DNA Quadruple Helices in Nanotechnology

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

DNA Quadruple Helices in Nanotechnology Jean-Louis Mergny*,†,‡,§ and Dipankar Sen*,∥,⊥

Chem. Rev. Downloaded from pubs.acs.org by UNIV OF BRITISH COLUMBIA on 01/04/19. For personal use only.

†

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing 210023, China ‡ ARNA Laboratory, Université de Bordeaux, Inserm U 1212, CNRS UMR5320, IECB, Pessac 33600, France § Institute of Biophysics of the CAS, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic ∥ Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada ⊥ Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada ABSTRACT: DNA has played an early and powerful role in the development of bottomup nanotechnologies, not least because of DNA’s precise, predictable, and controllable properties of assembly on the nanometer scale. Watson−Crick complementarity has been used to build complex 2D and 3D architectures and design a number of nanometer-scale systems for molecular computing, transport, motors, and biosensing applications. Most of such devices are built with classical B-DNA helices and involve classical A-T/U and G-C base pairs. However, in addition to the above components underlying the iconic double helix, a number of alternative pairing schemes of nucleobases are known. This review focuses on two of these noncanonical classes of DNA helices: G-quadruplexes and the imotif. The unique properties of these two classes of DNA helix have been utilized toward some remarkable constructions and applications: G-wires; nanostructures such as DNA origami; reconfigurable structures and nanodevices; the formation and utilization of hemin-utilizing DNAzymes, capable of generating varied outputs from biosensing nanostructures; composite nanostructures made up of DNA as well as inorganic materials; and the construction of nanocarriers that show promise for the therapeutics of diseases.

CONTENTS 1. Foreword 2. Introduction 2.1. G-Quadruplexes 2.2. i-Motif 2.3. Different Rules 2.3.1. Cation Sensitivity of G-Quadruplexes 2.3.2. Exquisite pH Sensitivity of the i-Motif 2.4. Structural Polymorphism 2.5. Different Properties of G-Quadruplexes and i-Motifs 2.5.1. Electrostatics 2.5.2. Stiffness 2.5.3. Resistance to Denaturing Conditions 2.5.4. Electrical Conductivity 2.5.5. Alternative Backbones and Base Modifications 2.6. Kinetics and Thermodynamics 2.6.1. Formation of G-Quadruplexes 2.6.2. i-Motif Formation 2.6.3. Kinetically Trapped Species 2.6.4. G-Quadruplex Dissociation 2.6.5. Strand Invasion 2.7. Ligands 2.7.1. Specific Proteins (G4) 2.8. Homorecognition Problem and How To Circumvent It 2.8.1. Guanines are a Quartet’s Best Friends © XXXX American Chemical Society

2.8.2. Non-G-Quartets 2.8.3. Combining Duplexes with G-Quadruplexes 2.9. Quadruplexes in Nanotechnologies 3. G-Wires 3.1. Definition 3.2. Higher-Order Structures Formed by G-Rich Oligonucleotides 3.2.1. Early Studies 3.3. Guided G-Wires 3.3.1. G-C Base-Pairing as a Guide 3.3.2. Extended Watson−Crick Duplexes Aiding G-Wire Assembly 3.3.3. Synapsable Motifs 3.4. G-Wires Formed by Poly d(G) 3.5. G4 Ligands Binding to G-Wires 3.6. Reversible G-Wires that Incorporate NonDNA Functionalities 3.7. C-Wires 4. Quadruplexes in Nanostructures 4.1. Simple DNA-Based Nanostructures 4.2. i-Motif-Based Supramolecular Assemblies 4.3. Quadruplexes as Partners for Larger Nanostructures

B B B C D D D E F F F F F G G G G G H H H I

I I I J J J J K K K K K L M M M M N N

Special Issue: Nucleic Acid Nanotechnology

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Received: October 19, 2018

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DOI: 10.1021/acs.chemrev.8b00629 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews 4.3.1. Supramolecular Quadruplex-Based Dendrimers 4.4. Observing/Modulating G4 Stability with DNA Origami 4.5. DNA Origamis Can Be Used To Modulate the Stability of G-Quadruplex Structures 4.6. Applications 5. Reconfigurable Structures and Nanodevices 5.1. General Considerations 5.2. Simplest Devices: Opening and Closing of a Quadruplex (G4 or i-Motif) 5.3. Quadruplex−Duplex Devices 5.4. Quadruplex−Quadruplex Devices 5.5. Applications 5.5.1. i-Motif Devices 5.5.2. DNA Walkers 5.5.3. G4-Based Molecular Beacons 5.6. DNA Logic Gates 5.6.1. G4 Logic Gates 5.6.2. Combining G-Quadruplexes and i-DNA 5.7. Nanopores and Nanochannels 5.8. Overview 6. Quadruplex DNA Nanostructures and Quadruplex−Inorganic Nanomaterial Composites for Analyte Sensing 6.1. General Considerations 6.2. Programmed Gold-Nanoparticle Aggregation as an Assay for G-Quadruplex Formation 6.3. Hemin·G-Quadruplex Complexes (Heme· DNAzyme) 6.4. Simple DNA Nanostructures for Biomolecular Detection 6.5. Inorganic Nanoparticle−DNA Quadruplex Composites for Biosensing 6.5.1. G4-DNA−Silver Nanoparticle Composites as Signal-Off and Signal-On Biosensors 6.5.2. Utilization of the Synthetic Capabilities of Heme·DNAzymes within DNA Nanostructures for Sensing 7. Nanocarriers and Therapeutics 7.1. Aptamers 7.1.1. AS1411, an Iconic G4-Forming Aptamer 7.1.2. i-Motif-Based Devices for the Controlled Release of a Cargo 7.2. Micelles 7.3. Hydrogels 7.4. Overview 8. Perspectives Author Information Corresponding Authors ORCID Notes Biographies Acknowledgments Abbreviations/Glossary: References

Review

complex 2D and 3D architectures and to design a number of systems for molecular computing, transport, motors, etc. DNA synthesis is now reasonably inexpensive, and DNA-based materials show good stability in aqueous solutions and in ionic liquids. Most of the above-mentioned kinds of DNA nanodevices are built with classical B-DNA helices and involve classical A-T/U and G-C base pairs. However, in addition to the iconic double helix, a number of alternative pairing schemes are known: “DNA comes in many forms” as written by A. Rich1 (for a review2). Indeed, these alternative structures may help to overcome some of the limitations of nanostructures composed solely of Watson−Crick base pairs, such as susceptibility to enzymatic degradation; low resistance to heat and to denaturing reagents; flexibility; deformability; and low sensitivity to chemical stimuli. In this review, we will focus on two of these oddities: Gquadruplexes and the i-motif. With a few exceptions, the results discussed here have been obtained with either short or long nucleic acid strands (mostly, but not exclusively DNA): notably, not with nucleosides, nucleotides, or isolated bases. For a review on the self-assembly of these latter entities and their applications in supramolecular chemistry, see ref 3.

N N N O O O O P Q Q R R R S S S T T

2. INTRODUCTION

T T

2.1. G-Quadruplexes

Nucleic acid sequences containing runs of guanines can form Gquadruplex structures, which are of special interest because of their high thermal stability, resistance to denaturing conditions, and stiffness. Research into quadruplexes has sustained a strong momentum because, contrary to many other alternative DNA or RNA conformations, G4 structures are stable under physiological conditions. G4-specific antibodies4 and in vivo NMR5 have also provided compelling data showing that this DNA structure is present in vivo. Guanine-rich RNA sequences also form stable G4 structures, which are often (but not always6) more stable than their DNA counterparts. Quadruplexes are composed of stacked nucleobase quartets (also called tetrads); in a G-quadruplex, each of the quartets arises from the planar association of four guanines by Hoogsteen hydrogen bonding (a network of (C2)NH2:N7 and O6:N1H hydrogen bonds). “Simple” quadruplexes are formed by 1−4 separate DNA strands, while higher-order G4-based structures (G-wires) may involve hundreds of individual strands. Quadruplexes are stabilized by intraquartet hydrogen bonds (each tetrad contains four guanines linked by eight hydrogen bonds), interquartet stacking (at least two quartets are present), and cation coordination. For a more detailed description of Gquadruplexes, one is referred to various review articles.7,8 A schematic drawing of a G-quartet is shown in Figure 1a, and a tetramolecular G-quadruplex structure is shown in Figure 2, left. Higher-order structures can also be obtained with DNAs incorporating guanine-like purine bases, such as isoguanine. Chaput and Switzer12 demonstrated the ability to expand DNA helix molecularity beyond quadruplexes by engineering nucleobases to fit dimensions required for a DNA pentaplex. They used 2′-deoxy-isoguanosine, for which the angle between hydrogen bond donor and acceptor groups is ∼67°, as compared as 90° for guanine. While 90° is a perfect angle to assemble a tetraplex, 67° is close to the ideal angle for a pentaplex (360/5 = 72°) (Figure 1b). Pentaplex formation was demonstrated most optimally in the presence of cesium cations while a more classical quadruplex is favored by the presence of potassium.12,13

U U V V

V

W W W X Y Y Z Z Z AB AB AB AB AB AB AB AB

1. FOREWORD Nucleic acids have emerged as powerful nanotechnological tools because of their predictable and controllable assembly. Watson−Crick complementarity has been used to build B


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Figure 1. (a) Guanine-quartet. (b) Isoguanine pentad. (c) GCGC quartet (other base arrangements are possible for these mixed quartets). (d) C−C+ base pair.

Figure 2. Presentation of tetramolecular G-quadruplex (left) and tetramolecular i-motif (right). The tetrameric d([TG4T]4) quadruplex depicted on the left comes from a crystal structure9 (PDB 2O4F). The i-motif structure is a tetramer formed by d(AACCCC), solved by NMR10 (PDB 1YBL). Pictures were generated with UCSF Chimera 1.12.11 Guanosines are depicted in brown, cytidines in pink, adenosines in blue, thymidines in green, phosphate backbones as white ribbons, and Na+ as purple spheres. Note the differences in length between these two structures which involve the same number of nucleotides (6 × 4).

later confirmed by crystallography.15 i-Motif structures are formed by one, two, or four individual C-rich strands. The geometry of the i-motif differs substantially from that of any other nucleic acid structure, including Watson−Crick duplexes and G-quadruplexes. The i-motif is actually more a “double duplex” (a composite of two equivalent parallel-stranded righthanded duplexes), rather than a real quadruplex. The two

Surprisingly, these modified nucleobases and pentaplex design have been underexploited in DNA nanotechnology despite the pentaplex’s unique structure and cation requirement/sensitivity. 2.2. i-Motif

Cytosine-rich DNA sequences may adopt an i-motif structure at mildly acidic pH. This was initially discovered by NMR14 and C


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interlocked duplexes within an i-motif are zipped together by the intercalation of hemiprotonated cytosine−cytosine (C·C+) base pairs (Figure 1d). Such systematic intercalation (the letter i in imotif stands for intercalation) means that each individual duplex is severely underwound and extended, as illustrated in Figure 2, right. The distance between two cytosine−cytosine base pairs in the i-motif is only 3.1 Å, which is shorter than the distance between base pairs or base quartets (around 3.4 Å). However, these two base pairs belong to two different duplexes, and the distance between two consecutive base pairs within the same duplex is actually doubled (6.2 Å): i-DNA is far more extended than the G-quadruplex (compare both structures in Figure 2). The i-motif is very flat, with two very wide grooves and two extremely narrow grooves: two strands belonging to two different duplexes are actually contacting each other. Since its discovery 25 years ago, interest in the i-motif did not grow as fast as it did for the G4 structure, given the former’s limited stability under physiological conditions (see Figure 3 for

Figure 4. Composite schematic of a DNA duplex, a G-quadruplex, and an i-motif. Adapted from ref 25. Copyright 2016 American Chemical Society.

2.3. Different Rules

The i-motif and the G-quadruplex rely on orthogonal pairing rules, leading to very different properties, which are summarized in Table 1. 2.3.1. Cation Sensitivity of G-Quadruplexes. Cation effects on G-quadruplexes differ significantly from those on duplexes, as direct cation coordination by guanine nucleobases is nearly always required for stable G4 formation. Potassium and strontium (K+ and Sr2+) function most optimally to stabilize Gquadruplexes, whereas Na+, NH4+, Rb+, Ca2+, and Ba2+ are less optimal, and Li+, Mg2+, and Cs+ are generally ineffective. Interestingly, and in contrast with a number of other DNA nanostructures, magnesium is not required for proper G4 folding. Largy et al.26 proposed the following general trend for G4 stabilization: Sr2+ > K+ > Ca2+ > NH4+, Na+, Rb+ > Mg2+ > Li+ ≥ Cs+ (alkali cations are shown in bold). Cations of larger ionic radii, such as potassium, locate between successive layers of quartets while smaller cations (sodium, for example) are also able to coordinate within the plane of individual tetrads or, indeed, localize at intermediate positions. Lithium ions have sometimes been considered as G4-destabilizing ions; they should, rather, be considered as “indifferent/neutral” toward quadruplex formation. In the presence of small amounts of potassium, a destabilization of the G4 by even large (up to 1 M) concentrations of LiCl was not observed. Cation binding to G4 structures actually involves two different modes: (i) nonspecific (diffuse), where the cations retain their outer-sphere hydration, and binding of nonspecific cations to the negatively charged backbone reduces the electrostatic repulsions and thus also promotes folding, as with double helices; and (ii) specific (sitebound) by coordination to the guanine O6. The abovementioned cations that participate in this matter are involved in electrostatic and donor−acceptor orbital interactions with the lone pair electrons of the O6 oxygen atom of guanine, yielding tight M+−O coordination bonds. A detailed discussion of cation effects on G-quadruplexes can be found in ref 26. 2.3.2. Exquisite pH Sensitivity of the i-Motif. The fundamental pairing scheme relies on the formation of intercalated, hemiprotonated C·C+ base pairs. As each base pair requires protonation of one cytosine at the N3 position (with a pKa between 4 and 5; its exact value depends on ionic strength, because of polyelectrolyte effects), pH plays a crucial role in the formation of this structure. Stability can be controlled by pH, and stability is optimal around the cytosine pKa.16 Increasing the solution pH by one unit (in the range 4.5−7.5) typically leads to a decrease in Tm of 15 °C or more (Figure 2). Increasing cytosine tract lengths results in increased thermal stability; sequences with at least five cytosines per tract fold into i-motif at room temperature and neutral pH.27 In parallel, Burrows and colleagues analyzed 10−30 nt long dC homooligonucleotides.28,29 Interestingly, the relation between C-tract and stability was not linear. The authors found that dCn strands

Figure 3. pH dependency of i-DNA stability as shown by UVabsorbance melting profiles at 260 nm of the intramolecular structure of the human telomeric sequence (CCCTAA)3CCC as a function of pH. Both heating and cooling profiles are shown; note the slight hysteresis at low temperature for pH 6.5 and higher. Melting may also be followed at 295 nm; in that case, inverted transitions are seen as absorbances decrease at this wavelength when i-DNA is denatured. UV melting curves were recorded on a Kontron Uvikon XL spectrophotometer with a temperature gradient of 0.2 °C/min: identical protocol as in refs 16 and 17).

an illustration of its pH dependency). Two very recent studies, however, argue that the i-motif is actually present within cells; this argument is based on staining data using specific i-motif antibodies18 and on in vivo NMR in human cells.19 Regardless, this limited stability at physiological pH is not necessarily a disadvantage for nanotechnology applications, and the extreme pH dependency of i-DNA can actually be an asset for pHresponsive devices (see below). In contrast to G-quadruplexes, for which both DNA and RNA can form stable G4 structures, RNA i-motifs are less stable than the corresponding DNA imotif,20,21 and application of i-motifs to nanotechnology has so far been limited to DNA oligomers. Interestingly, the sequence requirements somewhat mirror those for G4 formation; as a consequence, the complementary strand of a G4-forming sequence is itself prone to i-DNA formation, as depicted in Figure 4. Single-molecule studies suggest that the simultaneous formation of both structures on the same region is sterically disfavored. For a more detailed description of i-DNA one is referred to various review articles.22−24 D


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Table 1. Summary of Key Properties parameter

Watson−Crick duplex

G-quadruplex

i-motif

sensitivity to pH sensitivity to cation (Na, K) molecular crowding electrostatics

limited no

limited yes

extreme no

destabilizes polyanion

stabilizes extended polyanion with hemiprotonated bp

mechanical properties polymorphism “denaturing” conditions specific ligands type of pairing stability

flexible no sensitive yes; specific recognition is possible complementary base pairs predictable, depends on length and GC content yes

stabilizes polyanion with a central spine of positive charges variable parallel G4 are stiff important resistanta yes, for a given topology homorecognition GGGG short oligomers can form very stable G4

formation in cells

yes

stiff limited sensitive? a few homo base pair CC+ short oligomers can form very stable i-DNA... at acidic pH yes

a

Conditions considered to be denaturing for duplexes are not always denaturing for quadruplexes. Some G4 can resist formamide or urea and therefore still be observed on a denaturing gel. They are often thermally stable (see Table 2). Ethanol does not destabilize them. Molecular crowding stabilizes them; they are also formed in deep eutectic solvents and can still be formed in the gas phase (in ESI-MS experiments). The only reliable way to denature stable G4 is alkaline pH.

Figure 5. Intramolecular G-quadruplexes can adopt a variety of folds: antiparallel (A, C), parallel (B), or hybrid (D, E). Reproduced with permission from ref 33. Copyright 2015 Elsevier.

strand polarity, i.e., the relative arrangement of adjacent strands: parallel, hybrid, or antiparallel. In the latter case, two strands are oriented in one direction and the two others in the opposite way; these pairs may correspond to two adjacent or diagonally opposed strands, resulting in very different geometries of antiparallel quadruplexes (the “chair” and “basket” types, respectively). Different topologies available to monomeric, intramolecular G4-DNAs are presented in Figure 5. The detailed formalism of intramolecular G4 folding developed by Webba da Silva and colleagues allows the description of the relationships of type of loop and groove widths of a quadruplex stem.30−32 The folding topology and stability of intramolecular (and bimolecular) G-quadruplexes are determined to a large extent by their loops; both conformation and thermal stability are greatly dependent on loop permutation34 and loop length.35−38 These topologies affect the orientation of connecting loops (lateral, diagonal, or chain reversal/propeller) and the glycosidic torsion angle of each guanosine (e.g., anti or syn; parallel topologies

of length 15, 19, 23, and 27 nucleotides (i.e., 4n−1) have optimal stabilities, with pH of midtransition above 7.2 and thermal stabilities above 37 °C at pH 7.0.28 This ability to fine-tune imotif stabilities by the length of the strand should be useful for some nanotechnology applications. In any case, whenever imotif formation is considered at any solution pH value above the cytosine pKa, the formation is associated with a significant concerted proton uptake (around one per base pair formed16); this affects the spectroscopic and thermodynamics properties of the folded form, and it can also alter the pH of a solution if not properly buffered. 2.4. Structural Polymorphism

The structural diversity of G-quadruplexes results from a number of variables. One may first consider the number of quartets (typically three or more; but 2-quartet quadruplexes can also be encountered) and strand stoichiometry (one, two, three, or four strands). A major source of variation comes from E


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generally involve only syn guanosines). Finally, additional features such as unusual quartets (such as GCGC quartets;39,40 Figure 1c), capping base pairs or base triads (as in refs 41 and 42), or other stacking entities (such as pentads or hexads43), bulges,44 and snapbacks may also be involved. Structurally, Gquadruplexes are best considered to manifest complex shapes rather than being purely linear polymeric assemblies. Such an intrinsic structural diversity is potentially a problem for nanotechnology, since a single sequence can adopt different quadruplex folds depending on the experimental conditions. The iconic human telomeric motif d-(GGGTTA)n is arguably the most complex polymorphic G4 structure known to date.45 RNA quadruplexes, by contrast, exhibit a lower diversity of strand orientations as (with the notable exception of GFP-like in vitro selected RNA aptamers) all RNA strands in an RNA G4 run in a parallel orientation. Intramolecular G4 species are believed to be more biologically relevant while assemblies composed of two or more interacting species should be of interest for supramolecular chemistry. Tetramolecular quadruplexes allow the assembly of four identical strands in a generally predictable and controlled manner. Like G4-DNA, i-DNA is also polymorphic, but the variety of the latter’s structures is more limited given that, in contrast to Gquadruplexes, strand orientation is severely imposed (the two diametrically distant strands are always parallel to each other).14 Gel electrophoresis of i-motifs sometimes reveals the presence of more than one band, even from simple sequences. Guéron and colleagues noted that an intramolecular structure formed by four repeats of the human telomeric motif can adopt four configurations, in which all cytosines are base-paired and with minimal energetic differences.17 Correspondingly, spectroscopic measurements often reveal a multistep denaturation/renaturation process.27,29

length of B-DNA is significantly longer than that of singlestranded oligomers, such duplexes are too flexible to allow the building of rigid and stable nanostructures. A solution to this problem makes use of “double crossover” structures (topologically constrained adjacent pairs of four-way helical junctions), which alleviates in part the relative flexibility of individual double helices. i-DNAs and G-quadruplexes, however, are good alternative candidates to improve rigidity: the geometrical constraint imposed by the extended backbone (in the case of the i-motif) and/or larger stacking surface between successive quartets (for G-quadruplexes) enable both helices to be stiffer than the classical B-DNA double helix (see section 3 on Gwires). Initial predictions to this effect have been verified using a variety of techniques. The apparent molecule height reduction in scanning tunneling microscopy is much lower for quadruplexes than for duplexes; this is likely due to the Gquadruplex’s increased stiffness relative to the duplex.47 2.5.3. Resistance to Denaturing Conditions. Typical conditions that are expected to denature DNA, such as the one typically chosen for denaturing gel electrophoresis (incubation in 40−50% formamide, heating, and migration in 7−8 M urea) are not always sufficient to denature G-quadruplex structures.48 This property is an advantage for DNA-based nanotechnology, as structures relying on G4 formation will tolerate a larger range of temperatures and experimental conditions as well as the presence of denaturants. It creates problems during sample preparation, however, as undesired kinetically trapped species may be formed immediately following chemical synthesis of Grich oligonucleotides. In this case, a short (several minutes) incubation at alkaline pH is sufficient to destroy all competing structures. G-quadruplexes are still formedand actually stabilizedin the presence of high concentrations of ethanol46,49 that would denature classical duplexes. Electrospray-compatible organic cosolvents (methanol, ethanol, isopropanol, or acetonitrile) actually increase the stability and rate of formation of G-quadruplexes and cause structural transitions to strand-parallel conformers.50 Solvent effects influence the structure of G-quadruplexes formed in dehydrated and molecularly crowded environments, by the nature of the cosolvent and by the time scale of the reaction, with >200-fold acceleration of formation of bimolecular G-quadruplex in the presence of 60% cosolvent.51 Both G452 and i-DNA53 are favored by crowding conditions, in contrast with B-DNA which is destabilized by crowding.54 Molecular crowding can also induce a structural transition from antiparallel to parallel conformation of G-quadruplexes52 and enables G-quadruplex formation under salt-deficient conditions.55 2.5.4. Electrical Conductivity. DNA is the only type of biopolymer for which substantial electrical conductivity is observed in individual molecules over a length-scale of several nanometers, because of base stacking in a π-orbital system. The overlap of the π orbitals depends on the relative orientation of the base planes and thus on the specifics of the DNA structure. Thus, quadruplexes, being stiffer and more resistant to surface forces than double-stranded DNA, are inherently more likely to enable charge transport. Porath and colleagues reported clear evidence of polarizability of long G4-DNA molecules, measured by electrostatic force microscopy (EFM), while coadsorbed dsDNA molecules on mica were electrically silent, suggesting that G4-DNA could potentially be better than dsDNA as a conductive molecular wire.56 This finding was later confirmed by the same teams in 2014, which reported reproducible charge

2.5. Different Properties of G-Quadruplexes and i-Motifs

2.5.1. Electrostatics. Nucleic acids are polyanions, and as expected for a structure involving four negatively charged strands, electrostatics play a fundamental role in the stability of G-quadruplexes.46 Sufficient ionic strength is required to compensate for the electrostatic repulsion between the phosphate oxygens of four strands in a G4 structure. The quadruplex core, with its central spine of positive charge in its middle and four negatively charged phosphate-sugar backbones, offers interesting differences from a classical B-DNA double helix. Interestingly, if most quadruplex ligands are, expectedly, positively charged, a few G4 ligands, such as hemin or NMM, are either neutral or negatively charged. The electrostatics of the i-motif are even more unusual. While it also involves four negatively charged strands, this structure has two unique features that decrease its net linear charge: (i) Each CC+ base pair is protonated and bears a positive charge. (ii) The backbone is extended as a result of intercalation (see Figure 2, right). As a result, the overall net charge density along the imotif’s axis is actually lower than in a classical B-DNA duplex! Increasing the ionic strength or adding dications has limited, if any, effect on i-motif stability: when working at pH 5.5 or higher, stability is actually higher with no salt added,16 as the apparent pKa of cytosine is increased at low ionic strength because of a polyelectrolyte effect. The protons tend to condense around the negatively charged DNA backbone. 2.5.2. Stiffness. Stiffness is a highly desirable trait for DNA nanotechnology; unfortunately, even though the persistence F


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transport in G-quadruplexes adsorbed on a mica substrate.57 Currents ranging from tens of picoamperes to more than 100 pA were measured in quadruplexes over distances of 100 nm or more. The transport mechanism proposed involves thermally activated hole hopping from one multitetrad subsection of the DNA to the next, in a manner similar to what is found in conductive polymers. Interestingly, EFM measurements showed a stronger signal for tetramolecular G4-DNA than for monomolecular G4-DNA, while no polarizability was detected for the poly d(G)−poly d(C) duplex.47 Conductive AFM measurements on G-wires fit well with a model that combines fast coherent transport within short segments of the complex, with a slower hopping between the individual segments. 2.5.5. Alternative Backbones and Base Modifications. Most of the reports taking advantage of G4 or i-motif for nanotechnology use classical DNA (and more rarely RNA), because of cost, ease of synthesis, and tools available. Quadruplex formation, however, is not restricted to regular nucleic acids; indeed, modified backbones and bases can be accommodated into G4 structures.58,59 While substituting guanine by another nucleobase is often, but not always,59 detrimental to G4 stability (see below), G4 assembly tolerates a number of backbone modifications. Among oligonucleotide analogues, one of the most radical changes to the natural structure is in peptide nucleic acids (PNAs) where the entire sugar−phosphate backbone is replaced by a pseudopeptide. Homologous G-rich PNA and DNA oligomers, however, hybridize to form a hybrid PNA−DNA quadruplex of high thermodynamic stability.60

involved in this rate-limiting step and participate in the early stages of the quadruplex stem assembly. Substituting sodium by potassium affords a large (>10-fold) increase in association constants.63 Overall, the folding landscape of a G4 is rugged, and misfolded or partially folded structures may be present. The folding of DNA quadruplexes is best described by a kinetic partitioning mechanism, known also as multiple-pathway or multiple-funnel folding process.65−67 Non-native or imperfect quadruplexes can act as deep offpathway kinetic traps;68 kinetic partitioning is the only known folding mechanism that can explain very long folding times. The melting of tetramolecular G-quadruplexes is kinetically irreversible when working with dilute DNA solutions (μM strand concentration range).63 This allows the independent study of association and dissociation processes (for a review, see ref 69). A number of guanine base substitutions have been experimentally assessed: in general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, as shown by a reduced quadruplex lifetime/thermal stability andmore surprisinglya decrease in association kinetics.58 Extremely large differences (up to 109-fold!) were found between the association constants of these quadruplexes depending on modification position and nature as well on cation identity (sodium, potassium, or ammonium). These results demonstrate that most guanine substitutions are tolerated, at best: guanines remain a quartet’s best friends!58 2.6.2. i-Motif Formation. Folding and unfolding of the intramolecular i-motif follow relatively fast kinetics under mildly acidic conditions, as expected for a monomolecular process. However, this is no longer the case at pH 6.8 or higher, where a hysteresis phenomenon occurs, leading to observed large differences (>20 °C) in apparent Tm upon heating and cooling at pH 7.2 of an intramolecular process.70 While slow kinetics seem surprising for intramolecular folding, one should keep in mind that this single-strand i-DNA equilibrium also involves the net uptake of protons (≈one per base pair when pH ≫ pKa) which become vanishingly rare at neutral or basic pH. 2.6.3. Kinetically Trapped Species. The formation of imperfect or mismatched G-quadruplexes, particularly at low temperature and in the presence of potassium, can impede the formation of the canonical G-quadruplex by kinetic trapping. These unproductive intermediates can play an important role when doing structural studies or prepare higher-order structures such as G-wires (see below). Long G-runs are especially difficult to deal with. In contrast to the common belief that d(TG(n)T) DNA sequences would form tetramolecular G-quadruplex assemblies, mass spectrometry reveals that these sequences tend to fold into G-quadruplex strand−trimers, dimers, and eventually monomers as the G-tract length increases.71 Electrospray mass spectrometry revealed the existence of long-lived misfolded structures (off-pathway compared to the most stable structures) and enabled counting of the number of cations and quartets present in these individual species.72 For (TGnT)4 quadruplexes, kinetically trapped, mismatched structures can make the canonical strand−tetramer quadruplex practically inaccessible at low temperature in KCl.61 In the case of TG5T, tetramers do not have their four strands perfectly aligned to give five G-quartets: the structures contain one ammonium ion too few. At low temperature, the rearrangement of the kinetically trapped tetramers with presumably slipped strand(s) into the perfect G-quadruplex structure is extremely slownot even complete after 4 months.73 Interestingly, the addition of methanol to the

2.6. Kinetics and Thermodynamics

2.6.1. Formation of G-Quadruplexes. While intramolecular quadruplex formation tends to be relatively fast (except for kinetically trapped species; see below) intermolecular G-quadruplex formation is generally slow, especially for tetramolecular complexes.58 Stable complexes resisting boiling can be formed with relatively short G stretches (Table 2). Table 2. Illustration of Tetramolecular G4 Thermal Stability DNA/RNA sequence

G-track (nucleotides)

apparent Tma (Na+) (°C)

d-TGGGT d-TGGGGT r-UGGGGU d-TGGGGGT

3 4 4 5

16 54−75b 89 c

apparent Tma (K+) (°C) 48 c c c

a Apparent Tm depends on temperature gradient, and melting profiles exhibit a strong hysteresis. bDepending on temperature gradient and ionic strength. cResists boiling, at least for a few minutes.

Unsurprisingly, the association rate of bi- and tetramolecular complexes is concentration-dependent: for tetramolecular complexes, the association is third to fourth order in monomer strand.61,62 Counterintuitively, the association rate constant decreases with increasing temperatures, in accordance with a nucleation-zipping model.63 The association rate constant decreases with increasing temperature, reflecting a negative activation energy (Eon).63 Negative “apparent” activation energies for quadruplex formation are interpreted within the now so-called nucleation-zipping model, in a manner similar to what was proposed for duplex formation.64 The observation that ionic strength and nature of the monocation play an important role in the value of kon indicates that one or more cations are G


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Figure 6. Examples of G-quadruplex ligands. NMM and TMPyP4 are anionic and cationic porphyrin derivatives, respectively. 360A is a Cu(II) reconfigurable bis-quinolinium cationic ligand.83 BPEP 1 is a bis(phenyethynyl)pyridylcarboxamide compound.84 CV refers to Crystal violet; ThT to Thioflavin T. cex-NDI is an extended naphthalene diimide derivative specific for parallel G-quadruplexes.81 Hemin is a natural G-quadruplex ligand found in DNAzymes (see section 6.3). Hemin and NMM are negatively charged at neutral pH: a positive charge is not a requirement for selective G4 binding, and can actually be detrimental to selectivity.81

micromolecular or higher strand concentrations, an intermolecular duplex tends to be the predominant species when mixing a G-rich strand, susceptible to form a G4, with its complementary strand. 75 Mendoza et al. developed a fluorescence assay to study the kinetics of quadruplex-to-duplex conversion.76

monomer solution significantly accelerates the cation-induced G-quadruplex assembly. 2.6.4. G-Quadruplex Dissociation. While slow kinetics of association may be a problem for some applications, slow kinetics of dissociation can also become a nuisance. The lifetime of a G-quadruplex is much higher in potassium than in sodium and increases with the length of the guanine tract. Note that not all G-quadruplexes have extremely long lifetimes: if the half-life of the tetramolecular TGGGGT quadruplex is extremely long in 100 mM KCl (too long to be measured), the intramolecular thrombin-binding aptamer has a much shorter lifetime. Opening the quadruplex structure found in the stem of a G4 molecular beacon is also relatively slow74 and poses a problem for some applications. 2.6.5. Strand Invasion. Invading a G4 may be problematic/ extremely slow. The speed of the duplex−quadruplex exchange is a fundamental feature, in both biology and nanotechnology. A kinetic and thermodynamic study of this equilibrium is therefore necessary to fully optimize the properties of novel nanodevices. Interestingly, because of their orthogonal pairing rules, a number of parameters affect the thermodynamic stability of a quadruplex structure against a duplex motif. Unfortunately, few reports to date deal with kinetic studies on quadruplex structures. At

2.7. Ligands

Specific ligands: small compounds and light-up probes. Quadruplexes offer unique structural features for targeting by small molecules. A number of teams have developed G4-specific ligands; a thousand or so have been listed in the G4 ligand database (http://www.g4ldb.org/ci2/index.php),77 and a few examples are provided in Figure 6. Some of these compounds are light-up probes, with a strong fluorescence enhancement in the presence of G-quadruplexes. This allows the selective staining of a G4 within a larger structure, or in the presence of competing conformations. While some compounds such as Thioflavin T light up in the presence of all quadruplex structures,78 others are specific for a given topology (for a review, see ref 79). For example, N-methyl mesoporphyrin IX (NMM)80 and an extended naphthalene diimide derivative81 are specific for parallel G4 while crystal violet is a light-up probe for antiparallel H


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colleagues determined the structure of the intramolecular quadruplex formed by CTAGGG telomeric repeat variants in potassium. They observed the formation of an antiparallel Gquadruplex involving two G-quartets sandwiched between a GC base pair and a G-C-G-C quartet.40 A beautiful application of these mixed quartets to design an original G-wire will be presented in the next chapter.94 One can also play with purine analogues to design an artificial quartet involving the two “complementary” guanine derivatives xanthine and 8-oxo guanine.95 2.8.3. Combining Duplexes with G-Quadruplexes. In this case, base-pair complementarity between guide duplexes is used. There are distinct modes of duplex and quadruplex connectivity.96 Ideally, for nanotechnology, coaxial orientation of the duplex and quadruplex helices with continual base stacking should be desirable. The stability of these complexes can be substantially influenced by the base-pair steps proximal to the quadruplex−duplex junction. One design involved parallel duplexes. The design includes two elements: (i) several (3 or 4) parallel-stranded duplexes and a central parallel-stranded quadruplex.97 Formation was easya simple annealing with no special preparation protocol allowed the formation of the desired structure with good yields. Association was fast, thanks to the assistance provided by the bimolecular duplexes; otherwise formation of a tetramolecular quadruplex at micromolar concentrations can be exceedingly slow. Finally, thanks to the quadruplex core, the complex was robust, i.e., resistant to high temperatures and denaturing conditions.97 A more classical design involves regular Watson−Crick antiparallel duplexes, as shown over two decades ago.98 This design involves classical antiparallel duplexes; the resulting duplex is therefore expected to be antiparallel, but as shown by Mendoza et al.,99 the predominant structure often involves parallel quadruplexes. 5′−5′ or 3′−3′ linkages allow the formation parallel quadruplexes within these assemblies.100 Zhou et al. combined duplex and quadruplex parts to assemble a G-quadruplex structure from three different strands.101 What can be done with isolated strands may also be integrated into DNA origamis.102 Two duplexes with G−G mismatch repeats in the middle can be incorporated inside a DNA origami frame. Formation of a stable quadruplex is induced by addition of potassium. High-speed AFM enables the real-time analysis of the cation-induced formation of a tetramolecular G-quadruplex and its denaturation in salt-free conditions. Three consecutive guanines were sufficient to allow stable quadruplex formation. The origami frame and guide duplexes allow the precise control of strand stoichiometry and orientation.

structures: the presence of side loops in parallel G-quadruplexes does not protect the dye from the solvent, leading to a lower increase in fluorescence.82 In contrast, very few ligands are known for i-DNA, and the compounds often exhibit modest selectivity toward this structure.85 At first glance, the i-motif structure is so unique that one should be able to find compounds binding to it and no other structures. However, after screening a number of chemical libraries, it was found to be much harder to find i-DNA-specific as compared to G4-specific compounds (J.-L.M., unpublished observations). Waller and colleagues used a fluorescent intercalator displacement assay to screen for i-motif ligands.86 2.7.1. Specific Proteins (G4). Numerous proteins have been reported to bind to G-quadruplexes (for reviews, see refs 87−89). In addition, a number of aptamers recognizing proteins adopt a quadruplex fold (see section 7.1). This could be of interest for nanotechnology, as they may be used as reporters or provide new functionalities. Some polypeptides can recognize G4 structures and unfold them; they are called G4-helicases (for a review, see ref 90). While highly relevant for biology, these enzymes have rarely been used for nanotechnology applications. In a reciprocal fashion, some proteins not only bind to preformed G-quadruplexes, but also actively promote their formation. This is the case, for example, for Topoisomerase I,48 the yeast telomere-binding protein RAP191 and HIV nucleocapsid.92 The list of proteins binding to the i-motif is much shorter; even if some polypeptides are known to bind to cytosine-rich nucleic acids, it is often unclear whether they recognize the single-stranded or i-motif form of the nucleic acid. 2.8. Homorecognition Problem and How To Circumvent It

G-quartets (for G-quadruplexes) and CC+ base pairs (for imotif) are both based on homorecognition (rather than complementary or heterorecognition): these strands can therefore always bind to themselves! As a consequence, complex mixtures are obtained from self-assembly processes instead of well-defined objects. This fatal flaw would prevent the use of these motifs for most nanotechnology applications. 2.8.1. Guanines are a Quartet’s Best Friends. In general, the presence of a single nucleobase substitution within a guanine tract has a strong deleterious impact on the resulting quadruplex’s stability, with significantly reduced quadruplex lifetime/thermal stability as well as decreased association rate constants. Most guanine substitutions are deleterious to tetramolecular quadruplex structure, and most nonguanine quartets do not participate favorably in structural stability: these quartets are formed only by virtue of the docking platform provided by neighboring G-quartets.58 It is ironic that the Gquadruplex, which involves a single predominant building block, the G-quartet, is actually far more polymorphic than the DNA double helix, with its four canonical base pairs. 2.8.2. Non-G-Quartets. Notable exceptions to the “guanine-only” rule can be found, though. For example, guanine derivatives such as 8-bromo-guanine58 or 8-amino-guanine59 accelerate quadruplex formation. Even more interesting is 6methyl-isoxanthopterin, which can also form a stable quartet.58 This shows that the purine geometry is not an absolute requirement to form a stable quartet. All quartets discussed above were formed of four identical bases. This is not an absolute requirement, and mixed quartets have been experimentally observed39 (other mixed tetrads or hexads can be found in ref 93). For example, Phan and

2.9. Quadruplexes in Nanotechnologies

The following chapters present applications and illustrations of quadruplexes in nanotechnologies, in which G4 and i-motif structures are incorporated into a variety of objects (wires, origamis, gels, micelles, etc.) and used for a diversity of applications, such as biosensing or therapeutics, that take advantage of the unique properties of these fascinating structures. The literature on G-quadruplex-based nanotechnologies is currently extensive, and we have chosen to present representative examples rather than to try to be exhaustive. On the other hand, the number of articles dealing with nanotechnology applications of i-DNA is more limited; however, because of its unique pH dependency, this structure can add interesting functionalities to larger structures. I


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Figure 7. G-wire assembled from the Tetrahymena telomeric DNA sequence: 5′-G4T2G4. Reproduced from ref 110. Copyright 1994 American Chemical Society.

collectively, as “G-wires”. These include, additionally, structures formed from d(G4T2G4)110−112 (Figure 7); dC4T4G4T2G4;113 d(CGG)4;114 d(GGCGTTTTGCGG);115 d(G4);116 and d(G11T).117 Interestingly, even a minor change in the sequence of the G-rich sequence has been found to often lead to a drastic change in its self-assembling property. The G-wire formed by d(G4) was used to assemble metal phthalocyanine complexes to mediate electron transfer processes leading to electrocatalytic reduction of CO2. The binding of metal complexes on the Gquadruplex wire and electron transfer mediated by nanowire allowed efficient redox cycling of catalytic centers on the electrode.116 Phan and colleagues obtained high-resolution AFM images of G-wires formed with d(G4T2G4) in aqueous solution.112 Well-defined periodic structures allowed them to propose the formation of various types of G-wires: “(2,2) Adjacent”, “(2,2) Diagonal”, and “(3,1)”, all based on slipped strands models.112 G-wires are therefore polymorphic, and this polymorphism has important implications in nanotechnology involving DNA self-assembly. Kankia has made some interesting observations on guaninerich DNA oligonucleotides of the form 5′-(G3N)3G3, where “N” represents any of the four DNA bases. This motif is known to be the minimal one for folding to an intramolecular, all-parallel Gquadruplex. Kankia observed that the above motif, initially 5′(G3T)3G3, folds and assembles into a highly regular “infinite” Gwire with “unprecedented speed and stability”, indeed, melting above 100 °C.118 Other, related, repeat sequences, such as 5′(G2T)3G6(G2T)2G and 5′-(G3T)3G6(G3T)2G do not form Gwires, notably, because these oligonucleotides are inclined to fold into antiparallel G-quadruplexes, which do not appear to be suitable for further assembly into G-wires.119 Two other features of this work are noteworthy: first, the observation that the folding of 5′-(G3T)3G3 oligonucleotides into G-quadruplexes is highly exergonic has been exploited to devise both linear and exponential isothermal amplification methodologies for DNA (QPA, or “quadruplex priming amplification”), capable of an impressive 1010-fold amplification of a target sequence in less than 40 min.120,121 The second is the observation that 5′(G3G)3G3 (which is nothing other than the homopolymer oligodG) forms G-wires in the uniform way that all 5′-(G3N)G3 appear to do, by forming overlapping, intramolecular, wholly parallel-stranded G-quadruplexes in which the “N” base constitutes the propeller loop linking G-quartets.119 This last observation has potentially significant bearing on the question of the precise structure of the enzyme-generated, long poly d(G) wires innovated by Kotlyar and colleagues (see below). A pattern that emerges from all of the above examples of successful G-wire-forming sequences is that they must present guanine bases at either one of both their (5′ and 3′) termini. This is a necessary but not sufficient condition, given that 5′(G2T)3G6(G2T)2G and 5′-(G3T)3G6(G3T)2G do not form Gwires (vide supra). Is the presence of non-G bases at both ends of a sequence sufficient to prevent G-wire formation? The evidence to date is that such an absence does act as a powerful brake on the ability of that sequence to generate G-wires. As an initial

3. G-WIRES 3.1. Definition

A “G-wire”, for the purposes of this review, can be defined as an extended DNA nanostructure in 1-dimension, formed by the self-assembly of one or more individual DNA oligonucleotides by way of G-quadruplex formation. The key to the formation of a “wire” by numerous individual oligonucleotides can be on three different structural bases: the pure π−π stacking of terminal Gquartets of individual intramolecularly folded G-quadruplexes;93,103 the formation of G-quartets between “slipped” Grich strands from more than one oligonucleotide; and via the binding together of sticky ends from individual intramolecular G-quadruplexes to form composites bound, for example, via GC base pairs and G-C-G-C base quartets.93,94 The structural expectation of all G-wires is that they must present an overall structural homogeneity over the scale of their lengths as well a certain mechanical stiffness. In this respect, a G-wire is clearly distinguished from linear strings of individual G-quadruplexes; a G-wire is not simply the juxtaposition of several intramolecular quadruplexes, much as beads on a string or a pearl necklace. An example of such as structure has been described by Phan and colleagues.104 In that paper, the G-rich motifs are embedded into a relatively long (50 nt or so) motif, which allows each individual G4 to be independent. 3.2. Higher-Order Structures Formed by G-Rich Oligonucleotides

3.2.1. Early Studies. In the 1970s, fiber diffraction studies were carried out on poly d(I), as well as on gel- and fiberlike aggregates formed by guanine nucleosides and nucleotides (reviewed by Davis3). These early structures first revealed the base-pairing arrangements now known as hypoxanthine and guanine quartets, respectively. However, in the late 1980s, there was a resurgence of interest in noncanonical folding of guaninerich single-stranded DNAs.105−107 These studies on the formation of two-stranded and four-stranded DNA Gquadruplexes (“G4-DNA”) paved the way for discovery of larger, multistranded assemblies (for instance, containing, 8, 12, and even higher numbers of strands), “superstructures” or nanostructures that could be formed by DNA oligonucleotides containing stretches of at least three contiguous guanines. Thus, the sequence 5′-T8G3, incubated in high salt (e.g., 1 M KCl) at 20 °C at relatively high (∼1 mM) DNA concentrations, formed G-quartet-containing superstructures of 8, 12, or more DNA strands.108 These assemblages were early versions of what would later be called “frayed wires” generated from incubation of, for instance, dA15G15.109 The superstructures/nanostructures formed from TnG3 had their component DNA strands arranged in a wholly parallel orientation. As for the frayed wires formed by dA15G15, however, Raman spectroscopy found the presence of syn-guanosine within them, suggesting at least a partial presence or toleration of antiparallel G-quadruplexes within those wires. Since then, a large number of studies in the general area of selfassembling nanostructures from short G-rich DNA oligonucleotides have described a family of structures that can be described, J


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example, while 5′-T8G3 formed a G-wire, T8G3T did not.108 Chiorcea-Paquim et al. have also reported that while dG10 easily forms continuous, infinite G-wires and frayed wires, dTG9 forms limited G-wires containing a maximum of 18 G-quartets.122 By contrast to these, dTG8T, under comparable experimental conditions, does not form G-wires at all and only forms the standard, 8-quartet G-quadruplex. Are there any common features in the preparation protocols for G-wires? Given the highly intermolecular nature of G-wires, the formation of which requires the association and hydrogen bonding of multiple molecules of one or more DNA strands, high oligonucleotide concentrations (0.1−1.0 mM) typically lead to favorable outcomes, with most incubations to generate G-wires carried out around 20 °C. Incubation times reported by the various groups are variable; Webba da Silva, Spindler, and colleagues, for instance, have used slow dialysis to generate Gwires more efficiently than any thermal annealing protocol.123 Curiously, self-assembling G-wires formed from RNA Gquadruplexes have not been reported to date, although that is likely a matter of establishing the right experimental conditions for their formation. Because of the bulk of the 2′-hydroxyl within the constituent ribose sugar of RNAs, and the strong preference of nucleotides within RNA for the anti-glycosidic conformation, RNA, unlike DNA, forms only parallel quadruplexes. Comparably to DNA, however, RNA forms intramolecular Gquadruplexes from both a single-strand of RNA containing four guanine-rich motifs, as well as intermolecular quadruplexes involving four distinct strands containing a single guanine-rich motif. Since it is parallel-stranded G-quadruplexes (with the sequence of the oligonucleotide terminating in a guanine on at least one end) that most easily form G-wires, it appears likely that RNA should indeed be able to form G-wires.

G-wires. Sugimoto and colleagues125 prepared G-wires using a single canonical Watson−Crick duplex of substantial length to stack with and bridge together successive antiparallel Gquadruplexes. Using a distinct design, Yatsunyk, Mergny, and colleagues97 have combined unusual parallel-stranded duplexes to initially control the assembly of a G-quadruplex core. This approach was then successfully extended to generate a onedimensional assembled nanostructure (Figure 8a).

Figure 8. (a) Duplex-guided G-quadruplex. (Adapted from ref 97. Copyright 2013 American Chemical Society.) (b) Twisting G4-based DNA switch, composed of a G-quadruplex topologically constrained by four duplexes. (Adapted with permission from ref 126. Copyright 2014 John Wiley and Sons).

3.3.3. Synapsable Motifs. “Synapsable DNA” is a term used to describe duplex DNA constructs incorporating a central motif of G-G mismatch base pairs, flanked on either side by conventional Watson−Crick base pairs.98 Synapsable DNAs were designed to enable the side-by-side joining, via Hoogsteen base-pairing, of two DNA duplexes via formation of G-quartets involving guanines located in both participating duplexes. The G-G mismatch domains therefore function as Velcro strips to bind the two participating duplexes together side-by-side, rather than end-to-end (Figure 9), although synapsable DNA, first

3.3. Guided G-Wires

Several experimental approaches have been attempted to “guide” the assembly of G-wires. 3.3.1. G-C Base-Pairing as a Guide. Webba de Silva, Spindler, and colleagues have reported, in a series of papers, their approach of using short stretches of Watson−Crick base-pairing to assemble together parallel-stranded G-quadruplex modules capable of forming “guided” G-wires.94,124 Thus, the oligomer 5′-(GCGGAGGCG), dialyzed against a 100 mM sodium chloride buffer, generated extensive (∼900 nm long) and straight (as viewed using the atomic force microscope) G-wires, assembling an uninterrupted quadruplex in which successive layers of purely guanine quartets are held together by two short duplex stems consisting of two GC base pairs each, bridging the G-quadruplexes. In this arrangement, side-by-side interaction and hydrogen bonding between GC base pairs from their major groove sides generate G-C-G-C quartets, structurally isomorphous with their neighboring G-quartets. One curious property of this GC base-pair “guided” G-wire is that its CD spectrum is “inverted” relative to the spectra typically seen for B-form DNA duplexes as well for parallel- and anti-parallel-stranded Gquadruplexes. The authors reason that this unusual CD spectrum for their G-wire arises from the alternation of segments of parallel-stranded G-quadruplex and antiparallel GC-G-C quartets occurring within this particular wire. 3.3.2. Extended Watson−Crick Duplexes Aiding GWire Assembly. Two quite different sets of methodologies have been reported, on the use of extended duplex elements (both the standard antiparallel and parallel duplexes) to control and orient G4 assemblies in a manner favorable for generating

Figure 9. Synapsable DNA. (Adapted with permission from ref 98. Copyright 1996 Elsevier.)

reported in 1996, had been used successfully to generate DNAbased intramolecular switches.127 Mendez and Szalai showed, for the first time, that G-wire-like nanofibers of 250−2000 nm length could be generated via self-assembly of synapsable duplexes containing sticky ends on either side.128 3.4. G-Wires Formed by Poly d(G)

G-wires made of short G-rich oligonucleotides have the potential shortcoming as nonuniform polymers with gaps. K


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Figure 10. Generation of pure long G-wires. An innovative strategy involving end-biotinylated DNA strands and their immobilization on streptavidin beads to generate long G-wires. (Adapted with permission from ref 47. Copyright 2018 John Wiley and Sons.)

assembly by those oligonucleotides inclined to form these structures, it is reported that decreasing the annealing rate as well as raising either the salt or the oligonucleotide concentration tends to favor the formation of wires relative to that of merely intramolecular, monomeric G-quadruplexes. In agreement with prior observations on the proclivity of different oligonucleotides to form intramolecular G-quadruplexes of distinct topology and strand orientation, these authors confirm that oligonucleotides that prefer to form antiparallel or hybrid parallel−anti-parallel-stranded (as opposed to parallelstranded) G4 monomeric folds do not easily aggregate further to form G-wires, and that the substitution of loop nucleotides with abasic sites enhances the formation of G-wires.

Such a shortcoming may limit or reduce their mechanical stability and stacking properties of the resultant wire, impacting in turn on its ability to conduct an electrical current and thereby limiting its application in nanoelectronics. G-wires may also be formed with very long poly d(G) produced enzymatically. In 2005, Kotlyar and co-workers described how the Klenow exo-fragment of DNA polymerase I can be exploited to generate long poly d(G)−poly d(C) molecules of controlled lengths, starting from short dG10−dC10 duplexes.129 Following strand separation, this method enables the purification of long poly d(G) strands, which self-assemble into mono- and tetramolecular wires containing hundreds of stacked G-quartets. Such a controlled production of clean poly d(G) constituted a major improvement for the generation of long pure G-wires (Figure 10). The enzymatic procedures for the production of long (from tens of nanometers to microns) double-stranded poly d(G)−poly d(C) and quadruple-helical G4-DNA have recently been reported in refs 130−132. High-resolution scanning tunneling microscopy (STM) revealed the periodic structure of these wires, which behave in agreement with the expected helical morphology of Gquadruplexes.133 It has been proposed that the G-wires produced by this method are long, antiparallel G-quadruplexes formed from the long, single-stranded DNA that has folded back three times upon itself. However, other data suggest that these wires, like other G-wires, may also be parallel-stranded. Such data include their CD spectra, which resemble those of standard parallel G-quadruplexes; also, the key observation that these Gwires are 1/5 (rather than 1/4) the length of a poly d(G)−poly d(C) duplex formed from the same length of the G-rich strand.134 Kankia has proposed, on the basis of his own observations, that oligo-d(G) itself folds into seamless, parallelstranded G-wires of the class formed by oligonucleotides of the sequence 5′-(G3N)3G3 (vide inf ra), that G-wires formed from very long poly d(G) strands are of the same structural type as the “infinite” G-wire formed by 15-nt poly d(G) (in which precisely 3 nucleotides out of the 15, i.e., 1/5 of the total, participate in loops119). An in-depth and systematic recent experimental study of the requirements for forming G-wires has been reported by Varizhuk et al.,93 and provided key insights into the formation of these nanostructures. In terms of protocols for G-wire

3.5. G4 Ligands Binding to G-Wires

Mostbut not allG4 ligands stack on terminal tetrads. This end binding mode is less effective for long, perfect, Gquadruplexes: one cannot decorate a G-wire with such compounds. Intercalation, on the other hand, is believed to be disfavored (if not impossible) when cations are present within a G4’s core. One of the interesting properties of poly d(G) is its ability to form G-wires even in the absence of cations, in contrast with shorter sequences. Spectroscopic data suggest an intercalative mechanism of cationic porphyrin binding to potassium-free G-wires; such a binding mode, expectedly, is absent when dealing with the potassium form of this G-wire.135 Das et al.84 have reported interesting data showing that certain end-stacking bis(phenyethynyl)pyridylcarboxamide (BPEP) ligands assist the formation of G-wires by mixed-topology intramolecular G4 units formed by the human/vertebrate telomeric repeat (T2AG3)4 (h-TELO). The authors hypothesized that these ligands end-stack at either end of individual intramolecular h-TELO quadruplex units and help to join together such individual units to generate long wires. Interestingly, different G-wire morphologies are obtained depending on which isomer of a particular bis(phenyethynyl)pyridylcarboxamide (BPEP) ligand is present during G-wire growth. Thus, more uniform wires are generated via incubation with the compound BPEP 2 relative to incubation with the compound BPEP 1. Additionally, different levels of G-wire branching can be seen with different ligand-to-G4 ratios during incubation. L


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3.6. Reversible G-Wires that Incorporate Non-DNA Functionalities

mobile; in this chapter we will focus on defined static structures, while the next chapter will be dedicated to devices capable of structural switching in response to an environmental change.

Can the formation of G-wires be controlled to take place on demand? Also, can such wires, once formed, be reversed? Two distinct approaches to this problem have made use of G-rich DNAs either incorporating non-DNA components, or using an accessory, G-quadruplex-capable DNA-like polymer (PNA) to facilitate the wished-for morphological transition. Sugimoto and colleagues136 have devised a modified DNA oligonucleotide, based on d(G4T4G4), in which the thymines have been substituted with 2,2′-bipyridine moieties linked to DNA segments on either side via oligoether linkers. This design anticipated that, in the presence of soft metal ions (such as Ni2+), capable of coordinating well with the bipyridine units, the antiparallel-stranded G-quadruplex formed in the absence of Ni2+ would switch to a parallel-stranded G-quadruplex. Indeed, this is found to occur. The nickel-bound parallel-stranded Gquadruplex is able to undergo further, higher-order assembly to form long G-wires (∼200 nm), whereas the nickel-stripped DNA (achieved by addition of EDTA) no longer shows the morphology of a G-wire. Successive Ni2+ and EDTA treatments can cycle this modified oligonucleotide back and forth between G-wire and “disorganized” states. Using a distinct approach, Usui and co-workers137 have used a G-rich PNA, which incorporates an internal protease-susceptible site, to control the assembly of a G-wire from a G-rich, purely DNA oligonucleotide derived from the MYC oncogene. In this paradigm, in the first morphological state, the guaninecontaining PNA participates together with the MYC-derived Grich DNA to form hybrid PNA−DNA G-quadruplex “particles”, which do not display the morphology of a G-wire. However, addition of the protease capable of digesting a susceptible site within the PNA liberates the G-rich DNA from the above particles and enables the concomitant assembly of the second morphological state, the G-wire, assembled now from the wholly DNA oligonucleotide.

4.1. Simple DNA-Based Nanostructures

Quadruplexes and i-motif can both be used as non-Watson− Crick interaction modules to build nanostructures. Sen and colleagues connected two DNA double-strands via a G-G mismatch region, devising a structure called a ‘‘DNA synapse”98 (Figure 9). G-G mismatches within a Watson−Crick duplex confer a stickiness, or tendency to dimerization, to these sites within the duplexes and were called “sticky guanine domains”. These regions allow DNA double helices to stably bind one another at specific sites. Synapsis itself is straightforward and requires no prior duplex denaturation; simple mixing of the duplexes under physiological conditions is sufficient. Interestingly, the kinetics followed the order Na+ ≫ K+ > Li+ which does not correspond to the classical order found for G4 stability (K+ > Na+ ≫ Li+). Such a preference for sodium has also been reported for G-wire formation (see above). However, as discussed before,99 parallel-stranded quadruplexes are so stable that this topology predominates for long G-runs. Fahlman and Sen demonstrated that 5′−5′ or 3′−3′ linkages enable control of wholly parallel strand orientation within the quadruplex:100 G-G mismatch base pairs can be put in a parallel orientation within the duplex: the corresponding synapsed complex was expected to have parallel quadruplex strand orientations. In the presence of a highly favorable cation such as Sr2+, an unexpected “pinched” structure occurred within a single duplex when long G-runs where considered: formation of quadruplex occurs within the G-G mismatch region and did not require the association with another duplex. “Patterning” G-G mismatches by introducing thymine−thymine mismatches within the G-G domains allows the self-selective dimerization of a given domain: it dimerizes with another identical domain and not with duplexes possessing other G-G mismatch patterns:127 “self ” is preferred over “non-self ”. Interestingly, the G4-based synapse embedded within four duplex arms can be seen as a more constrained and less dynamic alternative to the classical DNA four-way junction. Double crossovers (topologically constrained and structurally rigid adjacent pairs of four-way junctions) have been used with great success to build DNA 2D structures; it would be interesting to see if one can substitute some or all of these crossovers by synapsable G-quadruplexes. Three- or four-way G4 junctions have been designed by Seeman and co-workers: a G4 motif is used as a connector element for a multiended DNA junction. Charges can enter the structure from one end of a three-way G4 motif, and can exit from the other end with minimal carrier transport attenuation.139 These results are in agreement with the earlier data of Huang and Sen,126 working on G4 4-way junctions (see below). The authors tested two different configurations, in which the G4 core can be either parallel or antiparallel. A spacer was inserted between the duplex and quadruplex regions. Parallel directionality can be realized by using chemically modified DNA with 3′−3′ and 5′−5′ linkages inserted at the G4 duplex junction. As the design of the antiparallel four-way G4 junction is virtually identical to the one we studied a few years ago, it would be interesting to check if the G4 core remains antiparallel, as long G-runs (G5 or G6) tend to adopt a more stable parallel fold, potentially losing a few base pairs or quartets in the process.99 In any case, optimization of stacking at the duplex−quadruplex should be key for optimal

3.7. C-Wires

Long poly d(C) molecules (hundreds or thousands of bases) of specific and uniform length can also be prepared. 138 Interestingly, these long C-strands exhibited a typical i-motif CD signature, and AFM analysis revealed the formation of homogeneous, compact, spherically shaped nanostructures at mildly acidic pH. These nanostructures presented two interesting and surprising properties: (i) the spherical structures have improved pH stability and were preserved even at neutral pH, in contrast to shorter C-runs; and (ii) the spherical shape was unexpected, as i-motif structures are rigid and characterized by an extreme helical stretching.

4. QUADRUPLEXES IN NANOSTRUCTURES DNA nanotechnology allows the organization of nucleic acids with exquisite spatial resolution. These structures can find applications in a number of fields (e.g., photonics, nanomechanics, sensing) because of the programmable control of their shape and size, addressability, ease of preparation, and biocompatibility. DNA-based nanostructures (origami, Lego) can adopt virtually any 2D or 3D shape and can be functionalized with a variety of groups such as DNAzymes, aptamers, fluorophores, or even larger elements such as metal nanoparticles. While the fundamental building block of these structures is the regular double helix, quadruplexes may well be accommodated. DNA nanostructures may be either static or M


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transport: a “floppy” junction may compromise overall positioning of such a device. Quadruplexes and duplexes can be combined in a variety of ways. Sugimoto and colleagues combined the G4T4G4 Oxytricha G-rich motif with duplex modules to design higher-order structures visible by AFM.125 Mendez and Szalai fabricated DNA-based nanofibers (up to 2 μm long) containing quadruplex and duplex regions.128 The “duplex” precursor DNA in their design includes a long run of guanines in each strand, sequences flanking the G-rich region that are complementary to another strand, and single-stranded overhangs. In this hybrid design, a G4-specific fluorescent light-up probe such as NMM may be used to demonstrate that the quadruplex is present in the structure. Increasing the annealing temperature increases fiber formation, in agreement with the proposed assembly model, which involves partial melting of some duplex regions. Unfortunately, control is not perfect, as different duplex arrangements are possible. Xu et al.140 demonstrated the cyclization of DNA based on the formation of a G-quadruplex structure. One can then obtain a relatively large and stable lariat based on G-quadruplex “foldback” structure. Sannohe and Sugiyama141 constructed a topologically complex structure in which a DNA catenane is obtained through the formation of a G-quadruplex structure triggered by the addition of potassium ions. As the resulting structure does not require the formation of covalent bonds, it is heat-sensitive and can be resolved by heat treatment. Heckel and colleagues used a slightly different concept, in which 168 bp long DNA minicircles equipped with G-rich appendixes can self-hybridize into intermolecular G-quadruplexes.142 In this case, the G-rich sequence acts as glue. Two different designs were compared, involving either a hairpin with a terminal G-rich loop, or a duplex ending with two singlestranded G-rich toeholds, each containing one G-run. Interestingly, formation of a diagonally looped G-quadruplex requires interlocking of the two strands and is not topologically possible when the DNA minicircles are assembled before G4 formation. This limitation is alleviated when working with the “tuning fork” minicircles.

because of the molecular recognition capacity of G-quadruplex aptamers: Liu et al. used the thrombin-binding aptamer as a robust platform to link proteins to periodic sites of a selfassembled DNA array.146 TBA is one of the smallest intramolecular quadruplexes; this 15-nucleotide long sequence was used to tether thrombin to a DNA lattice based on a triplecrossover (TX) DNA tile. Such an approach allowed the organization of proteins into 1D periodic arrays. Periodicity could be adjusted by incorporating TBA at every tile, every other tile, or every third tile. Thrombin was easily made evident by AFM as regularly spaced brighter spots. 4.3.1. Supramolecular Quadruplex-Based Dendrimers. Guanine-rich strands can be linked to the focal point of a dendron, and quadruplex formation drives the selfassembly process in the presence of proper metal ions.147 These noncovalent assemblies exhibit thermoresponsive behavior, and their dynamic nature allows the creation of combinatorial libraries of dendrimers. 4.4. Observing/Modulating G4 Stability with DNA Origami

The direct observation of DNA structural changes at the singlemolecule level is possible with an atomic force microscope (AFM). Sugiyama, Endo, and colleagues designed a clever 100 × 80 nm2 rectangular DNA origami frame with an inner vacant space148 to follow a number of phenomena (e.g., B−Z transitions, switching of photoresponsive oligonucleotides; some of these are detailed in the next chapter). In this case, the origami frame is a rigid, passive part, which positions strands at defined positions, allowing specific interactions to occur. This system was applied to G4 formation or disruption, either alone, or in the presence of ligands149 or proteins.92 G4 within an origami frame can be induced by both unprocessed NCp15 and matured NCp7 HIV nucleocapsid proteins, confirming the molecular chaperone activity of these NCp’s. An origami frame may also be used to follow a duplex to G4+imotif transition.150 Double-stranded DNA can be resolved into single-strands, G4, and i-motif by the addition of K+ under mildly acidic (pH 5.5) conditions. This construct may be used to study this transition at the single-molecule level, which may be relevant for the regulation of gene expression at promoters. High-speed AFM allows one not only to make evident the final folded state of G-quadruplexes, but also to make evident possible quadruplex intermediates, such as G-hairpin and G-triplexes.151

4.2. i-Motif-Based Supramolecular Assemblies

The high stability of the i-motif at acidic pH allows this structure to serve as the stem of one-dimensional nanowires, and a fourstrand stem can provide a new basis for three-dimensional DNA structures such as pillars. On the other hand, i-motif stability at neutral or alkaline pH is low, hampering a number of potential applications. Interestingly, mesoporous silica nanochannels can instigate the formation and stabilization of i-DNA even in neutral and alkaline media.143 This strategy offers an alternative way to control i-motif formation other than by using pH or thermal annealing.

4.5. DNA Origamis Can Be Used To Modulate the Stability of G-Quadruplex Structures

Olejko and colleagues designed an origami as a platform that suppresses the formation of a G-quadruplex152,153 (an oligonucleotide mimicking the human telomeric repeats (GGGTTA)4 or the variant motif (GGGATT)4) in the presence of sodium, allowing the selective detection of potassium ions by FRET. This sensing systems enables selective potassium sensing at concentrations from about 0.5 to 50 mm, even in the presence of high concentrations of sodium ions. A four-color FRET photonic wire was designed on a 2D DNA origami over a total distance of 10 nm; its functionality was shown to be dependent on G4 formation.153 Shrestha et al. and Jonchhe et al. analyzed the effect of confined space on the property of quadruplexes using DNA origami nanocages.154,155 When folded, the intramolecular quadruplex structure formed by the human telomeric motif (5′-(TTAGGG)4TTA) was put into nanocages of different sections (6 × 6 to 15 × 15 nm2). The authors found that the stability of G-quadruplexes increases when decreasing cage size,

4.3. Quadruplexes as Partners for Larger Nanostructures

Li and Mirkin used short tetramolecular G-quadruplexes to assemble nanoparticles.144 Highly cooperative transitions occur thanks to the concerted melting of multiple G4 structures connecting the nanoparticle (NP). The melting temperature was highly dependent on G-track length with Tm of 35, 53, and 87 °C for runs of 3, 4, and 5 G, respectively. NP aggregation is not always a desirable outcome: in that case, avoiding potassium salts and runs of consecutive guanines is suggested. Ren et al. grafted G-quadruplexes onto one-dimensional DNA nanostructures with precise positioning.145 This concept allows the use of DNA nanostructures as scaffolds for other partners, N


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Li et al. recently provided a masterful application of G4− origami complexes in vivo. They designed a 90 × 60 nm rectangular DNA origami sheet, which was then loaded with four thrombin proteins anchored on the surface. This flat surface was then converted into a hollow tube (with a diameter of ∼19 nm and a length of 90 nm) by fastening the thrombin-loaded DNA origami sheet along its long sides. As the four thrombin molecules are fastened to the inner surface and end up in the inner cavity, this conformational change shields them. Fastening is achieved by six pairs of fastener strands consisting of the G4forming AS1411 oligonucleotide and a partially complementary strand. In the absence of nucleolin, a duplex is formed, closing the tube. When nucleolin is present, AS1411 switches to a quadruplex state; the duplex is disrupted and tube opened, exposing thrombin161 and activating coagulation at the tumor site. As this device was found to be safe and immunologically inert in mice and miniature pigs, it represents the first use of a DNA nanorobot in vivo (see following chapters).

as showed by 100-fold faster folding rate. This is the result of a confined volume effect, in which the entropy of unfolded biopolymer decreases relative to the folded conformation.154 Jonchhe et al. found that water activity in small nanocages (with cross sections of 9 × 9 nm) decreased beyond the reach of regular cosolutes such as polyethylene glycol (PEG). The overall loss of water drove the folding of G-quadruplex or i-motif in nanocages.155 Incorporation of a G4 within a DNA minicircle may also affect its stability: Klejevskaja et al. found that the Gquadruplex unfolding kinetics within a circle were 10-fold slower than for the same “isolated” G4 motif.156 This illustrates that flanking DNA and topological restrictions can have an important impact on G4 stability, and this observation is relevant not only for biology but also for nanotechnology, as G4-based nanostructures are nearly always embedded within larger DNA constructs. 4.6. Applications

Incorporating quadruplexes into origami not only permits the more facile study and modulates the stability of these unusual structures, but also opens up specific applications that are made possible by the functionalities provided by the G-quadruplex portion of the origami. A few very recent examples are discussed below (two of them actually rely on G4 dynamic switching, which will be discussed in more details in the next chapter). The acquisition of single-wall nanotubes (SWNTs) with narrow length distributions has attracted much interest, given that the optical, electrical, and mechanical properties of SWNTs depend on their length. Being able to generate SWNTs of defined lengths is therefore highly desirable. Atsumi and Belcher developed a clever platform combining DNA origami and a G4heme complex (known as the heme·DNAzymesee below) to achieve the size-controlled cutting of SWNTs.157 Cutting is performed via a chemical oxidation system comprising hydrogen peroxide and hemin to produce radical species. Hemin, being a natural G4 ligand, is prepositioned in close proximity to the SWNT thanks to the use of a trifunctional DNA strand, which provides a (i) SWNT binding platform, (ii) a G4 core (to bind and activate hemin), and (iii) a “staple” domain, to be inserted at a specific position in a DNA origami. The Hemin-SWNT proximity enhances the biological activation of hydrogen peroxide by hemin within the heme·DNAzyme and accelerates SWNT cutting which would take weeks otherwise. Overall, this work demonstrates that a DNA origami allows for the transfer of spatial information to inorganic materials (SWNT in this example) and reveals the possibility of controlling chemical reactions at a predetermined location. This approach enables the adjustment of the lengths of SWNTs from a broad distribution of lengths. Torelli et al. designed a DNA nanorobot with a switchable flap, which opens in the presence of an input signal and exposes a heme·DNAzyme which would be buried and catalytically inactive otherwise.158,159 The size of traditional DNA origami is limited by the length of the M13 phage circular scaffold. To build larger structures, one needs to self-assemble several origami units into highly ordered superstructures. This can be achieved by classical DNA sticky-ended cohesion. By attaching specific functional oligonucleotides that may undergo a cationinduced conformational change between G-quadruplex and single-strand, Wang and colleagues built a cation-responsive bridge for the self-assembly of highly ordered DNA superstructures.160 This was illustrated by the conditional dimerization of cross-shaped DNA origami tiles.

5. RECONFIGURABLE STRUCTURES AND NANODEVICES 5.1. General Considerations

DNA nanostructures are not merely passive structural elements and are capable of controlled conformational changes. While the previous chapter mostly dealt with “static” structures, we will now explore how quadruplexes can be useful for dynamic devices. Exploiting the dynamic function of DNA sequences allows the conversion of DNA structures into DNA nanodevices with precisely controlled motions. When harnessed, these controlled motions may be used to generate molecular motors and other mechanical devices. While most DNA-based devices are based on duplex−hairpin/single-strands equilibria, some switchable structures involve unusual nucleic acid motifs, as discussed below. In the case of reconfigurable DNA nanostructures, an external trigger or fuel (e.g., another DNA strand, a change in pH or temperature, addition of cations, ligands, or proteins) induces a global and dynamic conformation change. This reconfiguration may be either permanent or reversible, depending on design; it generally relies on the integration of one or more elementary motifs, which are switchable depending on an external signal. These elements induce a structural transition provided they are located at suitable positions in the DNA nanostructure. Some of these switchable elements will be discussed in the “sensors” chapter, as the conformational change may be used to monitor the input signal: the quadruplex is not the switch, but the output as, for example, in “split quadruplexes”, in which a conformational change allows two G-rich regions to be in close proximity and form an intermolecular quadruplex. Formation of this G4 is then revealed by its DNAzyme activity in the presence of hemin, or by a fluorescence signal. A typical switchable element is a hairpin−duplex system, but the design may also involve unusual nucleic acid structures such as Z-DNA.162 We will present in more detail quadruplex switchable systems; an overview of the earlier attempts may be found in ref 163. Table 3 illustrates simple devices based on G4 or i-motif formation. 5.2. Simplest Devices: Opening and Closing of a Quadruplex (G4 or i-Motif)

Calling a denaturation/renaturation of a quadruplex a nanodevice may seem an overstatement, as any conformational change of a biomolecule would then qualify as a nanodevice. O


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conformations (free or Cu-complexed). The switch, which is fueled by alternate addition of small inorganic and organic compounds, is fast and reversible.83

Table 3. Simple Quadruplex-Based Devices “closed state” G4 i-motif G4 i-motif i-motif G4

“open state”

fuel

waste

(a) Single-Strand to Quadruplex single-strand cation/chelator cation−chelator complex single-strand pH change salt (b) Double-Strand to Quadruplex duplex single-strand duplex duplex single-strand duplex duplex pH change salt (c) Quadruplex to Quadruplex G4 cationa

5.3. Quadruplex−Duplex Devices

In contrast with the above-mentioned G4/single-strand switching devices, quadruplex−duplex devices involve two welldefined states. Devices incorporating duplexes with G4 were described first. They correspond to nanomachines that are capable of an extension−contraction movement based on a duplex−quadruplex equilibrium, which is fueled by the addition of DNA single-strands, generating duplexes as waste products.169,170 The interconversion between two well-defined topological states (an elongated double helix and a tightly coiled quadruplex) induces a linear motor-type movement. The switching time for this machine may be modulated by a number of factors, such as temperature, nature of the monovalent cation (Na+, K+), ionic strength, presence of divalent cations, sequence and chemical modification of the strand(s), as well as strand concentration. A simple design involved a single 21-base G4prone core (d-GGGTTAGGGTTAGGGTTAGGG), which switches between two states: an elongated double-strand of DNA and a tightly coiled quadruplex.170 This switch implies two transitions (Figure 11):

a

The structural conversion from one topology to another is generally easier to achieve in one direction; the reverse pathway being thermodynamically disfavored.

Nevertheless, these processes have some interesting characteristics, as follows: • The i-motif/single-strand interconversion corresponds to a concerted pH-driven conformational change. The open state is actually ill-defined, as it corresponds to a 21 nucleotide long single-strand, which should be considered as random coil (single-stranded DNA has a shorter persistence length as compared to dsDNA). Cycling is achieved by additions of base or acid, which generate salt as a waste product. These fuels are inexpensive, can freely diffuse, and produce waste products (water and KCl or NaCl), that minimally affect device operation. The imotif, because of its extreme pH dependency, has been used for proton-driven chemical oscillators,164 cellular pH indicators (see sensors, ref 165) or nanomotors.166 • The G4/single-strand device is slightly less straightforward: closing the device is achieved by addition of a cation promoting G4 formation (potassium, for example) while the reverse reaction is performed by addition of a chelator able to trap the G4-promoting cation. • Combining i-motif and G-quadruplex on the same device is even less straightforward, as these two modules have “mirror” requirements (runs of C vs runs of G) and therefore allow the formation of a competing duplex with multiple, contiguous GC base pairs. As G4 formation requires the presence of a G4-compatible cation, whereas the i-motif demands acidic conditions, one can construct a NOTIF logic gate based on this dual design.167 A more elaborate single-strand/G4 device was designed by Sen and colleagues.126 Four duplexes were assembled together, creating a central G-rich core that can either adopt a G4 conformation or be left unstructured with loose (but close) single-strands (Figure 7b). Switching between these two conformations is achieved through addition of G4-promoting ions or a chelator. The quadruplex−single-strand equilibrium may also be controlled by other ions. Yang et al. grafted a G4-forming sequence on the surface of mesoporous silica nanoparticles. In the presence of Ag+, silver ions interact with G bases and unfold the G-quadruplex, which subsequently opens the pores. These opened pore mouths can be closed again by adding glutathione: this system can therefore switch reversibly by the alternate addition of Ag+ ions and glutathione,168 allowing the on-demand release of a cargo loaded in the NP. Monchaud et al. designed a device involving a copper-mediated conformational switch controlling the G-quadruplex binding affinity of a structurally flexible ligand, 360A, that can adopt two well-defined

Figure 11. Principle of a duplex−quadruplex interconversion. Input signals for opening and closing the device are DNA strands; cycling of the device leads to an accumulation of a duplex as a waste product. Opening and closing can be followed by FRET provided that the G4forming motifs are labeled with appropriate fluorescent markers (shown as triangles). Adapted from ref 170. Copyright 2003 National Academy of Sciences.

(i) Step 1 is the opening of the quadruplex into a duplex conformation. For the quadruplex-to-duplex conversion, in the case of the human telomeric motif, quadruplexes cannot efficiently compete with duplex formation around neutral pH75 but can delay the association of two strands, especially at low temperature. (ii) Step 2 is the opening of the duplex with consequent liberation of the G4-prone strand, thus allowing for its intramolecular quadruplex refolding. Reformation of the quadruplex requires the addition of the “antifuel” strand to form a duplex byproduct: the progressive accumulation of the waste duplex may eventually poison the system, depending on the relative thermodynamic stabilities of the different structures.171 P


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in a few seconds at room temperature by addition of low (mM) KCl amounts to a sodium-rich sample.176 This conversion can be controlled by a number of parameters, including the cations’ nature and concentration, the strand concentration and sequence, and the temperature.

Both steps involve strand invasion processes; in one direction, formation of base pairs disrupts G-quartets, while for the reverse process, formation of base pairs disrupts another duplex. Strand invasion is facilitated by the presence of a toehold, which allows single-stranded DNA binding to the device before invasion: the formation of these few (typically 4−8) initial base pairs does not require unfolding or the disruption of other hydrogen bonding interactions. This initial step is generally fast (s time scale) at μM strand concentration. Once an initial, toehold-based short duplex is formed, extension of the duplex takes place by strand invasion. Interestingly, single-strand invasion of a quadruplex is fundamentally different from invasion of another duplex: once the first block of guanines is invaded by a neighboring, encroaching duplex, the whole G4 structure should collapse, and the full duplex formation should proceed rapidly. Under optimal conditions, switching from the closed to the open state takes less than 30 s, while the reverse process takes 3 s.171 One of the problems with such devices is that they require a precise amount of fuel for proper operation over multiple cycles. (The fuel and antifuel strands must be in stoichiometric amounts; this is easier said than done knowing imprecision in extinction coefficients when calculating DNA concentration!) An i-motif/double-strand interconversion is also possible;172 it corresponds to a concerted pH-driven conformational change. At basic pH, the duplex form is formed, while at acidic pH (5.0) the C-rich oligonucleotide folds into an intramolecular i-motif. As for G4-duplex devices, this reversible process can be monitored by fluorescent/quencher label pairsusing pHinsensitive probes! While the examples above involve relatively short strands, one may incorporate duplex−quadruplex devices into larger assemblies. Cao et al. built one-dimensional DNA nanostructures based on “slipped four-way junctions” which can either form Watson−Crick base pairs only, or alternating quadruplex and duplex DNA structures, depending on solution pH value and the nature of the monovalent cations present in solution.173

5.5. Applications

To exploit the potential of these devices, it is often necessary to attach them to a solid surface or a larger DNA nanostructure such as a DNA origami, provided that this linkage does not alter their function. Once anchored, DNA nanomachines can act cooperatively, moving beyond nanoscale effects. As a further bonus, immobilization allows the possibility of “washing” the solution, allowing the removal of waste products (such as salts or duplexes) generated by a cycling device. These waste products would eventually poison the device otherwise. When attached to a support, the stimulus-induced molecular-level motions of individual devices can be collectively translated to changes on a surface at larger length scales. However, as interfacial environments are more complex, grafting creates experimental and fundamental challenges for both planar and curved surfaces. Incorporating such small devices into larger structures also allows their direct observation by AFM, as the change resulting from an intramolecular G4 to duplex transition with a short (20−30 nt) oligonucleotide is very difficult to make evident given the small size of the objects considered. In contrast, when incorporated into a DNA origami frame, one can easily reveal this transition within this nanoscaffold at the single-molecule level. Yang et al. designed a nanoframe DNA origami system containing three pairs of connection sites to integrate photoresponsive ODNs and G-telomeric repeats together. Photoresponsive oligonucleotides bearing azobenzene groups were incorporated; these sequences can hybridize in the trans-form and dissociate in the cis-form of azobenzene upon photoirradiation at different wavelengths. The system can actually “choose” between intermolecular G4 formation and duplex formation on the basis of azobenzene conformation and cation concentration.177 Endo and co-workers150 demonstrated that a topologically controlled double-stranded region can be resolved into single-stranded DNA, G-quadruplex, and i-motif components by the addition of K+ and operation in acidic conditions. This process can be monitored by high-speed atomic force microscopy. Such a system is of interest not only for nanotechnology, but also for biology, as the sequence chosen corresponds to a motif found in the promoter of a human gene: the nanomachine can act as a structural model for studying the dynamics of duplex−quadruplex equilibrium which can play a role in the regulation of gene expression. A G4-duplex device may also be immobilized at the surface of a gold nanoparticle: the DNA conformational change varies the exposed active area on metal nanoparticles; this in turn induces a reversible change of the glucose oxidation activity of the Au NP,178 allowing a fine control of the catalytic properties of this system. This inducible “nanoshield” concept is especially interesting, as it remains challenging to regulate the catalytic activity of synthesized catalysts in a dynamic and reversible fashion. Simmel and co-workers designed a G4-duplex device based on the thrombin-binding aptamer (TBA) sequence; this device allowed the release or binding of a thrombin molecule.179 Maiolo et al. measured, for the first time, the surface work performed by TBA when switching from the linear to the Gquadruplex conformation upon K+ or Na+ cation binding.180

5.4. Quadruplex−Quadruplex Devices

In addition to the ssDNA−quadruplex and dsDNA−quadruplex devices described above, other clever devices can be designed on the basis of G4 formation. Karimata et al. introduced 2,2′bipyridine units within a DNA chain. This module provides Ni2+-responsiveness, and a controllable switch between a parallel G-wire and a bimolecular antiparallel quadruplex can be controlled by nickel ions or EDTA.174 This allows control of G-wire assembly: in the presence of Ni2+, an anti-parallelstranded G-quadruplex can switch to a parallel-stranded Gquadruplex which allows higher-order assembly and the formation of long G-wires.136 Clever and colleagues synthesized a chiral, glycol-based pyridine ligand within G-quadruplex-forming oligonucleotides.175 One G-quartet can be replaced by four pyridine ligands that are preorganized to coordinate to a copper ion. This modified sequence allows the Cu(II)-triggered switching from a hybrid mixture to an antiparallel topology, which controls a protein−G-quadruplex interaction (Thrombin−TBA in this example). Taking advantage of G-quadruplex polymorphism, it is possible to design G4−G4 devices, in which a change in ionic conditions induces an important structural rearrangement, even with unmodified DNA. Largy et al. screened a number of DNA sequences to find motifs that adopt different folds in sodium and in potassium. The anti-parallel-to-parallel conversion takes place Q


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They showed that K+ addition triggers a macroscopic surface work of about 70 pN nm/molecule. Saccà et al. patterned a G4forming oligonucleotide on a quasiplanar DNA array; switching of the devices is triggered by addition of DNA sequences and translated into linear extension/contractile movements that can be followed by AFM or FRET.181 These nanostructured devices are still functional when spatially confined within the scaffold, even if the crowded molecular surroundings affect the kinetics of DNA hybridization. Alternatively, the coordinated action of multiple hairpins can be used to mechanically unfold and refold a G-quadruplex motif bridged into the inner cavity of an origami.182 Tethering hairpins to the DNA origami architecture alters the energy landscape of the motifs, and this effect must be considered when incorporating these actuators within the system. Sen and colleagues showed that the reversible, intramolecular “pinching” of a duplex with a central G-G domain can be initiated and reversed by the binding and dissociation of certain specific cations. G-G domains incorporated into larger DNA assemblies could in principle serve as devices for inducing regulated structural changes in a contractile fiber or a contractile sheet.183 Huang et al. designed a “twisting electronic nanoswitch”. Different conformations are obtained upon addition and removal of K+ or Sr2+ ions, and these alternative conformers have strikingly distinct electronic properties.126 This switch can “twist” and “untwist” between distinct conformational states of differing electronic conductivity. Last but not least, Li et al. used this simple duplex−quadruplex equilibrium to control the opening/closing of a DNA origami.161 In this case, the conformational change is triggered by the presence of a G4-binding protein, nucleolin, which is expressed on tumor-associated endothelial cells. Control cells for which surface nucleolin expression is downregulated did not cause the DNA nanorobot to open. 5.5.1. i-Motif Devices. The unique pH dependency of the imotif enabled the design of DNA molecular motors ore sensors driven by pH changes165 (for a review, see ref 184). The output of these types of motors was combined to drive micrometersized cantilevers to bend. Such a pH dependency has proven useful for investigating pH-related processes within living cells, as shown by Krishnan and colleagues (see section 6.5 on sensors). The i-motif may also be immobilized on a surface. By immobilizing a DNA nanodevice onto microcantilevers, Shu et al. demonstrated that it is possible to convert the conformational change of the i-motif into cantilever bending.164 This immobilization can create a “smart” surface that changes its wettability in response to pH.185 Single-walled carbon nanotubes (SWNTs) can selectively induce human telomeric i-motif DNA formation at pH 7.0. Zhao et al. took advantage of this observation to design a DNA nanomachine induced by SWNTs on a gold surface: hybridization between the telomeric C-rich and G-rich strands can be modulated by the nanotubes while maintaining neutral pH.186 This is one of the few examples of an i-motif-based device that works efficiently and reversibly at a fixed pH. Liu et al. attached an i-DNA oligonucleotide tagged with a rhodamine green fluorophore to a gold surface: when folded, the terminal fluorophore was quenched because of its close proximity to the gold surface. Increasing the pH allowed unfolding of the structure and hybridization to a complementary strand, lifting the fluorophore away from the gold surface, greatly reducing quenching efficiency.187

5.5.2. DNA Walkers. Walking nanomotors are fascinating systems capable of transporting an object from one location to another on a nanometer scale. DNA walkers may also involve unusual DNA structures. Wang et al. described a pH-responsive DNA walker, which can reversibly transport specific molecules along an assembled track under environmental stimuli (proton concentration).188 Under slightly acidic conditions, the DNA folds into an i-motif structure, and the designed walker can take one step along the track each time when the environment is switched between acidic and alkaline: it can move back and forth between two destinations concomitant with pH variations. Wang and colleagues designed a DNA walker with an optically powered engine motif that reversibly extends and contracts via a G4-duplex conformational change.189 5.5.3. G4-Based Molecular Beacons. An original application of a duplex-G4 device is a G4-based molecular beacon. Molecular beacons are nucleic acid probes with a hairpin-shaped structure in which the 5′ and 3′ ends are selfcomplementary and form a classical antiparallel Watson−Crick duplex. Molecular beacons become fluorescent upon binding of the loop sequence to a complementary target (Figure 12). The

Figure 12. Principle of a G4-based molecular beacon. Hybridization of a complementary DNA or RNA target to the loop of the G4-beacon opens the quadruplex stem, leading to an increase in fluorescence as the fluorescent report group is longer in close proximity to the quencher. Adapted from ref 74. Copyright 2006 American Chemical Society.

beacon’s own complementary ends (the stem) can be replaced by a G-quadruplex motif (G4). Detection is then feasible by FRET (donor is quenched when the quadruplex is formed; addition of a sequence complementary to the loop restores its fluorescence).74 In the case of a G4-based molecular beacon one can play with a larger number of optimizing parameters: the intrinsic feature of the quadruplex motif facilitates the design of functional molecular beacons by independently varying the concentration of monovalent or divalent cations in the medium. This freedom provides versatility to optimize target recognition and mismatch discrimination and illustrates how having orthogonal pairing rules may provide additional levels of freedom when dealing with nucleic acid structures. A G4based molecular beacon can also be seen as a “split-quadruplex”, R


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created the output signal,195 a principle used multiple times in DNA-based calculations (for recent examples, see refs 196 and 197) as this constitutes a label-free and enzyme-free platform. Guo et al. employed stem-loop probes and toehold-mediated strand displacement to design a full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two-layer logic cascade. The principle is, once again, based on exposing or blocking (by Watson−Crick pairing) a Gquadruplex domain; the fluorescent output signal results from the light-up properties of a fluorescent G4 ligand, NMM.198 The design is different for Bader and Cockroft,199 who recently reported three G4-based logic gates (YES, OR, and AND) that operate simultaneously in a single test tube; these gates respond to unique Boolean DNA inputs by undergoing a G4-to-duplex topological conversion, which is revealed by Thioflavin T fluorescence. G4-based logic gates may also involve enzymes, as recently shown by Debnath et al.200 This device selectively can be used to detect the enzymatic activity of DNase I as well as perform logic operations and combinatorial logic systems; it can be implemented using different combinations of nucleases as inputs. The authors demonstrated that this platform can execute programmed function; as a proof of principle they encoded every integer (up to 16) into a four-bit binary number by different combination of inputs (four different nucleases) and showed that this device allows the identification of nonzero square numbers (1, 4, 9, 16). The split G-quadruplex molecular beacon proposed by Wang190 allowed the design of various logic gates. For example, an OR gate can be created when considering addition of sequences partially complementary to different regions of the beacon, if any of them are sufficient to open the structure. An AND logic gate was based on different input elements. This study culminated in the design of combinatorial logic gates capable of handling three and even four different inputs. As an example of a practical application, this system was used to discriminate multiples of three from natural numbers less than ten. By combining G-quadruplexes and silver nanoclusters, Gao et al.201 constructed a series of multifunctional, label-free, and multioutput logic circuits to perform nonarithmetic functions. These logic circuits share the same building blocks, indicating the high versatility of these biochemical logic devices. G4 formation was revealed by N-methyl mesoporphyrin IX (NMM) fluorescence enhancement. Another type of G4-based output signal for DNA calculation was proposed by Ge et al.: output single-strands released by a calculation can self-assemble into Gquadruplexes, which provide an amplified homogeneous electrochemical readout signal thanks to preferential binding to methylene blue.202 5.6.2. Combining G-Quadruplexes and i-DNA. Famulok and colleagues combined G4- and i-motif-forming oligonucleotides to build logic gates, taking advantage of the K+/H+ sensitivity of these two elements. The structural conversion from a bimolecular duplex to two intramolecular quadruplexes allows the building of molecular NOR, INH, and AND logic gates.203 Interestingly, DNA and RNA behaved differently: while a C-rich DNA strand readily forms an i-motif at acidic pH, RNA is far more refractory to adopting this conformation.20 The Famulok team also reported a reversible logic circuit built on the programmable assembly of a double-stranded DNA pseudocatenane, that serves as a rigid scaffold to position two separate branched-out head-motifs (a bimolecular i-motif and a Gquadruplex).204 The G-quadruplex only forms when preceded

which binds hemin and allows photoinduced electron transfer when closed.190 This device can execute computing functions: depending on the input, a series of logic gates (OR, AND, INHIBIT, and XOR) can be constructed (see section 5.6 below). 5.6. DNA Logic Gates

DNA’s predictable base-pairing properties, parallel processing capabilities, and ability to be recognized by specific molecules (ions, small compounds, other nucleic acids and proteins) constitute an attractive base to perform molecular computations. In these circuits, DNA sequences can be used to store state and monitor it via strand displacement reactions and to design logic circuits based on Boolean logic. Ideally, DNA gates should involve modular and mutually compatible DNA inputs and outputs. When equipped with appropriate fluorescent labels or other indicators, the response of nucleic acids to external stimuli can be transduced into a Boolean logic operation. The presence or absence of an input (e.g., a cation, a certain pH) and the increase or decrease of the output signal are related to a 1/0 event. The processing of binary and multivalued information may find a number of applications in the life sciences, for example, in biosensing. The unique properties of G4 and imotifs allow them to be used as sensors responsive to cations and pH, respectively. We will present below some examples of DNA-based logic gates incorporating these motifs. 5.6.1. G4 Logic Gates. Dong and colleagues described a G4based logic gate based on cation-tuned ligand binding and release.191 This gate is based on a cation-driven allosteric Gquadruplex DNAzyme which adopts a different conformation in the presence of potassium or lead: in this case, G4 polymorphism is actually an asset! The parallel G4 potassium form favors the binding of hemin, thereby promoting the DNAzyme activity. In contrast, the antiparallel Pb2+ G4 has a lower affinity for hemin, deactivating the DNAzyme. This device functions as a two-input INHIBIT logic gate. Zhao et al. also took advantage of the cation-dependent (K+ or Pb2+) configuration conversion of a G4 probe to design a label-free fluorescent AND logic gate.192 These two ions were also utilized in the design of an INHIBIT logic gate,191 based on the observation that Pb2+ has a higher efficiency for stabilizing G-quadruplexes and that a stable Gquadruplex does not always favor ligand binding. Dash and colleagues used a different quadruplex sequence and two Gquadruplex ligands to compute a wide variety of logic operations (XNOR, NOR, AND, NAND, NOT) based on differential combinatorial recognition.193 Reversible logic circuits integrated into functional nanodevices may allow the execution of cascade reactions. The implementation of DNA circuits in bulk reactions faces several difficulties, which include slow and diffusion-limited reactions as well as unwanted cross-talk reactions. Constraining distances between different components of a circuit solves these difficulties: when tethered to nanoplatforms such as a DNA origami, these devices can perform cascade reactions for signal amplification. To prevent the undesired, spontaneous activation of the cascade reaction, Mendoza and colleagues used Gquadruplex DNA structures to stabilize localized DNA circuits.194 Zhu et al. designed a four-way DNA junction-driven, toeholdmediated strand displacement method. In this case, the output strand drew the G-rich segments together to form a split Gquadruplex, which bound a ligand and enhanced its fluorescence: the G4 was not part of the logic circuit itself, but S


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K+ and H+-responsive nanochannel.213 They grafted these two motifs onto the top and bottom side of a cigar-shaped nanochannel. This channel is now double-gated, as each end may be independently and reversibly opened or closed depending on pH and potassium concentration.

by the assembly of the i-motif. The formation of the latter, in turn, requires acidic pH and unhindered mobility of the headmotif containing double-stranded DNA nanorings with respect to the central ring to which they are interlocked. This process is triggered by release of oligodeoxynucleotides. The authors employed these features to convert the structural changes into Boolean operations with fluorescence labeling. The nanostructure behaves as a reversible logic circuit consisting of tandem YES and AND gates.204 Xu et al. combined G-quadruplexes with another pH-sensitive unusual structure, a pyrimidine triplex. This nanostructure acts as an AND logic gate for H+ and K+ input, an INHIBIT gate for H+ and Ag+ (silver ions hinder the formation of Hoogsteen hydrogen bonding involved in G-quadruplex structures but do not prevent triplex formation), and a NOR gate for Ag+ and Cu2+ (G4 structures being disrupted by both ions).205 Guo et al. used silver ions and cysteine to build an IMPLICATION logic gate with triphenylmethane (TPM) fluorescence readout:206 Ag+ quenches TPM fluorescence only if cysteine is absent. Other cations may participate as well: Moshe et al. constructed supramolecular complexes between the Mg2+- and UO22+dependent DNAzymes, enabling the design of the OR and AND logic gates, using Mg2+ and UO22+ as inputs.207

5.8. Overview

Overall, quadruplex-based devices are capable of integrating various types of input, as follows: (i) H+ (for i-motif based systems which are exquisitely sensitive to pH changes). (ii) Specific cations promoting (e.g., K+, Sr2+) or disrupting (e.g., Ag+) G4 structures. (iii) Changes in temperature. (iv) DNA/RNA single-strands capable of inducing a conformational change via Watson−Crick pairing. DNA nanomachines are often set in motion by the addition of complementary strands, and quadruplexes offer similar, if not extended, possibilities. (v) Light. In this case, a caged residue can be unblocked by a light signal, which cleaves a protecting group. This uncaging is generally irreversible and would not allow reprotection. Such a procedure was proposed by Heckel and colleagues for the conditional formation of an intermolecular quadruplex.214 This caging/uncaging procedure offers interesting possibilities to gain more control over the construction of nanostructures, allowing a controlled stepwise assembly. (vi) All the inputs above are controlled by an operator. The first autonomous DNA device was powered by the pH changes generated by an oscillatory chemical reaction.164 Although the DNA response itself was fast, the oscillation reaction time period was relatively slow. The result of this switching can be the following: (i) A spectroscopic signal (generally fluorescence), which can result from a structure-sensitive fluorescent ligand, or direct labeling of one oligonucleotide. (ii) An enzymatic reaction, when using a G4-based DNAzyme (such as the heme·DNAzyme). (iii) A change in current/charge transport properties. (iv) A movement/global structural change (when embedded in a larger structure) resulting in a microscale mechanical work. Various types of mechanical motions can be observed: extension/contraction, twisting, rotation, or translation of the device or its attached cargo. (v) The conditional release of a cargo. This latter possibility will be developed in the chapter dedicated to nanocarriers and therapeutics. (vi) A phase transition or a deep modification of the mechanical properties of hydrogels (which are described in more details in the Nanocarriers and Therapeutics chapter). This variety of possible inputs and outputs explains why G4 and i-DNA have been extensively used as nanodevices.

5.7. Nanopores and Nanochannels

Artificial ion channels may find applications in biosensing or sequencing, or as antibacterial agents. G-quadruplexes and iDNA offer additional functionalities for these channels. In most, if not all, cases described so far, the quadruplex-forming sequence is added because of its ability to respond to an environmental change. An interesting example of a system combining G4 and nanochannels was provided by Bayley and colleagues. They found out that a protein (α-hemolysine) nanopore can be used as a sensor for the detection of a G4binding protein, thrombin.208 The pore is modified with the quadruplex-forming thrombin-binding aptamer (TBA) by forming a disulfide bond to a single cysteine residue near a mouth of the pore. One can then follow the binding of thrombin to TBA as it alters the ionic current through the pore. Nanomolar concentrations of thrombin can be detected. Hou et al. described a biomimetic nanochannel system based on quadruplexes (corresponding to the human telomeric motif) immobilized onto the inner surface of a single nanopore.209 This system becomes responsive to K+ in a 0−1.5 M concentration range, as a result of the conformational change of the G4-prone sequence, which induces a change in the effective pore size. Yu et al. designed a nanochannel-based electrochemical platform based on the covalent assembly of a G4-prone motif onto the inner wall of porous anodic alumina (PAA) nanochannels.210 Formation or disruption of the G-quadruplex modulates the steric hindrance of the channel, affecting the flux of indicator molecules inside the nanochannels. This system may be used for the sensitive detection of potassium and ATP. Xu and colleagues described an artificial potassium-gated ion channel based on titanium nanotubes loaded with Au nanoparticles and involving a G-quadruplex component. Potassium promotes the formation of the quadruplex, which blocks the transport of anions through the channels. These channels have both switchable permeability and ion selectivity.211 i-DNA and nanochannels may also be combined. This can be used to design single synthetic nanopores which are gated by proton-driven DNA molecular motors.212 Finally, Jiang and colleagues combined G4 and i-DNA structures to design a clever

6. QUADRUPLEX DNA NANOSTRUCTURES AND QUADRUPLEX−INORGANIC NANOMATERIAL COMPOSITES FOR ANALYTE SENSING 6.1. General Considerations

An important dimension of the design, synthesis, and testing of quadruplex DNA nanomachines (see Reconfigurable Structures and Nanodevices, above) is the utility of such mobile structures T


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Figure 13. Reproduced from ref 219. Copyright 2018 American Chemical Society.

6.3. Hemin·G-Quadruplex Complexes (Heme·DNAzyme)

in bioanalytical/analyte sensing applications. Nearly all the systems presented in this chapter are based on G4 formation, not i-DNA. This is a vast field, which takes advantage of Gquadruplexes, in particular, functioning as the sensing moiety itself (as in aptamers that fold into G4s), as the moving part of a shape/structure changing DNA sensing machine, or as the signal output deviceamong the most widely applied of which are G4hemin complexes, or heme·DNAzymes. i-DNA is not often encountered in aptamers; an interesting use of i-DNA in aptamer design was recently proposed by Tan and colleagues.215 They designed a structure-switchable aptamer (SW-Apt) making use of a split i-motif. The pH dependency of the i-DNA stem allows modulation of binding affinity in accordance with the microenvironment of target cells (folding is favored at acidic pH). This smart aptamer is therefore capable of distinguishing a target cell receptor from the same receptor on the membranes of nontarget cells. Some of the more conceptually simple, yet elegant, designs for quadruplex-based analyte sensing machines necessitate conformational changes of a monomeric quadruplex unit, in itself (for instance, a fluorophore- and quencher-labeled molecular beacon, that makes use of a conformationally plastic G4 aptamer; or, a split-G4 beacon whose loop constitutes an aptamer, and whose signal output is provided by the binding of hemin to the G4 in its undisrupted state), are not discussed in this chapter; they have been capably reviewed elsewhere.216 Instead, here we will touch on either sensors that incorporate multiple/functionally additive G4 modules or, more importantly, novel complexes that bring together quadruplex DNA motifs with metal and/or other inorganic surfaces and nanocomposites; crucially, we will focus on systems in which the analyte binding-generated output signal arises precisely from the juxtaposition of the nucleic acid and inorganic material components of the overall composite.

A convenient and extremely versatile reporter, specific to Gquadruplexes and to no other secondary structure of DNA and RNA, is the tight complex that hemin [Fe(III)-protoporphyrin IX] forms with G-quadruplexes, particularly with quadruplexes of all-parallel or mixed strand polarity. Such hemin-G4 complexes are often referred to as the “horseradish-mimicking DNAzyme” or, more generally, “heme·DNAzyme” (Figure 13). Sen and colleagues reported in 1998 that hemin, or Fe(III)protoporphyrin IX, complexes tightly and exclusively to Gquadruplexes.220 Such a complexation concomitantly activates the bound hemin toward oxidative catalysis, using hydrogen peroxide as oxidant. This unusual and unique catalytic system, with its likely relevance to primordial chemistry as well as to extant biology, has proven to be a versatile reporting system for DNA-based nanoscience and nanotechnology, as well as a reporter for the “on” state of a variety of DNA machines and nanosensors.216,221−224 Heme·DNAzymes exhibit a significant number of properties that make for useful signal “outputs”. In addition to the intrinsic spectral, catalytic, and electrochemical outputs that the DNAzyme itself is able to provide, by clever combining of the DNAzyme’s properties with those of protein enzymes such as glucose oxidase, by localization of the DNAzyme upon gold or other conductive surfaces, or by forming composite nanomaterials using the DNAzyme and quantum dots with gold/silver nanoparticles, a versatile palette of output signals can be achieved. Thus, the DNAzyme’s intrinsic oxidative activity can use chromogenic reducing substrates (such as ABTS or dimethylbenzidine) or fluorogenic substrates (such as Amplex Red) to produce a signal; a chemiluminescence output can be had using luminol as substrate. Synthesis of oxidized products with desirable properties (such as the oxidative production of conductive polyaniline from aniline base) is also within the range of the DNAzyme’s catalytic properties. In addition, the electrodeloaded DNAzyme is capable of electrocatalytically reducing hydrogen peroxide, which can be monitored using cyclic voltammetry; photoinduced electron transfer between the DNAzyme’s heme and proximally bound quantum dots or silver nanoparticles can be exploited via observed reduction or enhancement of the nanoparticle’s luminescence. In addition, more complex assemblages involving the DNAzyme can be used for yet more extensive and sophisticated signal output generation. Interestingly, the initial demonstration of oxidative catalytic activity of heme·DNAzymes, which is dependent on extrinsically added hydrogen peroxide, has since been enlarged by Willner and colleagues in remarkable demonstrations that heme·DNAzymes are also able to utilize dioxygen (O2) and different reducing substrates (NADH, ascorbate, cysteine, or glucose in conjunction with the glucose oxidase enzyme) to generate the same output signals, by way of generation of hydrogen peroxide in situ.225−227 Willner and colleagues have also reported that, in the presence of hydrogen peroxide but

6.2. Programmed Gold-Nanoparticle Aggregation as an Assay for G-Quadruplex Formation

As referred to earlier, spherical nucleic acids (SNAs) describe monodisperse gold nanoparticles that have been densely surface-derivatized with oligonucleotides.217 Seow et al.218 have used the general concept of SNAs to improvise a methodology for detecting G-quadruplex formation by given G-rich sequences. Indeed, they used a vertebrate telomeric sequence and one with a TnG5 sequence. Both sets of composites gave characteristic dynamic light scattering profiles under the conditions appropriate for intermolecular G-quadruplex formation, and this could be counteracted by the presence of free G-rich oligonucleotides. Further, the authors were able to tune the size of the nanoparticle assemblies by colocalizing a different sequence, capable of forming a hairpin, upon the gold nanoparticles, and by controlling the secondary structure of this second oligonucleotide using molecular beacons. U


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Figure 14. PET from a heme·DNAzyme to a silver nanoparticle. Reproduced from ref 234. Copyright 2013 American Chemical Society.

platforms take advantage of photoinduced electron transfer (PET) from either CdSe/ZnS quantum dots233 or silver nanoparticles234,235 to proximal, G4-bound heme irons. Thus, the de novo synthesis of G4-folded guanine-rich telomeric DNA polymerized by the enzyme telomerase (a reliable marker of cancer) from a DNA template bound to the surface of the quantum dots leads to the localization of heme upon the G4s, and a concomitant decrease in the fluorescence of the quantum dots. This methodology enables the detection of telomerase enzyme originating from as few as ∼270 human cells/μL. In the signal-off methodology of Wang and co-workers234 a simple DNA machinea DNA hairpin localized by one of its ends to a silver nanoparticleundergoes hybridization and structural changes in response to either a test DNA oligonucleotide of interest, or ATP as analyte, which binds to a DNA aptamer disguised within the hairpin (Figure 14 shows this approach). These structural changes liberate a previously sequestered Grich sequence within the nanoparticle-localized DNA hairpin, and this is now able to bind hemin and reduce the fluorescence of the silver nanoparticle via PET to newly nanoparticleproximal hemin residues. By contrast, an alternatively designed DNA machine bound to silver nanoparticles has preformed G4s as its default, analyte-free state; the binding of adenosine or RNA analytes to this latter DNA machine destroys the G-quadruplex and leads to a concomitant enhancement of silver nanoparticle fluorescence in response to analyte binding.235 Willner and colleagues have utilized yet another aspect of the photophysics of quantum dots, in tandem with the oxidative catalytic properties of heme·DNAzyme integrated into an analyte-sending DNA nanomachine, which is tethered by one of its ends to the surface of the quantum dot. Freeman et al.236 describe a signal-on platform for a broad range of sensing capabilities, of DNA, metal ions, as well as for analytes for which DNA receptors (aptamers) have been reported. The functional principle is to exploit a change in the distance separating a hemin-bound DNAzyme from its tethering quantum dot, triggered by analyte binding to the overall DNA machine. The heme·DNAzyme oxidizes a luminol substrate, whose oxidation leads to chemiluminescence, i.e., rapid emission of a photon. Chemiluminescence resonance energy transfer (CRET) to CdSe/ZnS quantum dots (QDs), whose efficiency strongly depends on the distance separation of the oxidized luminol and the quantum dot, can thus be used to monitor and quantitate analyte binding (Figure 15). In an interesting analytical application of the capability of heme·DNAzymes to utilize dioxygen and a reducing agent (in this instance, L-cysteine),226 telomeric G4s, folded (as in ref 233,

lacking a reducing substrate, electrode-immobilized heme· DNAzymes can reduce the hydrogen peroxide electrocatalytically, with the output being measured by cyclic voltammetry.228,229 Indeed, in a tour-de-force demonstration of the various output possibilities of the heme·DNAzyme, Willner and colleagues described variants of a fluorescently labeled (with a quantum dot or organic fluorophores) DNA machinea “walker”whose forward and reverse walking states generated, variously, chemiluminescence, chemiluminescence resonance energy transfer, electrochemical, or photoelectrochemical outputs.230 6.4. Simple DNA Nanostructures for Biomolecular Detection

One classic example of the triggered assembly of a relatively simple DNA nanostructure by an analyte (in this case, a specific microRNA, the miR-141 from prostate cancer cell lines) is a system where the presence of even very low concentrations (0.5 pM) of the miRNA led to the assembly of a DNA nanostructure that incorporated a large number of heme·DNAzymes.231 A detector DNA hairpin was opened, by means of strand invasion, by the analyte miRNA, which led to an isothermal “chain reaction” of DNA hairpin openings, culminating in the assembly of a DNA “Ferris wheel”, whose every spoke terminated in a Gquadruplex capable of binding hemin and forming a signalgenerating heme·DNAzyme. The color output generated by this innovative approach, measurable by the naked eye, corresponded to a sample consisting of as few as ∼280 human prostate cancer cells. In another intriguing example of this approach, Willner and colleagues232 described a detection methodology that depended on a DNA oligonucleotide (corresponding to a sequence from the BRCA1 oncogene) triggering assembly of an extended and repeating DNA duplex nanowire incorporating numerous, regularly spaced heme· DNAzyme appendages. This platform enabled a DNA detection limit of 1 × 10−13 M. 6.5. Inorganic Nanoparticle−DNA Quadruplex Composites for Biosensing

6.5.1. G4-DNA−Silver Nanoparticle Composites as Signal-Off and Signal-On Biosensors. Heme·DNAzymes, aside from their useful oxidative catalytic properties, show interesting photophysical properties by virtue of the ironcontaining heme within them. The groups of Wang, Xie, and Willner, respectively, have reported the design of relatively simple DNA G4/inorganic nanoparticle composites that are capable of signal-off233,234 and signal-on235 detection of a variety of analytes. These conceptually straightforward detection V


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exploit multiple recognition events for a given cell-surface epitope to immobilize the specific cells to be detected onto the surface of a gold electrode. The means for immobilization was multiple DNA tetrahedron nanostructures, conceptually similar to those used by Fan et al.,237 which were stably immobilized onto the electrode surface via three sulfhydryl groups (one per apex for three DNA tetrahedron apexes in contact with the electrode). Gold nanobeads, surface-derivatized with DNA duplex nanowires which incorporated both numerous pendant heme·DNAzymes as well as DNA aptamers for another specific cell-surface marker, were now localized onto the distal surface (i.e., away from the electrode) of the immobilized cell. From such a sandwich-like composite of electrode/immobilized cell/ heme·DNAzyme-derivatized gold nanoparticles, a very sensitive color signal was generated. Indeed, the authors report that this platform enabled a very broad dynamic range of detection of cells in culture, ranging from 5 cells to 107 cells/mL. Another elaborately designed but sensitive platform for visual detection of HIV DNA has been reported by Long et al.239 Sensitivity here was achieved by what the authors refer to as “multiamplification”, and this platform enabled eye-detection of HIV DNA in actual physiological media to a lower limit of 4.8 pM. Fundamentally, the authors set up a nanofibrous platform, upon which were immobilized streptavidin as well as the glucose oxidase (GOx) enzyme. Glucose oxidase is able to utilize glucose and dioxygen as substrates to generate hydrogen peroxide as one of its products.240 First, a biotinylated DNA hairpin duplex that incorporated a single-stranded toehold was immobilized onto the nanofibrous platform via the immobilized streptavidin. The analytein this case, single-stranded HIV DNAwas capable of hybridizing to the DNA toehold and thereby inserting itself into the hairpin, via strand displacement. This in turn freed up a single-stranded stretch of DNA, capable of folding autonomously to a G-quadruplex and binding hemin. In summary, the above composite platform synergistically utilized in situ GOxderived hydrogen peroxide, strand-displacement cascades, as well as multiple copies of heme·DNAzyme to achieve not only high colorimetric detection sensitivity but also exquisite sequence discrimination of analyte, capable of detecting a single base mismatch. As a final example, Famulok and colleagues designed an interlocked DNA nanostructure that is able to fine-tune the oxidative catalytic activity of a split DNAzyme. DNA rotaxanes have the ability to undergo programmable and predictable conformational changes: the catalytically inactive DNA rotaxane can be converted to a catalytically active conformation when a macrocycle is hybridized to an active position, allowing the formation of the catalytic G4 structure, a G4-based DNAzyme.241

Figure 15. CRET from a heme·DNAzyme to a CdSe/ZnS quantum dot. Reproduced from ref 236. Copyright 2011 American Chemical Society.

above) from G-rich repeating DNAs synthesized by the telomerase enzyme, bind hemin and oxidize cysteine that is present in the reaction solution. As a thiol-containing compound, cysteine stimulates the aggregation of gold nanoparticles, with such aggregates showing a blue color of the colloidal solution; however, oxidation of cysteine to the thiollacking cystine by the de novo synthesized (and heminsequestering) telomeric G4s results in disaggregation of the cysteine-aggregated gold nanoparticles, with a concomitant presentation of a red color to the altered colloidal solution. This methodologyby way of contrast with the earlier methodology (described above) from the Willner group for detection of telomerasepresents a highly sensitive detection of the enzyme, amounting to that amount obtained from as few as ∼27 cells/ μL.227 6.5.2. Utilization of the Synthetic Capabilities of Heme·DNAzymes within DNA Nanostructures for Sensing. An interesting analytical application of the purely synthetic capabilities of the heme·DNAyme, in combination with the properties of a distinctive DNA nanostructure (a tetrahedron made up of DNA duplexes), was reported by Fan et al.237 The tetrahedral DNA nanostructure had three of its four apexes labeled with sulfhydryl groups, which enabled the nanostructure to bind stably to the surface of a gold electrode. The distal, fourth apex (which was not in contact with the gold surface) had a built-in DNA extension with the potential to fold into an 8hydroxyguanine aptamer (an incomplete G-quadruplex). The presence of free 8-hydroxyguanine in the solution structurally completed the G-quadruplex, which then bound hemin, formed the DNAzyme, and was able to oxidize aniline present as a reducing substrate in the solution. The oxidation product of the aniline is a highly conductive polymer, which efficiently coated the DNA tetrahedron. Differential pulse voltammetry was now carried out using the same hemi, that had acted as the oxidative catalyst within the DNAzyme, except now acting as the redox label for electrochemical measurements. This system provided a label-free and highly sensitive (1 pM) detection of 8hydroxyguanine. Utilizing a similar DNA tetrahedron as used in the above report from Fan et al., an intriguing, if baroque, sensing platform for detection of whole cancer cells (HepG2 cells) was reported by the group of Y. Zhang.238 The goal of this platform was to

7. NANOCARRIERS AND THERAPEUTICS DNA can be considered a biocompatible biomaterial; several groups are working on DNA-based nanostructures that would confer targeted and efficient drug delivery to target cells. In this section, we will present different strategies in which quadruplexes are used as versatile tools to target specific cells and achieve controllable drug delivery. These structures can have a variety of functions, as summarized in Table 4. 7.1. Aptamers

A number of aptamers adopt a quadruplex fold, the most famous one being the thrombin-binding aptamer.242 Other aptamer sequences bind to small compounds such as NMM243 or hemin: W


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identified on the basis of its antiproliferative activity on cancer cells and subsequently determined to bind to nucleolin;251 this particular sequence is now under intensive study as there are hundreds of articles each year referring to AS1411 (for a review, see ref 252). G4 formation was shown to be necessary, but not sufficient, for antiproliferative effects. It is still unclear to what extent binding to nucleolin is responsible for quadruplexselective cytotoxicity or, indeed, accumulation in cancer cells. Several groups are developing nanoformulations of AS1411 to improve its efficacy. AS1411 maintains its quadruplex structure when conjugated to quantum dots, gold nanospheres, nanostars, and nanoclusters. Attaching AS1411 to gold nanospheres increased its cellular accumulation, antiproliferative activity, and cytotoxicity, both in cell lines and in in vivo models.253 AS1411-coated gold nanostars had enhanced cellular internalization compared to the same gold nanobjects linked to a nonG4-forming single-strand.254 Such an enhanced uptake results in an increase in cell death, as shown in pancreatic cancer and fibrosarcoma cells. Other G4-forming aptamers may also be conjugated to nano-objects. For example, Shiang et al. functionalized gold nanoparticles with a DNA sequence targeting the human immunodeficiency virus type 1 reverse transcriptase with IC50 in the low nM range.255 Both grafting density and linker length played a role in the inhibition activity of these Au NP. Musumeci et al. found that conjugating a quadruplex-forming sequence to an NP stabilizes it against enzymatic degradation.256 The affinity of AS1411 for nucleolin was used to shift the balance of a duplex−quadruplex equilibrium toward the quadruplex form to induce the opening of a tube-shaped DNA origami and deliver active thrombin solely to tumor sites in a highly controlled way.161 This exposed thrombin payload can induce coagulation at specific tumor sites; thrombosis was specific to the tumor vasculature, with a therapeutic effect in inhibiting tumor growth. Bare nanotubes rolled without the targeting aptamer strands did not bind to the surface of cells expressing nucleolin, and an antinucleolin antibody abolished binding as well. The nanorobots progressively accumulated in the tumor in nude mice, reaching a maximal accumulation at 8 h after injection. As noted by Tasciotti,257 Li et al. have succeeded in making thrombin an actual drug, which would otherwise be excluded from cancer therapies because of its pleiotropic effects. This seminal work may inspire the design of novel environmentally responsive cancer therapeutics to mediate delivery of different biologically active cargoes, such as short interfering RNA (siRNA), cytotoxic compounds, or peptides. Spherical nucleic acids (SNAs) typically consist of a gold nanoparticle core that is densely functionalized with oligonucleotides. Chinen et al. showed that the protein-binding properties of SNAs depend on the structure of these oligonucleotides.216 This protein corona alters SNA uptake in a phagocytic macrophage cell line, suggesting that G-rich sequences may exhibit altered biodistribution and pharmacokinetic profiles in vivo. This is a double-edged sword: even if G-rich sequences may lead to greater cellular internalization, they also lead to greater macrophage uptake, which could cause increased clearance from the bloodstream. While the approaches above were aimed at improving AS1411 uptake and efficacy, one can also envision the use of this DNA sequence for cancer-selective targeting and imaging. The idea is to take advantage of AS1411-selective accumulation in cancer cells to drive another cytotoxic drug to its intended target. AS1411 has indeed been used as a targeting agent to deliver

Table 4. Applications of Quadruplexes (G4 and i-DNA) in Therapeutics use

example

applications/comments

drug cap targeting agent marker

AS1411 signal-mediated opening AS1411-nucleolin

G4 has antiproliferative properties controlled release of a cargo specific targeting of cancer cells

fluorescent G4 ligand

matrix carrier

nanogels quadruplex ligand protein, small compound

following the fate of the nanocarrier holding together the nanomaterial drug delivery of the G4 ligand, photodynamic therapy

Hemin/G-quadruplex DNAzymes (heme·DNAzymes) have been discussed at length above. These DNAzymes are often used as reporter elements capable of producing an output signal such as chemiluminescence. In this section, we will describe the use of cell-specific aptamers for biological applications. Cellspecific aptamers are folded nucleic acid molecules that bind to specific cellular targets. Aptamers are taken into cells typically by clathrin-mediated endocytosis or macropinocytosis. Many of the aptamers selected against biologically relevant protein targets are G-rich sequences that can fold into stable G-quadruplex structures.244,245 Discussing all cellular applications of aptamers is beyond the scope of this review; one can refer to a recent review by Yoon and Rossi.246 We will restrict this review to cases in which (i) aptamers involve a G4 core or are linked to a quadruplex, and (ii) this system is used for the targeted delivery of a cytotoxic agent, which may be the aptamer itself. A significant number of aptamers against important targets adopt a G4 fold, as the G4 core provides a platform from which loops, overhangs, and/or bulges can establish specific interactions with their target. The first and best-known example is the thrombin-binding aptamer242 (for a review, see ref 247), which consists of highly compact and symmetrical antiparallel quadruplex structure involving two G-quartets. For aptamers, G4 polymorphism is not a problem but an asset, as very diverse quadruplex structures may be built, depending on number of strands, quartets, loop type, bulges, etc., explaining how this motif may be used for the specific recognition of a variety of targets. For example, Zhao et al. developed a DNA aptamer specific for the biomarker CD117, which is highly expressed on AML cells.248 This aptamer selectively binds to AML cells with high affinity (Kd < 5 nM), and is specifically internalized into CD117-expressing cells. Aptamer−methotrexate conjugates specifically inhibited AML cell growth, triggered cell apoptosis, and induced cell cycle arrest with little effect on CD117-negative cells. Unfortunately, specificity of the G4-forming aptamers is not always perfect, and cross-binding to several different proteins may limit their utility. For example, the G4-forming DNA aptamer against HIV-integrase also binds to interleukin-6.249 7.1.1. AS1411, an Iconic G4-Forming Aptamer. AS1411 (initially known as AGRO100) is not strictly speaking an aptamer, as it did not result from an in vitro selection procedure. This sequence, originally designed as a triplex-forming motif, is a 26-mer G-rich DNA oligonucleotide (d-GGTGGTGGTGGTTGTGGTGGTGGTGG) which is susceptible to G4 formation. It reached phase II clinical trials for treatment of renal cell carcinoma and acute myeloid leukemia. This sequence, which is well-tolerated in patients (at doses up to 40 mg/(kg day)) actually forms a variety of monomeric or dimeric G-quadruplex structures, as shown by Trent and colleagues.250 This motif was X


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As shown in the examples above, G4-forming DNA shows promises as a drug delivery vehicle (for a review, see ref 216). Its drug delivery generally relies on a structural change as the trigger releasing the drug. Aptamer-based gates may overcome major challenges associated with drug delivery, as shown by Hernandez et al., who used AS1411 switchable aptamer nanovalves on mesoporous silica nanoparticles;269 this constitutes a self-contained system with design features that can combine specific delivery to breast cancer cells with controlled drug release. 7.1.2. i-Motif-Based Devices for the Controlled Release of a Cargo. Chen et al. designed a proton-fueled molecular gatelike delivery system for controlled cargo release. They used i-motif-forming oligonucleotides as caps onto pore outlets of mesoporous silica nanoparticles. This i-motif cap, because of its pH dependency, can open and close the pores in response to a pH stimulus. This opening/closing is reversible and allows on-demand molecular transport.264

nanoparticles, oligonucleotides, and small molecules into cancer cells, and AS1411-linked materials can accumulate selectively in tumors following systemic administration, although the mechanisms mediating this effect are not completely understood (for a review, see ref 252). This G-quadruplex-forming aptamer can be used to incorporate a photosensitizer for tumor-targeted delivery.258 This concept is not restricted to the AS1411 sequence: other G4-forming motifs (which are also recognized by nucleolin) can be used as well. G4-ligand complexes can act as synergistic platforms for targeted photodynamic therapy (PDT). This was achieved for a protoporphyrin IX complex: the porphyrin derivative binds to the G4 core (the so-called T30695 sequence) and is selectively incorporated into cancer cells.259 A similar result may be obtained with AS1411-functionalized fluorescent gold nanoparticles260 or a silica matrix incorporating Fe3O4 and a fluorescent derivative (Ru(bpy)32+).261 In this report, Qu and colleagues designed photosensitizer-incorporated G-quadruplex DNA-functionalized magnetic nanoparticles to obtain a multifunctional platform for simultaneous magnetofluorescent imaging and targeted cancer photodynamic therapy. The photosensitizer is a well-known quadruplex ligand, the cationic porphyrin derivative TMPyP4. These nanoparticles exhibited an excellent biocompatibility, specific cellular uptake, and a remarkably enhanced efficiency in killing human breast MCF7 cancer cells. In a reciprocal fashion, i-DNA may be used to achieve the reversible pH-regulated control of photosensitized singlet oxygen production.262 Qu and colleagues used mesoporous silica nanoparticles as drug delivery systems for the light- or pH-induced release of a drug. A porphyrin derivative bound to a G-quadruplex was used as a photosensitizer to cleave DNA, thus opening the gate of the nanopores and releasing the doxorubicin cargo.263 Another interesting system involved a proton-fueled molecular gatelike device based on i-motif formation: an i-motif DNA cap can reversibly open and close a pore in response to pH changes.264 An alternative strategy was recently proposed by Oh and colleagues,265 in which the drug-loading stem part and the target-recognizing aptamer are two different entities, allowing greater versatility. The authors linked a protein tyrosine kinase (PTK) 7-specific DNA aptamer to a motif capable of adopting a quadruplex fold (15 consecutive guanines). The G4 moiety acts as a loading platform for a photosensitizer, methylene blue (MB), given that the binding affinity of MB to G-quadruplex DNA is much greater than that to single-stranded DNA. This MB-loaded Aptamer-G4 showed specific and enhanced uptake to cells overexpressing PTK7 with concomitant photodynamic effects. While this study represents a proof of concept (methylene blue can hardly be considered a therapeutic agent) other drugs can be loaded to a DNA nanostructure. G4 aptamers may also be linked to MRI contrast agents;266 this opens new possibilities for the targeted delivery of these imaging agents in vivo to detect specific cells or tissues, even behind intact blood vessels. Liu et al. conjugated a double-stranded tail to a G4-forming sequence. While the latter allows targeting of cancer cells via its interaction with nucleolin, the double-stranded region can act as a loading platform for a cytotoxic drug such as doxorubicin.267 Quadruplex motifs incorporated into supercoiled plasmids do not cause spontaneous deletions or decrease in plasmid yield.268 As quadruplexes tend to be taken up by cells, a simple idea was to improve plasmid delivery thanks to this quadruplex region. To our knowledge, this concept has not been experimentally tested.

7.2. Micelles

DNA-lipid conjugates have been studied for decades. Lipidbased DNA micelles are spherical nanoparticles self-assembled from amphiphilic DNA-lipid conjugates, in which the DNA strands are located in the corona, and hydrophobic lipid tails are in the core. These micelles are generally easy to prepare, and are biocompatible and programmable. Quadruplexes offer interesting new properties for these micelles. Hydrophobic interactions among lipid groups lead to the stabilization of quadruplexes.270 Such lipid stabilization then enables the fine-tuning of the stability of the micellar assemblies; for instance, the presence of an alkyl chain drastically accelerated the kinetics of tetramolecular parallel G4 formation.271 Not only do micelles favor G4, but the reciprocal effect is true as well: assembly into G4 structures may be a critical factor for the formation and stabilization of micelles. This is critically important, as lipid micelles are unstable in serum because of interactions with proteins such as albumin. G-quadruplex stabilizes micelles against disruption by serum albumin,272−274 and G4 structures actively favor micelle formation, as the critical micelle concentration (CMC) is lowered by G4 formation.275 This effect can be obtained with regular G-rich DNA oligomers or analogues capable of forming stable quadruplexes, such as 2′OMe.273 A very short 3-quartet core (UGGGU) is sufficient, as G-quadruplexes formed with 2′-OMe oligonucleotides are thermodynamically stable and resistant to nucleases. Irvine and colleagues designed “amph-vaccines”, which are amphiphiles composed of three distinct segments: an immunostimulatory CpG sequence, a central region containing up to 10 guanines followed by noninteracting thymidines, and a diacyl lipid tail.272 Altering the length of the guanine repeat modulates the stability of the micelles; in this case the oligonucleotide had a phosphorothioate modification which also allows G4 formation. G-quadruplex DNA structures can also be fused to cholesterol. Diez and colleagues characterized the anti-HCV inhibitory capacity of lipid-G-quadruplex conjugate structures, which were called “lipoquads”. The anti-HCV potency of these lipoquads was correlated with their ability to form stable quadruplexes.276 Interestingly, G4-induced micelle stabilization is tunable by hybridization-based molecular engineering. One can disrupt the G4 core by using “antisense” oligonucleotides designed to infiltrate the G-quadruplex. The destabilization of the Gquadruplex causes in turn a decrease in the stability of the micelles and eventually their disruption. To achieve this Y


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destabilization, one can add a complementary oligonucleotide which would hybridize to a complementary toehold (a singlestranded extension at one end of the G4-prone region), leading to the unfolding of the tetramolecular quadruplex, which destabilizes the micelles and release their cargo.275 This design may be adapted to a more complex system, in which the micelles become responsive to an external trigger (ATP); this involves an ATP-induced conformational switch of a DNA sequence complementary to the G4 part. In this case cargo release occurs selectively when ATP is present. Wilner et al. engineered lipid micelles with modified RNA sequences (2′OMe) forming Gquadruplex structures. These micelles enable the controlled release of a cargo upon G4 destabilization with an antisense oligonucleotide.272 Rather than adding an external strand, one can also use a caged hairpin DNA that can block the formation of a Gquadruplex only after exposure to UV-light,274 which releases a C-rich sequence, allowing its hybridization to the G-rich G4prone DNA strand. As a result, these micelles are destabilized by bovine serum albumin (BSA) which leads to an increased cellular uptake.

hydrogel that undergoes reversible transitions between solution−hydrogel−solid states and catalyzes the oxidation of aniline by H2O2 to form polyaniline, eventually leading to an electrically conducting matrix after proton doping.284 • Switchable DNA-based hydrogels can also be prepared on surfaces,285 allowing the surface to control the interfacial electron transfer properties and the functions of the hydrogel-modified electrodes. G4-based hydrogels can be reversibly switched between less stiff and stiff networks upon treatment with potassium or crown ethers.286 This example illustrates how quadruplex formation can affect the properties of hydrogels, but the inverse approach is possible as well: hydrogels can affect the properties of quadruplexes. Hasuike et al. took advantage of the space inside thermoresponsive poly(N-isopropylacrylamide) gels to control the stability of G-quadruplex reversibly via variations in temperature (25−45 °C).287 This could find applications for the controlled denaturation of an aptamer, releasing its protein cargo upon a change in temperature. 7.4. Overview

7.3. Hydrogels

The different systems (aptamers, micelles, and hydrogels) described above illustrate how DNA quadruplexes allow the design of new carrier materials with tunable properties. The programmable regulation of DNA-based micelles or hydrogels should allow the development of biocompatible DNA materials for therapeutics application, as these systems allow the controlled release of an active substance such as a small cytotoxic drug or an aptamer upon a specific trigger (e.g., a surface marker specific for a cancer cell).

Although hydrogels are mostly (generally >90−95%) composed of water, they behave like (soft) solids because of 3D networks of cross-links within them. Cross-linking is achieved by covalent or noncovalent (van der Waals; hydrogen bonding) interactions. The flexibility and high water-content of hydrogels enable them to be biocompatible, and nanogels have been innovated as drug delivery system carriers. 5′GMP and guanosine derivatives have been known to form gels for decades;277 hydrogels formed with nucleosides or nucleotides will not be considered here, and we will focus this section on gels incorporating DNA motifs. G4 or i-motif-forming DNA motifs can be incorporated into acrylamide polymer chains. These hydrogels can undergo a signal-triggered hydrogel-to-solution transition or a signalcontrolled change in stiffness (for a review, see ref 278). Copolymers consisting of acrylamide units and G4-forming oligonucleotides can yield a hydrogel in the presence of K+, and this hydrogel can be dissociated upon addition of 18-crown-6 ether, that efficiently chelates the K+ ions.279 Stimulusresponsive hydrogels are particularly attractive for drug delivery. These polymer chains can be bridged or dissociated depending on the external signal (e.g., pH changes, appropriate cations), acting as “smart” or stimulus-responsive materials which can be used for a number of applications, such as carriers for controlled release of drugs or enzymes, sensors, switchable catalytic materials, or as shape-memory hydrogels. A few examples are discussed below: • Cheng et al. designed a pH-responsive hydrogel which is capable of trapping a cargo and releasing it when the pH is increased.280 • Sadler and colleagues have shown that a photoactivatable dopamine-conjugated platinum(IV) anticancer complex can be incorporated into a quadruplex hydrogel, exhibiting selective phototoxicity against cancer cells.281 • Given that a number of proteins bind to G-quadruplexes, G4-based hydrogels may be used for the selective incorporation and release of G4-binding proteins such as thrombin via recognition of the thrombin-binding aptamer.282,283 • Willner and colleagues incorporated hemin into a Gquadruplex-cross-linked hydrogel to obtain a catalytic

8. PERSPECTIVES The last decades have seen a flurry of DNA-based nanotechnologies involving unusual nucleic acid structures. In this review, we have tried to demonstrate how quadruplexes enable a greater diversity of DNA-based building blocks and offer advantages over classical B-DNA. Nanoscale robots have potential as intelligent drug delivery systems that respond to molecular triggers. However, applications in nanomedicine for delivery, imaging, and diagnostics are still relatively rare (less so for diagnostics), and noncanonical base-pairing schemes are not often exploited to create DNA-based materials; even then, the quadruplex part is often a minor component of a much larger device: “quadruplex-only” structures are only observed in the case of G-wires formed with poly d(G). The use and versatility of controlled individual multimerization of G- or C-rich sequences should be particularly useful in the construction of complex objects: as stated before, the true power of using these “nano-oddities” comes from combining them with existing nanomaterials and integrating them into existing devices.2 Throughout this review, we tried to convey the message that quadruplexes offer interesting advantages over canonical nucleic acid structures, both for in vitro and cellular applications: • High stability: under ideal conditions, both G-quadruplexes and i-motif are thermally stable, and this stability is even higher in confined environments such as nanosized water pools288 or within an origami cage. Both structures are strongly enthalpy-driven (ΔH < 0), which means that their stability will be temperature-dependent. G4 is also preserved under denaturing conditions. Interestingly, G4 formation is compatible with physiological conditions, Z


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simple one- or two-dimensional shapes represents a novel approach that could transform how we think about drug delivery”.257 Despite these assets, there is still a relative paucity of studies taking full advantage of these unusual structural motifs in nanotechnologies. In order for the field to fully blossom, one would need to solve some of the following issues:

allowing these nanostructures or devices to be stable in cells. In addition, quadruplexes are resistant to nucleases. • Tunability: the high stability mentioned above can be modulated by external factors (pH, ionic conditions), and these structures can be opened by complementary strands to form a duplex. In other words, quadruplexes can act not only as passive structural elements, but also as active partners triggered by external signals. Perhaps even more interestingly, because of their orthogonal pairing rules, one can modulate the stability of a quadruplex with minimal effect on a surrounding larger duplex-based nanostructure. • These structures can be selectively recognized by small compounds or proteins. Some ligands are actually fluorescent, or else allow enzymatic activity (hemin). These compounds may be used to reveal the presence of this structure for sensor applications. • Interestingly, a number of modification/backbone analogues support G4 formation such as DNA, RNA, 2′OMe, PNA, or phosphorothioates289this is probably less true for i-DNA, as RNA and 2′OMe tend to disfavor i-motif formation.20 For nanotechnology purposes, the G- and C-quadruplexes are not symmetrical. Some of these differences explain why Gquadruplexes have been used more often than i-DNA for these applications: • Both structures are polymorphic (a single sequence can adopt several conformations), but polymorphism is more limited in the case of i-DNA, as strand orientation is fixed with two strands pointing in one direction and the two others in the reverse orientation. • The H+ vs K+ response is different: in the case of i-DNA folding, a higher number of protons are taken up (one per base pair). In the case of the human telomeric motif, six hemiprotonated base pairs or three G-quartets are formed with the C-rich strand and G-rich strands, respectively (and two potassium ions are typically taken up between the 3 quartets). As a consequence, i-DNA’s pH sensitivity is exquisite, allowing stability to flourish or collapse over a relatively narrow pH range. • Their stability under physiological conditions is clearly different: while G4 formation is compatible with physiological salt concentrations, i-motif is marginally stable, at best, at physiological pH.19 • A number of base analogues support G4 formation and may be used to improve G4 properties. For example, isoguanine pentaplexes can be used to bind heme and enhance its oxidative activity.290 On the other hand, the register of modifications allowing i-DNA formation is more limited. • The library of chemical compounds and proteins able to recognize the i-motif is limited, while a flurry of G4binding compounds exists. • No DNAzyme activity nor aptamer have been reported so far with an i-DNA core; we currently lack a small convenient protein (such as thrombin) binding to folded C-rich motifs. It is gratifying to observe that the first in vivo application of a DNA origami nanoparticle for cancer therapy involves a DNA G-quadruplex.161 This reconfigurable DNA nanorobot exhibited promising antitumor efficacy and, as noted by Tasciotti, “Creating complex, dynamic, three-dimensional structures from

• Because of the intrinsic “self-recognition” of G by G and C by C in G-quadruplexes and i-motif, respectively, this controlled assembly is far from trivial, even if partialbut not universalsolutions have been found. It is also difficult to use more than one G4 (or i-motif) device in a nanostructure, as assigning a specific strand to a given quadruplex is not straightforward. A few solutions exist though. For example, Tran et al. showed that L- and Dstrands self-exclude when mixed together, allowing the controlled parallel self-assembly of different G-rich strands through L-DNA use.291 • Heterogeneity and imperfections: Most of the concepts proposed in this review assume the correct assembly of the nanostructure. Yields of preparation may vary, however, and purifications steps are often required to obtain a sufficient level of homogeneity. Imperfections may have different impacts on single-molecule and bulk applications; their detection and removal can be more or less easy. These imperfections are especially detrimental to systems involving the multimerization of an element, as imperfections are likely to contaminate these higher-order structures. • For stimulus-responsive devices, even if a variety of stimuli have been described (e.g., cation, pH, temperature, light, ligands, DNA strands, etc.), an even larger choice would be welcome for some applications. For example, it would be interesting to have quadruplexes responding differently to cations or specific ligands, or different i-motif responding to pH changes within different pH ranges. This can be achieved by using cytosine analogues or a phenoxazine 2′-deoxynucleotide clamp for iM stabilization.292 • Structural polymorphism is a double-edged sword! Structural polymorphism complicates the design of “clean” G4-based nanodevices and the analysis of their kinetics and thermodynamics. It is interesting to note that our understanding of this polymorphism is currently improving, and that one can now accurately predict the topology of some G4 based on its primary sequence,293 thereby facilitating the design of G4-based nanoassemblies. • One of the Achilles’ heels of these devices is to rely on strand invasion for conformational changes. DNA branch migration is often initiated by DNA sticky end pairing. Toehold-mediated strand displacement has enabled the construction of sophisticated circuits, motors, and molecular computers. However, this process can be relatively slow (pH-driven devices are much faster), and this will impact the speed of processing. DNA catalysts may be used to speed up the hybridization kinetics of complementary strands. Maruyama and colleagues demonstrated that a comb-type cationic copolymer can accelerate G4 formation and improve the performance of a G4-duplex nanodevice.294,295 AA


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• The hemin/G4 complex (heme·DNAzyme) is a wonderful tool for nanotechnology, mostly as an output signal. Other label-free and enzyme-free systems would be welcome to build advanced logic devices. Ideally, different quadruplexes should lead to different fluorescent or enzymatic outputs. Some ligands such as NMM offer a fair level of selectivity toward one quadruplex topology: this may eventually allow the independent detection of different G4 outputs in the same test tube. In summary, G4 and i-motif offer physicochemical properties that are unique, rendering them suitable for application to a significant portion of the body of DNA-based nanotechnology. How might quadruplex-based DNA (or RNA) nanotechnology progress over the next 5−10 years? What are certainly possible to identify at the present moment are developments that will likely have a strong impact on near-term future development. For one, the predictability of G4 folding is starting to become accessible. Further, it can be anticipated that more and more excellent tools will increasingly become availablehigh-quality small-molecule ligands and antibodies, in particular. Diagnostics and therapeutics remain very promising areas for quadruplexbased DNA nanotechnology and will likely bear fruit in the near future. What about nanoelectronics? Making DNA conductive is an attractive goal;296 bottom up construction of very small circuits, that can compete or even have an advantage over silicon, remains a challenge to be demonstrated. The current state of the art allows the construction of linear or branched G-wires297 (which can be further decorated with gold298,299) but often with limited control over branching. Understanding the factors that stabilize these wires will be essential;300 in addition, as natural sequences can actually adopt a compact beaded filament structure,301 they may well be relevant for biology as well. To be successfully useful, all DNA-based structures must be able to scale up from the nm to the mm scale. Costs of construction and of raw materials remain attractive features for all DNA-based nanotechnology. A further benefit in this area will undoubtedly come from improvements in cost and from the ever-increasing versatility in oligonucleotide synthesis, as well as the coopting of structurally and functionally diverse analogues. As a final word, we feel that Richard Feynman’s 1959 statement “There is plenty of space at the bottom” remains completely current and apropos for nanostructures built using or incorporating DNA oddities such as G-quadruplexes and i-DNA.

He became interested in quadruplex nucleic acids in the 90’s, and his primary research interests lie in the structural characterization of various unusual DNA conformations involved in cancer. He received the INSERM Award in Therapeutical Research, is the “Key foreign scientist” of a Czech-based project on “DNA gymnastics”, and is also recipient of the Recruitment Program for Foreign Experts (1000plan) of China in Nanjing University. Dipankar Sen graduated with a B.A. (Hons.) from Cambridge University, and completed his Ph.D. in Chemistry at Yale University, with Donald M. Crothers. He then did postdoctoral work in Cellular and Developmental Biology at Harvard University with Walter Gilbert. He has been on the faculty at Simon Fraser University in British Columbia, Canada, since 1991 and is currently Professor of Chemistry and also Professor of Molecular Biology and Biochemistry. His research interests focus on the fundamental chemical and physical properties of DNA and RNA, nucleic acid catalysis, biosensors, and G-quadruplexes.

ACKNOWLEDGMENTS We thank J. Sponer, V. Gabelica and J. Zhou for helpful discussions as well as J. Chen, E. Largy. and M. Cheng for help in preparing the manuscript. This article is dedicated to the memory of Dr. Emil Paleček (1930-2018), a pioneer in the field of electrochemistry of nucleic acids. J.-L.M. is the recipient of the Recruitment Program for Foreign Experts (1000plan) of China [WQ20163200397] and acknowledges funding from Nanjing University [020514912216] and Fundamental Research Funds for the Central Universities (020514380144) and from the SYMBIT project (reg. no. CZ.02.1.01/0.0/0.0/15_003/ 0000477) financed by the ERDF. D.S. acknowledges funding from the Natural Sciences and Engineering Research Council of Canada (NSERC). ABBREVIATIONS/GLOSSARY: C·C+ Hemiprotonated cytosine−cytosine base pair found in an i-motif. DNAzyme Complex of G-quadruplex with hemin, showing peroxidase and peroxygenase activities. GRO G-rich oligonucleotide. G4 G-quadruplexes. G-quartet Planar arrangement of four guanines (also called a G-tetrad) G-wire Higher-order structure formed by G-rich oligonucleotides or by poly d(G) or poly r(G); one may also encounter the term “Frayed wire”, which supposes the presence of dangling ends protruding from the G-wire core. i-DNA Alternative name for the i-motif. NMM N-methyl mesoporphyrin IX (a fluorescent G4 ligand). NP Nanoparticle. Quadruplex Used here for G4 and i-DNA, as both structures involve four strands. TBA Thrombin-binding aptamer (a short 15 nt long DNA sequence forming an intramolecular G4 structure).

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jean-Louis Mergny: 0000-0003-3043-8401 Dipankar Sen: 0000-0003-0803-3002 Notes

The authors declare no competing financial interest. Biographies Jean-Louis Mergny received his Ph.D. (Pharmacology) in Prof. Claude Hélène’s laboratory in Paris, France (1991) followed by Postdoctoral training in Switzerland at the Biozentrum in Basel with Walter Gehring, where his work focused on Drosophila early development. He accepted a position in the Institut National de la Santé et de la Recherche Médicale (INSERM) in 1993, to head up a Group working on DNA structures.

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