Estimation of nucleic acid mimics (NAMs) diffusion in bacteria with fluorescence recovery after photobleaching (FRAP) and PyFRAP

The antibiotic resistance crisis urges novel drugs against bacterial infections. Nucleic acids can serve as such drugs by inhibiting vital bacterial genes, provided their diffusion within bacterial cells is understood. We adapted PyFRAP, originally for eukaryotic cell protein diffusion analysis based on Fluorescence Recovery After Photobleaching (FRAP), to assess nucleic acid diffusion in bacteria. Validating the method, FRAP assays were conducted in E. coli expressing enhanced Green Fluorescent Protein (eGFP), yielding a diffusion coefficient (DC) of 1.25 (0.38) µm²·s⁻¹, consistent with the literature. We then investigated the internalization and diffusion of a 14-mer nucleic acid mimic (NAM) sequence in live bacterial cells, employing four fitting models. NAMs exhibited DCs (1.90 x 10⁻³ – 6.98 x 10⁻³ µm²·s⁻¹) aligning with theoretical values. This study validates PyFRAP for bacterial FRAP analysis, facilitating the understanding of nucleic acid diffusion and bacterial internalization and efflux mechanisms.