Functionalizing DNA origami to investigate and interact with biological systems

Abstract

DNA origami has emerged as a powerful method to generate DNA nanostructures with dynamic properties and nanoscale control. These nanostructures enable complex biophysical studies and the fabrication of next-generation therapeutic devices. For these applications, DNA origami typically needs to be functionalized with bioactive ligands and biomacromolecular cargos. Here, we review methods developed to functionalize, purify, and characterize DNA origami nanostructures. We identify remaining challenges, such as limitations in functionalization efficiency and characterization. We then discuss where researchers can contribute to further advance the fabrication of functionalized DNA origami.

ToC blurb

DNA origami nanostructures are useful constructs for biophysical and therapeutic studies. In this Review, we discuss how these nanostructures are functionalized with bioactive conjugates, purified, and characterized, and we compare the advantages and limitations of these methods in the context of different applications.

Introduction

The DNA origami method enables high-yield fabrication of DNA nanostructures at the 1–100 nm length scale^1,2. In this method, a long single-strand DNA (ssDNA) ‘scaffold’ is folded into a desired shape using short oligonucleotide ‘staple’ strands via Watson-Crick base pairing. The DNA origami method can now generate 2D and 3D bricklike geometries^3–5, intricate wireframe assemblies^6,7, and higher-order superstructures^8–10. For challenging sequence designs, top-down design algorithms have enabled rapid prototyping of nanostructures and democratized this technique to a broader range of scientists and engineers^6,7,11–15. Fabrication advances in custom scaffolds^16–21, biological²² and enzymatic^23,24 staple synthesis, and scaled-up thermal annealing²⁵ now allow for large-scale production of DNA origami. With several bottlenecks in DNA origami nanostructure design and fabrication overcome, DNA origami is widely applied to probe biological systems and has potential as a translational biomaterial^26–28.

DNA origami is particularly useful when applied to interrogate and interface with biological systems owing to its ability to position bioactive moieties at the nanometer length scale^29–31. For example, functionalized DNA origami nanostructures (Figure 1a) have been used to determine the nanoscale properties governing immune receptor activation^32–36 and enzyme activities^37–39. Enzyme cascade systems can be enhanced by programming molecular motion into nanoscale enzyme arrays⁴⁰. DNA origami nanostructures functionalized with small molecule antigens at different nanoscale spacings are able to determine the spatial tolerances of antibody-antigen interactions⁴¹, and functionalized icosahedral nanostructures have revealed spatial rules of B cell receptor activation³².

Figure 1. Functionalized DNA origami nanostructures interact with biological systems.

a | Functionalized DNA origami nanostructures have conjugates of interest attached to DNA origami nanostructures using specific functionalization methods. b | These functionalized nanostructures can interact with biological systems at different length scales to enable various biological experiments. c | Examples of functionalized DNA origami nanostructures utilized in each investigation type: determining the spatial tolerance of antibody-antigen recognition⁴¹; determining the spacing rules of B-cell receptor signaling³²; and investigating a cancer vaccine in vivo⁴⁵. Panel C (Left) adapted with permission from ref.⁴¹, Springer Nature Limited. Panel C (Center) adapted with permission from ref.³², Springer Nature Limited. Panel C (Right) adapted with permission from ref.⁴⁵, Springer Nature Limited.

DNA origami also has the ability to compute, performing Boolean logic in therapeutic applications to deliver small molecules, nucleic acids, and therapeutic proteins in vitro⁴² and in vivo^43,44. For example, in animal models, a modular DNA origami nanotube delivered therapeutic proteins to tumors⁴³, antigenic peptides and adjuvants to dendritic cells⁴⁵, and siRNA and chemotherapeutic drugs to tumors⁴⁴. These delivery studies highlight that a single DNA origami nanostructure can be utilized in widely different applications, depending on what it is functionalized with. This modularity positions DNA origami as an emerging disease-agnostic biomaterial for a broad range of therapeutic delivery and vaccine applications.

All of the aforementioned examples can broadly be classified as molecular biophysics studies, cellular biophysics studies, or cargo delivery studies (Figure 1b). For each of these applications, the DNA origami nanostructure needs to be functionalized with a wide range of bioactive moieties, from small molecules to large proteins. Each type of study also requires different functionalization techniques, fabrication scales, stabilities, and costs, leading to a specific set of suitable methods (Figure 1c).

In this Review, we describe how DNA origami is functionalized, purified, and characterized, and we discuss advantages and limitations of each approach in the context of different applications. We identify where innovation in this space is needed to further advance the field, as well as areas of investigation that would help facilitate the translation of DNA origami towards clinical applications.

Functionalizing DNA origami

DNA origami nanostructures are fabricated, as reviewed elsewhere⁴⁶, by designing staple sequences, manually or algorithmically, for a given scaffold; producing the scaffold and staple strands; combining the scaffold and staple strands in buffer and annealing the mixture; and purifying and characterizing the resulting DNA nanostructures.

There are two typical workflows to functionalize DNA origami. Individual components of the DNA nanostructure can be functionalized before the nanostructure is self-assembled (Figure 2), or the entire nanostructure can be functionalized following its self-assembly (Figure 3a). For the scope of this Review, we define ‘functionalization’ as the attachment of distinct chemical moieties onto the DNA nanostructure.

Figure 2. A typical workflow for pre-assembly functionalization of DNA origami.

Staples are modified before assembly with the desired conjugate, either through solid-phase or liquid-phase chemistry. Modified staples are then assembled with the scaffold and other staples to yield a functionalized DNA origami nanostructure.

Figure 3. A typical workflow for post-assembly functionalization of DNA origami.

a | Staples with addressable functionalization sites are assembled with the scaffold and other staples into DNA nanostructures. The conjugate of interest is then incorporated into the nanostructure through non-covalent or covalent methods. b | Non-covalent strategies attach conjugates onto DNA nanostructures through non-covalent molecular interactions. c | Covalent strategies rely on the formation of a covalent bond between the nanostructure and the conjugate.

Pre-assembly functionalization

In pre-assembly functionalization, conjugates of interest are covalently bound to staple strands that are then integrated into the DNA nanostructure during self-assembly (Figure 2). One advantage of this strategy is that the valency of conjugates on the designed DNA nanostructure is known and coverage is quantitative per conjugation site, given the purity of the staple conjugates and the quantitative integration of staples into the nanostructure. In addition, the covalent attachments between the conjugates and DNA nanostructure potentially benefit the stability of the assembly. Pre-assembly functionalization can be performed in either the solid or liquid phase, providing far greater control over reaction conditions (such as concentration or co-solvents) than post-assembly functionalization methods.

Pre-assembly techniques have been used to conjugate fluorophores^{35,36,47–86}, lipids^{33,50,56,57,60,66,80,87–89}, small molecules^{35,36,41,63,85,90–100}, aptamers^84,101–104, peptides^56,105,106, and polymers¹⁰⁷ to DNA origami. These techniques are especially common for incorporating fluorophores, as they are attached easily to staples during solid-phase synthesis of the oligonucleotides using phosphoramidite chemistry. Such constructs have been used as a biosensor⁷², to investigate cellular uptake⁴⁸, and to silence genes in plants⁷⁷. Lipids attached to DNA origami displaying stimulatory ligands with specific spatial arrangements have been used to investigate intracellular signaling of T cells³³. By incorporating the lipids into the DNA nanostructures prior to assembly, the nanostructures could later be anchored to a lipid bilayer and used to determine the minimal signaling unit promoting efficient T cell activation. Aptamers can also be integrated into the scaffold of the nanostructure through scaffold sequence design, for example for targeted cancer therapy¹⁰⁸. Scaffold functionalization offers the potential for high conjugate density on nanostructures; this is an underexplored area that we believe has potential to expand the DNA origami functionalization toolkit.

Although useful in numerous applications, pre-assembly functionalization has two major disadvantages. First, it is incompatible with conjugates that are sensitive to the high temperatures and high salt concentrations needed for DNA nanostructure self-assembly, such as proteins that can denature and aggregate. The second disadvantage is that functionalized staples need to be individually synthesized, purified, and characterized. This bottleneck can limit the throughput and thus ability to prototype nanostructures through the generation of libraries, a common technique to explore promising therapeutic delivery nanotechnologies such as lipid nanoparticles^109,110. Now that other workflow bottlenecks in DNA origami structural design and fabrication have been overcome, functionalization workflows that afford the ability to generate material libraries are particularly important to enable the rapid exploration of DNA origami chemical design space.

Post-assembly functionalization

As an alternative, conjugates of interest can be attached to the nanostructure after it is self-assembled, either through non-covalent or covalent approaches. Non-covalent approaches, such as the popular DNA-DNA hybridization method, were initially developed for their ease of implementation and today are the most commonly reported methods to functionalize DNA origami (Figure 3b). They do suffer from limitations in select applications, however, and covalent approaches can help fill in these gaps.

Non-covalent functionalization

DNA-DNA hybridization is the most common non-covalent approach. This method relies on ssDNA overhangs, typically 15–25 nucleotides long, which are extended from the DNA nanostructure. The desired conjugate of interest is chemically attached to another ssDNA handle that has a complementary sequence to the overhangs. After the nanostructure is assembled, it is incubated with the conjugate-DNA molecule to produce the functionalized nanostructure. This technique can attach proteins^{34,37–40,42,43,54,55,59,61,73,75,78,79,89,92,104,107,111–152}, fluorophores^{43,79,100,114,115,122,130,131,136,142,143,151,153–169}, aptamers^81,170–178, RNA123,179–181, small interfering RNA^{44,77,160,182}, short hairpin RNA⁶⁷, antisense oligonucleotides¹⁷¹, peptides^{32,44,45,152,156,183-187}, lipids^{62,82,89,141,148,157,165,167–169,188–194}, metals^{42,59,75,103,122,124,126,150,151,162,182,195–201}, quantum dots²⁰², genomic DNA²⁰³, polymers¹⁰⁷, and branched oligonucleotides²⁰⁴ to DNA nanostructures. The sequence-programmable, heterovalent nature of the method made it possible to orthogonally attach antigenic peptides, adjuvanting nucleic acids, and fluorophores to DNA nanostructures to fabricate an environmentally-responsive cancer vaccine⁴⁵. DNA-DNA hybridization suffers from loss of nanoscale addressability, however, as hybridization sites can span several nanometers in length, and risks DNA-DNA dissociation and loss of the conjugate. These design limitations are in competition because increasing hybridization length to increase thermodynamic stability correspondingly decreases nanoscale spatial resolution. Additionally, exposed duplexes generate additional susceptibility to nuclease degradation.

A technique similar to DNA-DNA hybridization is DNA-X hybridization, in which specific DNA-recognizing moieties, such as peptide nucleic acids (PNAs) and DNA-binding proteins, are used as handles to attach to ssDNA overhangs. DNA-PNA hybridization attached a clinically relevant HIV antigen to virus-like wireframe DNA origami particles, which were then used to study antigen-B-cell receptor interactions at the nanoscale³². DNA-binding protein interactions also enable site-specific protein positioning. In the initial report of this strategy, nanostructures incorporated with zinc-finger-binding DNA motifs could then conjugate with zinc-finger proteins of interest²⁰⁵. This technique has been extended to other DNA-protein binding systems²⁰⁶, and was utilized for structural biology^65,207,208, such as for single particle cryogenic electron microscopy of the DNA-binding proteins themselves²⁰⁹. DNA-X hybridizations have stronger interaction strengths than DNA-DNA hybridization, leading to higher fidelity bonds and more efficient fabrication protocols. However, DNA-X systems are often more expensive and require more expertise to implement.

Biotin-streptavidin functionalization relies on the high affinity non-covalent interaction between the streptavidin protein and the biotin small molecule. This method is easily accessible due to the ease of installing biotin onto staples, and the ease of engineering streptavidin systems for many purposes. It is often used to functionalize nanostructures with proteins^{33,47,57,58,89,96,105,119,185,210–219}, but can also attach peptide²²⁰ and metal²²¹ conjugates. However, streptavidin-based functionalization requires modifying the streptavidin protein itself with a conjugate in a site-selective, stoichiometric manner, which can be challenging for non-protein conjugates. Additionally, the large size of streptavidin itself limits nanoscale addressability. Finally, when used in cargo delivery investigations, streptavidin can also cause undesired immunogenicity^222,223.

The choice of functionalization strategy can influence the outcome of mechanistic studies in which DNA origami is used. In a comparative analysis of DNA-DNA hybridization, DNA-X hybridizations, and streptavidin-biotin systems, each method was evaluated for its efficiency in attaching a TCR-activating ligand to DNA origami, and for the functionalized construct to activate T cells⁸⁹. Although the functionalization efficiencies were similar between the methods, importantly, activation levels differed significantly. Specifically, the DNA-DNA hybridization method that is commonly used to functionalize nanostructures with proteins decreased the functionality of the TCR-activating ligand once attached, attributed to the negatively charged DNA hybridization linker interfering with TCR binding. In contrast, decreases in activity were not observed with the biotin-streptavidin and the DNA-PNA hybridization systems.

There are several other methods to non-covalently functionalize DNA origami. Metal-nitrilotriacetic acid-His coordination bonds can pattern proteins onto DNA nanostructures^224,225. Intercalation between DNA duplexes is a common and simple way to load small molecules in nanostructures, particularly chemotherapeutic drugs such as doxorubicin and daunorubicin^{44,67,83,163,164,174,182,200,203,221,226–236}. However, researchers have identified doxorubicin aggregation mechanisms and spectral dynamics that complicate this functionalization strategy²³⁶. Complexation methods utilizing physical interactions between the negatively charged phosphate backbones of DNA duplexes and the conjugate allow for highly dense functionalization with proteins^49,237–242, fluorophores²⁴³, polymers^{100,151,168,202,244–249}, peptides^86,250, lipids²⁵¹, and small molecules²⁵². However, complexation and intercalation strategies typically lack nanoscale spatial control of conjugate positioning and stoichiometric control of conjugate copy number per nanostructure, which are two of the key advantages of DNA origami nanotechnology.

Covalent functionalization

Various bioconjugation and general covalent chemistries have been adopted to covalently functionalize DNA origami nanostructures (Figure 3c). Protein tags are specific peptide and protein motifs that rapidly and efficiently form covalent bonds with a small molecule²⁵³. SNAP^{217,253–257}, HALO^{166,217,253,255,256,258–260}, and CLIP^255,260 tags, as well as more efficient, rationally engineered tags²⁶¹, have conjugated proteins to DNA origami. Combining these protein tags with DNA-protein binding systems can create orthogonal covalent chemistries with enhanced kinetics and functionalization efficiencies^255,257,260. Protein tag functionalization strategies employ mild conditions beneficial to both the nanostructure and the conjugate. However, it requires additional protein engineering of the conjugate and is limited in conjugate scope. Additionally, these tags could potentially lead to undesired immunogenicity in cargo delivery investigations, as noted for streptavidin systems.

N-Hydroxysuccinimide (NHS) esters react with a primary amine to form an amide bond. They are common bioconjugation reagents²⁶² owing to the prevalence of primary amines in biomacromolecules, such as the lysine residue. Amine groups are easily integrated into staple strands, and a wide range of NHS-functionalized molecules are commercially available. This reaction chemistry is more general than protein tags and has been used to conjugate proteins²⁶³, metals¹¹², and small molecules²⁶⁴. Still, this method suffers from poor functionalization efficiency in aqueous environments and has thus rarely been implemented in the DNA origami literature since its initial demonstration.

Click chemistries²⁶⁵ are well-suited to address the above limitation owing to their superior reaction thermodynamics and kinetics in aqueous environments. Click moieties, such as azides and alkynes, are readily incorporated into staple strands and desired conjugates. Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry was first demonstrated on a DNA origami sheet to attach a small molecule²⁶⁴, and has since been extended to attach proteins¹²² and to stabilize nanostructures through staple-staple conjugations²⁶⁶. Strain-promoted azide-alkyne cycloaddition (SPAAC) has slower reaction kinetics than CuAAC, but benefits from not requiring the copper catalyst, which is harmful to biological systems²⁶⁷ and is complicated to implement in DNA-based systems²⁶⁸. SPAAC has been used to polymerize monomeric origami units²⁶⁹, densely decorate nanostructures with fluorophores²⁷⁰, and functionalize wireframe DNA origami nanostructures with proteins, peptides, polymers, carbohydrates, and fluorophores²⁷¹.

Compared to non-covalent methods, covalent methods address two key fabrication challenges. First, because covalent bonds are more stable than non-covalent bonds, covalently functionalized DNA nanostructures are less likely to lose conjugates of interest. Conjugation stability and biodegradability are likely to become critical design parameters as these nanostructures are increasingly utilized for therapeutic payload delivery in vivo. Second, covalent strategies may enable conjugates to be spaced more precisely on nanostructures; high spatial resolution is often a critical design parameter, in particular in molecular and cellular biophysics investigations. A disadvantage of covalent methods is that they require a large number of staples containing functionalization sites, which may either be expensive to purchase or require in-house equipment and technical expertise to prepare. Additionally, the kinetics of some covalent reactions, such as SPAAC²⁷², are slower than those of non-covalent reactions^273,274, making it more challenging to achieve high functionalization efficiency.

Considerations and analysis

Depending on the investigation type, each functionalization method has different advantages and disadvantages (Table 1). When choosing which method to use for a given investigation, the following considerations are important: the compatibility of the workflows with the conjugate of interest (for example, can the conjugate survive self-assembly conditions?); whether achieving quantitative functionalization efficiency is essential; whether the long-term and/or biological integrity of the conjugate-nanostructure bond are important; and the amount of the functionalized DNA origami nanostructure to be fabricated. From a manufacturability perspective, short reaction times and low required equivalents of conjugate may benefit DNA origami as a translational biomaterial.

Table 1.

DNA origami functionalization strategies.

Functionalization technique		Advantages	Disadvantages	Conjugate scopea
Pre-assembly
Covalent		Simple synthesis Simple characterization High functionalization efficiency Broad conjugate scope Simple heterovalent display	Not implementable with protein conjugates Inefficient workflow if many staples need to be functionalized Inefficient workflow if many different conjugates need to be functionalized	Fluorophores Lipids Small molecules Aptamers Peptides Polymers
Post-assembly
Non-covalent	DNA-DNA	Broadest conjugate scope Most method development	Possible site of nuclease degradation Less spatial control	Proteins Fluorophores
		Simple heterovalent display		Lipids Small molecules Aptamers Peptides Polymers siRNA ASO Metals Misc. oligonucleotides
	DNA-X	Stronger non-covalent bonding than DNA-DNA High functionalization efficiency	Minimal conjugate scope Expensive, or requires technical expertise	Proteins
	Biotin-Streptavidin	Extremely strong non-covalent bond	Minimal conjugate scope Large tag incorporated onto conjugate of interest	Proteins Peptides
	Metal-NTA	Orthogonal to other methods	Minimal conjugate scope Minimal development	Proteins
	Intercalation	Simple synthesis Dense functionalization	Lack of stoichiometric control Lack of spatial control Minimal conjugate scope	Small molecules
	Complexation	Simple synthesis Dense functionalization Broad conjugate scope	Lack of stoichiometric control Lack of spatial control Minimal conjugate scope	Proteins Fluorophores Lipids Small molecules Peptides Polymers
Covalent	Protein Tags	High functionalization efficiency Heterovalent display	Minimal conjugate scope Requires expertise in protein engineering Large tag incorporated onto conjugate of interest	Proteins
	NHS-ester	Simple synthesis	Low functionalization efficiency Minimal development Difficult to implement heterovalent display	Proteins Small molecules Metals
	Click chemistry	High functionalization efficiency	Expensive, or requires technical expertise	Proteins Fluorophores
	Broad conjugate scope	Difficult to implement heterovalent display	Small molecules Peptides Polymers Carbohydrates

^aConjugate scope is not the theoretical conjugate scope, but rather the scope that has been demonstrated in the literature. ASO: antisense oligonucleotide. siRNA: small interfering RNA.

Nanoparticles that are heterovalent—that is, nanoparticles displaying more than one type of molecule on their surface—are useful in many areas, including vaccinology^275,276. Pre-assembly functionalization methods, and the post-assembly non-covalent DNA-DNA and DNA-X hybridization methods, can easily produce nanostructures with heterovalency. In the case of pre-assembly methods, each staple can be functionalized with a different conjugate. For the DNA-DNA and DNA-X hybridization methods, the sequence-programmable nature of DNA allows multiple different conjugates to be attached to specific parts of the nanostructure based on unique DNA overhang sequences. Introducing heterovalency through covalent methods is more challenging, as orthogonal, efficient reactive groups have to be installed onto both the staples and conjugates. Heterovalency has been demonstrated with protein tag functionalizations^253,255, but not with other covalent chemistry systems, or for conjugates other than proteins.

Purifying functionalized DNA origami

Once DNA origami nanostructures are functionalized, they typically need to be purified (Figure 4). This purification step is required even when conjugates are attached with high functionalization efficiency, because conjugates are added in excess. With pre-assembly functionalization strategies, excess staples, excess functionalized staples, non-folded scaffolds, and misfolded nanostructures are purified away from functionalized nanostructures. When post-assembly strategies are implemented, excess conjugates are purified away from functionalized nanostructures. In both cases, methods typically used to purify non-functionalized DNA origami nanostructures, as reviewed elsewhere^46,277, are employed.

Figure 4. Methods to purify functionalized DNA origami nanostructures.

Purification methods aim to separate functionalized nanostructures from other components in the reaction mixture. In pre-assembly workflows, these methods purify away unfolded scaffolds, excess staples, excess functionalized staples, and misfolded nanostructures. In post-assembly workflows, excess conjugates, and possibly catalysts or side products, are purified away from the functionalized nanostructure. a | Affinity purification methods rely on the affinity between a specific tag on nanostructures and an external material, either column walls or magnets. b | Membrane purification methods utilize membranes that separate reaction mixtures components by a size cut-off. c | Sedimentation purification methods utilize the difference in sedimentation rates between components in the reaction mixture during centrifugation, either in buffer or glycerol solutions. d | Size purification methods rely on different retention times of reaction mixture components in different media, such as agarose gels and SEC beads.

Affinity purification

Affinity purification techniques rely on attractive interactions between the functionalized DNA origami, which has been labeled with affinity tags, and the purification equipment to separate the nanostructure from the untagged conjugates (Figure 4a). In its first implementation, nanostructures with His-tag-modified staple strands were purified on a cobalt-based metal affinity resin, owing to the affinity between the His-tag and metal ions¹⁰⁵. His-tags are commonly used for the purification of proteins themselves, and therefore may not be suitable for purifying functionalized DNA nanostructures when the conjugate of interest is a protein.

Magnetic beads can also provide the affinity for this type of purification. In the most common version of this approach, biotinylated staples are first incorporated into the nanostructure. After functionalization, the nanostructures are incubated with, and become affixed to, streptavidin-coated magnetic beads. Nanostructures can then be pulled from the rest of the solution containing excess conjugates with a magnet^{73,133,146,147,219,261}. Finally, the functionalized nanostructures are released from the magnetic beads through competitive binding that displaces the biotin-streptavidin bond. This strategy has also been performed using DNA hybridization between a DNA origami gene logic chip tagged with ssDNA overhangs and complementary ssDNA-labelled magnetic beads²⁵⁴. This technique was extended further to perform solid-phase synthesis of DNA origami-protein nanostructures; the nanostructure is first attached to magnetic beads through biotin-streptavidin interactions, then functionalized with protein, and then purified from excess protein before being removed from the magnetic beads²⁵⁸. Although magnetic purification has mainly been used to separate protein conjugates from functionalized DNA origami, they are implementable with other conjugate classes such as fluorophores¹³¹.

Membrane purification

Membrane purification techniques utilize a membrane with a size-based cut-off to separate large nanostructures from small conjugates. Centrifugal filtration, which couples such a membrane with centrifugation to speed up the process (Figure 4b), is by far the most common approach to purify functionalized DNA origami. It is suitable for nanostructures functionalized with proteins^{32,33,42,43,89,96,104,111,112,128,131,148,152,206,213,215,216,224,271}, peptides^{32,44,45,152,187,220,271}, metals^{42,112,182,198,200}, fluorophores^{43,82–85,131,158,165,167,168,243,270,271}, lipids^{62,82,88,89,148,165,167,168,189}, small molecules^{83,85,182,228,234,271}, polymers^168,244,271, and nucleic acids^{44,45,84,108,170,171,175,177,178,182,278}. When purifying protein-functionalized nanostructures, using protein-^61,131 or surfactant-coated^32,131,271 membranes can increase the recovery yield. Centrifugal filtration is viable for preclinical in vivo investigations because the filters are commercially available at large volumes^43,45. However, as the conjugates get larger, more purification rounds are required due to less efficient separations, possibly leading to more impurities or lower purification recovery yields. Centrifugation-based methods also have limitations when approaching clinical or industrial scales.

Dialysis is a membrane-based method that separates excess conjugates into a reservoir buffer through diffusion. Because it limits local concentration gradients and centrifugal forces, dialysis is milder than centrifugal filtration and is feasible at larger scales, although the speed of purification is substantially slower. It has been used to separate nanostructures functionalized with proteins²⁵³ and lipids^{188–190,192}.

Sedimentation purification

Centrifugation can separate functionalized nanostructures from conjugates by taking advantage of their different rates of sedimentation (Figure 4c). The most basic method, often employed to separate small molecule drugs like doxorubicin from the functionalized nanostructures^{164,174,227,233,236}, is to simply centrifuge the sample in aqueous buffer, discarding the conjugates remaining in the supernatant¹³⁷. However, centrifugation suffers from low molecular weight resolution, leading to limited applicability.

Rate-zonal centrifugation has a broader range of applicability. It was initially developed as a scalable, non-laborious method to purify DNA nanostructures after self-assembly with high separation resolution²⁷⁹. In a typical implementation, reaction mixtures containing functionalized nanostructures and excess conjugates are incubated in glycerol gradient solutions and then centrifuged, resulting in separated fractions of nanostructures and conjugates. This separation occurs because the different components sediment through the gradient at different rates during centrifugation. For functionalized DNA origami nanostructures, this method can purify away protein^{117,123,126,131,134,140,141,143,150}, lipid^141,189,191, nucleic acid^123,126, metal^126,150, and fluorophore¹⁴³ conjugates, showcasing its broader scope compared to centrifugation. A drawback is that it struggles to obtain high concentrations of pure functionalized DNA origami samples. High concentrations of nanostructures can be necessary, for example during structural characterization with cryo-EM, and for administration in animal models where there are upper limits of injection volumes.

PEG precipitation can overcome this limitation by giving high recovery yields of the purified nanostructure²⁸⁰. Large molecular weight nanostructures are separated from lower molecular weight conjugates due to excluded volume effects from the addition of PEG molecules. Nanostructures are precipitated from solution due to PEG crowding, and then resuspended into a buffer of choice at a desired concentration. It can purify DNA origami nanostructures functionalized with proteins^{59,65,79,122,127,130,131,149,206}, fluorophores^{35,36,48,56,79,81,122,130,131,153,154}, metals^59,122, peptides⁵⁶, aptamers^81,173,176, small molecules^35,36,97,98, and lipids⁵⁶. However, this method leaves residual PEG molecules in samples⁴⁶, potentially disrupting some investigations, for example due to the possible immunogenicity of PEG.

Size purification

Size purification techniques, like membrane purification, rely on a material’s ability to separate molecules based on size (Figure 4d). Size-exclusion chromatography (SEC) uses a column packed with porous beads to separate smaller and larger molecules from each other due to different size-dependent retention times within the pores. SEC can purify DNA origami nanostructures functionalized with protein^{34,38,49,131,255,257}, fluorophore^{80,131,154,163}, and lipid80 moieties. SEC is able to purify large amounts of material in principle, and SEC-based purification technologies have scaled to industry in multiple biotechnological contexts^281,282.

Gel filtration is similar to SEC, except it uses centrifugation rather than pump- or gravity-driven flow to send sample through the column. Gel filtration has purified DNA origami nanostructures functionalized with proteins^{131,132,166,205,218,260}, fluorophores^{71,72,131,161,166}, peptides¹⁸⁶, and polymers²⁴⁹. An advantage of gel filtration over SEC is that it requires no specialized chromatography instrumentation; however, purification scales are limited by the volume capacities of the gel column and centrifuge.

Gel purification relies on electrophoresis to sort materials through a gel, commonly agarose-based. As an applied electric field drives the sample through the gel, materials of different size and charge migrate differently in the media, forming bands of pure substances that can be collected by excising the band, dissolving the gel, and exchanging the buffer to yield a pure sample. Gel purification has higher resolution than SEC, and has been utilized to purify functionalized nanostructures with polymers¹⁰⁷, fluorophores^{50,52,67,78,131,162}, lipids^50,199, metals^{151,162,197,199}, small molecules^155,203, nucleic acids^67,179,203, and proteins^{78,118,131,144,151,263}, demonstrating its broad utility. One limitation of this approach is scalability. It is typically challenging to obtain sufficient quantities of functionalized nanostructure for preclinical in vivo investigations.

Free-flow electrophoresis purification uses similar principles for separation as gel purification, but occurs in a matrix-free (buffer only) environment. It was introduced to overcome limitations of other methods, namely speed and chemical/mechanical stress, in purifying protein-functionalized DNA origami nanostructures²¹⁷. With this method, purified functionalized DNA origami can be obtained rapidly (~10–15 minutes) under mild physicochemical conditions with moderate recovery yields (50–75%), but it is unknown how it would perform with nanostructures functionalized with non-protein conjugates.

Considerations and analysis

Considerations of conjugate type, fabrication scale, equipment requirements, purity requirements, and end-use concentration requirements are important to identify an appropriate purification method. When deciding, one might consider whether purification after functionalization is necessary, whether impurities introduced during purification (e.g., PEG purification) are acceptable, what scale of functionalized DNA origami nanostructure needs to be purified, and what the end-use concentration requirement of functionalized nanostructure is.

A comparative study examined the ability of seven purification methods—centrifugal filtration, gel filtration, rate-zonal centrifugation, PEG precipitation, gel purification, magnetic purification, and SEC—to purify fluorophore- and protein-functionalized nanostructures¹³¹. Although for most methods the recovery yield depended on the conjugate, the recovery yield for magnetic bead capture was independent of the conjugate’s properties, suggesting its potential as a universal purification method. Methods that tend to concentrate nanostructure samples, such as PEG precipitation, led to aggregation of the nanostructures.

Characterizing functionalized DNA origami

One important characteristic of DNA origami that distinguishes it from other soft-material nanotechnologies is its ability to control the stoichiometry and nanoscale organization of conjugates. However, in order to realize this control, characterization methods need to distinguish between fully functionalized and partially functionalized nanostructures. Functionalization efficiency that is lower than desired may influence the outcome of mechanistic studies, as well as the efficacy of therapeutics. Additionally, the absence of adequate characterization may lead to the misinterpretation of investigative results.

Generally, there is a lack of suitable characterization methods to analyze the functionalization efficiency of DNA origami²⁸³, in part because of nanostructure concentration ranges that result from fabrication. Typical reaction characterization techniques from other soft-matter nanotechnology or organic chemistry contexts, such as infrared spectroscopy, Raman spectroscopy, and nuclear magnetic resonance spectroscopy, are not suitable for characterizing functionalized DNA origami. Lipid nanoparticles have had similar characterization challenges during their developmental arc²⁸⁴.

There are three general classes of practical characterization for functionalized DNA origami: visual characterization, spectroscopic characterization, and separation-based characterization (Figure 5).

Figure 5. Characterization techniques used to quantify the functionalization of DNA origami nanostructures.

a | Visual characterization techniques rely on a physical probe (light or matter) to create a visual reconstruction of the nanostructure. The images are then used to quantify functionalization efficiency. b | Spectroscopic characterization techniques rely on a spectral fingerprint of the conjugate. UV or fluorescence spectroscopy determines the concentration of conjugate in a sample, which is compared to the concentration of nanostructures for functionalization efficiency quantification. c | Native separation-based characterization utilizes differences in retention time of functionalized nanostructures and unfunctionalized nanostructures to resolve, and in some cases quantify, functionalization efficiency. Denatured separation-based characterization relies on the physical separation and subsequent identification of the nanostructure components. Comparing the amount of reacted and unreacted functionalization sites allows for functionalization efficiency quantification.

Visual characterization

In visual characterization, a probe generates images of the nanostructures that are then analyzed to count the number of conjugates on the nanostructures (Figure 5a). Early in the development of DNA origami, which began with 2D and bricklike assemblies, atomic force microscopy (AFM) was the standard characterization tool given its ease of use. Thus, AFM was also adopted to characterize nanostructure functionalization, where functionalization sites are visualized through differences in height from the nanostructure. The earliest reports of functionalizing DNA origami with biological conjugates^210,211,225, covalent reactions on DNA origami²⁶⁴, and covalent protein functionalization²⁵³ relied on AFM to verify the functionalized structure. It has since been extensively used to quantify functionalization with proteins^{34,37–39,47,58,104,116,119–121,125,128,130,135,137,139,142,166,185,205,207,217,218,224,254,255,257,258,260,261,263} as well as metals201 and nucleic acids^176,181.

Transmission electron microscopy (TEM) can also characterize functionalized nanostructures by visualizing the functionalization sites via the difference in contrast between the DNA nanostructure and conjugate. It can quantify protein^{59,79,131,148,206,209,212,214}, metal^{59,122,150,198,202}, and lipid¹⁹¹ functionalization.

Both visual characterization methods determine structural information about the nanostructure and conjugates, include single-particle information not easily attainable by other methods, and require low sample amounts. However, they, have important limitations^46,283: they are insensitive to small molecule conjugates such as fluorophores or peptides, have inherent resolution limits, and cannot easily measure the functionalization of 3D nanostructures. Additionally, these techniques are low throughput, limiting their ability to characterize libraries of nanostructures.

Spectroscopic characterization

Spectroscopic characterization relies on ratiometric analysis of the number of conjugates and number of nanostructures in the sample, as determined by spectroscopic fingerprints (Figure 5b). In a typical UV or fluorescence spectroscopy-based experiment, the conjugate of interest either inherently has a spectroscopic fingerprint (either ultraviolet (UV) absorbance or fluorescence) or is engineered to have such a fingerprint (usually by attaching a fluorophore). The conjugate concentration is determined via an external standard, and the DNA nanostructure concentration is measured directly through UV absorbance measurements. The ratio of conjugate concentration to nanostructure concentration gives the functionalization efficiency. Spectroscopic techniques have quantified the functionalization of nucleic acids^44,45,182, peptides^44,45, fluorophores²⁷¹, small molecules²⁵², and proteins^{32,33,43,61,73,89,117,141,144,271} on nanostructures.

Spectroscopic characterization can be implemented simply if the conjugate of interest has an inherent spectroscopic fingerprint (for example, fluorescent tryptophan residues in proteins). However, this technique also suffers from several drawbacks. Many conjugates of interest in biological applications, including most small molecule targeting ligands, have no spectroscopic fingerprint; incorporating one, such as a fluorophore, though commonly performed, can change the biophysical and biochemical nature of the conjugate. Using fluorophores can also be problematic as commonly used cyanine dyes have DNA sequence-dependent fluorescent brightening and quenching²⁸⁵. In addition, the DNA nanostructure concentration measurement is approximate, as molar extinction coefficients are empirical estimates, and researchers often vary in how they measure this concentration. Nanostructure concentration measurements can be improved by incorporating reference fluorophores into the nanostructure prior to assembly, improving the accuracy of nanostructure concentration measurements,⁷⁹ or by using quantitative polymerase chain reaction assays to provide an unbiased, absolute measure of total nanostructure amounts⁷¹. Finally, these ratiometric methods are inaccurate in the presence of conjugate impurities that would lead to the over-estimation of the functionalization efficiency.

Separation-based characterization

Separation-based characterization relies on the physical separation of nanostructures to determine how many sites were functionalized (Figure 5c). Native agarose gel electrophoresis (AGE), a common method to characterize the self-assembly of DNA origami, has also been used to assess DNA nanostructure functionalization with proteins^{80,115,118,132,149,152,259}. The nanostructure band may shift when partially or fully functionalized, depending on conjugate charge, size, and functionalization density. Although an intrinsically qualitative assay, the use of an external reference can produce semi-quantitative results. An advantage of native AGE is that it requires low sample amount, similar to AFM and TEM. It is also simple to implement and can be applied as an initial characterization tool when assessing sample quality, or to screen for optimal reaction conditions. Once an external reference is established, we find this technique especially useful for quality control when producing multiple batches of sample material for preclinical in vivo investigations.

Some separation-based methods use denatured nanostructures to quantitatively determine functionalization efficiencies. After functionalization, the DNA nanostructure is denatured into its scaffold and staple strands, which contain conjugation sites that may or may not be successfully functionalized. Electrophoresis and chromatography separate the conjugated and unconjugated staples from each other and the rest of the DNA nanostructure components via differences in retention times, enabling the functionalization efficiency to be quantified. Electrophoresis-based assays rely on differences between the size and charge of nanostructure components; they were able to quantify functionalization efficiency with small molecule²⁸⁶, and protein¹²³, and peptide conjugates¹⁸⁷. In contrast, chromatography-based assays rely on differences in hydrophobicity between nanostructure components. Our group reported a method based on liquid chromatography that affords quantitative functionalization efficiency determination²⁷¹. Functionalized nanostructures are denatured on a liquid chromatography column, and conjugated staples separate from both non-conjugated functionalization site staples and the rest of the nanostructure components through differences in hydrophobicity to facilitate analysis. We used this technique to monitor the functionalization efficiency of a model DNA origami nanostructure with proteins, peptides, polymers, carbohydrates, and fluorophores. This technique is agnostic to conjugate type and purity, and can be extended to non-origami DNA nanostructures. Electrophoresis- or chromatography-based methods may have better resolution of conjugated and non-conjugated staples, depending on conjugate type and functionalization method employed.

Considerations and analysis

This functionalization efficiency analysis is often skipped when pre-assembly functionalization methods are used, because it is assumed that all staples with desired conjugates are incorporated into the nanostructure. This assumption may not be accurate, however, as staple incorporation into bricklike origami nanostructures is often less than 100%²⁸⁷; it is currently unknown how wireframe objects perform in this regard. The scarcity of facile techniques to determine staple incorporation also contributes to the lack of characterization.

Combining multiple characterization methods to confirm ensemble measurements of functionalization efficiency together with single particle information about nanostructure integrity and population distribution will give the most descriptive information about a functionalized DNA origami nanostructure. When fabricating materials libraries, assay throughput is an additional factor to consider; spectroscopic and separation-based characterization are positioned to handle these high-throughput requirements. Finally, the possibility of nanostructures folding without the entire set of staples²⁸⁷ can become a confounding factor in characterizing functionalized nanostructures.

Almost all characterization of functionalized DNA origami investigates functionalization efficiency. An additional, crucial aspect of their functionalization is ensuring that the attached conjugate is still bioactive, rather than only present. For example, before introducing a delivery vehicle into animal models, it is expected that the attached targeting ligand’s functionality will have been characterized using an in vitro assay to ensure native receptor recognition. B cell receptor³² and T cell receptor⁸⁹ reporter lines have been used to do just this, and we believe that this type of functional characterization will be an essential and eventually routine step in the fabrication pipeline of functionalized DNA origami in the future.

Outlook

The past 16 years have witnessed substantial advances in what DNA origami can be functionalized with, how DNA origami can be functionalized, and how functionalized DNA origami can be purified and characterized. With these functionalized nanostructures, researchers have conducted impactful investigations in therapeutic delivery, immunology, vaccinology, biological catalysis, and molecular biophysics.

Interesting trends are observed from the history of DNA origami publications in these areas (Supplementary Figure 1, Supplementary Methods), which we analyze below. Now that several bottlenecks in the design and fabrication of DNA origami nanostructures have been overcome, we anticipate that more studies functionalizing DNA origami nanostructures to investigate biological systems will disseminate. It will be interesting to note whether the historically dominant methods in this area will continue to be used, or whether less common techniques will emerge as more powerful methods for specific applications.

Functionalization

Proteins and fluorophores are the dominant conjugates of interest in these fields, and in the past five years, nucleic acids, such as siRNAs, have been increasing in popularity (Supplementary Figure 1a). The post-assembly DNA-DNA hybridization method outpaced the initially popular pre-assembly and post-assembly biotin-streptavidin methods to become the most common functionalization method — presumably owing to its facile implementation (Supplementary Figure 1b). Post-assembly covalent functionalization methods still lag in development and adoption.

From the available reaction details in the literature, many hybridization protocols (Supplementary Figure 2a) operate in the high functionalization efficiency, low equivalents zone, whereas covalent protocols often require large conjugate equivalents to obtain high functionalization efficiency. It appears that the chosen conjugation method, not the conjugate type, determines the functionalization efficiency and conjugate equivalents required (Supplementary Figure 2b). Depending on the method, DNA origami can be functionalized with protein with high or low functionalization efficiency, and the conjugate equivalents required to achieve these conversions span orders of magnitude. Visualizing reported functionalization efficiency and conjugate equivalents over time shows that these important metrics are trending slightly towards improvement, but there is still room for progress (Supplementary Figure 2c and Supplementary Figure 2d). We note that using fewer conjugate equivalents is not only economically desirable; conjugates like proteins have upper concentration limits that restrict the amount of excess conjugate that can be dissolved into the reaction mixture.

We believe there are several factors hindering future progress in functionalization, including insufficient rigor in reporting and characterizing functionalization reactions, the use of unique nanostructures and conjugates by different research groups, and a lack of systematic studies on functionalizing DNA origami across different nanostructure and conjugate types.

Of the publications we analyzed (Supplementary Figure 2), one-quarter did not include information on the equivalents of conjugates used, and one-half did not include information on functionalization efficiency. Though we recognize that this information may not be necessary for accomplishing immediate experimental goals, we posit that reporting detailed procedures and characterization protocols may help to promote development in the field and facilitate reproducibility of experimental results.

Researchers can not only improve existing functionalization methodologies, but also devise new ones. Drawing inspiration from a strategy that incorporates both DNA-protein binding and covalent methods^205,255,257, one can envision other approaches to template catalyzed covalent functionalization to improve reaction rates and conversion. To build on progress made in orthogonal covalent reactions of protein conjugates^253,255, researchers could extend orthogonal covalent reactions to other conjugate systems, such as by implementing orthogonal click chemistries like thiol-X, Diels-Alder, and tetrazine chemistries to complement the established alkyne-azide chemistries.

Purification

Membrane-based methods have been and still are the most common purification technique, followed by size-based, sedimentation-based, and then affinity-based methods (Supplementary Figure 1c). Although affinity-based magnetic purification has the potential to be a universal purification technique¹³¹, it is currently utilized infrequently.

Additional development of purification methodologies may be beneficial for translational prospects. Within membrane methods, centrifugal filtration is the most common technique to purify these functionalized nanostructures. As therapeutic investigations require larger scales, dialysis—which is more scalable than centrifugation-based methods—should be thoroughly investigated as an alternative membrane purification method.

Following up on the earlier comparative analysis of two conjugate types and seven purification techniques¹³¹, purification methods should be evaluated over additional parameter spaces: broad conjugate scope, purification recovery yields at different scales of fabrication, and different assembly types such as brick and wireframe structures. Such a systematic study could help researchers identify which purification method will be optimal for their individual investigation.

Finally, all current purification methods focus on separating excess conjugates from functionalized DNA nanostructures. An outstanding, and formidable, technical challenge is how to purify fully functionalized from partially functionalized DNA origami nanostructures.

Characterization

The visual characterization techniques, AFM and TEM, continue to be by far the dominant methods to characterize functionalized DNA origami; there is slow growth in adoption of spectroscopic and separation-based methods (Supplementary Figure 1d). As more of the DNA origami nanostructures used in biophysical or cargo delivery investigations become three-dimensional and functionalized with small molecules, these techniques may no longer be amenable. Spectroscopic and separation-based techniques should be further adopted and advanced to enable more thoroughly characterized systems.

Adapting other techniques may offer additional tools for characterizing functionalization efficiency. Cryogenic electron microscopy (Cryo-EM) is capable of resolving fine structural details of DNA origami objects. For example, recently cryo-EM successfully resolved DNA origami nanostructures functionalized with a poly(Lys)-PEG copolymer, revealing differences between the functionalized and native forms of the nanostructure²⁸⁸. However, cryo-EM is seldom used to characterize functionalized DNA origami. Challenges include the high level of technical expertise needed, advanced instrumentation access, large material amounts and concentrations required, and sufficient contrast of functionalized conjugates. Even in ideal cases, site-specific defects may be difficult to characterize due to limited resolution, and sample averaging performed for reconstruction limits insight into heterogeneous structural features. Applying this advanced technical capability to functionalized DNA origami systems may eventually offer useful insight into how functionalization changes both native nanostructures and conjugates.

Additionally, mass spectrometry, either at the molecular or nanostructure²⁸⁹ levels, could enable new ways to quantify functionalization efficiency. Finally, chemical and related footprinting techniques, applicable to both DNA²⁹⁰ and RNA^291,292, may offer new inroads to study functionalized nanostructures.

Final Remarks

Major progress in fabrication methods have enabled DNA origami nanostructures to become a state-of-the-art material platform for probing biological systems. Seminal contributions to the field have expanded the scope of conjugates that can be attached to DNA origami nanostructures, increased the efficiency and effectiveness of these conjugations, developed and scaled purification methods, and developed quantitative and rapid characterization methods. As DNA origami trends towards clinical translation, we believe an emphasis on efficient reaction conditions that achieve quantitative functionalization efficiency; scalable, high-yield purification; and multiplexed, quantitative characterization of functionalized DNA nanostructures will enable this powerful nanotechnology to meet its full potential.