Programming designer DNA nanostructures for blocking enveloped viral infection

Project Summary/Abstract SARS-CoV-2, the etiological pathogen of COVID-19, has resulted in a pandemic. There remains an urgent need for innovative technologies which facilitate the development of affordable antiviral precision medicine. SARS-CoV-2 is an enveloped virus, and the structure of the trimeric spike protein clusters on the virion has been solved. To develop innovative, affordable, and biocompatible antiviral candidates against SARS-CoV-2 infection and transmission, we exploited the structural characteristics of viral surface proteins that can be matched at nanoscale precision by engineered DNA nanostructure platforms. Based on the structure of the SARS-CoV-2 virion and surface spike trimer layout, we have synthesized a designer DNA nanostructure (DDN) that takes the form of a macromolecular 'net' whose vertices are a precise mechanical match to the spacing and positioning of the spike protein matrix displayed on the virus outer surface. We hypothesize that the structural properties and the layout patterns of SARS-CoV-2 spike proteins can be exploited to design DDNs with nanoscale precision which are capable of matching and capturing intact SARS-CoV-2 virions with ultrahigh binding avidity and selectivity, thereby blocking SARS-CoV-2 infection. We have screened and found DNA aptamers and nanobodies that are specific for the spike receptor-binding domain (RBD). These spike binders can be incorporated into the 'knots' of the DDN net to allow the simultaneous binding of multiple DNA aptamers with multiple spikes on the viral surface in a polyvalent, pattern-matching fashion. The DNA 'net'-aptamer prototype construct has afforded dramatic increase in SARS-CoV-2 binding avidity. This construct can work as a decoy to block viral entry into host cells and is about 1,000-fold more potent than the free aptamer. In this R21 proposal, we aim to extend this technology to enable the incorporation of multiple types of probes against spike RBD and to validate the safety and effectiveness of DDNs in antiviral therapy in vitro and in vivo. We propose two specific aims: to (1) design, synthesize, validate, and further optimize the virus-capturing avidity against various SARS- CoV-2 variants of concern (VOCs); and (2) to determine the antiviral potency and cytotoxicity of the designed DDNs during SARS-CoV-2 infections in vitro in human lung epithelial cells and in vivo in human ACE2-knockin mice. Completion of this work will help us define the antiviral potency and safety of the DNA nanostructures that are designed to perfectly match epitope layouts on the viral surface to capture and wrap live viruses. The estimated cost of DDN treatment is approximately $10/dose (a price that likely decreases at large-scale synthesis), making it an affordable therapy. This DDN platform may further contribute to the rapid development of antiviral precision medicine against emerging SARS-CoV-2 VOCs, as well as other enveloped viruses such as influenza and HIV.