# Peptide nucleic acids (PNAs) control function of SARS-CoV-2 frameshifting stimulatory element trough PNA-RNA-PNA triplex formation

## TL;DR

This study shows that peptide nucleic acids can inhibit SARS-CoV-2 RNA function, with unexpected results about their effectiveness and mechanism.

## Contribution

The study reveals that antisense PNAs are more effective than triplex-forming PNAs in inhibiting SARS-CoV-2 frameshifting.

## Key findings

- Antisense PNAs inhibited frameshifting more effectively than PNA tail-clamps.
- Cationic triplex-forming PNAs caused sequence non-specific inhibition of frameshifting.
- Thermal stability of PNA-RNA complexes did not correlate with frameshifting inhibition.

## 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.

•Binding of peptide nucleic acids (PNAs) inhibited protein translation and frameshifting of SARS-CoV-2 RNA.•Surprisingly, duplex-forming antisense PNAs were stronger inhibitors of frameshifting than triplex-forming PNA tail-clamps.•Frameshifting inhibition did not correlate with PNA affinity and PNA-RNA complex thermal stability.•Sequence non-specific inhibition of frameshifting was observed for cationic triplex-forming PNAs.

Binding of peptide nucleic acids (PNAs) inhibited protein translation and frameshifting of SARS-CoV-2 RNA.

Surprisingly, duplex-forming antisense PNAs were stronger inhibitors of frameshifting than triplex-forming PNA tail-clamps.

Frameshifting inhibition did not correlate with PNA affinity and PNA-RNA complex thermal stability.

Sequence non-specific inhibition of frameshifting was observed for cationic triplex-forming PNAs.