SARS-CoV-2 RNA G-quadruplex-templated synthesis of a small molecule to regulate Nsp10 expression

<h2>Summary</h2> The SARS-CoV-2 genome harbors potential non-canonical RNA G-quadruplex (rG4)-forming sequences RG-1, RG-2, and RG-3. In this work, SARS-CoV-2 rG4s are employed for the first time as templates to guide <i>in situ</i> click chemistry via a 1,3-dipolar cycloaddition between alkyne and azide fragments. Using this bioorthogonal strategy, both RG-1 and RG-2 quadruplexes direct the synthesis of <b>T5</b>, a thiazolyl peptidomimetic compound. Biophysical studies reveal that <b>T5</b> displays high binding affinity for both RG-1 and RG-2 structures, with a clear preference for RG-2 over RG-1. Subsequently, treatment with <b>T5</b> results in reduced expression of nucleocapsid (N) and nonstructural protein 10 (Nsp10) proteins in mammalian cells transfected with GFP-conjugated RG-1 or RG-2 sequences. <b>T5</b> also co-localizes with the G4-specific antibody BG4 and modulates CoV-2 Nsp10 and N expression by interacting with non-canonical rG4 structures, as demonstrated by luciferase assays. These findings provide valuable insights into developing therapeutic strategies against highly infectious viruses such as COVID-19, even without access to specialized laboratory facilities.