Branching Crisscross Polymerization of Single-Stranded DNA Slats.

Controlling where and when self-assembly happens is crucial in both biological and synthetic systems as it optimizes the utilization of available resources. We previously reported strictly seed-initiated linear crisscross polymerization with alternating recruitment of single-stranded DNA slats that are aligned in a parallel versus perpendicular orientation with respect to the double-helical axes. However, for some applications, it would be advantageous to produce growth that is faster than what a linear assembly can provide. Here, we implement crisscross polymerization with alternating sets of six parallel slats versus six perpendicular slats and use this framework to explore branching behavior. We present architectures that, respectively, are designed to exhibit primary, secondary, and hyperbranching growth. Thus, amplification via nonlinear crisscross polymerization can provide a route for applications such as low-cost, enzyme-free, and ultrasensitive detection.

Enzyme-Free Exponential Amplification via Growth and Scission of Crisscross Ribbons from Single-Stranded DNA Components.

The self-assembly of DNA-based monomers into higher-order structures has significant potential for realizing various biomimetic behaviors including algorithmic assembly, ultrasensitive detection, and self-replication. For these behaviors, it is desirable to implement high energetic barriers to undesired spurious nucleation, where such barriers can be bypassed via seed-initiated assembly. Joint-neighbor capture is a mechanism enabling the construction of such barriers while allowing for algorithmic behaviors, such as bit-copying. Cycles of polymerization with division could accordingly be used for implementing exponential growth in self-replicating materials. Previously, we demonstrated crisscross polymerization, a strategy that attains robust seed-dependent self-assembly of single-stranded DNA and DNA-origami monomers via joint-neighbor capture. Here, we expand the crisscross assembly to achieve autonomous, isothermal exponential amplification of ribbons through their concurrent growth and scission via toehold-mediated strand displacement. We demonstrate how this crisscross chain reaction, or 3CR, can be used as a detection strategy through coupling to single- and double-stranded nucleic acid targets and introduce a rule-based stochastic modeling approach for simulating molecular self-assembly behaviors such as crisscross-ribbon scission.

Mapping Single-Molecule Protein Complexes in 3D with DNA Nanoswitch Calipers.

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples is a challenging goal that would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices, we call DNA nanoswitch calipers, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement with previously reported structures.

Three-phase DNA-origami stepper mechanism based on multi-leg interactions.

Nanoscale stepper motors such as kinesin and dynein play a key role in numerous natural processes such as mitotic spindle formation during cell division or intracellular organelle transport. Their high efficacy in terms of operational speed and processivity has inspired the investigation of biomimetic technologies based on the use of programmable molecules. In particular, several designs of molecular walkers have been explored using DNA nanotechnology. Here, we study the actuation of a DNA-origami walker on a DNA-origami track based on three principles: 1) octapedal instead of bipedal walking for greater redundancy; 2) three pairs of orthogonal sequences, each of which fuels one repeatable stepping phase for cyclically driven motion with controlled directionality based on strain-based step selection; 3) designed size of only 3.5 nm per step on an origami track. All three principles are innovative in the sense that earlier demonstrations of steppers relied on a maximum of four legs on at least four orthogonal sequences to drive cyclic stepping, and took steps much larger than 3.4 nm in size. Using gel electrophoresis and negative-stain electron microscopy, we demonstrate cyclic actuation of DNA-origami structures through states defined by three sets of specific sequences of anchor points. However, this mechanism was not able to provide the intended control over directionality of movement. DNA-origami-based stepper motors will offer a future platform for investigating how increasing numbers of legs can be exploited to achieve robust stepping with relatively small step sizes.

Thermal cycling of DNA devices via associative strand displacement.

DNA-based devices often operate through a series of toehold-mediated strand-displacement reactions. To achieve cycling, fluidic mixing can be used to introduce 'recovery' strands to reset the system. However, such mixing can be cumbersome, non-robust, and wasteful of materials. Here we demonstrate mixing-free thermal cycling of DNA devices that operate through associative strand-displacement cascades. These cascades are favored at low temperatures due to the primacy of a net increase in base pairing, whereas rebinding of 'recovery' strands is favored at higher temperatures due to the primacy of a net release of strands. The temperature responses of the devices could be modulated by adjustment of design parameters such as the net increase of base pairs and the concentrations of strands. Degradation of function was not observable even after 500 thermal cycles. We experimentally demonstrated simple digital-logic circuits that evaluate at 35°C and reset after transient heating to 65°C. Thus associative strand displacement enables robust thermal cycling of DNA-based devices in a closed system.

Rapid in vitro production of single-stranded DNA.

There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40-50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 µl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.

Glutaraldehyde Cross-Linking of Oligolysines Coating DNA Origami Greatly Reduces Susceptibility to Nuclease Degradation.

DNA nanostructures (DNs) have garnered a large amount of interest as a potential therapeutic modality. However, DNs are prone to nuclease-mediated degradation and are unstable in low Mg2+ conditions; this greatly limits their utility in physiological settings. Previously, PEGylated oligolysines were found to protect DNs against low-salt denaturation and to increase nuclease resistance by up to ∼400-fold. Here we demonstrate that glutaraldehyde cross-linking of PEGylated oligolysine-coated DNs extends survival by up to another ∼250-fold to >48 h during incubation with 2600 times the physiological concentration of DNase I. DNA origami with cross-linked oligolysine coats are non-toxic and are internalized into cells more readily than non-cross-linked origami. Our strategy provides an off-the-shelf and generalizable method for protecting DNs in vivo.

Modulation of the Cellular Uptake of DNA Origami through Control over Mass and Shape.

Designer nanoparticles with controlled shapes and sizes are increasingly popular vehicles for therapeutic delivery due to their enhanced cell-delivery performance. However, our ability to fashion nanoparticles has offered only limited control over these parameters. Structural DNA nanotechnology has an unparalleled ability to self-assemble three-dimensional nanostructures with near-atomic resolution features, and thus, it offers an attractive platform for the systematic exploration of the parameter space relevant to nanoparticle uptake by living cells. In this study, we examined the cell uptake of a panel of 11 distinct DNA-origami shapes, with the largest dimension ranging from 50-400 nm, in 3 different cell lines. We found that larger particles with a greater compactness were preferentially internalized compared with elongated, high-aspect-ratio particles. Uptake kinetics were also found to be more cell-type-dependent than shape-dependent, with specialized endocytosing dendritic cells failing to saturate over 12 h of study. The knowledge gained in the current study furthers our understanding of how particle shape affects cellular uptake and heralds the development of DNA nanotechnologies toward the improvement of current state-of-the-art cell-delivery vehicles.

Force Spectroscopy and Beyond: Innovations and Opportunities.

Life operates at the intersection of chemistry and mechanics. Over the years, we have made remarkable progress in understanding life from a biochemical perspective and the mechanics of life at the single-molecule scale. Yet the full integration of physical and mechanical models into mainstream biology has been impeded by technical and conceptual barriers, including limitations in our ability to 1) easily measure and apply mechanical forces to biological systems, 2) scale these measurements from single-molecule characterization to more complex biomolecular systems, and 3) model and interpret biophysical data in a coherent way across length scales that span single molecules to cells to multicellular organisms. In this manuscript, through a look at historical and recent developments in force spectroscopy techniques and a discussion of a few exemplary open problems in cellular biomechanics, we aim to identify research opportunities that will help us reach our goal of a more complete and integrated understanding of the role of force and mechanics in biological systems.

DNA-Corralled Nanodiscs for the Structural and Functional Characterization of Membrane Proteins and Viral Entry.

Here we present a modular method for manufacturing large-sized nanodiscs using DNA-origami barrels as scaffolding corrals. Large-sized nanodiscs can be produced by first decorating the inside of DNA barrels with small lipid-bilayer nanodiscs, which open up when adding extra lipid to form large nanodiscs of diameters ∼45 or ∼70 nm as prescribed by the enclosing barrel dimension. Densely packed membrane protein arrays are then reconstituted within these large nanodiscs for potential structure determination. Furthermore, we demonstrate the potential of these nanodiscs as model membranes to study poliovirus entry.

Single-Molecule Clocks Controlled by Serial Chemical Reactions.

Chemical clocks usually achieve well-defined temporal delays through concentration thresholding coupled to the production, degradation, activation, or inhibition of downstream effectors. In this way, the stochastic dynamics of many individual molecules yield essentially deterministic bulk behavior through ensemble averaging. As a result, their temporal evolution is governed by ensemble dynamics rather than by the behavior of an individual molecule or complex. Here, we present a general approach for the design of single-molecule clocks that permits quasi-deterministic control over the lifetime of single molecular interactions without any external synchronization. By coupling the dissociation of a bimolecular complex to a series of irreversible chemical steps, we interpose a well-defined time delay between binding and dissociation. The number and speed of irreversible steps can be varied to systematically tune both the lifetimes of complexes and the precision of the time delay, raising the prospect of localized timekeeping in nanoscale systems and devices.

Selective Nascent Polymer Catch-and-Release Enables Scalable Isolation of Multi-Kilobase Single-Stranded DNA.

Scalable methods currently are lacking for isolation of long ssDNA, an important material for numerous biotechnological applications. Conventional biomolecule purification strategies achieve target capture using solid supports, which are limited in scale and susceptible to contamination owing to nonspecific adsorption and desorption on the substrate surface. We herein disclose selective nascent polymer catch and release (SNAPCAR), a method that utilizes the reactivity of growing poly(acrylamide-co-acrylate) chains to capture acrylamide-labeled molecules in free solution. The copolymer acts as a stimuli-responsive anchor that can be precipitated on demand to pull down the target from solution. SNAPCAR enabled scalable isolation of multi-kilobase ssDNA with high purity and 50-70 % yield. The ssDNA products were used to fold various DNA origami. SNAPCAR-produced ssDNA will expand the scope of applications in nanotechnology, gene editing, and DNA library construction.

Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT.

Super-resolution fluorescence microscopy is a powerful tool for biological research, but obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed super-resolution imaging that achieves sub-10-nm spatial resolution in vitro on synthetic DNA structures. We also report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-color super-resolution imaging in vitro on synthetic DNA structures as well as four-color two-dimensional (2D) imaging and three-color 3D imaging of proteins in fixed cells.

Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching.

Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential. A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix. This leakage, or 'uncoupling' of the proton electrochemical potential, is mediated primarily by uncoupling proteins. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination, combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1-4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.

Programming Self-Assembly of DNA Origami Honeycomb Two-Dimensional Lattices and Plasmonic Metamaterials.

Scaffolded DNA origami has proven to be a versatile method for generating functional nanostructures with prescribed sub-100 nm shapes. Programming DNA-origami tiles to form large-scale 2D lattices that span hundreds of nanometers to the micrometer scale could provide an enabling platform for diverse applications ranging from metamaterials to surface-based biophysical assays. Toward this end, here we design a family of hexagonal DNA-origami tiles using computer-aided design and demonstrate successful self-assembly of micrometer-scale 2D honeycomb lattices and tubes by controlling their geometric and mechanical properties including their interconnecting strands. Our results offer insight into programmed self-assembly of low-defect supra-molecular DNA-origami 2D lattices and tubes. In addition, we demonstrate that these DNA-origami hexagon tiles and honeycomb lattices are versatile platforms for assembling optical metamaterials via programmable spatial arrangement of gold nanoparticles (AuNPs) into cluster and superlattice geometries.

Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

Self-assembly of DNA into nanoscale three-dimensional shapes.

Molecular self-assembly offers a 'bottom-up' route to fabrication with subnanometre precision of complex structures from simple components. DNA has proved to be a versatile building block for programmable construction of such objects, including two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra. Templated self-assembly of DNA into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase 'scaffold strand' that is folded into a flat array of antiparallel helices by interactions with hundreds of oligonucleotide 'staple strands'. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helices constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes-monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross-with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concentrations. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manufacture of sophisticated devices bearing features on the nanometre scale.

Purification of DNA-origami nanostructures by rate-zonal centrifugation.

Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1-1 µg purified DNA nanostructures, obtaining >100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40-80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1-100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.

Multilayer DNA Origami with Terminal Interfaces That Are Flat and Wide-Area.

DNA origami is a popular nanofabrication strategy that employs self-assembly of a long single scaffold strand, typically less than 10 kilobases in length, with hundreds of shorter staple strands into a desired shape. In particular, origami arranged as a single-layer rectangle has proven popular as flat pegboards that can display functionalities at staple-strand breakpoints, off the sides of the constituent double helices, with a ∼5.3 nm rhombic-lattice spacing. For applications that demand tighter spacing, functionalities can be displayed instead on the termini of helices of multilayer DNA origami. However, pegboards with the greatest addressable surface area are often found to be the most versatile. Given the practical limitations of the length of the scaffold that can be easily realized, designs that minimize the length of each helix would have advantages for maximizing the number of helices and therefore the number of addressable pixels on each terminal surface. Here we present an architecture for multilayer DNA origami displaying flush terminal interfaces from over 200 helices that each are only 5.3 turns in length. We characterize an example using cryo-EM imaging paired with single-particle analysis for further analysis of the global structure.

DNA origami vaccine (DoriVac) nanoparticles improve both humoral and cellular immune responses to infectious diseases.

Current SARS-CoV-2 vaccines have demonstrated robust induction of neutralizing antibodies and CD4+ T cell activation, however CD8+ responses are variable, and the duration of immunity and protection against variants are limited. Here we repurposed our DNA origami vaccine platform, DoriVac, for targeting infectious viruses, namely SARS-CoV-2, HIV, and Ebola. The DNA origami nanoparticle, conjugated with infectious-disease-specific HR2 peptides, which act as highly conserved antigens, and CpG adjuvant at precise nanoscale spacing, induced neutralizing antibodies, Th1 CD4+ T cells, and CD8+ T cells in naïve mice, with significant improvement over a bolus control. Pre-clinical studies using lymph-node-on-a-chip systems validated that DoriVac, when conjugated with antigenic peptides or proteins, induced promising cellular immune responses in human cells. These results suggest that DoriVac holds potential as a versatile, modular vaccine platform, capable of inducing both humoral and cellular immunities. The programmability of this platform underscores its potential utility in addressing future pandemics.