Fig. 1: Hydrolysis of RNA and not DNA results in loss of eDNA fibres and P. aeruginosa biofilm dissolution.

Nuclear magnetic resonance (NMR) of extracted nucleic acid (NA) gel isolate and confocal laser scanning microscopy of Pseudomonas aeruginosa biofilms. a NMR ¹H-¹³C heteronuclear single quantum (HSQC)-total correlation spectroscopy (TOCSY) spectrum of extracellular nucleic acid (NA) gel isolate dissolved in 0.1 M NaOD, (10 mg ml⁻¹) at 25 °C showing the C1’-H1’ cross peaks of RNA (blue ovals) and DNA (red rectangles) and their correlations to the neighbouring carbon C2’-H1’. Molecule structures (right) illustrate correlations. b 1-D ³¹P NMR of NA isolate gel and crude P. aeruginosa biofilm with proton decoupling showing the presence of monoesterified (i.e., monoribonucleotides) and diesterified (i.e., DNA) phosphate peaks (i), and 2-D ¹H-³¹P heteronuclear correlation (HETCOR) spectrum of extracellular NA showing the ³¹P-¹H cross-peaks of monoribonucleotides and DNA (ii). Coupling of monoesterified phosphates to H2’ or H3’ of eight monoesterified monoribonucleotides (from left to right: 3’ AMP/3’ GMP, 3’ CMP/3’ UMP, 2’ CMP/2’ UMP, 2’ AMP, 2’ GMP); and diesterified phosphate to DNA H3’ and H5’/H5” protons, are denoted by the dashed lines. There is a discontinuity (vertical wavy line) in the ³¹P axis due to the different thresholds required to illustrate the ³¹P-¹H correlations in the monoesterified and diesterified regions. All samples were prepared in 0.1 M NaOD, (10 mg ml⁻¹) at 25 °C. c Confocal micrographs of five-day P. aeruginosa biofilms at pH 7 and d following alkaline transesterification (37 °C for 16 h; n = 3), stained with TOTO-1 showing eDNA fibres (green). Scale bars represent 10 µm. e Rheogram of five-day Pseudomonas aeruginosa PAO1 wildtype biofilm and alkalinised wildtype biofilm (0.3 M KOH at 37^oC for 16 h; n = 3) in frequency sweep at 25 °C, 0.026 mm gap, 0.3 amplitude. A tan δ > 1 represents fluid behaviour, while tan δ < 1 indicates gel behaviour. For e, the biological triplicates are averaged for both conditions and plotted against frequency. Relevant source data for Fig. 1e is provided as a source data file.