Optimizing the stability of a DNA origami snub cube inhibitor

Description

With climate change threatening to increase the frequency of global pandemics, the need for quick and adaptable responses to novel viruses will become paramount. DNA nanotechnology offers a highly customizable, biocompatible approach to combating novel outbreaks. For any DNA nanotechnology-based

With climate change threatening to increase the frequency of global pandemics, the need for quick and adaptable responses to novel viruses will become paramount. DNA nanotechnology offers a highly customizable, biocompatible approach to combating novel outbreaks. For any DNA nanotechnology-based therapeutic to have future success in vivo, the structure must be able to withstand serological conditions for an extended time period. In this study, the stability of a wireframe DNA snub cube with attached nbGFP used to bind a nonessential viral epitope on Pseudorabies virus is evaluated in vitro both with and without one of two modifications designed to enhance stability: 1) the use of trivalent spermidine cations during thermal annealing of the nanostructure, and 2) the introduction of a polylysine-polyethylene glycol coating to the conjugated nanostructure. The design, synthesis, and purification of the multivalent inhibitor were also evaluated and optimized. Without modification, the snub cube nanostructure was stable for up to 8 hours in culture media supplemented with 10% FBS. The spermidine-annealed nanostructures demonstrated lesser degrees of stability and greater degradation than the unmodified structures, whereas the polylysine-coated structures demonstrated equivalent stability at lower valencies and enhanced stability at the highest valency of the snub cube inhibitor. These results support the potential for the polylysine-polyethylene glycol coating as a potential method for enhancing the stability of the snub cube for future in vivo applications.

Date Created
2023-05
Agent

Exploration of Aggregation and Multivalency as Viral Inhibition Strategies

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Description
Scientists are entrusted with developing novel molecular strategies for effective prophylactic and therapeutic interventions. Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their

Scientists are entrusted with developing novel molecular strategies for effective prophylactic and therapeutic interventions. Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their transformative potential in healthcare, to date, antivirals have been clinically approved to treat only 10 out of the greater than 200 known pathogenic human viruses. Additionally, as obligate intracellular parasites, many virus functions are intimately coupled with host cellular processes. As such, the development of a clinically relevant antiviral is challenged by the limited number of clear targets per virus and necessitates an extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. Therefore, a means to develop virus- or strain-specific antivirals without detailed insight into each idiosyncratic biochemical mechanism may aid in the development of antivirals against a larger swath of pathogens. Such an approach will tremendously benefit from having the specific molecular recognition of viral species as the lowest barrier. Here, I modify a nanobody (anti-green fluorescent protein) that specifically recognizes non-essential epitopes (glycoprotein M-pHluorin chimera) presented on the extra virion surface of a virus (Pseudorabies virus strain 486). The nanobody switches from having no inhibitory properties (tested up to 50 μM) to ∼3 nM IC50 in in vitro infectivity assays using porcine kidney (PK15) cells. The nanobody modifications use highly reliable bioconjugation to a three-dimensional wireframe deoxyribonucleic acid (DNA) origami scaffold. Mechanistic studies suggest that inhibition is mediated by the DNA origami scaffold bound to the virus particle, which obstructs the internalization of the viruses into cells, and that inhibition is enhanced by avidity resulting from multivalent virus and scaffold interactions. The assembled nanostructures demonstrate negligible cytotoxicity (<10 nM) and sufficient stability, further supporting their therapeutic potential. If translatable to other viral species and epitopes, this approach may open a new strategy that leverages existing infrastructures – monoclonal antibody development, phage display, and in vitro evolution - for rapidly developing novel antivirals in vivo.
Date Created
2022
Agent