Ponente
Descripción
Introduction: Regulatory small RNAs have emerged as promising therapeutic targets because they control essential bacterial stress-response pathways; however, their systematic identification remains limited by incomplete annotations and the lack of robust discovery strategies. Objective: To develop a bioinformatic workflow for identifying evolutionarily conserved antisense regulatory RNAs with therapeutic potential and demonstrate its application in Klebsiella pneumoniae. Materials and Methods: A computational pipeline was developed to analyze fifty complete K. pneumoniae genomes using genomic coordinates and conserved gene organization instead of previously annotated RNAs. The workflow identified conserved genomic neighborhoods associated with persistence-related genes, evaluated intergenic distance, strand orientation and syntenic conservation, and prioritized candidate regulatory RNAs. Selected candidates were characterized through promoter prediction, secondary structure and accessibility analyses using ViennaRNA, followed by three-dimensional structural validation with AlphaFold 3. As proof of concept, a peptide-conjugated phosphorodiamidate morpholino oligomer was rationally designed against the highest-ranked target. Results: The analysis identified a previously unprioritized cis-antisense RNA completely overlapping the persistence toxin gene GhoT in all fifty genomes analyzed, demonstrating complete evolutionary conservation of this genomic architecture. Structural analyses revealed an accessible apical loop suitable for antisense recognition, and three-dimensional modeling supported the formation of a stable RNA-oligonucleotide heteroduplex. Conclusions: This workflow enables the systematic identification of conserved regulatory RNAs independently of existing annotations and provides a reproducible strategy for prioritizing RNA-based therapeutic targets against bacterial persistence. The proposed approach may be extended to other multidrug-resistant bacterial pathogens.