Abstract
The highly structured nature of the SARS-CoV-2 genome provides many promising antiviral drug targets. One particularly promising target is a cis-acting RNA pseudoknot found within a critical region called the frameshifting stimulatory element (FSE). In this study, peptide nucleic acids (PNAs) binding to stem 2 of FSE RNA inhibited protein translation and frameshifting, as measured by a cell-free dual luciferase assay, more effectively than PNAs binding to stem 1, stem 3, or the slippery site. Surprisingly, simple antisense PNAs were stronger disruptors of frameshifting than PNA tail-clamps, despite higher thermal stability of the PNA-RNA-PNA triplexes formed by the latter. Another unexpected result was a strong and sequence non-specific enhancement of frameshifting inhibition when using a cationic triplex-forming PNA in conjunction with an antisense PNA targeting key regions of the frameshifting element. Our results illustrate both the potential and the challenges of using antisense PNAs to target highly structured RNAs, such as SARS-CoV-2 pseudoknots. While triplex forming PNAs, including PNA tail-clamps, are emerging as promising ligands for RNA recognition, the binding affinity enhancements when using cationic modifications in triplex-forming PNAs must be carefully balanced to avoid loss of sequence specificity in complex biological systems.