Supplementary Material for: Target based biofilm inhibition and antibiotic enhancement strategy by miR.101.3p using DNA tetrahedrons

Cystic Fibrosis is an inherited disease caused by mutations in the CF transmembrane conductance regulator gene. It is characterized by progressive decline in lung function, often driven by chronic respiratory infections, particularly with Staphylococcus aureus and Pseudomonas aeruginosa. MicroRNAs, small, non-coding RNAs that negatively regulate protein expression by binding to mRNA, are altered in people with CF, and contribute to the pulmonary manifestations of CF. The management of CF lung infections is complicated by the formation of bacterial biofilms and antimicrobial resistance. In silico analysis identified hsa.miR.101.3p as a miRNA potentially targeting genes associated with β-lactam resistance and biofilm formation in P. aeruginosa, as well as genes involved in the overall growth of S. aureus. To facilitate delivery, miRNA-mimic DNA oligonucleotides were conjugated to DNA tetrahedrons. The structural integrity of the DNAtd-miRNA complexes was confirmed via transmission electron microscopy, characterized by nanoparticle tracking analysis, and successful bacterial uptake was verified using fluorescence microscopy. DNAtd-miR.101.3p significantly reduced the viability of S. aureus and P. aeruginosa. DNAtd-miR-101-3p enhanced the activity of the beta-lactam antibiotic cefotaxime against planktonic and biofilm-forming P. aeruginosa. The mechanisms involve DNAtd-miR.101.3p targeting of ampC, fleN and pslK. MiR.101.3p, which is expressed in CF bronchial epithelial cells, displays unique inhibition properties against P. aeruginosa and S. aureus in vitro and increases the rate of the bactericidal activity of cefotaxime against P. aeruginosa.