Development of Selective Antiviral RNA-Degrading Chimeras Mimicking Ribonuclease Activity

PROJECT SUMMARY Endogenous ribonucleases (RNases) are an important component of the host immune response to viral infections. Mimicking the RNase function, RNA-degrading chimeras (RDC) are a class of molecules that selectively bind RNA and recruit RNases, typically RNase L, to cleave the viral RNA. However, RNase L- dependent RDCs typically achieve only ~75% RNA knockdown, insufficient for complete viral clearance. To overcome this limitation, we propose to develop a novel class of direct-acting antivirals: RDCs that cleave RNA directly through general acid-general base catalysis, more faithfully mimicking the mechanism of natural ribonucleases. Additionally, we will improve the selectivity of RNA-binding scaffolds by targeting a conserved viral RNA structure and developing a high-throughput synthesis and screening pipeline. Importantly, we will implement a second-generation chemical-guided SHAPE sequencing (cgSHAPE-seq) technology for activity- based RNA profiling. This platform will enable competitive, transcriptome-wide evaluation of ligand binding specificity in live cells. We will demonstrate the effectiveness of this new approach to antiviral design in the context of Zika virus (ZIKV), a mosquito-borne flavivirus associated with severe neurological disorders, including Guillain-Barré syndrome and microcephaly. We hypothesize that our optimized RDCs will significantly reduce ZIKV RNA levels both in vitro and in ZIKV-infected human neural progenitor cells. This project represents the first application of RDCs to directly degrade ZIKV RNA and introduces a generalizable strategy for RNA-targeting antiviral development. By establishing robust methods for RNA binding optimization and transcriptome-wide selectivity profiling, this work has the potential to revolutionize antiviral drug discovery and provide a scalable approach to combat emerging RNA viral threats. 1