Project Summary/Abstract While the vast majority of antiviral efforts to combat severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) focus on essential viral proteins, emerging evidence shows that conserved viral RNA (vRNA) structural elements are compelling targets with the potential for pan-antiviral activity. Despite this promise, however, selective targeting of RNA using drug-like small molecules remains challenging. In particular, methodologies for screening small molecule libraries against RNA remain underdeveloped, and do not adequately address the central problem of target specificity. As a result, RNA-targeted screens often fail to yield efficacious compounds. The proposed study takes direct aim at this technological gap through the development of a novel RNA-targeted screening technology using L-aptamers composed of mirror-image L-DNA. The PI previously established that L- aptamers can be evolved to bind native D-RNA structures, including SARS-CoV-2 vRNAs, with high affinity and selectivity. He now proposes to develop L-aptamers into RNA-specific competitive displacement probes for identifying small molecules with analogous properties. The general utility of nucleic acid aptamers, combined with the unique RNA-binding properties of L-aptamers, impart the proposed L-aptamer-displacement assay with several advantages over current RNA-centric screening technologies, and is hypothesized to facilitate the discovery of small molecules with unprecedented RNA-binding capabilities. The PI has already prepared an L-aptamer targeting a conserved RNA element with the SARS-CoV-2 genome, which will be developed into a biochemical assay that couples competitive displacement of the L- aptamer from the vRNA target with an optical readout (Aim 1). Using this assay, the PI will initiate a high- throughput screen to identify potent ligands targeting the corresponding vRNA. The most promising lead compounds will be evaluated for antiviral activity against SARS-CoV-2 infected cells (Aim 2). Parallel efforts will be undertaken to generate L-aptamers against additional SARS-CoV-2 RNA structures (Aim 3), which will be shuttled through this same pipeline. Successful completion of this project will signify a major advance in the area of RNA-targeted drug discovery. While combatting SARS-CoV-2 is the immediate goal, technologies developed herein are readily adaptable to target any RNA virus. By targeting essential RNA structures that are conserved across β-coronaviruses, the PI envision that this approach will allow for identification of antiviral compounds with broad-spectrum activity that might quickly pivot to address future outbreaks.