No abstract

DNA origami is a powerful nanomaterial for biomedical applications due in part to its capacity for programmable, site-specific functionalization. To realize these applications, scalable and efficient conjugation protocols are needed for diverse moieties ranging from small molecules to biomacromolecules. Currently, there are no facile and general methods for in situ covalent modification and label-free quantification of reaction conversion. Here, we investigate the postassembly functionalization of DNA origami and the subsequent high-performance liquid chromatography-based characterization of these nanomaterials. Following this approach, we developed a versatile DNA origami functionalization and characterization platform. We observed quantitative in situ conversion using widely accessible click chemistry for carbohydrates, small molecules, peptides, polymers, and proteins. This platform should provide broader access to covalently functionalized DNA origami, as illustrated here by PEGylation for passivation and HIV antigen decoration to construct virus-like particle vaccines.

Viruslike particles (VLPs) fabricated using wireframe DNA origami are emerging as promising vaccine and gene therapeutic delivery platforms due to their programmable nature that offers independent control over their size and shape, as well as their site-specific functionalization. As materials that biodegrade in the presence of endonucleases, specifically DNase I and II, their utility for the targeting of cells, tissues, and organs depends on their stability in vivo. Here, we explore minor groove binders (MGBs) as specific endonuclease inhibitors to control the degradation half-life of wireframe DNA origami. Bare, unprotected DNA-VLPs composed of two-helix edges were found to be stable in fetal bovine serum under typical cell culture conditions and in human serum for 24 h but degraded within 3 h in mouse serum, suggesting species-specific endonuclease activity. Inhibiting endonucleases by incubating DNA-VLPs with diamidine-class MGBs increased their half-lives in mouse serum by more than 12 h, corroborated by protection against isolated DNase I and II. Our stabilization strategy was compatible with the functionalization of DNA-VLPs with HIV antigens, did not interfere with B-cell signaling activity of DNA-VLPs in vitro, and was nontoxic to B-cell lines. It was further found to be compatible with multiple wireframe DNA origami geometries and edge architectures. MGB protection is complementary to existing methods such as PEGylation and chemical cross-linking, offering a facile protocol to control DNase-mediated degradation rates for in vitro and possibly in vivo therapeutic and vaccine applications.

Multivalent antigen display is a well-established design principle to enhance humoral immunity elicited by subunit vaccines. Protein-based virus-like particles (VLPs) are an important vaccine platform that implements this principle but also contain thymus-dependent off-target epitopes, thereby generating neutralizing and defocused antibody responses against the scaffold itself. Here, we present DNA origami as an alternative platform to display the receptor binding domain (RBD) of SARS-CoV-2. DNA-based scaffolds provide nanoscale control over antigen organization and, as thymus-independent antigens, are expected to induce only extrafollicular B-cell responses. Our icosahedral DNA-based VLPs elicited valency-dependent BCR signaling in two reporter B-cell lines, with corresponding increases in RBD-specific antibody responses following sequential immunization in mice. Mouse sera also neutralized the Wuhan strain of SARS-CoV-2 - but did not contain boosted, DNA-specific antibodies. Thus, multivalent display using DNA origami can enhance immunogenicity of protein antigens without generating scaffold-directed immunological memory and may prove useful for rational vaccine design.

We demonstrate a promising approach towards designing force-responsive polymers. A thiocarbonylthio group exhibits amplified mechanochemical activity, triggering healing via crosslinking.