads to formation of an FCC-type lattice of AuNPs. When additional AuNPs are incorporated at the tetrahedron centers, a diamond-type lattice of AuNPs forms. The bottom inset shows cryo-STEM images of the diamond-type lattices. Scale bars: 500 and 50 nm, respectively. Adapted with permission from ref 427. Copyright 2016 American Association for the Advancement of Sciences. (B) Illustration of the setup used for obtaining more than 100-fold signal enhancement of the fluorescence from a small molecule dye positioned in the plasmonic hotspot between two 100 nm spherical AuNPs. Reprinted with permission from ref 429. Copyright 2012 American Association for the Advancement of Sciences.

recently, Liedl and co-workers formed large, rigid 3D DNA lattices by polymerization of tensegrity triangles built from 14helix bundles. AuNP−DNA conjugates of 10 or 20 nm were immobilized in the monomer structures, and upon polymerization, ordered 3D AuNP lattices were obtained.428 Another focus of functionalizing DNA nanostructures with AuNPs is to enhance fluorescent signals through the creation of plasmonic hotspots. To achieve this, the Tinnefeld group investigated the use of AuNPs positioned in 3D DNA origami also containing a small molecule fluorophore. They used a DNA AM


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Figure 46. Scheme showing the method developed in the Mirkin lab for the formation of QD−DNA conjugates. Hydrophobic QDs containing oleylamine or TOPO ligands are made hydrophilic by ligand exchange with MPA. Subsequently, the hydrophilic QDs are soaked in thiol-modified DNA to eventually provide QD−DNA conjugates that remain stable in 0.3 M NaCl.436

Figure 47. Formation of QD−DNA conjugates using histidine tags. (A) Scheme for the formation of QD−DNA conjugates by the use of ssDNA conjugated to a His-tag. The His-tag coordinates Zn2+-ions of the ZnS layer of the QD. (B) Illustration showing the formation of a DNA-templated photonic wire based on small molecule fluorophores and a QD. The QD was incorporated into the ZnS layer of a CdSe/ZnS core−shell QD using Histag-DNA binding. Reprinted from ref 81. Copyright 2010 American Chemical Society. (C) Immobilization of a QD in a DNA nanocage by His5-tag coordination. The His-tag DNA was either preincubated with the QD and subsequently incorporated in the cage, or alternatively, the His-tag DNA was incorporated into the DNA structure followed by incubation with hydrophilic QD. Adapted from ref 442. Copyright 2017 American Chemical Society.

reviewed by Jing et al.438 A wide range of QDs are commercially available today and for use in DNA nanotechnology, watersoluble QDs such as carboxyl- and amino-functionalized ones are of specific interest. Moreover, Streptavidin-coated QDs are also popular for use in DNA nanotechnology for their ability to directly bind biotinylated DNA. 5.4.2.2. QDs for DNA Nanotechnology. The formation of QD−DNA conjugates is an obvious route to employing QDs in DNA nanotechnology. Yan, Liu, and co-workers discussed methods for preparing QD−DNA conjugates in their review from 2013.439 The first preparation of QD−DNA conjugates

trioctyl phospineoxide (TOP/TOPO), and they are soluble in nonpolar solvents rather than water. However, for use in DNA nanotechnology, water-soluble QDs are required. Ligandexchange is therefore typically performed before DNA functionalization and mercaptopropionic acid (MPA) is widely used for exchanging the hydrophobic capping layer.436 Alternatively, QDs can be prepared in water to provide immediate access to hydrophilic QDs. In one example, glutathione (GSH) was used as stabilizer and hydrophilic ligand for preparation of CdTe QDs in water at around 100 °C from NaHTe and CdCl2.437 Aqueous synthesis of QDs was recently AN


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Figure 48. QD-DNA conjugates formed using amphiphilic polymers. (A) Hydrophobic QDs are incubated with amphiphilic polymers containing reactive handles for conjugation to DNA. (B) 1D SAXS data (left) of DNA-assembled QD-AuNP CsCl-type lattice shown in the structural illustration. The right panel shows an STEM image of the same lattice. Scale bar = 50 nm. Both AuNPs and QDs are functionalized with DNA using SPAAC conjugation between DBCO-modified DNA and an azide-containing amphiphilic polymer. Reprinted with permission from ref 70. Copyright 2013 Springer Nature. (C) Illustration of AuNP-QD satellite type structure formed through DNA assembly. The QD−DNA conjugates were prepared by peptide-bond formation between commercially available carboxylate-containing amphiphilic polymer-stabilized QDs and amino-modified DNA. The right panel shows interparticle distances as determined from a DNA model and by SAXS for constructs based on different lengths of ds spacer DNA. Adapted from ref 446. Copyright 2015 American Chemical Society.

was described by the Mirkin group in 1999 where they also demonstrated the assembly of multiple QDs by Watson−Crick base pairing.436 In the past, DNA-functionalization of QDs was not achieved due to challenges handling the hydrophobic TOP/ TOPO covered QDs in aqueous environments. The key to overcoming this was a ligand exchange with MPA to provide QDs soluble in water (Figure 46), a method previously described for the conjugation of proteins to QDs by Chan et al.440 Soaking MPA-QDs in solutions of thiol-modified DNA provided QD−DNA conjugates stable in aqueous buffers. Although the conjugate solutions showed decent stabilities, aggregation was an issue over time due to thiol oxidation into disulfides that dissociated from the particles.441 To overcome this, research focused on covalently attaching DNA to QD capping ligands, which was achieved via multiple different approaches. An important example is the use of histidine (His)tags that efficiently coordinate Zn2+-ions of Zn-containing QDs. The preparation of His6−DNA conjugates therefore provides efficient materials for making QD−DNA conjugates (Figure 47A). For this, Boeneman et al. attached a formylbenzoic acid NHS ester to amino-modified DNA and a His6-peptide was modified with a nicotinic hydrazide to enable formation of a His6-DNA conjugate via hydrazone formation. CdSe/ZnS core−shell QDs were rendered hydrophilic by incubation with a PEG-modified dihydrolipoic acid (DHLA) analogue. Upon hybridization to dye-modified DNA and a brief incubation with the hydrophilic QDs, DNA-templated photonic wires were formed based on a QD donor and several small molecule dye mediators and acceptors (Figure 47B).81 QDs have also been caged in DNA nanostructures using oligohistidine-DNA conjugates incorporated in DNA cages (Figure 47C),442,443

and different chemistries including disulfide formation and thioliodoacetamide coupling have been used for His6-mediated QD−DNA conjugation.56,439,444 Another method for covalently attaching DNA to the capping ligands of QDs is by introduction of an amphiphilic polymer modified with reactive handles. Mirkin’s group introduced a generalizable method for functionalizing nanoparticles based on this approach.70 The functionalized amphiphilic polymer was formed by reacting poly(maleic anhydride-alt-1-octadecene) (PMAO) with an oligoethylene glycol linker containing an amine in one terminus and an azide group in the other. When this amphiphilic polymer was added to nanoparticles, such as oleylamine-capped CdSe/ZnS core−shell QDs in chloroform, the polymer wrapped tightly around the particles with its hydrophobic parts interdigitated in the hydrophobic capping layer, thus forming stable, polymer-coated QDs. Due to the hydrophilic ethylene glycol-based linkers, the particles were soluble in water and they could react with DBCO-functionalized DNA in SPAACs (Figure 48A). The obtained QD-DNA conjugates were used for the creation of lattices characterized using SAXS and TEM (Figure 48B). QDs containing amphiphilic polymer layers are commercially available. For instance, they are offered with carboxylate and amino functionalities to allow further derivatization using amide bond formation. Sun and Gang developed a simplistic approach for DNA-functionalization of commercially available QDs capped with carboxylate-containing amphiphilic polymers in which amide-bond formation using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and amino-modified DNA was employed.445 The pH, the QD/DNA ratio, and NaCl effects were all optimized to provide a useful and simple procedure for AO


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DNA modification of commercially available QDs. The same lab later used conjugates prepared using this protocol for creation of satellite structures composed of a single AuNP surrounded by numerous QDs attached through DNA-linkers. The satellites were used for light-harvesting, and the optical output could be controlled by the length of its DNA-linkers (Figure 48C).446 Through bis-NHS linkers, QDs containing amino groups at the surface can also be coupled to amino-modified DNA. Hydrophobic QDs can be rendered hydrophilic by silanization, which at the same time enables incorporation of reactive handles such as thiols into the silanized shell.447 This facilitates DNA conjugation using for instance sulfo-SMCC (4-(Nmaleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester) coupling with amino-modified DNA. Sulfo-SMCC is also used for attaching thiol-modified DNA to amino-capped QDs and for modifying QDs stabilized by thiolcontaining amphiphilic polymers.448 In another important method for preparing QD-DNA conjugates, DNA-functionalization is carried out during QD synthesis. In pivotal work from the Kelley group, Ma et al. used chimeric nucleic acids based on phosphorothioate-DNA (psDNA) in one end and phosphodiester-DNA (poDNA) in the other end (ps-poDNA).449 The psDNA has much higher affinity for Cd2+-ions than the poDNA part, and when the chimeric nucleic acids were added to the synthesis solution containing NaHTe, CdCl2, and GSH, the psDNA was imbedded in the QDs while the poDNA remained largely accessible at the QD-surface (Figure 49A). By tuning the length of the psdomain, the DNA-valence of QDs was even controllable in this approach, which enabled DNA-templated assembly of complex, multi QD structures in a controlled fashion.450 The synthesis strategy was further developed by the Liu and Yan groups, who prepared core−shell QDs with ps-poDNA chimeras incorporated only into the shell. The cores were formed in an initial step, before a CdS or ZnS shell was grown in the presence of the ps-poDNA chimera. This provided a reliable method for making QD−DNA conjugates from various different QD precursors, and the particles could be efficiently positioned on triangular DNA origami by hybridization to extended staple strands.451 Later, the same groups used QD−DNA conjugates prepared in the same way to create a spectroscopic ruler by incorporating a QD and an AuNP on the same triangular DNA origami at various distances (Figure 49B). The AuNP-mediated quenching of the QD was distance dependent over a large range of distances and hence has the makings of a long-range spectroscopic ruler.452 A similar approach was used by Schreiber et al., who synthesized a ZnS layer on commercially obtained QDs. The obtained QDs were hydrophobic, and a ligand exchange with MPA was carried out prior to growing the ZnS layer. The obtained particles were used for creating AuNP clusters protruding from QD cores using DNA origami (Figure 49C).410 CdZnTeS QDs have also been modified with DNA during synthesis using the po-psDNA chimera approach where the DNA was modified with a Rhodamine fluorophore, and the construct could be used for visual detection of hydrogen peroxide as well as blood glucose.453 In a fascinating, alternative approach to control the orientation and number of modifications protruding from QDs, Krishnan and co-workers encapsulated a single QD inside DNA icosahedra.454 Two halves of the icosahedron were prepared containing 5′-phosphorylated sticky ends that were complementary to each other. Upon incubation with DHLAcapped QDs, the icosahedron assembled around the QD, and

Figure 49. Formation of QD−DNA conjugates using ps-poDNA chimeras. (A) Diagram showing in-synthesis DNA-functionalization of QDs through aqueous QD synthesis in the presence of ps-poDNA chimeras. The strong interaction of psDNA with Cd2+ yields QD− DNA conjugates with psDNA embedded in the QD and poDNA extending from it.449 This approach can be extended to a core−shell approach where the shell is synthesized on a commercial core in the presence of ps-poDNA chimeras. In this case, psDNA is only embedded in the shell.410 (B) A spectroscopic ruler created by immobilization of QD−DNA from in-synthesis functionalization on triangular DNA origami at five different distances from a AuNP. The bottom insets show a TEM image of 70 nm interparticle distance designs and a chart depicting the distance-dependent quenching of the QD emission by the AuNP. Scale bar = 100 nm. Adapted from ref 452. Copyright 2014 American Chemical Society. (C) Illustrations and TEM images of AuNP-QD nanoclusters and dye-QD nanoclusters assembled on threearmed DNA origami. The QD−DNA conjugates were prepared through in-synthesis DNA functionalization during the synthesis of a shell onto commercially obtained QDs. Scale bars = 100 nm. Adapted with permission from ref 410. Copyright 2014 Springer Nature.

functional handles were added on the icosahedra DNA template to allow further modification of the structure with endocytic AP


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apply this simple and popular system. Its simplicity, however, comes at the cost of reduced immobilization yield because the SA-QD samples may contain free SA, which takes up biotin binding sites. Moreover, multiple SAs are typically attached to each QD, thus making it a very sterically demanding modification that might prevent immobilization in close proximity to other nanoobjects. In 2004, SA-QDs were used by the Alivisatos group for assembling nanostructures composed of AuNPs and QDs.456 Later, SA-QDs were employed when Yan, Liu, and co-workers positioned QDs on DNA tile based assemblies.457 They were also used for immobilization of QDs with programmable periodicity on DNA origami nanotubes (Figure 51A),458 for immobilization of QDs on linear DNA arrays (Figure 51B),459 and for immobilization of QDs and AuNPs on the same DNA origami sheet.460 More recently, SAQDs have also been used for patterning QDs on sequentially grown DNA templates and in combination with focused ion beam lithography for patterning origami containing QDs over macroscopic length scales (Figure 51C).461,462 As described above, there are multiple methods for immobilization of QDs on DNA nanostructures. While SAQD conjugates are commercially available and readily applicable, they can suffer from suboptimal immobilization yields. Better yields can be obtained from particles prepared by conjugation of DNA to QDs stabilized by functional amphiphilic polymers, or from in-synthesis DNA modification of QDs. There should be a suitable method for any desired application, and in many studies, QDs should be considered as alternatives to small molecule fluorophores.

ligands (Figure 50A). In another attempt to control the valence of QD−DNA conjugates, the Fan lab employed ps-poDNA

6. PROTEINS IN DNA NANOTECHNOLOGY Through millions of years, proteins have evolved into extremely complex molecular machines handling a range of functions with amazing efficiency, and de novo design of nanomaterials with similar functionality is an immense challenge. It is, therefore, a highly appealing strategy to harness protein function from Nature’s repertoire when creating functional nanostructures, which may be achieved through immobilization of proteins in DNA nanostructures. This section describes chemical methods for incorporation of proteins in DNA nanostructures and provides a series of examples to highlight proteins at work in DNA nanotechnology.5,29 There are two main strategies for immobilizing proteins in DNA nanostructures. The first strategy involves covalently linking DNA to proteins, which can then be hybridized to DNA nanostructures (section 6.1). The second strategy does not require protein modification, since it takes advantage of noncovalent interactions between the protein and the DNA nanostructure (section 6.2).29

Figure 50. QD−DNA conjugates with controlled valence. (A) Illustration showing the assembly of two halves of a DNA icosahedron in the presence of QDs into the full icosahedron structure caging the QD. The structures can be further functionalized with endocytic ligands stoichiometry- and site-specifically. The bottom left panel shows a TEM image of the assembled icosahedron-QD structures. Scale bar = 200 nm. The bottom right inset shows an example of single particle tracking in live cells achieved using the icosahedron-QD structure. Adapted with permission from ref 454. Copyright 2016 Springer Nature. (B) Schematic illustration of the approach for controlling the valence of QD−DNA conjugates by using preassembled ps-poDNA chimeras. The bottom left inset shows an agarose gel with bands from the controlled-valence structures marked in yellow-border rectangles. Reprinted with permission from ref 455. Copyright 2017 John Wiley & Sons, Inc.

6.1. Covalently Linked Protein−DNA Conjugates

chimeras.455 The negatively charged MPA ligands of the QDs were partly exchanged for a neutral polyethylene glycol thiol to allow the ps-part (50 adenosines) of the chimeras to interact with the QD. The po-part of the chimeras acted as sticky ends for base pairing with other ps-poDNA chimeras. Through the design of different po-parts of the chimeras, premixing of these enabled high control over the DNA valence on the QDs (Figure 50B). For the immobilization of QDs on DNA nanostructures, the binding of Streptavidin-conjugated QDs (SA-QDs) to biotinylated DNA has so far been the most widely applied method. Both SA-QDs and biotinylated DNA are commercially available, and therefore, no further chemical derivatization is required to

Protein−DNA conjugates can be formed and subsequently hybridized to extended staple strands of DNA nanostructures, or alternatively, the conjugation reaction can be performed directly on a functional handle incorporated in the nanostructure. Formation of covalent bonds between protein and DNA is therefore crucial for immobilizing proteins in DNA nanostructures. 6.1.1. Conjugation Using Heterobifunctional Linkers. Numerous chemical methods exist for modifying proteins, and extensive research efforts have focused on the development of residue-specific conjugation reactions.463 In DNA nanotechnology, reactions with nucleophilic residues at the protein surface AQ


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Figure 51. Immobilization of QDs on DNA origami using Streptavidin−QD conjugates. (A) AFM images of QDs positioned in periodic patterns on 6helix bundle DNA origami. Scale bars = 100 nm. Right insets show the height-profile of a QD and the line profile of the construct imaged above. Adapted from ref 458. Copyright 2010 American Chemical Society. (B) Illustration of the assembly of QDs on long linear multiorigami constructs along with AFM images and line profile of the structures. Upper scale bar = 1 μm. Bottom scale bar unknown. Adapted with permission from ref 459. Copyright 2014 Royal Society of Chemistry. (C) Illustration of surface patterning by focused ion beam lithography followed by immobilization of triangular DNA origami-QD conjugates in the patches. Bottom left panel shows an AFM image of the patches with the inset highlighting the presence of DNA origami and QDs in the patches. Scale bar = 300 nm. Bottom right panel shows an epifluorescence image of the macroscopic pattern of QDs arranged over tens of micrometers. Scale bar = 2 μm. Adapted from ref 461. Copyright 2017 Springer Nature. Licensed under a Creative Commons agreement (https://creativecommons.org/licenses/by/4.0/).

protein conjugates using CuAAC or SPAAC (Figure 52, bottom). Linkers containing an azide-group in one end and an NHS ester or a maleimide in the other end are also popular reagents. All the reagents mentioned above are commercially available as are the modified oligonucleotides required for formation of protein−DNA conjugates.59,79,87 Protein−DNA conjugates formed using heterobifunctional linkers have been frequently used in DNA nanotechnology. The glucose-6-phosphate dehydrogenase (g6pDH)-DNA conjugate used by the Yan group to create an enzyme reactor based on a DNA-tweezer was prepared by first coupling the protein to SPDP before removing excess of the small molecule.464 Subsequently, thiol-modified DNA was reacted with the maleimide unit to provide the protein−DNA conjugate. The enzyme and its cofactor were incorporated on opposite arms of the nanotweezer that could be opened and closed via strand displacement reactions, hence providing an on−off switch for the enzyme reactor. Yan and collaborators also used SPDP to form the protein−DNA conjugates used in their seminal work describing the formation of a glucose oxidase (GOx) and horse radish peroxidase (HRP) enzymatic cascade on DNA origami.59 The same enzyme pair was used by Zhao et al. to assemble a DNA nanocage from two half-cages (Figure 53A).465 Each component of the enzyme pair was attached to DNA using SPDP coupling and then hybridized to their respective halfcages. When the cages were fully assembled, increased catalytic efficiency of the enzymes and improved stability toward proteases were observed. The Andersen group also investigated

remain highly popular. Cysteine and lysine residues are generally targeted using probes containing Michael acceptors (typically maleimides) and activated esters (typically NHS esters), respectively. Seemingly, the simplest approach for attaching oligonucleotides to proteins is through reaction with oligonucleotides containing such reactive handles. As discussed previously, the direct incorporation of NHS esters and maleimides during oligonucleotide synthesis is not straightforward and requires specialized reagents and precautions. The concequence of this is a widespread use of heterobifunctional linkers that allow coupling of oligonucleotides containing standard modifications such as thiols, amines, or alkynes to cysteine- or lysine-residues of proteins. Linkers containing an NHS ester or sulfo-NHS ester in one end and a maleimide group in the other end are widely used reagents. Prime examples of such reagents are succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and sulfo-SMCC that link thiol-modified DNA to lysines or amino-modified DNA to cysteines (Figure 52, top). Another popular class of reagents for linking amines to thiols involves an activated disulfide in one end and an NHS ester in the other end. The most frequently used reagent is 3-(2-pyridyldithio)propionic acid NHS ester (SPDP) (Figure 52, middle) where the pyridyl-activated disulfide enables thiol-exchange and hence disulfide formation with oligonucleotide thiols or cysteine-residues. Reagents containing the same reactivity, but with different linkers and solubilizing groups, are also readily available. Heterobifunctional linkers containing azides are also important for formation of DNA− AR


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Figure 52. Examples showing the use of commonly applied heterobifunctional linkers for preparation of protein−DNA conjugates. SMCC and sulfoSMCC are effective reagents for linking cysteine or lysine residues to amino- and thiol-modified DNA, respectively (upper panel). So is SPDP, but a cleavable disulfide bond is formed between the protein and the DNA when using this reagent (middle panel). NHS-azide heterobifunctional linkers facilitate reaction of lysine residues with alkyne-modified DNA via CuAAC or SPAAC (bottom panel). For simplicity, only one reactive handle is depicted on the protein surface.

enzyme caging in a study where α-chymotrypsin was reacted with an azide-NHS heterobifunctional linker to provide azidehandles at the protein surface (Figure 53B).87 Subsequently, azide−alkyne cycloaddition with a DBCO-modified staple strand positioned inside the DNA vault enabled precise positioning of the enzyme. Strand displacement reactions facilitated opening and closing of the vault, and only the open conformation of the vault allowed substrate diffusion to the enzyme and thereby increased enzymatic activity. In a study also described above (Figure 9A), the Fan lab used catalase−DNA conjugates and GOx−DNA conjugates that were both formed using SPDP-coupling to image enzymatic cascades at the single molecule level.158 Recently, Fisher et al. incorporated nucleoporins inside DNA origami nanorings to form organized nuclear pore complexes. Nucleoporins were expressed with a single C-terminal cysteine, and sulfoSMCC coupling with amino-modified DNA was employed to obtain nucleoporin−DNA conjugates.79 Rosier et al. used sulfoSMCC coupling to attach DNA to protein G, and the resulting products enabled immobilization of antibodies on DNA origami as described in more detail later in this section.78 Very recently, Nie, Yan, Ding, Zhao, and co-workers used sulfoSMCC coupling to attach thrombin to thiolated poly-T strands that were subsequently hybridized to rectangular DNA origami.466 The obtained structures could be rolled up into tubes and equipped with a nucleolin-recognizing aptamer which enabled selective binding at tumor cells. The system intriguingly provided a route to in vivo suppression of tumor growth by induction of thrombosis and hence blockage of blood-supply to the tumor tissue. Specialized kits based on heterobifunctional linkers for attaching DNA to proteins are also available. In the seminal

work by Douglas et al. describing the preparation of a smart, logic-gated DNA nanorobot, antigen binding fragments (Fabs) were incorporated into the DNA origami structure (Figure 53C).80 DNA−Fab conjugates were prepared using the commercially available HyNic/4FB kit which is based on two heterobifunctional linkers (Figure 53D). One of them contains an NHS ester in one end and a protected hydrazide in the other end to allow incorporation of hydrazide handles to the protein surface. The other heterobifunctional linker contains an NHS ester in one end and an aldehyde group in the other end, enabling functionalization of amino-modified DNA with aldehyde handles. Subsequently, the protein and DNA are coupled through formation of a highly stable hydrazone bond. Antibody−DNA conjugates have also been formed using inverse electron demand Diels−Alder (IEDDA) reactions for which the reactive handles were introduced using heterobifunctional linkers.467 Heterobifunctional linkers exist with a multitude of different reactive handles, and their ease of use has made them highly popular for preparing protein−DNA conjugates. However, since proteins typically have many reactive handles, the obtained products can be inhomogeneous, and the number and position of DNA strands on the protein may vary. In some cases, more homogeneous protein−DNA conjugates are desired, and the formation of such conjugates is the topic of the next sections. 6.1.2. Site-Specific Attachment of DNA to Proteins. Proteins are highly complex molecules that generally contain numerous reactive handles at their surface. Attaching molecular cargo such as a DNA strand to one specific site of a protein therefore seems a tremendous challenge. Protein engineering, however, has the power to enable site-specific functionalization of proteins. Whereas nucleophilic lysine residues are abundant AS


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Figure 53. Protein−DNA conjugates obtained using heterobifunctional linkers and their applications in DNA nanotechnology. (A) Illustration of nanocaging of an enzyme-pair mediating an enzymatic cascade. The protein−DNA conjugates were formed using SPDP conjugation. (B) Illustration of a 3D DNA nanovault containing α-chymotrypsin which was attached to the vault by SPAAC. Parts A and B are adapted from refs 465 and 87. Copyright 2016 and 2017 Springer Nature, under the creative commons agreement (https://creativecommons.org/licenses/by/4.0/). (C) Illustration of the well-known therapeutic DNA nanorobot described in ref 80. The purple shapes in the interior of the structure are antibody Fab domains attached to DNA using HyNic-4FB conjugation chemistry. Adapted with permission from ref 80. Copyright 2012 American Association for the Advancement of Science. (D) Scheme showing the formation of protein−DNA conjugates using the commercially available HyNic-4FB system used for preparing the Fab−DNA conjugates loaded in the above-mentioned nanorobot. Note that the acetone protecting group of the hydrazide is slowly removed under the conjugation conditions and no separate deprotection step is required.

subsequently immobilized in a rhomboidal DNA nanoactuator.469 Only when the two halves of the protein were brought close, GFP fluorescence was observed. Obviously, cysteine-incorporation for specific conjugation is not a generalizable tool since many proteins in fact contain several reactive cysteine residues. The search for more general tools to incorporate handles for site-specific protein conjugation has spurred the development of expanded genetic codes which allow incorporation of unnatural amino acids (UAAs).470,471 Here, one or more codons are recognized by alternative tRNAs (tRNAs) that specifically recognize the UAA codon which does not encode one of the canonical amino acids. An amino acyl tRNA synthetase (aaRS) designed to bind the UAA of choice and transfer it to the tRNA is also needed. Importantly, orthogonality to the existing tRNAs and aaRSs is required so that the UAA is neither attached to other tRNAs nor are canonical amino acids attached to the introduced tRNA. The most widely used strategy is the use of amber codon suppression pioneered by the Schultz lab (Figure 55).472 The amber codon is an Escherichia coli stop codon, but an orthogonal tRNA/aaRS

on protein surfaces, this is not the case for cysteine residues. Often, cysteine residues are involved in structural disulfide bonds and many proteins lack nucleophilic cysteine residues. Therefore, genetic engineering to incorporate a cysteine residue during protein expression may provide a straightforward approach for installing a reactive handle that can be specifically addressed. For instance, Fisher et al. and Ketterer et al. used this approach to attach DNA to nucleoporins at a specific cysteine residue incorporated at the protein’s C-terminus (Figure 54A).79,82 Both of these studies focused on investigating nuclear pore complexes formed inside ring-like DNA origami structures (Figure 54B−C). The specific attachment of DNA at the Cterminus of the nucleoporins allowed the full length of the disordered proteins to participate in multiprotein complexformation within the DNA rings. Cysteine-incorporation also facilitated attachment of biotin to zinc finger proteins (ZFPs), which were subsequently immobilized on origami and visualized by AFM upon incubation with Streptavidin.468 Furthermore, cysteine-incorporation allowed functionalization of the two halves of split green fluorescent protein (GFP) which were AT


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Figure 54. Incorporation of cysteine for site-specific conjugation of DNA to proteins. (A) Schematic showing the formation of a nuclear pore protein− DNA conjugate through an artificially incorporated C-terminal cysteine residue. (B) Illustration of nuclear pore protein (Nup) assembly inside DNA origami rings. The strategy provides control over the number of proteins making up the Nup-complex inside the DNA ring.82 Adapted from ref 82. Copyright 2018 Springer Nature. Licensed under a creative commons agreement (https://creativecommons.org/licenses/by/4.0/). (C) Illustration (top) and AFM topography image (bottom) of Nup-complexes formed inside DNA origami cylinders and immobilized on lipid bilayers.79 Adapted from ref 79. Copyright 2018 American Chemical Society.

residue to allow sulfoSMCC coupling to amino-modified DNA. The resulting protein-G−DNA conjugate bound noncovalently to antibodies, but photoactivation yielded covalent conjugates between the antibody and the BPA moiety of protein G. The resulting homogeneous antibody−DNA conjugates were efficiently immobilized on flat rectangular DNA origami (Figure 56). Amber codon suppression was also used by Stulz and coworkers to create homogeneous protein−DNA conjugates by incorporation of azidophenylalanine at various sites of superfolding green fluorescent protein (sfGFP) and TEM βlactamase.475 This allowed attachment of ssDNA modified with bicyclononyne at specific sites of the proteins by SPAAC. Measurements of FRET between different sfGFP−DNA conjugates and a Texas Red fluorophore revealed that the FRET efficiency depended on the protein−DNA attachment site. It was further demonstrated that the modification site of TEM β-lactamase significantly affected its enzymatic activity. Both of the proteins were further immobilized on flat DNA origami structures where the enzymatic activities and fluorescent properties of multiprotein assemblies were investigated (Figure 57A). The results highlight the importance of the attachment site to protein activity and illustrate the potential for tuning the function of protein−DNA nanoassemblies by rational design of the protein−DNA attachment site. Work from Smider, Schultz, and co-workers demonstrated that protein−DNA conjugates could be formed by oxime formation upon introduction of acetyl phenylalanine to proteins via amber codon suppression (Figure 57B).107 The same strategy was employed when acetyl phenylalanine was incorporated in Fab-domains of various antibodies to facilitate coupling to aminooxy-functionalized DNA and further use of the Fab−DNA conjugates to create Fab-dimers.476 Also, aminooxy-functionalized PNA was coupled to different Fab-

Figure 55. Amber codon suppression. Illustration of the principle behind amber codon suppression for installation of UAAs in proteins, and structures of typical UAA side chains (From left to right: azidophenylalanine, acetylphenylalanine, and propynyltyrosine) installed for orthogonal bioconjugation. Top panel adapted with permission from ref 470. Copyright 2017 Springer Nature.

pair originally derived from an archaebacterium enables UAA introcution in response to amber codons. Numerous UAAs with reactive handles such as ketones, azides, alkynes, and arylhalides have been incorporated using this strategy.473 Further expansion of the genetic code can be achieved by the introduction of 4letter codons as pioneered by the Chin group.474 In contrast to the 64 possible codons in the 3-letter genetic code, the 4-letter codons provide access to 256 unique codons. In DNA nanotechnology, amber codon suppression was used for incorporation of para-benzoylphenylalanine to protein G, which has high affinity for the Fc-domain of native antibodies.78 Protein G was further modified with a single N-terminal cysteine AU


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Figure 56. Site-specific formation of antibody−DNA conjugates using a protein G adaptor. Protein G was expressed with a benzoylphenylalanine cross-linker installed using amber codon suppression. A Cterminal cysteine-residue was also incorporated to allow site-specific sulfoSMCC coupling with amino-modified DNA. Site-specific, covalent antibody−DNA conjugates were obtained upon photo-cross-linking, and the products were successfully immobilized on DNA origami. Adapted from ref 78. Copyright 2017 Royal Society of Chemistry under a creative commons agreement (https://creativecommons.org/ licenses/by/3.0/).

Figure 57. Applications of amber codon suppression in DNA nanotechnology. (A) Illustration of work employing the incorporation of azidophenylalanine for site-specific SPAAC-mediated attachment of DNA to various locations on sfGFP and TEM β-lactamase. The dependence of protein activity on modification site was investigated for the proteins both in solution and on DNA origami. Adapted from ref 475. Copyright 2017 American Chemical Society. (B) Scheme showing the formation of a stable oxime bond between aminooxy-functionalized oligonucleotide and acetyl phenylalanine-modified protein. (C) PNAand DNA-directed formation of Fab-multimers. Fab-domains were conjugated to DNA or PNA through the incorporation of a single acetyl phenylalanine residue followed by reaction with aminooxy-functionalized oligonucleotides. The chart shows the severe T-cell mediated cytotoxicity of Fab heterodimers compared to unconjugated Fab domains. Reprinted from ref 476. Copyright 2013 American Chemical Society.

domains enabling the formation of various homo- and heteromultimers (Figure 57C). Interestingly, when Fabdomains derived from the antibody therapeutics Cetuximab and Rituximab were coupled to αCD3, which is known to bind T-lymphocytes, the bispecific constructs led to T-cell mediated killing of the targeted cancer cells. 6.1.3. Peptide Tags for Formation of Protein−DNA Conjugates. Incorporation of peptide tags that enable sequence-specific enzymatic ligation is also effective for sitespecific conjugation of DNA to proteins.202 This strategy requires modification of the polypeptide sequence with a peptide tag recognizable to a specific enzyme such as Sortase A, Formyl generating enzyme, Phosphopantetheinyl transferase, Farnesyl transferase, Transglutaminase, etc.477 As an example, Khatwani et al. added CVIA-tags to the C-terminus of enhanced GFP and mCherry. This tag is recognized by Saccharomyces cerevisiae Farnesyl transferase (ScFTase), and by adding azidofarnesyl as substrate, an azide handle was introduced enzymatically to the C-termini of the proteins. The azide handle then enabled site-specific attachment of ssDNA via CuAAC or SPAAC (Figure 58A).478 Duckworth et al. used this strategy for preparing DNA−GFP conjugates that were subsequently incorporated in DNA tetrahedrons.479 Sortase A catalyzes the ligation of molecules containing an oligoglycine sequence with ones containing an LPXTG sequence (Figure 58B), thereby providing another ligation method for the formation of protein−DNA conjugates. Belcher and co-workers attached maleimide-functionalized peptides with the sequence GGGK or LPETGG to thiol-modified DNA.480 The resulting peptide−DNA conjugates were subsequently ligated to phage-proteins containing the complementary peptide sequence to eventually allow the formation of phage multimers by DNA hybridization. Protein−DNA conjugates were prepared by Pippig et al. using Phosphopantetheinyl transferase (Sfp) that attaches substrates containing

coenzyme A (CoA) groups to proteins containing one of three different peptide tags (ybbR-, S6, or A1) (Figure 58C).477 For this experiment, they used the ybbR-tag (DSLEFIASKLA) attached to GFP further coupled to a peptide tag for binding to a single-chain antibody fragment.481 The protein was ligated to CoA-modified DNA and used in an example of so-called “single molecule cut and paste”. This was realized when an AFM cantilever was functionalized with a single-chain antibody specifically binding the GFP peptide tag which allowed single proteins to be moved around on a DNA-functionalized surface by the cantilever. 6.1.4. Self-Labeling Polypeptides for Formation of Protein−DNA Conjugates. Although peptide tags are very useful for formation of protein−DNA conjugates, it requires significant chemical manipulation of DNA-strands, the use of ligases, and expression of the tagged proteins. Thus, simpler approaches such as self-labeling polypeptides (SLPs) are often used for attaching DNA to proteins. SLPs are proteins or peptides that react substrate-specifically to form covalent bonds between the SLP and the substrate. In contast to the peptide tags described above, SLPs do not require a ligase to mediate the AV


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Figure 58. Protein−DNA conjugation using peptide tags. (A) Insertion of azide-handle using farnesyl transferase and a CVIA peptide tag to incorporate an azide-containing farnesyl-analogue. (B) Sortase A ligation for formation of a protein−DNA conjugate using an LPXTGG-tagged protein and a peptide−DNA conjugate with an oligoglycine peptide sequence. (C) Phosphopantetheinyl transferase recognizes the ybbR-tag and catalyzes the incorporation of CoA-modified DNA. Although the ybbR tag is shown as a C-terminal modification here, it can also be used for Nterminal and internal modification. DNA−CoA conjugates are commercially available but can also be readily formed using sulfoSMCC coupling between amino-modified DNA and CoA.

O6-alkylguanine-DNA transferase that reacts specifically with O6-alkylated guanine of DNA.483 Reaction with O6-benzylguanines is particularly rapid, and this functionality is generally employed for protein labeling using SNAP-tags (Figure 59, top). The CLIP-tag is derived from the SNAP-tag but has been engineered to react specifically with O2-benzylcytosine instead of O6-benzylguanine (Figure 59, bottom).484 Importantly, the CLIP tag is orthogonal to the SNAP-tag, enabling the use of the two tags in combination.

ligation and they may be attached to any protein of interest. The most famous examples of self-labeling proteins are the HaloTag and the SNAP-tag, but the CLIP-tag has also been investigated in DNA nanotechnology.482,483 The HaloTag is a Haloalkane dehalogenase tag that reacts with molecules containing chloroalkane groups, often 1-chlorohexyl or 4-chlorocyclohexyl. By expressing a protein of interest with the HaloTag, the protein can be linked to cargo molecules such as DNA functionalized with a chloroalkane (Figure 59, middle).482 The SNAP-tag is a 19.4 kDa polypeptide tag derived from the DNA-repair protein AW


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Figure 59. Self-labeling polypeptides (SLPs). (A) Schemes for the DNA labeling of SNAP-, Halo-, and CLIP-tag fusion proteins through reaction with DNA containing ligands for the respective SLPs. (B) Chemical structures of common commercially available NHS esters for introduction of SLPligands to amino-modified DNA.

The Niemeyer group made use of SNAP-tags and HaloTags for the specific incorporation of both an oxidoreductase (Gre2) and the monooxygenase P450 BM3 reductase domain (BMR) on flat DNA origami sheets.487 The DNA origami−protein complexes were purified by free flow electrophoresis, and the purified structures were investigated in an NADP+/NADPH cascade where Gre2 catalyzed the oxidation of butanol to butanal in an NADP+-dependent reaction. NADPH generated in the same reaction was required for cytochrome C-reduction mediated by BMR, which resulted in a measurable optical output. The same group also described the use of the optimized HaloTag, halo-based oligonucleotide binder (HOB).488 This tag was engineered to contain five positively charged lysine residues at its binding interface, thus improving interaction with DNA nanostructures and providing more efficient protein immobilization. Recently, the Morii group combined the use of SNAPtag, CLIP-tag, and HaloTag with zinc finger protein (ZFP)mediated DNA binding to achieve rapid, specific, and orthogonal attachment of proteins to dedicated sites on DNA origami (Figure 60B).489 This strategy enabled establishment of

Reck-Peterson, Shih, and co-workers fused SNAP-tags to dynein and kinesin motor proteins.485 An O6-alkylguanine NHS ester was coupled to amino-modified DNA enabling attachment of the DNA to the SNAP-tags. In this way, motor proteins with opposite polarity could be positioned on the same 12-helix bundle DNA origami. In the resulting “tug-of-war” between the two types of motor proteins (Figure 60A), the polarity of the complexes could be controlled through the introduction of photocleavable linkers between proteins and the DNA bundle thus enabling selective removal of one type of motor protein from the complex. The Shih lab performed further studies on motor proteins attached to DNA nanostructures.486 Myosin V and myosin VI were attached at opposite ends of a DNA nanospring after conjugation of the motor proteins to DNA using SNAP-tag and HaloTag methodologies. When positioned on an actin filament, the two motor proteins walked in opposite directions, which built up tension in the spring. Eventually, one motor protein would fall off the filament, thus contracting the DNA spring. AX


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Figure 60. Applications of self-labeling polypeptides (SLPs) in DNA nanotechnology. (A) Illustration (Lef t) of dynein (D) and kinesin (K) motor proteins functionalized with DNA using SNAP-tag ligation and immobilized on a 12-helix bundle. Upon binding to actin filaments, the two motor proteins walk in opposite directions. By cleaving off one species of motor proteins by 405 nm photoirradiation, the direction of walking can be changed as observed from the Kymograph (right). The purple lightning denotes the starting time of irradiation. Adapted with permission from ref 485. Copyright 2012 American Association for the Advancement of Science. (B) Illustration of the orthogonal positioning of three enzymes in three different positions on DNA origami using HaloTag, SNAP-tag, and CLIP-tag in combination with DNA-binding proteins. Reprinted from ref 489. Copyright 2017 American Chemical Society.

affinity-guided protein labeling can provide access to homogeneous protein−DNA conjugates from commercially available proteins. This potentially provides an entry point for a larger group of researchers seeking to harness the power of proteins within DNA nanotechnology. In an elegant example of DNA-templated protein−DNA conjugation, Li and collaborators used ssDNA functionalized with small molecule ligands for various proteins to siteselectively template protein−DNA conjugation (Figure 62).490 Upon noncovalent binding of the ligand−DNA chimera to the protein of interest, a complementary DNA-strand containing a diazirine-group hybridized to the probe. Photo-cross-linking between the diazirine−DNA and the protein of interest was then induced to provide site-selective attachment of DNA to the protein. This approach was later used for selection of DNA encoded libraries.495 Very recently, Yan et al. used a similar approach to siteselectively attach DNA to carbonic anhydrase II (CAII) and αthrombin.496 For CAII-labeling, a benzenesulfonamide ligand was attached to the DNA template and addition of complementary DNA activated with an NHS ester functionality readily reacted with lysine residues in proximity to the sulfonamide binding site. Since the benzenesulfonamide is an inhibitor of CAII, its enzymatic activity was inhibited as long as the template strand remained attached to the protein, but removal of the template strand by toehold-mediated stranddisplacement restored protein activity. To label α-thrombin, two

an enzyme cascade on DNA origami consisting of three enzymes catalyzing the xylose > xylitol > xylulose > xylulose P conversion. SLPs are very popular for ligating proteins to DNA. The polypeptide tags are incorporated during standard protein expression, and their ligands are easily introduced to aminomodified DNA using commercially available ligand-NHS esters. However, the bulkiness of the polypeptide tags limits the spatial control of the proteins on DNA origami and the addition of the large polypeptide tags might impede the folding, and thereby the function, of the protein of interest. Additionally, protein expression is required and the system cannot be adapted to commercially available proteins without tags. Therefore, methods providing homogeneous protein−DNA conjugates without the need for genetic engineering are highly attractive as described in the next section. 6.1.5. Affinity-Guided Attachment of DNA to Proteins. Methods that can provide homogeneous protein−DNA conjugates without the need for protein engineering are highly desired. Affinity-guided protein labeling employs a directing group for achieving site-selective labeling of proteins (Figure 61). The directing group can be a small molecule ligand or an aptamer for the protein of interest,490,491 but more promiscuous groups such as metal-coordinating groups are also used.492 Several methods have been developed based on this concept, and we have recently reviewed the site-selective labeling of native proteins with DNA.493,494 Although the method has not been widely adapted within the DNA nanotechnology field, AY


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Figure 61. Preparation of protein−DNA conjugates using affinity-guided labeling. The left scheme depicts the formation of a protein−DNA conjugate using a single probe containing both the directing group (DG, orange shape) and the reactive group (RG, blue box). Unless a cleavable linker is introduced, the directing group remains associated with the conjugation product. The right scheme depicts DNA-templated protein conjugation, where one DNA strand contains the directing group while the reactive group is located on a complementary strand. DNA hybridization mediates the reaction, and using appropriate purification, for instance by strand-displacement, the directing group is removed from the product.

immunoglobulin (IgE), and a cytoplasmic guanine nucleotide exchange factor (Cytohesin-2).491 Here, phenyl azides were attached to the aptamers to facilitate covalent photo-crosslinking between the aptamers and the protein. The labeling reactions were successfully performed both at cell-membranes and in cell lysates. Labeling sites for ligand-directed labeling are often close to the active site, which may disrupt the protein activity. Although aptamer-directed labeling circumvents this, it requires the selection of novel aptamers for every target. A more general class of directing groups relies on metal coordination. Allbritton and co-workers took advantage of the high affinity of Histidine tags for nitrilotriacetic acid (NTA) in the presence of certain cations such as Ni2+.492 A single probe containing the NTAdirecting group, a photoreactive benzophenone group, and an oligonucleotide was prepared (Figure 63). Following incubation with His6-tagged dihydrofolate reductase in the presence of Ni2+, the probe was covalently attached to the protein upon exposure to light. Only one DNA strand was attached per protein, and importantly, no labeling was observed in the absence of the His6tag. Recently, our group developed a method for DNA-templated protein conjugation (DTPC, Figure 64).494 Here, tris-NTA is used as a metal-coordinating directing group that is attached to a guiding oligonucleotide. A complementary oligonucleotide sequence functionalized with a lysine-reactive group is added before DNA-templated ligation can take place. The guiding DNA strand is removed during purification, and the desired protein−DNA conjugate is obtained (Figure 64A). In the initial studies, NHS esters were used as reactive groups, and siteselective labeling of several His6-tagged proteins including GFP,

Figure 62. DNA-programmed photoaffinity labeling. (A) Schematic showing the ligand-directed, DNA-templated formation of protein− DNA conjugates upon photoirradiation. (B) Gel analysis of conjugation reactions verifying that all components illustrated in (A) are required for formation of protein−DNA conjugates. Adapted with permission from ref 490. Copyright 2013 John Wiley and Sons, Inc.

aptamers with varying affinity for α-thrombin were successfully employed as directing groups. Aptamers were also employed by Vinkenborg et al. for siteselective labeling of a membrane protein (c-Met), an AZ


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DNA conjugate via the transferrin-receptor internalization pathway (Figure 64B). Several antibodies contain a histidine cluster at the Fc-domain that can also be targeted by metal coordination. We demonstrated that DTPC could, indeed, be used for site-selective conjugation of DNA to commercially available, murine monoclonal IgG1 antibodies although it required exchange of Ni2+ to Cu2+ to facilitate a stronger coordination between the tris-NTA directing group and the IgG1 histidine clusters. To avoid nontemplated labeling, a high NaCl concentration (600 mM) was used for antibody labeling in DTPC to antibodies. Later, the DTPC method was refined when the reactive NHS ester was exchanged for a more stable aldehyde unit allowing conjugation of DNA to the protein of interest via reductive amination.73 Furthermore, a 1,2-diol linker was incorporated between the DNA and the protein of interest. This linker is cleaved in the presence of periodate, thus providing an aldehyde handle on the protein for further conjugation with alkoxyamines. Very recently, Mortensen et al. developed a probe based on the same concepts. In this case, however, both the directing NTAgroups and the reactive aldehyde group were introduced to a single small molecule containing an azide handle for further functionalization such as site-selective conjugation to alkynemodified oligonucleotides.497 Serotransferrin-DNA conjugates prepared using DTPC were used by Schaffert et al. for internalization of DNA origami structures. The conjugates were immobilized on DNA origami by hybridization to extended staple strands, and the DNA structures were efficiently internalized by the transferrinreceptor pathway.498 Affinity-guided protein labeling thus provides a promising method for obtaining homogeneous protein−DNA conjugates without the need for proteinexpression.

Figure 63. Structure of a probe used in ref 492 for formation of protein−DNA conjugates. The probe contains an NTA directing group (circled in blue), a benzophenone reactive group (circled in red), and an oligonucleotide of interest.

6.2. Noncovalent Binding for Immobilization of Proteins in DNA Nanostructures

The previous sections described the formation of protein−DNA conjugates that could be immobilized on DNA nanostructures by Watson−Crick base pairing. Several methods for specific protein immobilization on DNA nanostructures through other high-affinity noncovalent interactions are described below. These methods circumvent the need for protein−DNA conjugation, and they include the use of sequence-specific DNA-binding proteins and high-affinity binding of proteins to small molecule ligands, aptamers, or metal-coordinating groups positioned in DNA nanostructures. Furthermore, less specific protein immobilization can be achieved through electrostatic interactions between positively charged proteins and negatively charged DNA nanostructures. 6.2.1. DNA-Binding Proteins. The most widely used method for immobilization of proteins in DNA nanostructures using DNA-binding proteins is based on the use of zinc finger proteins (ZFPs), naturally found as structural motifs in transcription factors or nucleases. Each zinc finger domain recognizes specific four base pair sequences of dsDNA through Cys2His2 domains interacting in the major groove, and proteins with three zinc finger domains recognize 10 base pair sequences with high affinity.499 The structural basis of the ZFP−DNA interaction was revealed for the ZFP, Zif268, by Pavletich and Pabo,500 and to some extent, modified ZFPs can be artificially generated to bind desired sequences.501 In DNA nanotechnology, the use of ZFPs for protein immobilization has been pioneered by the Morii group. Nakata

Figure 64. DNA-templated protein conjugation (DTPC) using NTAmediated metal coordination. (A) Illustration of the metal-coordination and subsequent DNA-templated conjugation leading to formation of a GFP-DNA conjugate via DTPC. (B) A Serrotransferrin-DNA conjugate loaded with a complementary, fluorophore-labeled strand was efficiently internalized in cells via the transferrin-receptor internalization pathway. Adapted with permission from ref 494. Copyright 2014 Springer Nature.

Interleukin-6, and Endoglycosidase H was demonstrated. Although many recombinant proteins are expressed with His6tags, these tags are not found in native proteins. Importantly, we found that several native metal-binding proteins, including Serotransferrin and Carboxypeptidase B, were also labeled using DTPC. The proteins remained active upon conjugation as illustrated by the cell internalization of the serotransferrin− BA


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et al. described the use of Zif268 and the artificially generated zinc finger protein, AZP4, for orthogonal immobilization of proteins on DNA origami (Figure 65) with both of the ZFPs

Figure 66. ZFP-fusion proteins mediating the formation of K+-channels in live cells. (A) Schematic representation of Kir3-zif268 tetrameric channel and its binding to DNA. (B) DNA origami designed for binding to zif268 and AZP4. (C) Illustration of whole-cell patch-clamp experiments performed to evaluate DNA origami-mediated K+-channel formation. (D) Chart showing K+ current amplitudes for cells expressing subunit-ZFP fusion proteins. The biggest signal is observed when the origami construct shown in (B), containing both the zif268 and AZP4 binging sites, is employed (red bar). Adapted with permission from ref 503. Copyright 2018 John Wiley and Sons, Inc.

Figure 65. Immobilization of proteins on DNA origami using zinc finger proteins (ZFPs). ZFPs conjugated to biotin immobilized specifically in locations containing the ZFP-binding sequence. The site-specific immobilization in one of the 5 possible binding cavities (bottom lef t) was visualized using AFM after incubation with Streptavidin (bottom right). Scale bar = 100 nm. Fusion proteins were also immobilized on DNA origami, thus making ZFPs a generally applicable tool for protein immobilization. Adapted with permission from ref 468. Copyright 2012 John Wiley and Sons, Inc.

was investigated in whole-cell patch clamp experiments on HEK293T cells (Figure 66C) where the cells expressed the subunit-ZFP fusion proteins while DNA origami was added to the cell medium. An increased K+-influx was only observed when specific DNA tags were present on the origami, thus illustrating that DNA origami can mediate assembly of functional membrane channel protein-multimers (Figure 66D). The specific binding of other proteins to DNA has also been investigated in DNA nanotechnology. For instance, Martin et al. demonstrated that p53, a dsDNA-binding transcription factor, was effectively immobilized inside a solid 3D DNA origami structure by sequence-specific binding to its DNA target (Figure 67A) to protect the protein during sample preparation for cryoelectron microscopy.504 Theoretically, fine-tuning of the DNA origami design should enable control over the protein orientation, which would provide an important tool for structural biology. This potential was, however, only partly realized, but still novel information about the symmetry of p53 tetramers bound to DNA was acquired at 15 Å resolution. In another approach to increase understanding of biological interactions, the Dietz group used a DNA origami spectrometer to measure the forces of nucleosome interactions.505 Histone octamers were immobilized on the DNA spectrometer through their binding to dsDNA containing ssDNA extensions. Furthermore, the mechanism of action of DNA-binding enzymes has been studied at the single molecule level using high-speed AFM imaging of DNA origami frames containing DNA substrates for various enzymes.33 Pioneered by Endo, Sugiyama, and co-workers, DNA methyl transferases, baseexcision repair enzymes, and DNA recombinases were investigated using this strategy.506−508

recognizing specific 10-base pair dsDNA sequences extending from DNA origami.468 Initially, the ZFPs were expressed with an N-terminal reactive cysteine residue that allowed conjugation to biotin units. Immobilization of the ZFPs on DNA origami could be visualized by AFM upon incubation with Streptavidin (Figure 65, bottom right). The authors then moved on to fuse fluorescent proteins to the ZFPs. Site-specific immobilization of the fusion proteins on DNA origami was visualized by AFM, and the fluorescent proteins also allowed characterization of the protein−origami construct by fluorescence scanning in agarose gel electrophoresis. Leucine zippers are another class of proteins that bind sequence-specifically to DNA. The Morii group combined the use of zif268 and the leucine zipper CGN4 to enable site specific immobilization of two sequential proteins from an enzymatic cascade.502 Recently, the same group used zif268 and AZP4 in combination with SLPs (HaloTag, SNAP-tag, and CLIP-tag) to orthogonally immobilize three proteins that all catalyzed individual steps in an enzymatic cascade leading to the formation of Xylulose P from Xylose (Figure 60B, section 6.1.4).489 Three distinct sites on the DNA origami were specifically addressed although only two DNA-binding proteins were employed. This addressability was driven by the specific formation of covalent bonds dictated by the respective SLP-ligands attached to the DNA-tags. In very recent work, Kurokawa et al. used ZFPs to direct the formation of Kir3 K+-channels on DNA origami (Figure 66).503 Both zif268 and AZP4 were used as adaptor proteins, to allow assembly of different heterotetrameric and homotetrameric K+-channels. The activity of the K+-channels BB


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Structures as Interface for Cells (MOSAIC) (Figure 68).510 Patterns of ssDNA were created on glass-slides by inkjet printing of amino-modified oligonucleotides to the epoxy activated surface. Subsequently, flat rectangular DNA origami structures containing capture strands were immobilized on the patterns by Watson−Crick base pairing. The opposite surface of the DNA structure contained biotinylated, extended staple strands, and incubation with fluorescently labeled Streptavidin therefore facilitated visualization of the DNA origami immobilized in desired surface patterns (Figure 68A). Biotinylated Epidermal growth factor (EGF) was further immobilized by binding to preimmobilized Streptavidin to study the interactions with live MCF7 cells. Importantly, the position and number of EGF proteins on the DNA origami surfaces significantly affected the activation of the EGF receptor (EGFR) in live cells (Figure 68B). Increasing the number of EGF proteins as well as the interprotein distances both increased EGFR activation. Ke et al. created a rhomboidal DNA origami structure with flexible joints where the angles were adjusted through a strut responding to base pairing with incoming “strut-locking” DNA strands (Figure 69A).469 The structure was further equipped with the two halves of a split enhanced GFP (eGFP). The two halves were both expressed with terminal reactive cysteine residues enabling coupling to biotin through disulfide formation with a pyridyldithiol-biotin compound (HPDP). A Streptavidin bridge facilitated protein immobilization via hybridization to extended staple strands, and the two halves of eGFP were positioned on opposing arms of the structure. The two nonfluorescent halves of eGFP only recombined to provide a fluorescent output when the structure was in its closed conformation.469 The Tinnefeld group very recently adopted the photosynthetic complex Peridinin-chlorophyll a protein (PCP) to their plasmonic antenna setup previously described (Figure 45B, section 5.4.1.3).511 A 500-fold fluorescence enhancement was observed after PCP immobilization in the plasmonic hotspot generated between two 100 nm AuNPs on a DNA nanopillar. For immobilization, the authors used a PCP-Streptavidin complex in combination with biotinylated DNA hybridized to extended staple strands of the structure. Ora et al. immobilized a commercially available Lucia Luciferase Streptavidin conjugate in a tubular 3D DNA origami containing biotinylated DNA in its interior. The DNA origami structure was used for transfection into HEK293 cells, where the luciferase activity was retained.512 Although the biotin−Streptavidin interaction has been widely used for protein-immobilization, other small molecule−protein interactions are also worth mentioning. In general, small molecule antigens can immobilize their antibodies on DNA nanostructures as demonstrated by He et al. for antifluorescein immobilization through interaction with fluorescein.282 Aptamers and peptide ligands are also useful for protein immobilization. For instance, Thrombin was immobilized on DNA lattices functionalized with Thrombin aptamers while Saccà and co-workers made use of a heptapeptide with high affinity for the serine protease DegP (Figure 69B).513,514 The peptide was functionalized with a maleimide group that enabled coupling to thiolated DNA. The resulting peptide−DNA conjugate hybridized to 18 staple strands extending into the cavity of a 3D DNA hexagonal cage where certain DegP oligomers were efficiently immobilized upon binding to the heptapeptides. 6.2.3. Electrostatic and Hydrophobic Interactions. The surface residues of proteins govern their electrostatic and

Figure 67. New applications of DNA-binding proteins in DNA nanotechnology. (A) Illustration of the 3D DNA origami structure used for immobilizing p53. The structure of p53 was determined using cryo EM. An example of a selected class of cryo EM images is shown in the upper right inset. Scale bar = 20 nm. The bottom right inset illustrates the possibility to control protein arrangement by altering the sequence of its dsDNA binding partner. Reprinted with permission from ref 504. Copyright 2016 National Academy of Sciences. (B) Conceptual drawing and EM micrographs showing the formation of DNA−protein hybrid nanostructures using double-TAL proteins and a dsDNA template. Adapted with permission from ref 509. Copyright 2017 American Association for the Advancement of Science.

Praetorius et al. used transcription activator like (TAL) effector proteins to guide the assembly of protein−DNA hybrid nanostructures (Figure 67B).509 Sets of double-TAL proteins recognizing two sequences of dsDNA were used to specifically connect regions of the dsDNA template. All components were genetically encoded, and the hybrid structures were successfully folded isothermally at room temperature into circular or square shapes of dsDNA−protein hybrids in a predesigned manner. In general, the sequence-specific binding of many DNA-binding proteins makes them excellent tools for site-specifically addressing DNA nanostructures. 6.2.2. Small Molecule−Protein Interactions. Another approach for immobilizing proteins on DNA nanostructures is based on protein binding to small molecule ligands conjugated to the DNA nanostructure itself. The by far most common small molecule−protein pair for this is biotin−Streptavidin, which features extremely high affinity binding. Proteins can be expressed with Streptavidin tags, and biotin is readily attached to DNA using standard conjugation chemistry or in-synthesis modification. The tetravalent binding of biotin to Streptavidin further enables the immobilization of biotinylated proteins on biotin-containing DNA nanostructures through a Streptavidin bridge. In a fascinating application of biotin−Streptavidin binding, the Niemeyer group described Multiscale Origami BC


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Figure 68. Multiscale Origami Structures as Interface for Cells (MOSAIC). (A) Illustration (top) of the concept behind MOSAIC. DNA origami structures with extended staple strands of orthogonal sequences recognize specific DNA-patterned surfaces as observed from fluorescence imaging (bottom). (B) Biotinylated DNA origami structures patterned with Streptavidin−EGF conjugates activate the EGF receptor (EGFR) when interacting with whole cells. The activated EGFR is specifically recognized by an antibody−Cy5 conjugate allowing imaging of activated receptors (red spots in the top left fluorescence image). The top right image shows fluorescence of enhanced GFP-EGFR fusion proteins and enables identification of cells. The chart (bottom right) shows the average number of red spots under individual cells which correlates to EGFR activation for different protein patterns as those illustrated and imaged by AFM in the bottom left inset (C = close, F = far, numerical = number of proteins per origami). In general, proteins positioned far from each other on DNA origami induce higher activation of EGFR. Adapted with permission from ref 510. Copyright 2015 John Wiley and Sons, Inc.

removal by dialysis, hydrophobic interactions between nonpolar protein regions and the hydrophobic DNA barrel interior led to the reconstitution of the membrane proteins. The 3D structure of α-hemolysin reconstituted within the DNA structure could be determined using cryo-EM, although with a poor resolution of around 30 Å. This strategy shows future promise for otherwise challenging structural characterization of membrane-associated proteins and again points to the importance of DNA nanotechnology for addressing biological challenges.520 As described above, applications of proteins in DNA nanotechnology are versatile and plentiful. Recent developments highlight how DNA nanotechnology can contribute to other research fields, but also how proteins can be used to refine DNA nanotechnology. Chemistry is at the core of integrating proteins with DNA nanostructures, and this section has provided an overview of methods for achieving this.

hydrophobic interactions with the surrounding environment. Hydrophilic areas enable coordination of metal-ions and water, while hydrophobic patches facilitate interactions with nonpolar environments such as cell membranes. As discussed in the section on affinity-guided attachment of DNA to proteins, metal-coordination can be used to selectively address certain regions of protein surfaces. Turberfield developed a tris-NTA modification of oligonucleotides described above (section 6.1.5) for DTPC.515 They later used this modification in a study of single molecule imaging of His6-tagged proteins immobilized on regular tris-NTA modified DNA arrays by cryo-electron microscopy.515,516 Norton and co-workers exploited NTA-modified DNA for immobilizing His6-tagged proteins through Ni2+-mediated coordination with NTA-tags positioned on DNA origami.517 We made use of the tris-NTA coordination to attract native antibodies to DNA origami, and if desired, the antibodies were covalently linked to amino-modified DNA strands in the nanostructure through the use of a bis-NHS linker.518 In a less specific use of electrostatic interactions, Mikkilä et al. immobilized positively charged virus capsid proteins on DNA origami to enable cellular delivery of the nanostructures (Figure 70A).361 In a very recent work by Dong et al., hydrophobic interactions were used for reconstitution of the membrane proteins α-hemolysin and trimeric envelope glycoprotein within 3D DNA origami structures (70B).519 This was achieved through the construction of a hydrophobic environment within the DNA structure by incorporation of lipid-conjugated oligonucleotides. Membrane proteins with hydrophobic regions for membrane association were mixed with the DNA origami barrel in buffer containing detergent. Following detergent

7. SURFACE-IMMOBILIZATION OF DNA NANOSTRUCTURES While DNA nanostructures are generally folded and modified in solution, surface-immobilization is required to characterize the structures using common techniques such as AFM and single molecule spectroscopy. Moreover, while the creation of higherorder structures can to some extent be achieved through lattice formation in solution followed by immobilization,521 this is frequently precluded due to degradation of the structures during immobilization. For this reason, lattice formation often requires growth of the structures on a surface, and it is therefore of great importance to understand the interactions of DNA nanostructures with surfaces commonly employed in DNA nanotechnology research. This section aims to provide a brief BD


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Figure 70. Protein immobilization in DNA nanostructures using hydrophobic and electrostatic interactions. (A) Positively charged virus capsid proteins bind electrostatically to negatively charged DNA origami. The resulting complexes are efficiently transfected into cells. Adapted from ref 361. Copyright 2014 American Chemical Society. (B) Illustration of the concept used for reconstitution of α-hemolysin in a DNA nanobarrel with a hydrophobic interior. When detergents are removed from the mixture of proteins and nanobarrels, α-hemolysin immobilizes in the interior of the nanobarrel due to hydrophobic interactions. Adapted with permission from ref 519. Copyright 2018 John Wiley and Sons, Inc.

Figure 69. Applications of small molecule protein interactions for protein immobilization in DNA nanostructures. (A) Incorporation of two halves of split eGFP in an adjustable rhomboidal DNA actuator. eGFP recombines only in the closed confirmation to emit fluorescence. Reprinted from ref 469. Copyright 2016 Springer Nature under a creative commons agreement (https://creativecommons.org/licenses/ by/4.0/). (B) Illustration of DegP immobilization in DNA nanostructures using binding to a peptide−DNA conjugate (top). The bottom panel illustrates a DegP complex immobilized within a 3D DNA hexagon and structures of two of the known DegP oligomers (6-mer and 12-mer, respectively). Adapted from ref 514. Copyright 2017 Springer Nature under a creative commons agreement (see (A)).

diffusion of DNA nanostructures immobilized on mica using divalent cations.523,524 In these cases, the structures remained associated with the surface, but due to competition between monovalent and divalent ions, the binding was sufficiently weak to allow diffusion of the structures on the surface which in turn mediated lattice formation. Various multivalent cations have been investigated for immobilization of DNA strands on mica in the presence of Na+, which provided valuable information on which ionconcentration ranges were suitable for imaging of the DNA for various multivalent cations.525,526 The data and guidelines provided in these studies, however, do not directly translate to larger nanostructures where the geometries and surface areas are significantly different, and in-depth studies for different types of DNA nanostructures will be required to develop general models for the interactions between DNA nanostructures and mica. Immobilization of DNA nanostructures on Si/SiO2 surfaces is highly important for the development of DNA nanostructured electronics. As for mica, this can be achieved using multivalent cations, although alkaline pH is generally required for rendering the silanols of the surface negatively charged.527 The use of high Mg2+-concentrations provides strong immobilization, but in general Mg2+- and Na+-salts are undesirable when making electronic devices due to deterioration of the electronic properties of the silicon substrate.405 Alternative strategies for immobilization include modification of the surface with positively charged amines.35,528 This can be achieved by treatment with aminosilanes such as (3-aminopropyl)triethoxysilane (APTES), which provides very uniform aminomodified surfaces enabling the immobilization of DNA

overview of the chemistry underlying surface-immobilization of DNA nanostructures on some of the most commonly encountered surfaces. 7.1. Electrostatic Interactions

Muscovite mica is the surface of choice for immobilization of DNA nanostructures due to its nearly perfect cleavage providing easy access to atomically flat surfaces.522 Due to the replacement of some silicon atoms with aluminum impurities in the tetrahedral mica sheets, the surface is overall negatively charged. Since DNA nanostructures are also highly negatively charged, cations are required to screen the charges and enable surface immobilization. Multivalent cations are highly efficient for this and, generally, immobilization is achieved using divalent cations such as Mg2+ or Ni2+ (Figure 71A). These ions bridge the anionic mica and DNA surfaces and provide strong immobilization whereas monovalent ions provide much weaker binding. Immobilization using Mg2+ and Ni2+ is a generally applicable approach suitable for all surfaces that can be rendered negatively charged. Interestingly, addition of Na+-ions was shown to enable BE


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Figure 71. Methods for immobilization of DNA nanostructures on surfaces. (A) Bridging between anionic surface and anionic DNA nanostructure using multivalent cations, typically Mg2+ or Ni2+, enables immobilization on various surfaces including mica and SiO2. (B) Surfaces rendered positively charged by treatment with aminosilanes enable direct immobilization of DNA nanostructures through electrostatic interactions. (C) Covalent binding of nanostructures to surfaces can be realized using amino-modified extended staple strands that react with activated surface groups such as NHS esters and isothiocyanates. (D) Avidin-bridging is typically used for immobilization on glass. Biotinylated BSA adsorbs to the surface, and addition of Streptavidin or Neutravidin. enables binding to DNA nanostructures containing biotinylated extended staple strands.

approach, the SiO2-surface was modified with cyano-groups by treatment with 1-cyano(4-dimethylamino)pyridinium tetrafluoroborate (CDAP). Reaction of amino-modified DNA origami with the cyano groups afforded isourea bonds that remained stable when kept in amine-free buffer.

nanostructures without addition of multivalent ions (Figure 71B).35,527−529 A similar strategy was used for introducing negative charge to gold surfaces. Carboxylate-terminated alkanethiols were reacted with the gold surface through gold− thiol bonding, and the carboxylate termini were used for DNA origami immobilization through Mg2+-bridging.530 Highly oriented pyrolytic graphite (HOPG) can also be rendered charged through reaction with carboxylate or amino-terminated alkane thiols.531 For instance, HOPG was treated with cysteamine which afforded a positively charged layer on the surface and enabled immobilization of DNA origami.532 Aminomodified silicon surfaces were also used for immobilization of graphene oxide whose carboxylic acid groups reacted in amidebond forming reactions with the surface-amines by annealing at 180 °C.533 This enabled the efficient immobilization of DNA origami on graphene oxide and on nitrogen-doped graphene oxide by Mg2+-bridging, whereas binding to reduced graphene oxide was less efficient.

7.3. Biotin−Avidin Interactions

DNA nanostructures have emerged as an attractive platform for single molecule fluorescence microscopy due to the unique positional control they provide over fluorescent molecules at the nanoscale. To carry out this type of experiments, immobilization of the nanostructures on glass slides is a requirement. This has been most widely achieved through adsorption of biotinylated BSA on the glass surface which enables immobilization of biotinylated DNA nanostructures by bridging with Streptavidin or Neutravidin. (Figure 71D).108,429,539−542 To increase control over the immobilization process, it is often carried out in a flow channel, and pretreatment of glass slides with hydrofluoric acid followed by thorough washing is typically employed.543 Streptavidin bridging was also used for immobilization of DNA origami in lipid bilayers, by introduction of biotinylated lipids and biotinylated DNA origami.544

7.2. Covalent Interactions

Another approach enabling the immobilization of DNA nanostructures on surfaces without introducing large amounts of salts involves covalent bonding to the DNA nanostructures. An appealing strategy making use of covalent binding is the immobilization of thiol-modified nanostructures on gold surfaces. This was for instance used by Fan and co-workers for binding a DNA tetrahedron to a gold surface with a specific face of the structure attached to the surface.534 The gold−thiol interaction was also used for bridging gold islands with DNA origami nanotubes,535 for trapping rectangular DNA origami with thiol-modified corners between gold islands on a SiO2surface,536 and for immobilization of origami using dielectrophoretic trapping between gold electrodes.537,538 Rothemund and co-workers have further devised a methodology for covalent immobilization of DNA origami on Si/SiO2 surfaces.527 Surface-treatment with a carboxylate-terminated silane (carboxyethylsilanetriol, CTES) on lithographically patterned domains enabled covalent binding via amide-bond formation using EDC and sulfoNHS in combination with amino-modified DNA origami (Figure 71C). In an alternative

7.4. Hydrophobic Interactions

As discussed previously, there has been an urge toward the development of higher-order structures formed via on-surface lattice formation. While addition of monovalent cations to samples immobilized using Mg2+-bridging facilitated surfacediffusion and hence to some extent enabled lattice formation,523,524 there is a demand for more efficient methods. In examples described previously in this review, cholesterol anchors were incorporated in DNA nanostructures to enable insertion in lipid membranes for various applications (See Section 2.4.3).135,155,158−161,169,170 The mobility of the structures in the lipid bilayers can be controlled through the number of incorporated cholesterol anchors, and in this way structures can be tuned to rapidly diffuse in the lipid bilayer. This enabled the efficient formation of lattices on lipid surfaces.156,157,545 Niemeyer and co-workers used dip-pen nanolithography employing phospholipids to generate lipid patches of arbitrary shapes and demonstrated that cholesterol-modified BF


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DNA origami could be immobilized selectively on the patches.544 Hydrophobic interactions have also been used for immobilizing DNA origami on Teflon amorphous fluoropolymer (Teflon AF).546 Here, one to five porphyrins were incorporated in the rectangular DNA origami to mediate binding to the hydrophobic surface. Interestingly, single molecule TIRF microscopy could be carried out directly on the Teflon surface.

Carbon nanotubes were immobilized in DNA nanostructures through noncovalent interactions with ssDNA although only with modest efficiency. This highlights that there is plenty of room for chemists to improve incorporation of CNTs and other carbon-based materials such as graphene sheets and graphene nanoribbons in DNA nanostructures. An important advantage of the functionalization of molecules and materials with one or more DNA strands is that otherwise insoluble species generally become soluble in water. Once dissolved in aqueous solution and equipped with specific DNA sequences, almost all such materials can be incorporated at specific locations in DNA nanostructures. The greatest asset offered by DNA nanotechnology is the ability to position molecules, biomolecules, and materials relative to each other with a resolution of a few nanometers or less.123 DNA nanotechnology offers a unique opportunity to investigate and take advantage of interactions between single entities. This has been demonstrated thoroughly for FRET interactions between dyes for studying distances, dynamics, and kinetics in DNA nanostructures but also to investigate light harvesting and directional energy transfer.126,554 The unique opportunity to organize metal nanoparticles in space enabled the study and design of plasmonic nanostructures with unique nonlinear optical behavior,24 while the ability to place dyes relative to metallic nanostructures opened new avenues in development and understanding of plasmonic nanoantennas.429 DNA nanostructures can be rendered conducting by metallization,317 or alternatively, localized electronic wires can be placed on DNA nanostructures. This was exemplified by the field-effect transistor created by arranging two carbon nanotubes in an orthogonal arrangement on DNA origami and subsequently connecting them to electrodes.246 Chemical modification of DNA has further enabled the positioning of a range of other electronically interesting materials on DNA origami, which for instance served to overcome challenges normally encountered in electrochemistry.199 Controlling chemical reactions by means of proximity is another facility offered by DNA nanotechnology. This has opened a new direction in synthesis, where molecules will only react if they are DNA-encoded to do so, even when present in complex chemical environments containing numerous potential reaction partners. This DNA-programmed synthesis strategy was initially used to prepare DNA encoded combinatorial libraries of organic compounds for drug discovery, which is now an area of increasing commercial importance, but the concept also spurred other advances in DNA nanotechnology.272 Macromolecular entities were assembled from molecular modules,50 polymers were formed on DNA scaffolds,182,308 and even the autonomous, multistep synthesis of sequence-controlled oligomers was realized.273,277 Furthermore, chemistries have been developed that allow internal cross-linking of DNA nanostructures to increase their stability and thereby make them more applicable for in vivo function.291,295 DNA nanostructures offer a unique opportunity to arrange enzymes relative to each other for studying enzymatic cascades and potentially create nanofactories.32 Furthermore, the positioning of protein ligands relative to each other in DNA nanostructures can be applied to study optimal distances for interactions with multiple surface receptors in cells and hence provide valuable biological insight.498,510,555 The advances in DNA nanostructure design enable the use of appropriately functionalized DNA-nanostructures as templates or molds for shaping other nanomaterials such as lip-

7.5. Other Methods for Surface-Immobilization

Another attractive strategy for surface-immobilization of DNA origami is via base pairing between extended staple strands and ssDNA protruding from the surface. In an elegant approach, Niemeyer and co-workers patterned amino-modified ssDNA on glass slides.510 The glass surface was initially washed and then reacted with the aminosilane, APTES. Subsequently, poly(bisphenol A-co-epichlorohydrin) was reacted with the surface to provide epoxy-terminated linkages that reacted with aminomodified DNA delivered via inkjet spotting. DNA origami equipped with extended staple strands complementary to the surface DNA patterns readily immobilized on the glass, as visualized by fluorescence microscopy. Alternatively, aminosilane modified glass could be treated with a bis-NHS linker or a bis-isothiocyanate to enable binding to amino-modified DNA.547 Of interest, a method based on spray-coating has emerged for immobilizing DNA origami on glass and silicon substrates without the need for any salts in the spraying buffers.548 This method can be of significant interest for large-area deposition and for immobilization on flexible substrates. Although the adsorption on mica using divalent cations remains the most popular method for surface-immobilization of DNA nanostructures, researchers will often benefit from considering alternative strategies. The behavior of surfaceimmobilized structures depends highly on the immobilization conditions as well as the surface employed and, therefore, the immobilization process should be given adequate consideration.

8. CONCLUSION AND OUTLOOK All artificial DNA nanostructures have their origin in chemical synthesis of oligonucleotides. Even for structures where the staple strands are made enzymatically,549,550 and for in vivo and in vitro RNA nanostructures,551,552 the designed genes of the expression systems were made by chemical synthesis. The advantage of using synthetic oligonucleotides is the ease of chemical modification during the automated synthesis as outlined in this review. Numerous phosphoramidite reagents are commercially available for functionalization with chemical handles, dyes, biotin, hydrophobic groups, etc., and oligonucleotides containing such modifications can even be obtained commercially. The only major drawback is that the modified sequences are significantly more expensive than unmodified DNA strands. Custom synthesis of phosphoramidites enables incorporation of basically all types of compounds into DNA, as long as they are compatible with DNA synthesis conditions. However, many compounds, in particular proteins and most peptides, are not compatible with the chemistry of DNA synthesis, and in these cases postsynthesis conjugation is the method of choice. Altogether, a wide range of nanomaterials have been functionalized with DNA and incorporated in DNA nanostructures. This includes polymers,353,355 dendrimers,553 nanodiamonds,363 proteins,5,29 metal nanoparticles,24,37 quantum dots,410 and a variety of other inorganic materials.368 BG


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central to drive the field forward. Equally important, DNA nanotechnology has provided chemists with an intriguing platform for doing chemistry with a unique degree of control. More and more chemists are seizing this opportunity, and we expect that new chemistries and increasingly useful materials will arise from this.

osomes,162,163,166,167 metal nanoparticles,328−330 polymer nanoparticles,182,300,306 and complex silica nanocomposites.366 All the numerous functions of DNA nanostructures discussed throughout this review are based on chemical engineering of the structures. The major focus was to provide an overview of the different chemistries employed and the tremendous opportunities provided by chemical modification of DNA nanostructures. Although most of the DNA nanostructures reported so far are fascinating and contribute to advancing the field, they mostly have little or no actual practical application. However, in recent years, the focus on practical applications of DNA nanotechnology has become much more pronounced and, in some areas, DNA nanostructures are becoming increasingly important. DNA nanotechnology and in particular functionalized DNA origami are emerging as valuable biophysical tools to study optical phenomena,24 protein structure and function,504,519 and interactions of proteins with cells.510 The vision of applying DNA nanostructures as a breadboard for nanooptical and nanoelectronics circuitry is still in its infancy, but the formation of increasingly efficient FRET cascades as well as optical resonators from coupled dyes shows promise for future applications.126,556,557 One of the major challenges for making molecular electronics on DNA origami is the connection to electrodes; however, with the increasing size of discrete nanostructures and advances in localized immobilization of DNA nanostructures; this is still an area of great potential.331,527 For development of novel methods in nanomedicine, DNA nanotechnology offers the opportunity to make discrete multifunctionalized structures with very high control of stoichiometry. This has potential for fabrication of highly advanced drug delivery vehicles and when combined with the mechanics of DNA nanotechnology, nanorobots with advanced responsive behavior can be fabricated.80,466 Chemical modification of such nanostructures will be crucial in terms of improving their in vivo stability which is a necessity if the structures are to find use as therapeutics. Similarly, the addressability of DNA nanostructures make them unique candidates for in vivo imaging and drug delivery, but also for these applications, the in vivo degradation of unmodified DNA nanostructures poses a major challenge. Implementation of known chemical modifications, such as nucleic acid analogues, is likely to partially solve this. Besides this, the development of new chemistries for stabilization and surface modification of DNA nanostructures will bring their application in medicine, drug delivery, and in vivo imaging a step closer. Apart from standard modifications of oligonucleotides, the majority of chemistries for DNA nanotechnology have been developed during the past 15 years, and this development is continuing. Many new methods for conjugation of molecules, including proteins, to DNA are currently being developed, which will make it significantly easier to incorporate proteins in DNA nanostructures. Recent advances in using DNA nanostructures to template the synthesis of other shape-specific nanomaterials is another area with great potential. This area will benefit from current developments in large scale DNA origami synthesis and new chemistries for incorporation of inorganic materials in DNA nanostructures. For application in nanomedicine we believe that new methods to load drugs in DNA nanostructures and development of new cleavable linkers to liberate the medicine under changing environments will be important future aspects. Predicting the future directions of DNA nanotechnology is challenging, but it is beyond doubt that chemistries will be

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Kurt V. Gothelf: 0000-0003-2399-3757 Notes

The authors declare no competing financial interest. Biographies Mikael Madsen is currently a postdoctoral researcher in the Gothelf lab. He obtained his Ph.D. degree from the Gothelf lab based on his work on conjugated polymers in DNA nanotechnology. In 2017, he worked with peptide chemistry in the lab of Bradley L. Pentelute at MIT, and his current research interests are within peptide and polymer chemistry for use in DNA nanotechnology. Kurt Gothelf obtained his Ph.D. degree from the lab of Professor K. A. Jørgensen at Aarhus University, Denmark, after studies in organic chemistry and asymmetric catalysis. Then he followed a postdoctoral stay in Professor M. C. Pirrung’s group at Duke University, USA, after which he joined the faculty at Aarhus University, working with surface chemistry and DNA nanotechnology. Since 2007, he has been Full Professor at Aarhus University and from 2007−2017 he was heading the DNRF Center for DNA Nanotechnology. He is currently director of the Novo Nordisk Challenge Center for Multifunctional Biomolecular Drug Design and coordinator of the Marie SkłodowskaCurie ITN network DNA-Robotics.

ACKNOWLEDGMENTS We thank Ditte Høyer Engholm for proofreading the manuscript. The work was funded by The Independent Research Fund Denmark (Grant Number DFF-7014-00382), The Danish National Research Foundation (Grant Number DNRF81), and the Novo Nordisk Foundation (Grant Number NNF17OC0028070). LIST OF ABBREVIATIONS A adenine aaRs amino acyl tRNA synthetase AFM atomic force microscopy AON antisense oligonucleotide APTES (3-aminopropyl)triethoxysilane ATRP atom-transfer radical polymerization AuNP gold nanoparticle AuNR gold nanorod Bhoc benzhydryloxycarbonyl BSA bovine serum albumin BSPP bis(p-sulfonatophenyl)phenylphosphine C cytosine CAII carbonic anhydrase II CD circular dicroism CE cyanoethyl CNT carbon nanotube CPG controlled pore glass CTAB cetyl trimethylammonium bromide BH


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STM SUV SWCNT T TAL TBDPS TdT TEG TEM TMS TIRF TOP TOPO tRNA U ZFP

Ctr CuAAC DA DBCO dBSA ddNTP DHLA DMT DNA dNTP DOPC ds DTPC DWCNT EDC EGF eGFP EGFR FRET Fmoc G GFP GOx GSH HI HOPG HPDP

citric acid copper-catalyzed azide−alkyne cycloaddition Diels−Alder dibenzocyclooctyne denatured bovine serum albumine dideoxynucleoside triphosphate dihydrolipoic acid dimethoxytrityl deoxyribonucleic acid deoxynucleoside triphosphate 1,2-dioleoyl-sn-glycero-3-phosphocholine double-stranded DNA-templated protein conjugation double-walled carbon nanotube 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide epidermal growth factor enhanced green fluorescent protein epidermal growth factor receptor Förster resonance energy transfer fluorenylmethyloxycarbonyl guanine green fluorescent protein glucose oxidase glutathione hot-injection highly oriented pyrolytic graphite N-[6-(biotinamido)hexyl]-3-(2-pyridyldithio)propionamide HRP horse radish peroxidase HU heat up IEEDA inverse electron-demand Diels−Alder LNA locked nucleic acid MNP metal nanoparticle MOSAIC multiscale origami structures as interface for cells MPA mercaptopropionic acid MWCNT multiwalled carbon nanotube NHS N-hydroxysuccinimide NTA nitrilotriacetic acid PNA peptide nucleic acid pNIPAM poly(N-isopropylacrylamide) PAINT points accumulation for imaging in nanoscale topography PANI polyaniline PCP peridinin-chlorophyll a protein PEG polyethylene glycol PMAO poly(maleic anhydride-alt-1-octadecene) po phosphodiester POPC palmitoyl-oleoyl-phosphatidylcholine PPV poly(p-phenylene vinylene) ps phosphorothioate QD quantum dot RNA ribonucleic acid SA-QD Streptavidin-coated quantum dot SAXS small-angle X-ray scattering sfGFP superfolding green fluorescent protein SLP self-labeling polypeptides SMCC N-(maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester SNARE soluble NSF attachment protein receptor SPAAC strain-promoted azide−alkyne cycloaddition SPDP 3-(2-pyridyldithio)propionic acid NHS ester ss single-stranded STEM scanning transmission electron microscopy

scanning tunneling microscopy small unilamellar vesicle single-walled carbon nanotube thymine transcription activator like tert-butyldiphenylsilyl terminal deoxynucleotidyl transferase triethylene glycol transmission electron microscopy trimethylsilyl total internal reflection fluorescence trioctylphosphine trioctylphosphineoxide transfer RNA uracil zinc-finger protein

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