2. Structural Characterization of Proteins on DNA Nanoscaffolds
The original goal of DNA nanotechnology, as conceived by Ned Seeman in 1982 [7
], was to generate a three-dimensional crystal from the self-assembly of individual DNA strands linked by immobile Holliday junctions. Such a crystal could, in turn, be used to scaffold proteins in a repeating lattice in space and solve their structure using X-ray crystallography without having to crystalize the proteins (Figure 2
]. Key to this endeavor is the rigid attachment of the proteins to a mechanically robust, 3D DNA latticework, since even slight variations in their orientation would result in poor diffraction and preclude high-resolution characterization. The first rationally designed, self-assembled DNA crystal was reported by Seeman, Mao and coworkers in 2009, based on a tensegrity triangle motif—a nanoscale analogue of a mechanically rigid macroscopic object—comprised of three DNA strands (Figure 2
]. This crystal was characterized to 4–14 Å resolution (depending on the design), with the largest cavities surpassing 1100 nm3
in volume. In 2016 and 2017, Yan, Seeman and coworkers reported a second DNA crystal design, once again employing three strands but relying on four stacked duplexes linked by Holliday junctions as a key motif (Figure 2
]. These motifs diffracted to ~3 Å, and a subsequent report that optimized the Holliday junction sequence yielded crystals that diffracted to 2.6 Å, which allowed for visualization of individual purine-pyrimidine base stacks (Figure 2
]. Critically, this latter design allowed for crystal cavities with ~50% larger edges (albeit at a reduced resolution) and volumes of 1250 nm3
, paving the way for incorporation of larger proteins.
The above examples demonstrated that mechanically rigid 3D lattices of DNA duplexes could form crystals with defined void spaces that could host proteins. A key next step to this work will be to demonstrate attachment of proteins, either by using DNA binding proteins, or through chemical conjugation (see Section 4
), in a rigid and predictable fashion, in order to solve their structure using the crystal with a known structure as a host. In 2006 Paukstelis demonstrated a different DNA crystal design could serve as a “molecular sieve”, allowing the incorporation of a small protein (GFP, 28 kDa) but not a larger one (MBP-RFP, 280 kDa) [13
]. This report was followed in 2014 with a demonstration that enzymes immobilized inside crystals were still catalytically active [14
]. Although these reports did not attempt to immobilize the proteins for structural solution, the fact that they could permeate the 3D volume of the crystal and retain their structure and function (as evidenced by the GFP fluorescence and enzyme activity) bode well for achieving structural solution in the future.
In recent years, rigid DNA nanostructures have also been applied to protein structural determination using cryoelectron microscopy (cryo-EM). Although cryo-EM methods are constantly improving, it is still difficult to characterize the structure of small proteins (<100 kDa) due to the lack of image contrast with the background. To circumvent this limitation, proteins can be complexed with larger species like antibodies to increase their effective size and make them easier to discern in the image. Inspired by these studies, several groups have explored using DNA nanostructures, which are readily visualized by cryo-EM due to their large size (~tens of nanometers) and high contrast relative to the background, as “markers” to find the protein. Furthermore, because DNA origami nanostructures can be highly anisotropic, they can serve as “nanoscale goniometers” [15
] that define the absolute orientation of the attached molecules, further facilitating class-averaging and solution of protein structure. Unlike DNA crystals, which, for the time being, are restricted to rather small proteins (~30–50 kDa) that can fit in their cavities, DNA origami nanostructures can be designed with much larger void spaces for hosting proteins. Indeed, work by Turberfield and coworkers demonstrated that simple 2D DNA lattices could host proteins like RuvA [17
] or G-protein coupled receptors (GPCRs) [18
] in their cavities to generate repeating arrays that could be visualized by cryo-EM. Although these approaches gave only low resolution (20–30 Å) reconstructions of the protein, they demonstrated the potential for a DNA scaffold as a “sample holder” to image proteins, while preventing aggregation or undesired surface adhesion effects.
Cryo-EM had been used to characterize the 3D structure of DNA cages and to demonstrate their three-dimensionality for many years [19
], but not until 2012 was the structure of a compact, rigid and block-like DNA nanostructure reported for the goal of protein structural characterization [21
]. Dietz, Scheres, and coworkers designed a block-like “pointer” object with densely packed helices on a square lattice, and solved its structure to a maximum resolution of 9.7 Å (for the more rigid core), and 14 Å at the more flexible outer periphery (Figure 3
A). This resolution, nonetheless, allowed clear discernment of the DNA strand routing in the object, including the crossover points, stacked Holliday junctions or deviations from the designed lattice geometry due to backbone repulsion. In 2016, these same groups applied this technique to solving the structure of a DNA-binding protein, the transcription factor p53 (Figure 3
]. The researchers designed a DNA origami cage to specifically sit perpendicular to, and vertically span the vitreous ice sheet containing the sample. The center of the structure was spanned by a DNA duplex bearing the binding site for the p53 protein. Changing the relative location of this binding site (relative to the helical axis of the duplex, Figure 3
C) resulted in a different viewing angle of the protein. In this way, the DNA origami served as a nanoscale goniometer, enabling different perspectives of the attached protein. The authors used the origami-enforced images to solve the structure of the protein to ~15 Å, and, thereby, elucidate new details about the symmetry of the oligomer. The ultimate resolution was limited, however, by the flexibility of the DNA origami construct, and specifically the single duplex spanning the cavity. Two years later, Mao and coworkers reported the reconstitution of the membrane protein α-hemolysin in a hydrophobic DNA origami cavity, and probed its structure by cryo-EM [22
]. They were able to observe the structure of the protein at a resolution of 30 Å, partly due to the absence of interactions that pinned it down in one conformation in the lipid cavity.
In 2020, Douglas and coworkers extended the nanogoniometer approach to solve the structure of BurrH (another DNA-binding protein) to a resolution of 6.5 Å (Figure 3
]. The DNA origami served as a barcoded, asymmetric object that could directly report the relative conformation of the protein, which was again attached to DNA duplex spanning a cavity in the nanostructure. The nanostructure “barcodes” on the origami specified the rotation angle of the protein on the bound duplex, and the rotational tilt of the duplex between two orientations. Compared with Dietz and Scheres’s work, Douglas and coworkers were able to obtain higher resolution by increasing the yield of origami structures bearing proteins (resulting in more particles per image) and tuning the origami aspect ratio to ensure proper orientation upon adhesion to the transmission electron microscopy (TEM) grid. Three key innovations will likely enable researchers to push the resolution limit of DNA-scaffolded proteins down to <3 Å, which would enable atomic resolution. The first is chemical conjugation to the scaffold in a rigid fashion, as outlined in Section 4
below. Interestingly, Douglas and coworkers proposed that highly rigid attachment of their protein was not strictly necessary and could even be detrimental, but chemical conjugation could also enable analysis of non-DNA binding proteins. The second innovation is increasing the resolution of the DNA scaffold itself to the atomic level. A third advancement would be to use computational simulation with atomic resolution to better fit cryo-EM maps, as also discussed in Section 4
In 2020, the Dietz and Scheres groups reported that by taking into account structural fluctuations in DNA origami, and interpreting the cryo-EM maps with molecular dynamics simulations, they could further improve the resolution of 3D origami nanostructures to ~4 Å [23
]. In this fashion, they could distinguish the fine features of the objects, including major and minor grooves, single-strand breaks and crossover positions. Importantly, this technique demonstrated the inherent flexibility and undesired or unintended deformations in DNA nanostructures, and allowed the authors to correct them through sequence design or covalent crosslinking. In this fashion, it should be possible to design a rigid and well-defined object with atomically-defined locations for protein attachment. By combining this advanced method with better chemical immobilization of arbitrary proteins on a DNA scaffold, as well as methods for covalently locking flexible points in the DNA structure itself [24
], the potential for structural solution of individual proteins becomes ever more tantalizing. We also highlight that while atomic resolution may be a worthwhile ultimate goal for these structures, even determining protein structure to 3–6 Å could be useful for many studies, especially in conjunction with mechanical force application as in Section 3
below. For example, the unfolding of a multidomain protein whose structure is already known could probably be probed sufficiently even at nonatomic resolutions.
3. DNA Nanoscaffolds for Interrogating Ligand Spacing, Valency, and Confinement
A second area where DNA nanomechanical devices have played an important role in biology is in probing the required distance and multivalency of proteins for effectively binding to cell receptors or triggering a biological effect. DNA origami nanostructures are particularly effective at this role for several reasons. First, they are rigid objects (especially if multilayer, block-like nanostructures are used), so thermal fluctuations do not change the distance between attached ligands much [25
]. Second, they typically span dimensions of tens to hundreds of nanometers, a size scale well matched to many biological receptor complexes on cells. Third, it is relatively straightforward to change the distance between ligands, or the number of ligands, by extending staple strands at arbitrary points on the structure (with a resolution of ~5–10 nm). Fourth, the shape of a DNA nanostructure can be tuned to match the application at hand, e.g., a linear rod to serve as a molecular caliper [26
], a wireframe polyhedron to mimic a virus [27
], or a ring to recapitulate the nuclear pore [28
Since the inception of DNA origami [1
], one of the most attractive applications for these structures was as a “molecular breadboard” to attach other species, like nanoparticles or proteins, with nanoscale control. Indeed, a vast and rich literature exists on the attachment of proteins to DNA nanoscaffolds, with particular interest in enzymatic cascades as molecular assembly lines. One potential limitation of the original, two-dimensional designs reported by Rothemund, and used by many others since, is that they are quite flexible. With the advent of three-dimensional origami comprised of several layers of stacked helices linked by multiple crossovers [2
], much more rigid objects can be obtained, which do not deform significantly and can thus enforce specific distances between bound ligands. Here we discuss several seminal examples of biological studies that would not be possible, or be much more difficult to carry out, without the unique properties of origami. Due to space limitations, we will not discuss the extensive work on enzymatic cascades, and instead direct the interested reader to several excellent reviews on this topic [30
In 2014, the Hogberg and Teixera labs used a 3D origami scaffold as a “nanocaliper” to position the ephrin-A5 protein ligand by attaching it to a single-stranded (ss) DNA handle complementary to extensions of staple strands from the nanostructure (Figure 4
]. In this way, the authors were able to control not only the number of ligands, but also the distance separating them (either 40 or 100 nm). This technique demonstrated that not only were two ligands necessary for receptor activation (compared with a monovalent system), but the sample with 100 nm spacing worked just as well as the one with 40 nm distance between proteins. Furthermore, using eight ligands spaced 14 nm apart did not yield any further improvements in bioactivity compared with the two ligands. A key to this approach’s success was the rigidity of the nanocaliper, which enforced the desired distances and allowed for a systematic study of both spacing and absolute number of receptors that would not be possible with other approaches (like antibody clustering of the receptors). In a follow-up study, Hogberg and coworkers were able to use a similar nanocaliper to immobilize small molecule antigens with controlled distances to probe the spatial tolerance of antibody binding [33
]. Once again, spacing the molecular species on a rigid and tunable scaffold allowed for a precise investigation of IgG bivalent binding not possible with traditional methods like surface plasmon resonance. The ability of DNA origami to control the nanoscale spacing and number of ligands can also be applied to study the proteins that must oligomerize to function, like the potassium channel Kir3 [34
], or caspase 9 [35
]. In the caspase 9 example, the addressable pegboard also allowed the authors to determine that clustering of the proteins enhanced activity; even though dimerization alone did activate the enzymes, spatially tunable clusters of three or four enzymes worked even better, but only if the proteins were also within a given distance.
Although the above example used linear templates to space proteins or ligands, a key advantage of DNA nanotechnology is the high degree of programmability in shape that it allows. In 2020, Irvine and Bathe and coworkers described a polyhedral DNA origami scaffold for presenting B-cell ligands in order to create a vaccine (Figure 4
]. This approach allowed the researchers to investigate the effect of protein number and spacing, as well as compare the spherical particle to a linear scaffold (akin to the Hogberg caliper). The authors found that increasing the ligand spacing resulted in more potent B-cell receptor activation, and the rigid segregation of ligands on the origami scaffolds, enhanced activity compared with more flexible ssDNA or polyethylene glycol (PEG) linkers. As with the nanocaliper, the programmability of DNA nanostructures enabled a direct, apples-to-apples comparison of various ligand presentations on a readily tunable scaffold, which would have been impossible with other approaches. Very recently, DNA origami scaffolds were also used to study the clustering of T-cell receptors (TCRs) by attaching binding antibody fragments to the DNA scaffold [36
]. Furthermore, the origami structures were modified with cholesterol and embedded in a supported lipid bilayer, facilitating the motion and clustering of the TCRs in a highly biomimetic fashion, allowing the authors to determine that T cell activation required at least two TCRs within a distance of 20 nm. The DNA origami thus enabled both nanoscale control of ligand spacing, but also served as a nanoscale “adapter” to mimic the biological arrangement by embedding the proteins in a lipid membrane.
Another powerful application of DNA nanostructure templates in biology is to probe the cooperative actions of multiple proteins working in tandem. In 2012, Reck-Peterson and colleagues used a DNA origami 12-helix bundle to position multiple copies of the motor proteins dynein or kinesin-1 (Figure 4
]. The rigid origami nanostructure thus served to place these proteins with controlled valence and spacing to probe their cooperativity, and found that increasing the number of motors did not increase the velocity of the origami on a microtubule-modified surface. However, the spatial addressability of the handles enabled a “tug of war” arrangement between the two types of proteins, which are opposite-polarity molecular motors. Furthermore, introducing photocleavable linkers in the DNA handles for one type of motor allowed their triggered “release” from the scaffold with light, resulting in the other protein dominating. In 2015, the Sivaramakrishnan lab used a DNA origami nanotube to mimic a myosin fiber, and used DNA handles to position myosin heads with the same pitch (14.3 nm) as in the natural filament (Figure 4
]. Once again, the authors were able to probe the synergistic effects of multiple myosin heads on nanotube motion on an actin-modified surface, and found that neither the number of myosin heads, nor their density, affected the gliding speed. In this way, the researchers validated a key biochemical mechanism for these molecular motors. In both of these examples, the DNA origami nanostructures provide an unparalleled tool for positioning these motor proteins with not just controlled spacing and valency but, as in the Reck-Peterson paper, precise stoichiometry between multiple types of proteins. The rigidity of these multihelical objects is critical to prevent spatial fluctuations that would perturb the desired distances or allow for undesired interactions between the proteins.
A third distinct area of biological exploration enabled by DNA nanostructures is probing the function of proteins in confined nanoscale volumes. Two sets of researchers in 2018, one led by the Dietz and Dekker groups [29
], and one by the Lin and Lusk labs [28
], used a rigid DNA origami ring to mimic the nuclear pore complex. The goal of both teams was to understand the collective assembly and pore blocking function of proteins called FG-nups, by confining them to a cylindrical volume ~40 nm in diameter (Figure 4
E–G). The use of DNA origami also allowed for a tunable incorporation of proteins (from 32–48), and independent modulation of their orientation (i.e., pointing “in” vs. “out” of the ring, with only the former being mimetic of the nuclear pore). It is hard to envision another system that can control both the nanoscale area/volume available to proteins with high precision, but also the number of proteins attached to that volume, and the direction in which those proteins are oriented relative to it. The DNA origami rings balance flexibility (to create a circular structure) with rigidity (to prevent its deformation), while allowing for a stoichiometrically-defined number of proteins via the DNA handles used. Using a similar ring-like structure that encircled a liposome and could position soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, the Shih and Rothman groups were able to determine that only one to two SNAREs were sufficient for membrane fusion [39
]. This work was particularly notable because the DNA nanostructure controlled both the number of the proteins, but also their proximity to a liposome, helping recreate a complex biological arrangement that would not have been possible without the rigidity and shape control intrinsic to the origami, as well as the multiple chemical functionalities that the structure can present.
4. Measuring or Applying Forces with DNA Nanodevices
The third application of DNA nanostructures that we discuss is in measuring or applying forces on proteins at the single-molecule level. Although other methods, like optical tweezers or attachment to an AFM tip, exist for pulling on proteins with forces in the piconewton (pN) regime, these require specialized equipment and are limited to analyzing one protein at a time. DNA nanodevices have the advantage that they are relatively inexpensive and allow analysis of many proteins in parallel in a single sample. Furthermore, the biophysical properties of DNA are fairly well understood, or can be readily probed using computational simulations, as we discuss in Section 4
, and readily tunable with only minor changes to the DNA sequences that comprise the origami nanoobject. We also note that simple DNA duplexes are extremely promising as biophysical sensors, e.g., to probe the forces applied by cell integrins to the extracellular matrix [40
], but since these do not involve DNA nanostructures we will not cover them here.
The structural programmability of DNA origami structures led several groups to probe their use as nanoscale force-generating devices akin to calipers or mechanical jacks. An added advantage to these systems is that DNA duplexes could be used as “cranks” to apply a given force. Alternatively, purely nanoscale effects absent from the macroscale world (like electrostatic repulsion or entropic springs) can be employed as well. Funke and Dietz reported a hinged DNA origami nanostructure where the distance between the rigid arms could be tuned with several DNA “adjuster” duplexes, with adjustments tunable down to a single base pair (Figure 5
]. Remarkably, this device could achieve displacement steps of only 0.04 nanometers, slightly less than the Bohr radius, due to the lever-arm effect. Tinnefield, Liedl, and coworkers demonstrated a different nanomechanical device based on the entropic forces that oppose extension of a single strand of DNA [43
]. By reducing the number of nucleotides in these strands, the force could be readily tuned. As an alternative trigger for applying forces at the nanoscale, Stephanopoulos and coworkers described a DNA tweezer with a hairpin loop that could be triggered to open with a displacement strand. By incorporating the displacement strand into the nanostructure, but photocaging it, the authors were able to generate tens of piconewtons of force with a brief pulse of UV light that removed the cages and allowed the strand to open the tweezer (Figure 5
]. The Castro lab reported a host of nanoscale analogues to macroscale mechanical devices, like crank-sliders, pistons, Bennett linkages and a waterbomb base [45
], which can readily be extended to biophysical studies on proteins. Fygenson and Schulman described DNA “nunchuck” devices, comprised of two long DNA nanotubes linked by a flexible hinge that can mechanically magnify the angle of the hinge and measure single-molecule forces [47
]. All the above demonstrations relied intimately on both the rigidity of the DNA helices comprising them and the ability to use DNA hybridization or coiling to apply precise forces.
In two reports from 2016, these caliper- and jack-like structures were applied to measure the forces between double stranded DNA and nucleosomes, the protein complexes that wrap the genomic material in the nucleus. Both works, one by the Poirier and Castro groups [49
] and the other by the Dietz lab [50
], used a hinge-like structure to position double stranded DNA between the arms of the device and allow the nucleosomes to bind (Figure 4
C,D). By measuring the angle between the arms of the caliper, which could be closed by the nucleosome binding to and winding the DNA, various molecular details of these protein complexes could be determined such as the effect of salt on binding strength, the number and orientation of neighboring nucleosomes, the length of DNA or transcription factor binding on the nucleosome. In a related study that same year, the Dietz group used their caliper device to measure the forces between two different nucleosomes (as opposed to between nucleosomes and DNA), which is key for understanding the higher-order organization of chromatin into structures in the nucleus (Figure 4
]. Both the relative force applied on the nucleosomes, as well as their distance, orientation and chemical modification (e.g., acetylation) could be probed to yield an energy landscape as a function of internucleosome distance. These examples all demonstrate the great potential in DNA nanodevices for studying DNA-binding proteins with single-molecule resolution, with an emphasis on controlling their displacement and energy landscape, or other molecular properties of the proteins and their substrates. As we discuss in Section 4
below, using site-specific bioconjugation methods would extend these studies to arbitrary proteins, and enhancing the rigidity of attachment would enable greater control over protein orientation for measuring protein-protein interaction energies.
An alternative design for applying and measuring forces on proteins was reported by Iwaki and coworkers in 2016, and relied on a DNA nanostructure “spring” to tug on the motor protein myosin VI (Figure 4
]. Unlike the caliper-like designs mentioned above, this spring consisted of a two-helix DNA bundle with negative superhelical strain (following design rules for curved and twisted origami) [53
] to give a structure similar to a macroscopic spring. The authors first characterized the force-extension curve of this spring using an optical trap and demonstrated that the DNA nanostructure had a roughly 10-fold lower spring constant than double-stranded (ds) DNA. As a result, their design matched the stall force of the myosin motors (~2 pN) at a much shorter length than dsDNA, which in turn allowed for shorter run lengths (~700–800 nm). The nanostructure could thus precisely “match” the intrinsic biophysical properties of the motor, whereas simpler force-applying devices like dsDNA were unsuited to this particular protein. Using this device, the authors were able to demonstrate that at higher forces the mechanism of myosin VI motion switched (from hand-over-hand to inchworm motion), as well as set up a tug of war between myosin VI and myosin V by attaching them to opposite ends of the spring. The programmable mechanical properties of DNA nanostructures played a key role in the ultimate application, and although optical tweezers were used to calibrate the device it could be used thereafter to probe protein function without a complicated experimental setup. The application of DNA origami to single molecule biophysics also presents challenges in the correct interpretation of the measured force-extension properties, as one needs to correctly interpret measured values of a statistical ensemble of forces and extensions [54
], as well as take into account the mechanical properties and extensions of double-stranded and single-stranded segments under tension in the DNA origami constructs that are used to apply force [55