Structure-Dependent Electrical Conductance of DNA Origami Nanowires.

Exploring the structural and electrical properties of DNA origami nanowires is an important endeavor for the advancement of DNA nanotechnology and DNA nanoelectronics. Highly conductive DNA origami nanowires are a desirable target for creating low-cost self-assembled nanoelectronic devices and circuits. In this work, the structure-dependent electrical conductance of DNA origami nanowires is investigated. A silicon nitride (Si3 N4 ) on silicon semiconductor chip with gold electrodes was used for collecting electrical conductance measurements of DNA origami nanowires, which are found to be an order of magnitude less electrically resistive on Si3 N4 substrates treated with a monolayer of hexamethyldisilazane (HMDS) (∼1013 ohms) than on native Si3 N4 substrates without HMDS (∼1014 ohms). Atomic force microscopy (AFM) measurements of the height of DNA origami nanowires on mica and Si3 N4 substrates reveal that DNA origami nanowires are ∼1.6 nm taller on HMDS-treated substrates than on the untreated ones indicating that the DNA origami nanowires undergo increased structural deformation when deposited onto untreated substrates, causing a decrease in electrical conductivity. This study highlights the importance of understanding and controlling the interface conditions that affect the structure of DNA and thereby affect the electrical conductance of DNA origami nanowires.

Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching.

DNA-based FluoroCubes were recently developed as a solution to photobleaching, a ubiquitous limitation of fluorescence microscopy (Niekamp; ; Stuurman; ; Vale Nature Methods, 2020). FluoroCubes, that is, compact ∼4 × 4 × 5.4 nm3 four-helix bundles coupled to ≤6 fluorescent dyes, remain fluorescent up to ∼50× longer than single dyes and emit up to ∼40× as many photons. The current work answers two important questions about the FluoroCubes. First, what is the mechanism by which photostability is enhanced? Second, are FluoroCubes compatible with Förster resonance energy transfer (FRET) and similar techniques? We use single particle photobleaching studies to show that photostability arises through interactions between the fluorophores and the four-helix DNA bundle. Supporting this, we discover that smaller ∼4 × 4 × 2.7 nm3 FluoroCubes also confer ultraphotostability. However, we find that certain dye-dye interactions negatively impact FluoroCube performance. Accordingly, 4-dye FluoroCubes lacking these interactions perform better than 6-dye FluoroCubes. We also demonstrate that FluoroCubes are compatible with FRET and dark quenching applications.

Stress in DNA Gridiron Facilitates the Formation of Two-Dimensional Crystalline Structures.

Programmable DNA nanotechnology has generated some of the most intricate self-assembled nanostructures and has been employed in a growing number of applications, including functional nanomaterials, nanofabrication, biophysics, photonics, molecular machines, and drug delivery. An important design rule for DNA nanostructures is to minimize the mechanical stress to reduce the potential energy in these nanostructures whenever it is possible. This work revisits the DNA gridiron design consisting of Holliday junctions and compares the self-assembly of the canonical DNA gridiron with a new design of DNA gridiron, which has a higher degree of mechanical stress because of the interweaving of DNA helices. While the interweaving DNA gridiron indeed exhibits lower yield, compared to its canonical counterpart of a similar size, we discover that the mechanical stress within the interweaving gridiron can promote the formation of the two-dimensional crystalline lattice instead of nanotubes. Furthermore, tuning the design of interweaving gridiron leads to the change of overall crystal size and regularity of geometry. Interweaving DNA double helices represents a new design strategy in the self-assembly of DNA nanostructures. Furthermore, the discovery of the new role of mechanical stress in the self-assembly of DNA nanostructures provides useful knowledge to DNA nanotechnology practitioners: a more balanced view regarding mechanical stress can be considered when designing future DNA nanostructures.

DNA nanotechnology assisted nanopore-based analysis.

Nanopore technology is a promising label-free detection method. However, challenges exist for its further application in sequencing, clinical diagnostics and ultra-sensitive single molecule detection. The development of DNA nanotechnology nonetheless provides possible solutions to current obstacles hindering nanopore sensing technologies. In this review, we summarize recent relevant research contributing to efforts for developing nanopore methods associated with DNA nanotechnology. For example, DNA carriers can capture specific targets at pre-designed sites and escort them from nanopores at suitable speeds, thereby greatly enhancing capability and resolution for the detection of specific target molecules. In addition, DNA origami structures can be constructed to fulfill various design specifications and one-pot assembly reactions, thus serving as functional nanopores. Moreover, based on DNA strand displacement, nanopores can also be utilized to characterize the outputs of DNA computing and to develop programmable smart diagnostic nanodevices. In summary, DNA assembly-based nanopore research can pave the way for the realization of impactful biological detection and diagnostic platforms via single-biomolecule analysis.

Programming Dynamic Assembly of Viral Proteins with DNA Origami.

Biomolecular assembly in biological systems is typically a complex dynamic process regulated by the exchange of molecular information between biomolecules such as proteins and nucleic acids. Here, we demonstrate a nucleic-acid-based system that can program the dynamic assembly process of viral proteins. Tobacco mosaic virus (TMV) genome-mimicking RNA is anchored on DNA origami nanostructures via hybridization with a series of DNA strands which also function as locks that prevent the packaging of RNA by the TMV proteins. The selective, sequential releasing of the RNA via toehold-mediated strand displacement allows us to program the availability of RNA and subsequently the TMV growth in situ. Furthermore, the programmable dynamic assembly of TMV on DNA templates also enables the production of new DNA-protein hybrid nanostructures, which are not attainable by using previous assembly methods.

Programming DNA Tube Circumference by Tile Offset Connection.

DNA tubes with prescribed circumferences are appealing for numerous multidisciplinary applications. The DNA single-stranded tiles (SSTs) assembly method has demonstrated an unprecedented capability for programming the circumferences of DNA tubes in a modular fashion. Nevertheless, a distinct set of SSTs is typically required to assemble DNA tube of a specific circumference, with wider tubes requiring higher numbers of tiles of unique sequences, which not only increases the expense and design complexity but also hampers the assembly yield. Herein, we introduce "offset connection" to circumvent such challenges in conventional SST tube assembly. In this new connection scheme, the boundary SST tiles in an SST array are designed to connect in an offset manner. To compensate for the offset, the SST array has to grow wider until the array can close to form a wide tube with a tolerable degree of twist. Using this strategy, we have successfully assembled DNA tubes with prescribed circumferences consisting of 8, 12, 14, 16, 20, 24, 28, 32, 36, 42, 56, or 70 helices from two distinct sets of SSTs composed of 19×4 or 19×14 tiles.

Dynamic DNA Structures.

Dynamic DNA structures, a type of DNA construct built using programmable DNA self-assembly, have the capability to reconfigure their conformations in response to environmental stimulation. A general strategy to design dynamic DNA structures is to integrate reconfigurable elements into conventional static DNA structures that may be assembled from a variety of methods including DNA origami and DNA tiles. Commonly used reconfigurable elements range from strand displacement reactions, special structural motifs, target-binding DNA aptamers, and base stacking components, to DNA conformational change domains, etc. Morphological changes of dynamic DNA structures may be visualized by imaging techniques or may be translated to other detectable readout signals (e.g., fluorescence). Owing to their programmable capability of recognizing environmental cues with high specificity, dynamic DNA structures embody the epitome of robust and versatile systems that hold great promise in sensing and imaging biological analytes, in delivering molecular cargos, and in building programmable systems that are able to conduct sophisticated tasks.

Programmable DNA Hydrogels Assembled from Multidomain DNA Strands.

Hydrogels are important in biological and medical applications, such as drug delivery and tissue engineering. DNA hydrogels have attracted significant attention due to the programmability and biocompatibility of the material. We developed a series of low-cost one-strand DNA hydrogels self-assembled from single-stranded DNA monomers containing multiple palindromic domains. This new hydrogel design is simple and programmable. Thermal stability, mechanical properties, and loading capacity of these one-strand DNA hydrogels can be readily regulated by simply adjusting the DNA domains.

Synthetic approaches to RBC mimicry and oxygen carrier systems.

Whole blood or red blood cell (RBC) transfusions are highly significant, clinically, for blood replacement therapies in traumatic injuries, presurgical conditions, and anemias. However, natural RBC-based products suffer from limited shelf life due to pathological contamination and also present risks of refractoriness, graft-versus-host disease, immunosuppression, and acute lung injury. These issues can be only partially resolved by pathogen reduction technologies, serological blood testing, leukoreduction, and specialized storage; hence, they severely affect the efficacy and safety of the blood products. Consequently, there is a significant interest in synthetic RBC analogues that can mimic its oxygen-transport properties while allowing convenient manufacture, reproducibility, long shelf life, and reduced biological risks. To this end, the current Review provides a comprehensive description and discussion of the various research approaches and current state-of-the-art in synthetically mimicking RBC's oxygen-carrying biochemical properties, as well as the biophysical parameters (shape, size and mechanical modulus) that influence RBCs' hemodynamic transport properties in blood flow.

A platelet-mimetic paradigm for metastasis-targeted nanomedicine platforms.

There is compelling evidence that, beyond their traditional role in hemostasis and thrombosis, platelets play a significant role in mediating hematologic mechanisms of tumor metastasis by directly and indirectly interacting with pro-metastatic cancer cells. With this rationale, we hypothesized that platelets can be an effective paradigm to develop nanomedicine platforms that utilize platelet-mimetic interaction mechanisms for targeted diagnosis and therapy of metastatic cancer cells. Here we report on our investigation of the development of nanoconstructs that interact with metastatic cancer cells via platelet-mimetic heteromultivalent ligand-receptor pathways. For our studies, pro-metastatic human breast cancer cell line MDA-MB-231 was studied for its surface expression of platelet-interactive receptors, in comparison to another low-metastatic human breast cancer cell line, MCF-7. Certain platelet-interactive receptors were found to be significantly overexpressed on the MDA-MB-231 cells, and these cells showed significantly enhanced binding interactions with active platelets compared to MCF-7 cells. Based upon these observations, two specific receptor interactions were selected, and corresponding ligands were engineered onto the surface of liposomes as model nanoconstructs, to enable platelet-mimetic binding to the cancer cells. Our model platelet-mimetic liposomal constructs showed enhanced targeting and attachment of MDA-MB-231 cells compared to the MCF-7 cells. These results demonstrate the promise of utilizing platelet-mimetic constructs in modifying nanovehicle constructs for metastasis-targeted drug as well as modifying surfaces for ex-vivo cell enrichment diagnostic technologies.

Adhesive Dynamics Simulations of Highly Polyvalent DNA Motors.

Molecular motors, such as myosin and kinesin, perform diverse tasks ranging from vesical transport to bulk muscle contraction. Synthetic molecular motors may eventually be harnessed to perform similar tasks in versatile synthetic systems. The most promising type of synthetic molecular motor, the DNA walker, can undergo processive motion but generally exhibits low speeds and virtually no capacity for force generation. However, we recently showed that highly polyvalent DNA motors (HPDMs) can rival biological motors by translocating at micrometer per minute speeds and generating 100+ pN of force. Accordingly, DNA nanotechnology-based designs may hold promise for the creation of synthetic, force-generating nanomotors. However, the dependencies of HPDM speed and force on tunable design parameters are poorly understood and difficult to characterize experimentally. To overcome this challenge, we present RoloSim, an adhesive dynamics software package for fine-grained simulations of HPDM translocation. RoloSim uses biophysical models for DNA duplex formation and dissociation kinetics to explicitly model tens of thousands of molecular scale interactions. These molecular interactions are then used to calculate the nano- and microscale motions of the motor. We use RoloSim to uncover how motor force and speed scale with several tunable motor properties such as motor size and DNA duplex length. Our results support our previously defined hypothesis that force scales linearly with polyvalency. We also demonstrate that HPDMs can be steered with external force, and we provide design parameters for novel HPDM-based molecular sensor and nanomachine designs.

Shaped DNA origami carrier nanopore translocation influenced by aptamer based surface modification.

DNA origami is widely used as a translocation carrier to assist solid-state nanopore analysis, e.g., soft linear origami carrier and special-shaped origami structures. In the linear origami carriers based nanopore sensing, molecular modifications induced tiny structural and charge changes, can result in significant variations on translocation signals to facilitating single-molecule sensing. However, an understanding on the influences of surface modifications on special-shaped DNA origami structures during solid-state (SS) nanopores translocation is still far elusive. Herein, we reported a surface modification strategy using aptamer/target-binding to influence the translocation of the shaped origami ribbon carrier through SS-nanopore. Our measurements indicate that the translocation signal variations can respond to ATP/aptamer binding on the carrier surface, even to the surface modifications induced by spatial distributions and enzyme catalysis. Meanwhile, the results also suggest a possibility to identify small spatial and electronic changes on DNA origami by using SS-nanopore. We envision that the surface aptamer-binding influenced origami translocation strategy could find more applications in origami carrier assisted SS-nanopore sensing and detection.

Cooperative binding of T cell receptor and CD4 to peptide-MHC enhances antigen sensitivity.

Antigen recognition by the T cell receptor (TCR) of CD4+ T cells can be greatly enhanced by the coreceptor CD4. Yet, understanding of the molecular mechanism is hindered by the ultra-low affinity of CD4 binding to class-II peptide-major histocompatibility complexes (pMHC). Here we show, using two-dimensional (2D) mechanical-based assays, that the affinity of CD4-pMHC interaction is 3-4 logs lower than that of cognate TCR-pMHC interactions, and it is more susceptible to increased dissociation by forces (slip bond). In contrast, CD4 binds TCR-pre-bound pMHC at 3-6 logs higher affinity, forming TCR-pMHC-CD4 tri-molecular bonds that are prolonged by force (catch bond), and modulated by protein mobility on the cell membrane, indicating profound TCR-CD4 cooperativity. Consistent with a tri-crystal structure, using DNA origami as a molecular ruler to titrate spacing between TCR and CD4 we show that 7-nm proximity optimizes TCR-pMHC-CD4 tri-molecular bond formation with pMHC. Our results thus provide deep mechanistic insight into CD4 enhancement of TCR antigen recognition.

Monochromatic Fluorescent Barcodes Hierarchically Assembled from Modular DNA Origami Nanorods.

With the rapid advancement of fluorescence microscopy, there is a growing interest in the multiplexed detection and identification of various bioanalytes (e.g., nucleic acids and proteins) for efficient sample processing and analysis. We introduce in this work a simple and robust method to provide combinations for micrometer-scale fluorescent DNA barcodes of hierarchically assembled DNA origami superstructures for multiplexed molecular probing. In addition to optically resolvable dots, we placed fluorescent loci on adjacent origami within the diffraction limit of each other, rendering them as unresolvable bars of measurable lengths. We created a basic set of barcodes and trained a machine learning algorithm to process and identify individual barcodes from raw images with high accuracy. Moreover, we demonstrated that the number of combinations can be increased exponentially by generating longer barcodes, by controlling the number of incorporated fluorophores to create multiple levels of fluorescence intensity, and by employing super-resolution imaging. To showcase the readiness of the barcodes for applications, we used our barcodes to capture and identify target nucleic acid sequences and for simultaneous multiplexed characterization of binding kinetics of several orthogonal complementary nucleic acids.

Accurate genotyping of fragmented DNA using a toehold assisted padlock probe.

Fragmented DNA from blood plasma, i.e., cell-free DNA, has received great interest as a noninvasive diagnostic biomarker for "point-of-care" testing or liquid biopsy. Here, we present a new approach for accurate genotyping of highly fragmented DNA. Based on toehold-mediated strand displacement, a toehold-assisted padlock probe and toehold blocker were designed and demonstrated with new controllability in significantly suppressing undesired cross-reaction, promoting target recycling and point mutation detection by tuning the thermodynamic properties. Furthermore, toehold-assisted padlock probe systems were elaborately designed for 14 different single-nucleotide variants (SNVs) and were demonstrated to be able to detect low concentration of variant alleles (0.1%). In addition, a target, spanning a narrow sequence window of 29 nucleotides on average is sufficient for the toehold-assisted padlock probe system, which is valuable for the analysis of highly fragmented DNA molecules from clinical samples. We further demonstrated that the toehold-assisted padlock probe, in combination with a unique asymmetric PCR technique, could detect more target SNVs at low allele fractions (1%) in highly fragmented cfDNA. This allows accurate genotyping and provides a new commercial approach for high-resolution analysis of genetic variation.

Programmable assembly of gold nanoparticle nanoclusters and lattices.

Deterministic assembly of metallic nanoparticles (e.g. gold nanoparticles) into prescribed configurations has promising applications in many fields such as biosensing and drug delivery. DNA-directed bottom-up assembly has demonstrated unparalleled capability to precisely organize metallic nanoparticles into assemblies of designer configurations. However, the fabrication of assemblies comprising delicate nanoparticle arrangements, especially across large dimensions (e.g. micron size), has remained challenging. In this report, we have designed DNA origami hexagon tiles that are capable of assembling into higher-order networks of honeycomb arrays or tubes with dimensions up to several microns. The versatile addressability of the unit tile enables precise and periodic positioning of nanoparticles onto these higher-order DNA origami frame structures. Overall, we have constructed a series of 9 gold nanoparticle architectures with programmable configurations ranging from nanometer-sized clusters to micrometer-sized lattices. We believe these architectures shall hold great application potential in numerous biomedical fields.

Design and operation of reconfigurable two-dimensional DNA molecular arrays.

Information relay and cascaded transformation are essential in biology and engineering. Imitation of such complex behaviors via synthetic molecular self-assembly at the nanoscale remains challenging. Here we describe the use of structural DNA nanotechnology to realize prescribed, multistep, long-range information relay and cascaded transformation in rationally designed molecular arrays. The engineered arrays provide a controlled platform for studying complex dynamic behaviors of molecular arrays and have a range of potential applications, such as with reconfigurable metamaterials. A reconfigurable array consists of a prescribed number of interconnected dynamic DNA antijunctions. Each antijunction unit consists of four DNA domains of equal length with four dynamic nicking points, which are capable of switching between two stable conformations through an intermediate open conformation. By interconnecting the small DNA antijunctions, one can build custom two-dimensional (2D) molecular 'domino' arrays with arbitrary shapes. More important, the DNA molecular arrays are capable of undergoing programmed, multistep, long-range transformation driven by information relay between neighboring antijunction units. The information relay is initiated by the trigger strands under high temperature or formamide concentration. The array's dynamic behavior can be regulated by external factors such as its shape and size, points of transformation initiation, and/or any engineered information propagation pathways. This protocol provides detailed strategies for designing DNA molecular arrays, as well as procedures for sample production, purification, reconfiguration, and imaging by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The procedure can be completed in 4-7 d.

Approaches to synthetic platelet analogs.

Platelet transfusion is routinely used for treating bleeding complications in patients with hematologic or oncologic clotting disorders, chemo/radiotherapy-induced myelosuppression, trauma and surgery. Currently, these transfusions mostly use allogeneic platelet concentrates, while products like lyophilized platelets, cold-stored platelets and infusible platelet membranes are under investigation. These natural platelet-based products pose considerable risks of contamination, resulting in short shelf-life (3-5 days). Recent advances in pathogen reduction technologies have increased shelf-life to ~7 days. Furthermore, natural platelets are short in supply and also cause several biological side effects. Hence, there is significant clinical interest in platelet-mimetic synthetic analogs that can allow long storage-life and minimum side effects. Accordingly, several designs have been studied which decorate synthetic particles with motifs that promote platelet-mimetic adhesion or aggregation. Recent refinement in this design involves combining the adhesion and aggregation functionalities on a single particle platform. Further refinement is being focused on constructing particles that also mimic natural platelet's shape, size and elasticity, to influence margination and wall-interaction. The optimum design of a synthetic platelet analog would require efficient integration of platelet's physico-mechanical properties and biological functionalities. We present a comprehensive review of these approaches and provide our opinion regarding the future directions of this research.