Unmasking the diversity of extracellular nucleic acids in the biofilm matrix using nucleic acid-binding dyes

Extracellular nucleic acids (eNA) are central components of bacterial biofilms, contributing to structural integrity, antibiotic tolerance, and emerging functions such as extracellular electron transfer and peroxidase-like catalysis. While extracellular DNA has traditionally been assumed to adopt the canonical B-DNA conformation, biofilms are now known to contain non-canonical structures, including Z-DNA/RNA (Z-NA), G-quadruplex DNA/RNA (G4-NA), and substantial amounts of extracellular RNA. Conventional nucleic acid-binding dyes are widely used for rapid eNA detection, yet their specificity for these diverse structures has not been systematically evaluated. Here, we compare the fluorescence properties of eleven cyanine monomer and dimer dyes (TOTO™, BOBO™, YOYO™, and POPO™ series, SYTOX™ Green, SYTOX™ Red, and propidium iodide) against synthetic B-DNA, Z-DNA, G4-DNA, A-RNA, Z-RNA, and G4-RNA oligonucleotides, with Z-NA stabilised through brominated guanosine analogues synthesised in-house. A clear pattern emerged: green-fluorescent dyes preferentially bound canonical B-DNA, whereas red-fluorescent counterparts displayed broader specificity that extended to non-canonical structures. TOTO™-3 and SYTOX™ Red bound G4-NA with higher fluorescence than B-DNA, and propidium iodide showed an unexpected preference for A-RNA over B-DNA. These observations were validated in Staphylococcus aureus biofilms by parallel immunolabelling with structure-specific antibodies. TOTO™-3, YOYO™-3, BOBO™-3, POPO™-3, and propidium iodide reproduced the eNA distribution at the bacterial cell surface. Finally, we introduce poly-A tailing with fluorescently labelled ATP as a stringent, RNA-specific imaging method for biofilms. Together, these results provide practical guidelines for visualising the structural diversity of eNA in biofilms.