Analyzing DNA Nanotechnology: A Call to Arms For The Analytical

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Analyzing DNA Nanotechnology: A Call to Arms For The Analytical Chemistry Community In order to fully realize the potential of DNA nanotechnology, it is crucial to overcome the lack of robust analytical techniques that continue to hinder the purification and characterization of DNAbased structures. In this Feature, we provide a snapshot of the current state of metrological techniques in DNA nanotechnology and look forward to emerging technologies that may offer new ways to probe and visualize these complex structures. Divita Mathur†,‡ and Igor L. Medintz*,‡ †

College of Science, George Mason University, Fairfax, Virginia 22030, United States Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Code 6900, Washington, D.C. 20375, United States have made DNA nanotechnology widely adopted in research areas that desire designer constructs in this size regime; these are highlighted in recent reviews.2,3 This field has attracted scientists from multiple disciplineschemists, physicists, engineers, computer scientists, and biologistsas a tool to unravel single-molecular interactions,4 develop sophisticatedmultiplexed sensors,5 along with creating nanopatterns,6 plasmonic complexes,7 energy transfer substrates,8 delivery nanobots,9 molecular programming circuits,10 molecular walkers,11 and mechanical actuators,12 among a host of other nanodevices and potential utilities.13 The functional application of DNA nanostructures is preceded by scientific procedures for the purification and characterization of these structures. Purification and characterization are measures imperative to yield optimally pure and well-defined/verified synthetic DNA-based products before deploying them in subsequent applications that span from in vitro to in vivo. However, the current state of primitive and finite metrologies is a major limitation to the field and offers an unprecedented opportunity for analytical chemistry to make significant enabling contributions. Additionally, readily available go-to resources, which have systematically surveyed the available protocols and highlighted best practices in this field Ella Maru Studios in order to assist scientists in the deliberate selection of the most suitable technique on the basis of the properties of their DNA nanosystems, are scarce at the moment. he field of DNA nanotechnology has enabled scientists to We begin by noting an important overarching common realize and rapidly expand the ability to “build” objects at the nanoscale. This building of nanoscale objects, such as theme, namely, that the processes involved in purifying and blocks, platforms, cages, and flasks is analogous to building a analyzing DNA nanostructures are often interchangeable, normal-sized machine with metal parts or raising a building wherein the former provides vital information for the latter with bricks and mortar. Moreover, DNA architecture at such a or a single analytical technique can simultaneously serve both small scale, on the order of 10−9 m, is programmable, selfpurposes. In this Feature, we discuss the available techniques assembling, and completely biocompatible in nature. Since the and their suitability for some of the most common types of inception of this field from a DNA tile derived from a simple DNA products as well as aim to pique the interest of the 1 Holliday Junction more than 3 decades ago (Figure 1), its analytical community in order to pave the way toward new versatility, structural diversity, and applicability have grown applications and enabling technologies. exponentially and, for the moment, seem limitless. The cumulative properties, especially that of programmable nanoscale self-assembly of almost any static or dynamic architecture ‡

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Figure 1. Basic overview of DNA-based structures. (a) Structure of a DNA double helix. A double-stranded DNA (dsDNA) is formed via base pair hybridization between two complementary single-stranded DNA (ssDNA) molecules, where adenine (A) binds to thymine (T) with 2 hydrogen bonds, and guanine (G) binds to cytosine (C) with 3 hydrogen bonds. A dsDNA molecule typically has a diameter of 2 nm and height of 0.34 nm/ base and it forms the basic assembly unit in DNA-based structures. (b) More than one ssDNA can hybridize using base pair complementarity to form a Holliday Junction (right) or single and double crossover motifs (left). These are the most elementary DNA architectures that are prevalent in DNA nanostructures. (c) One of the most successful methods of creating DNA structures is called DNA origami, in which a long ssDNA (called a “scaffold”) is folded into a predetermined shape using short oligonucleotides (called “staples”). Shown here are some of the earliest two-dimensional structures made using DNA origami. Scale bar = 100 nm. Reprinted by permission from Macmillan Publishers Ltd.: Nature, Rothemund, P. W. Nature 2006, 440, 297−302 (ref 15). Copyright 2006. (d) Left side: Another technique is tile self-assembly, in which a set of short oligonucleotides self-assemble to form DNA-based lattices and arrays. Shown here are two-, three-, and four-branched DNA tiles assembling into arrays. Reprinted with permission from Macmillan Publishers Ltd.: Nature, Winfree, E.; Liu, F.; Wenzler, L. A.; Seeman, N. C. Nature 1998, 394, 539−544 (ref 80). Copyright 1998. Reprinted with permission from He, Y.; Chen, Y.; Liu, H.; Ribbe, A. E.; Mao, C. J. Am. Chem. Soc. 2005, 127, 12202−12203 (ref 137). Copyright 2005 American Chemical Society. From Yan, H.; Park, S. H.; Finkelstein, G.; Reif, J. H.; LaBean, T. H. Science 2003, 301, 1882− 1884 (ref 43). Reprinted with permission from AAAS. Right side: Short oligonucleotides can also be designed to mimic DNA “bricks” that selfassemble into a molecular canvas or block to create a multitude of structures. Shown here are DNA bricks self-assembling to form some letters. From Ke, Y. G.; Ong, L. L.; Shih, W. M.; Yin, P. Science 2012, 338, 1177−1183 (ref 23). Reprinted with permission from AAAS.

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COMPLEXITIES OF DNA-BASED STRUCTURES

entirely driven by the inherent property of self-assembly via thermal annealing of oligonucleotides in the presence of a suitable buffer and typically requires no other external regulatory measures. There are numerous strategies for developing DNA-based nanostructures. A summary of some commonly used strategies can be found in Table 1, where, for each kind of technique, different parameters are given in order to provide a perspective on the potential challenges of customizing metrological approaches. Each strategy contributes uniquely to the arsenal of tools available in DNA nanotechnology, in the form of the permissible size-range of the resulting nanostructures, their flexibility, addressability, and dynamic behavior, which are

To appreciate the requirements of such DNA structures, some background on their design, assembly, properties, and components is warranted. DNA-based structures are two- and three-dimensional architectures created by the self-assembly of an ensemble of synthetic and natural DNA (see Figure 1). On the basis of the principles of Watson-Crick base pairing, singlestranded DNA (ssDNA) segments are designed with complementary domains (or subsequences) to enable their collective hybridization into a predetermined nanoscale architecture, with a typical collective goal of good yield and high concentration of product structure(s). The process is B

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Analytical Chemistry Table 1. Representative DNA Structural Assembly Strategiesa

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Yield values are only an estimate; actual values vary by example. 2D DNA origami: Reprinted by permission from Macmillan Publishers Ltd.: Nature, Rothemund, P. W. Nature 2006, 440, 297−302 (ref 15). Copyright 2006. 3D DNA origami: From Dietz, H.; Douglas, S. M.; Shih, W. M. Science 2009, 325, 725−730 (ref 29). Reprinted with permission from AAAS. DNA gridiron: From Han, D. R.; Pal, S.; Yang, Y.; Jiang, S. X.; Nangreave, J.; Liu, Y.; Yan, H. Science 2013, 339, 1412−1415 (ref 30). Reprinted with permission from AAAS. DNA wireframe: Reprinted by permission from Macmillan Publishers Ltd.: Nature Nanotechnology, Zhang, F.; Jiang, S.; Wu, S.; Li, Y.; Mao, C.; Liu, Y.; Yan, H. Nat. Nanotechnol. 2015, 10, 779−784 (ref 31). Copyright 2015. Polyhedral mesh: From Veneziano, R.; Ratanalert, S.; Zhang, K.; Zhang, F.; Yan, H.; Chiu, W.; Bathe, M. Science 2016, 352, 1534 (ref 21). Reprinted with permission from AAAS. DNA SST/brick/scaples: Ke, Y. G.; Ong, L. L.; Shih, W. M.; Yin, P. Science 2012, 338, 1177−1183 (ref 23). Reprinted with permission from AAAS. “Origami” of DNA origami: Reprinted with permission from Zhao, Z.; Liu, Y.; Yan, H. Nano Lett. 2011, 11, 2997−3002 (ref 32). Copyright 2011 American Chemical Society. DNA origami-tile assembly: Wang, P.; Gaitanaros, S.; Lee, S.; Bathe, M.; Shih, W. M.; Ke, Y. J. Am. Chem. Soc. 2016, 138, 7733−7740 (ref 22). Copyright 2016 American Chemical Society. Tile self-assembly: Reprinted with permission from Rothemund, P. W.; Papadakis, N.; Winfree, E. PLoS Biol. 2004, 2, e424 (ref 33). Copyright 2004 Rothemund et al.

reviewed in depth in other publications.3,14 There are two overarching categories into which these design strategies can be classified. The first are based on the seminal technique called DNA origami, in which a long ssDNA, called a scaffold, is folded into a predetermined shape using a pool of short oligonucleotides, called staples. DNA origami, invented by Paul Rothemund in 2006,15 uses the genomic DNA of the filamentous E. coli bacteriophage m13mp18 (7 249 bases in length) as the scaffold strand and can create two- and threedimensional nanostructures on the order of ∼100 nanometers (nm) per dimension. Other strategies have been introduced that either address the sequence and scalability limitations of DNA origami by introducing alternative scaffolds16−18 or enhance the properties of DNA origami structures using different folding schemes.19−22 The second kind of assembly technique uses an ensemble of short DNA oligonucleotides to create two- and threedimensional arrays, lattices, and nanostructures. The DNA brick23 and single-stranded tile (SST)24 techniques consist of a two- or three-dimensional DNA “canvas” containing hundreds

of oligonucleotides, from which desirable structures can be “carved” out by selecting an appropriate subset of strands. The “scaples” technology allows users to exploit the ease of a DNA origami design scheme but substitute the scaffold strand with staple mimics, called scaples (scaffold + staples), in order to build any DNA origami-like structure with custom-built nonbiological sequences instead of the more limited viral m13 sequence.25 DNA tile assembly, which originally sowed the seeds of DNA nanotechnology, is the creation of DNA tiles, arrays, and lattices made from a far smaller set of DNA oligonucleotides.26,27 While the latter technique offers scalability and sequence variability, it can suffer from poor yields. Despite their different approaches to designing a given DNAbased structure, all follow a common route of structure assembly, namely, identifying the requisite set of oligonucleotides, creating a reaction mixture with the oligonucleotides in suitable buffer and salt (e.g., Mg2+, Na+, Ni2+), and lastly, initiating self-assembly using an optimal thermal annealing program. Computational resources are often recruited to assist in designing the DNA constructs and their constituent C

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Analytical Chemistry Table 2. Some Representative Modifications Applied onto DNA Constructsa

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AuNP: Reprinted by permission from Macmillan Publishers Ltd.: Nature Nanotechnology, Kershner, R. J.; Bozano, L. D.; Micheel, C. M.; Hung, A. M.; Fornof, A. R.; Cha, J. N.; Rettner, C. T.; Bersani, M.; Frommer, J.; Rothemund, P. W.; Wallraff, G. M. Nat. Nanotechnol. 2009, 4, 557−561 (ref 48). Copyright 2009. AgNP: Reprinted with permission from Lubitz, I.; Kotlyar, A. Bioconjug. Chem. 2011, 22, 482−487 (ref 37). Copyright 2011 American Chemical Society. QD: Reprinted with permission from Bui, H.; Onodera, C.; Kidwell, C.; Tan, Y.; Graugnard, E.; Kuang, W.; Lee, J.; Knowlton, W. B.; Yurke, B.; Hughes, W. L. Nano Lett. 2010, 10, 3367−3372 (ref 39). Copyright 2010 American Chemical Society. Carbonaceous nanomaterials: Reprinted with permission from Mangalum, A.; Rahman, M.; Norton, M. L. J. Am. Chem. Soc. 2013, 135, 2451−2454 (ref 40). Proteins: Reprinted with permission from Shen, W.; Zhong, H.; Neff, D.; Norton, M. L. J. Am. Chem. Soc. 2009, 131, 6660−6661 (ref 51). Copyright 2009 American Chemical Society. Other materials: Reprinted with permission from Stephanopoulos, N.; Liu, M. H.; Tong, G. J.; Li, Z.; Liu, Y.; Yan, H.; Francis, M. B. Nano Lett. 2010, 10, 2714−2720 (ref 42). Copyright 2010 American Chemical Society.

synthetic DNA sequences.19,28 In DNA origami and derivatives thereof, the staple strands are present in 5- to 10-fold excess compared to the scaffold strand, a critical stoichiometric step used to drive the reaction in the forward direction and improve overall yields. The final annealing product contains primarily excess staples, partially formed or incorrectly incorporated scaffold particles, fully formed desired structures, and higher order aggregates. Much of the undesired species are believed to originate from cross-hybridization between structures. In strategies involving ensembles of short oligonucleotides, such as the bricks, scaples, and tile-assembly techniques, the final annealing output is also a heterogeneous pool of the desirable architecture, partial structures, chimeric shapes, concatamers, and unbound ssDNA strands. The unfavorable byproducts and excess DNA strands, if not separated from the correct nanostructure, can and will adversely affect the functionality of the nanostructure either by participating in undesired or side chemical reactions, or by compromising the structural integrity of the nanostructure. From just this brief tutorial, the need for most DNA structures to undergo both purification and enrichment for most subsequent applications becomes readily apparent. DNA is also highly amenable to chemical conjugation with a host of different kinds of molecules, such as proteins, fluorescent dyes, nanoparticles (NPs), and inorganic substrates using well-established generic chemistries. With the help of the spatial information on each participating DNA strand within a

nanostructure (by virtue of the design), it is possible to immobilize proteins, other nucleic acids, and NPs onto these structures with nanometer precision and high efficiency. This broad propensity of DNA to be conjugated with other chemicals in an addressable manner has been harnessed to couple a host of different molecules onto DNA nanostructures, see for example Table 2 where, for each modification, the most commonly used DNA substrate as well as some applications are included. They are quite frequently modified with dyes,8,35,50 various nanomaterials such as gold/silver NPs (AuNP, AgNP),6,36,37,49 quantum dots (QD),38,39 carbon NPs,40 and other molecules41−43 by chemically altering a subset of constituent DNA strands with the desired modification. In post-assembly reactions, the unbound modified strands present the biggest impediment to direct characterization and application of DNA structures by contributing to false positive signals or decreasing the signal-to-noise ratio, thereby negating the original purpose of developing the nanostructure. Directly related to this, due to a host of still unresolved issues, considerable research continues on developing a toolbox of easily implemented, site-specific, and bioorthogonal chemistries for modifying DNA with nanomaterials and other biologicals to facilitate bionanotechnology in general.44−47 In light of these factors, it becomes crucial that DNA-based structures be treated with suitable purification protocols to eliminate unwanted materials along with byproducts and obtain highly homogeneous, concentrated, and pure samples for D

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Figure 2. Schematic highlighting the separation range for some of the predominant purification techniques in DNA nanotechnology. The legend illustrates some of the most commonly found ingredients in a completed assembly reaction. Each purification technique is efficient in separating the desirable structure (a small structure such as a tetrahedron, DNA origami based structures such as a smiley face, or higher order structures excluding concatamers) from other constituents of an assembly reaction in a specific size range. In some cases (such as PEG purification) the purification techniques have a lower cutoff limit, designated by the flat end on the lower side of an arrow. Some techniques (such as electrophoresis) are able to isolate the DNA product from larger as well as small contaminants, shown as lines with two flat ends. The biggest challenge lies in isolating the 100% completely modified constructs, for example, a correct smiley DNA origami structure with 1 orange “nose” and 2 yellow “cheek” labels from the incomplete products that lack one or more modifications, as well as from concatamers and aggregates.

• Can the target structure be tested and improved before final application? Moreover, once a nanostructure is produced with sufficient purity and yield, it remains of no use unless it is characterized in order to validate its correct formation, dimensions, concentration, and secondary (base pairing complementarity) and higher-level (e.g., linear versus twisted) structural properties. If the nanosystem is decorated with dyes, other biomolecules, and NPs, questions arise on whether their spatial orientation relative to each other as well as the DNA substrate agrees with the predicted architecture as well as what fraction of the DNA

subsequent characterization and then application. Inevitably, scientists seek to answer some or all of the following questions about their synthetic DNA construct: • Was the target structure made? • How much was made? • What is the purity? • Are there any imperfections in the final structure? • What else was made? • Can the target structure be purified from other structures? • Are the modifications at the desired locations? E

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techniques and discusses their common advantages and disadvantages. Size-Based Separation. Electrophoresis is the most widely used method for purification as well as characterization of DNA nanosystems. The technique separates DNA-based structures from other unwanted species using the core principle of electrophoretic mobility of objects in a resistive medium. Samples are allowed to migrate through a matrix of specific density, typically an agarose or polyacrylamide gel, within an electric field. The different contents of the sample move through the gel at different velocities as a function of their charge per unit mass, the end result of which in the case of nucleic acids is separation of DNA products according to their size into bands along the migration lane. The band representing the DNA structure is then excised and treated to extract pure sample using spin columns,52 electroelution,36 sucrose wells,53 etc. The gel density in electrophoresis is a function of the size of the nanostructure, wherein 0.3−1% agarose gels are used for larger structures34 and 1.5−3% agarose gels otherwise.19,52 Gel electrophoresis is useful for separating desirable structures from smaller as well as larger byproducts and raw ingredients, which is something not easily accessed with other size-based separation strategies. However, it is fraught with difficulties and issues such as low recovery, gel residue contamination, and the inability to purify large amounts of sample. Additionally, sample heating due to the electrophoretic resistance of the gel is a major problem, which leads to denaturation of the structures. To mitigate this, gel electrophoresis is typically run at 4 °C or on ice, which is only partially effective. Mg2+ (or another cation), which is crucial for the stability of DNA structures, gradually precipitates on the anode during electrophoresis, thereby reducing its soluble concentration and compromising DNA structural integrity. To add to this, the electrophoresis buffer has to be connected with a peristaltic pump or a salt bridge in order to compensate for salt precipitation. It is also possible to separate particles using free-flow electrophoresis (FFE), which is a matrix-free method of separating biomolecules as a function of their electrophoretic mobility in an aqueous medium. Particles separate out based on their total charge and are collected as fractions when exiting the chamber. FFE is frequently used for the separation of minute amounts of sample and is a powerful approach for separating proteins, subcellular components, and cells owing to its liquidbased medium.72 It, therefore, is useful for the separation and purification of DNA structures that are functionalized with protein, enzymes, or cellular molecules.73 The speed of the entire process is highly competitive (