Targeting SARS-CoV-2 RNA Pseudoknots Using Triplex-Forming Peptide Nucleic Acids - SARS-CoV-2, the causative agent of COVID-19, is a positive-sense single-stranded RNA virus in the Coronaviridae family. The RNA of SARS-CoV-2 folds into complex double-helical structures that open the door for novel anti-viral approaches targeting RNA instead of proteins. Positive-sense RNA viruses use the specifically folded RNA structures to highjack the host cell's machinery, regulate viral replication and mRNA translation, evade antiviral responses, and produce more viral particles. The mechanisms by which SARS-CoV-2 and other coronaviruses control these processes are not well understood. However, highly conserved structural motifs of viral RNA, such as pseudoknots and hairpins, are critical for replication and translation. These motifs act as functional switches of the viral life cycle and are critical for SARS-CoV-2 pathogenesis. In this application, we propose to use triplex-forming peptide nucleic acids (PNAs) to control the conformation and biological function of SARS-CoV-2 pseudoknots. The long- term goals are to (1) better understand SARS-CoV-2 biology and pathogenesis and (2) develop new and broad-spectrum therapeutics against multiple coronavirus strains. The specific aims are to (1) study the conformational control of pseudoknot RNA switches with triplex-forming PNA and (2) study how the PNA binding and RNA switching affects coronavirus biology. We are well positioned to achieve these Aims because our research group has been working on sequence-specific recognition of biologically significant RNA molecules using triplex-forming PNAs for more than a decade. The proposed research is significant and innovative because the biology of dynamic viral RNA switches is poorly understood and have not been widely targeted in antiviral therapy. Coronaviruses can mutate their RNA genome and cross species boundaries to infect humans. Therefore, future emergence of new human coronaviruses is almost guaranteed. Mutated genomes can be rapidly sequenced, allowing us to understand how the mutations change the structure of regulatory RNA motifs. Novel therapeutic agents targeting structured RNA motifs will be applicable against multiple strains of coronaviruses and will be easily adjustable for mutated viruses, which is a key bottleneck for traditional antiviral therapeutics. If SARS-CoV-2 (or another coronavirus) mutates the PNA target site, all that is needed is to sequence the mutated genome and change the sequence of therapeutic triplex-forming PNA. RNA is emerging as a major regulatory molecule in wide variety of biological processes and as a promising novel therapeutic target. Better understanding of the biological role of viral RNA switches holds tremendous promise to result in innovative antiviral approaches to combat SARS-CoV-2 and other coronaviruses. If successful, the proposed research will develop new tools for studying the biology of complex regulatory viral RNAs, which will have broad and sustained impact on our ability to combat the current and future pandemics.
