Hepatitis B Virus Neutralization with DNA Origami Nanoshells.

We demonstrate the use of DNA origami to create virus-trapping nanoshells that efficiently neutralize hepatitis B virus (HBV) in cell culture. By modification of the shells with a synthetic monoclonal antibody that binds to the HBV envelope, the effective neutralization potency per antibody is increased by approximately 100 times compared to using free antibodies. The improvements in neutralizing the virus are attributed to two factors: first, the shells act as a physical barrier that blocks the virus from interacting with host cells; second, the multivalent binding of the antibodies inside the shells lead to stronger attachment to the trapped virus, a phenomenon known as avidity. Pre-incubation of shells with HBV and simultaneous addition of both components separately to cells lead to comparable levels of neutralization, indicating rapid trapping of the virions by the shells. Our study highlights the potential of the DNA shell system to rationally create antivirals using components that, when used individually, show little to no antiviral effectiveness.

Antigen-Triggered Logic-Gating of DNA Nanodevices.

Synthetic nanoscale devices that reconfigure dynamically in response to physiological stimuli could offer new avenues for diagnostics and therapy. Here, we report a strategy for controlling the state of DNA nanodevices based on sensing antigens with IgG antibodies. To this end, we use IgG antibodies as structural elements to kinetically trap reconfigurable DNA origami structures in metastable states. Addition of soluble antigens displace the IgGs from the objects and triggers reconfiguration. We demonstrate this mechanism by antigen-triggered disassembly of DNA origami shells for two different IgGs and their cognate antigens, and we determined the corresponding dose response curves. We also describe the logic-gated actuation of DNA objects with combinations of antigens, as demonstrated with AND-type shells that disassemble only when two different antigens are detected simultaneously. We apply our system for the antigen-triggered release of molecular payload as exemplified by the release of virus particles that we loaded into the DNA origami shells. We expect our approach to be applicable in many types of DNA nanostructures and with many other IgG-antigen combinations.

Programmable icosahedral shell system for virus trapping.

Broad-spectrum antiviral platforms that can decrease or inhibit viral infection would alleviate many threats to global public health. Nonetheless, effective technologies of this kind are still not available. Here, we describe a programmable icosahedral canvas for the self-assembly of icosahedral shells that have viral trapping and antiviral properties. Programmable triangular building blocks constructed from DNA assemble with high yield into various shell objects with user-defined geometries and apertures. We have created shells with molecular masses ranging from 43 to 925 MDa (8 to 180 subunits) and with internal cavity diameters of up to 280 nm. The shell interior can be functionalized with virus-specific moieties in a modular fashion. We demonstrate this virus-trapping concept by engulfing hepatitis B virus core particles and adeno-associated viruses. We demonstrate the inhibition of hepatitis B virus core interactions with surfaces in vitro and the neutralization of infectious adeno-associated viruses exposed to human cells.

Revealing the structures of megadalton-scale DNA complexes with nucleotide resolution.

The methods of DNA nanotechnology enable the rational design of custom shapes that self-assemble in solution from sets of DNA molecules. DNA origami, in which a long template DNA single strand is folded by many short DNA oligonucleotides, can be employed to make objects comprising hundreds of unique DNA strands and thousands of base pairs, thus in principle providing many degrees of freedom for modelling complex objects of defined 3D shapes and sizes. Here, we address the problem of accurate structural validation of DNA objects in solution with cryo-EM based methodologies. By taking into account structural fluctuations, we can determine structures with improved detail compared to previous work. To interpret the experimental cryo-EM maps, we present molecular-dynamics-based methods for building pseudo-atomic models in a semi-automated fashion. Among other features, our data allows discerning details such as helical grooves, single-strand versus double-strand crossovers, backbone phosphate positions, and single-strand breaks. Obtaining this higher level of detail is a step forward that now allows designers to inspect and refine their designs with base-pair level interventions.

Single-Molecule Observation of the Photoregulated Conformational Dynamics of DNA Origami Nanoscissors.

We demonstrate direct observation of the dynamic opening and closing behavior of photocontrollable DNA origami nanoscissors using high-speed atomic force microscopy (HS-AFM). First the conformational change between the open and closed state controlled by adjustment of surrounding salt concentration could be directly observed during AFM scanning. Then light-responsive moieties were incorporated into the nanoscissors to control these structural changes by photoirradiation. Using photoswitchable DNA strands, we created a photoresponsive nanoscissors variant and were able to distinguish between the open and closed conformations after respective irradiation with ultraviolet (UV) and visible (Vis) light by gel electrophoresis and AFM imaging. Additionally, these reversible changes in shape during photoirradiation were directly visualized using HS-AFM. Moreover, four photoswitchable nanoscissors were assembled into a scissor-actuator-like higher-order object, the configuration of which could be controlled by the open and closed switching induced by irradiation with UV and Vis light.

Nanoscale rotary apparatus formed from tight-fitting 3D DNA components.

We report a nanoscale rotary mechanism that reproduces some of the dynamic properties of biological rotary motors in the absence of an energy source, such as random walks on a circle with dwells at docking sites. Our mechanism is built modularly from tight-fitting components that were self-assembled using multilayer DNA origami. The apparatus has greater structural complexity than previous mechanically interlocked objects and features a well-defined angular degree of freedom without restricting the range of rotation. We studied the dynamics of our mechanism using single-particle experiments analogous to those performed previously with actin-labeled adenosine triphosphate synthases. In our mechanism, rotor mobility, the number of docking sites, and the dwell times at these sites may be controlled through rational design. Our prototype thus realizes a working platform toward creating synthetic nanoscale rotary motors. Our methods will support creating other complex nanoscale mechanisms based on tightly fitting, sterically constrained, but mobile, DNA components.