Rational Design of Chimeric Antisense Oligonucleotides that Target FMN Riboswitch mRNAs and Inhibit the Growth of Methicillin-Resistant <i>Staphylococcus aureus</i>

Antimicrobial resistance poses a significant threat to medicine, exemplified by methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), which is resistant to nearly all antibiotics. We designed chimeric antisense oligonucleotides (ASOs) targeting the conserved flavin mononucleotide (FMN) riboswitch, a regulator of essential bacterial metabolic pathways. Three chemically modified ASOs were optimized for stability, binding affinity, and RNase H recruitment, and conjugated to the pVEC cell-penetrating peptide for uptake. Antibacterial activity was tested against MRSA ATCC 6538, with growth monitored by optical density. pVEC-ASO-3 showed the most potent effect, with MIC₅₀ and MIC₉₀ values of 1.25 and 7.5 nM, respectively. No inhibition was observed in <i>Escherichia coli</i>, which lacks the FMN riboswitch, confirming specificity. Cytotoxicity testing in human A549 cells revealed 95% viability at the highest concentration. These results demonstrate that riboswitch-targeted ASOs can selectively inhibit MRSA growth with minimal toxicity, supporting their potential as a new class of antibacterial agents against drug-resistant pathogens.