Abstract

Riboswitches are structured non-coding mRNA segments in which ligand binding to an aptamer domain induces conformational changes in a downstream expression platform to regulate gene expression, positioning them as attractive targets for antimicrobial therapy. The fluoride riboswitch, found in several pathogenic bacteria, is a promising yet underexplored target despite evidence of its role in bacterial defense through fluoride ion (F⁻) binding in the presence of Mg²⁺. In this study, we investigate the conformational stability of (i) the holo form of the Thermotoga petrophila fluoride riboswitch aptamer (RNA in the presence of F⁻+Mg²⁺+K⁺) relative to (ii) the apo form (RNA in the absence of F⁻+Mg²⁺+K⁺). Conformational thermodynamic analysis reveals that the holo riboswitch is stabilized by the Ion recognition site, the Pseudoknot, and Stem 1, whereas Stem 2, Loop 1, Loop 2, and most unpaired nucleotides exhibit pronounced disorder and destabilization.  Complementary docking studies identify these destabilized regions as putative binding pockets for non-cognate ligands. Together, these findings provide structural and thermodynamic insights into fluoride riboswitch–ligand interactions, guiding the design of nucleic acid–targeted therapeutics, including RNA-modulating drugs and engineered aptamers. Future in vitro and in vivo validation will be critical for translating these computational predictions into novel strategies to combat antimicrobial resistance.