Targeting bacterial TPP riboswitches using pVEC-conjugated chimeric antisense oligonucleotides.

Antisense oligonucleotides (ASOs) provide a sequence-specific approach for targeting bacterial RNAs and suppressing the expression of genes important for bacterial growth and proliferation, making them prominent agents in the design of a novel class of antibacterial agents. This protocol describes the design, preparation, and experimental validation of a chimeric ASO conjugated to the cell-penetrating peptide pVEC and directed against the thiamine pyrophosphate (TPP) riboswitch. The workflow includes bioinformatic identification of conserved and accessible regions within the TPP aptamer, design of a chemically modified ASO, preparation of an in vitro transcribed TPP aptamer RNA substrate, and assessment of ASO-dependent RNA cleavage using an RNase H assay. The protocol further describes bacterial growth-inhibition assays in Listeria monocytogenes and Bacillus subtilis, the use of Escherichia coli as a specificity control, and evaluation of pVEC-ASO-1 cytotoxicity in A549 human cells using an MTT-based viability assay. Although presented for the TPP riboswitch, the approach can be adapted to other bacterial mRNA regions. This protocol therefore provides a flexible experimental framework for evaluating riboswitches and other bacterial RNA elements as potential targets for ASO-based antibacterial development.