Introduction: The Flavin mononucleotide riboswitch (FMN riboswitch) is the second most prevalent riboswitch, presented in more than 4300 bacterial species. Sensing FMN, it regulates the synthesis of vital cellular metabolites in Staphylococcus aureus (S. aureus) and seven more pathogenic bacteria included in the priority list of the World Health Organization important for combating antimicrobial resistance. The following study presents the process of the rational design of an antisense oligonucleotide (ASO) with bacteriostatic effects targeting the FMN riboswitch in S. aureus.
Methods: The rational design of ASO, targeted to the multidrug-resistant bacterium S. aureus, is based on bioinformatics and genomic studies, encompassing the following: an analysis of international databases, Clustal X multiple alignments, a selection of appropriate motifs, BLAST analysis in human, probiotic, and other pathogenic bacteria, biochemical pathways, and RNA folding analysis (MFE and PF).
Results: The bioinformatics study results show how the FMN riboswitch is highly suitable for ASO design, as it controls the expression of the fmnP gene in S. aureus (FMN transporter) by translation prevention, and the ribD operon (enzymes for FMN synthesis) by transcription termination. This allows us to design an ASO, which is recognized by both the riboswitch responsible for FMN synthesis and the one for the transporter protein. Its bacteriostatic effect in S. aureus is observed in 700nM, 4.5 μg/mL. Cytotoxicity tests confirm that it is not inherent in the human cell lining of non-small cell lung cancer A549 at this concentration.
Conclusions: Since FMN is not presented in the human genome and regulates the synthesis and transport of essential proteins for the survival and division of bacterium metabolites, it provides the potential as target for designing antibacterial drugs against the most severe human pathogenic bacteria. Our proprietary bioinformatics protocols for suitability and for ASO design allow us to introduce novel ASO as potential effective antimicrobial therapeutic with a growth inhibitory effect in S. aureus.
