Target-Based Biofilm Inhibition and Antibiotic Enhancement Strategy by MiR.101.3p Using DNA Tetrahedrons

Introduction: Cystic fibrosis (CF) is an inherited disease caused by mutations in the CF transmembrane conductance regulator gene (CFTR). It is characterized by progressive decline in lung function, often driven by chronic respiratory infections, particularly with Staphylococcus aureus and Pseudomonas aeruginosa. MicroRNAs (miRNAs), small noncoding regulatory RNAs that negatively regulate protein expression by binding to mRNA, are altered in people with CF and potentially contribute to the pulmonary manifestations of CF. The management of CF lung infections is further complicated by the formation of bacterial biofilms and the emergence of antimicrobial resistance which renders conventional treatments ineffective.

In silico analysis identified hsa-miR.101.3p as a promising miRNA with potential targets including genes associated with beta-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 (DNAtds). 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 both S. aureus and P. aeruginosa. Furthermore, DNAtd-miR.101-3p enhanced the activity of the beta-lactam antibiotic cefotaxime against both non-mucoid and mucoid planktonic and biofilm-forming P. aeruginosa. The mechanisms involve DNAtd-miR.101.3p targeting of ampC, fleN, and pslK.

DNAtd-miR.101.3p displays unique inhibition properties against P. aeruginosa and S. aureus in the exponential phase of bacterial growth in vitro and increases the rate of the bactericidal activity of cefotaxime against P. aeruginosa.