A Structure-Guided Bifunctional Aptamer Mitigates <i>Pseudomonas aeruginosa</i> Virulence and Biofilms by Disrupting las and rhl Quorum Sensing Systems

Quorum sensing (QS) is a microbial communication system that mediates the virulence factor synthesis and biofilm formation through signaling molecules. QS-targeted antivirulence strategies have emerged as a new approach to avoiding antibiotic resistance by weakening bacterial pathogenicity instead of directly killing bacteria, thereby reducing the selection pressure caused by traditional antibiotics. However, the molecular mechanisms that disrupt QS by blocking the binding of signaling molecules to receptor proteins remain elusive. Here, we engineered a bifunctional aptamer Cb-2 targeting the QS system of <i>Pseudomonas aeruginosa</i>─a canonical QS model organism─through structure-guided rational design integrating molecular docking with progressive truncation and sequence optimization. This aptamer specifically targeted both 3O-C12-HSL (the las system signal molecule) and C4-HSL (the rhl system signal molecule), achieving a dual blockade of QS cascade signaling. The intervention resulted in significant suppression of environmental virulence determinants (51.76% inhibition of rhamnolipid synthesis; 66.28% reduction in pyocyanin production) coupled with 74.46% attenuation of biofilm formation. This aptamer-targeted intervention strategy on the QS system exhibits significant potential for developing low environmental toxicity and highly targeted antimicrobial alternatives.