Detecting extracellular G4 DNA motifs in biofilms through energy transfer between DNA-binding dyes TOTO™-1 and...

Secondary DNA structures in extracellular DNA are important for the function and resilience of bacterial biofilms. Non-canonical DNA structures are, however, challenging to localize and characterize. We demonstrate here a FRET-based approach to detect G-quadruplexes (G4s) in vitro and in situ in the extracellular matrix of bacterial biofilms by combining the DNA-binding dyes TOTO™-1 and SYTO™60. Upon addition of the dyes and excitation of TOTO™-1, we observed strong energy transfer (FRET) between the two dyes in synthetic G4 DNA structures. Subsequently, we demonstrate the application of FRET to visualize G4 DNA in the extracellular matrix of biofilms and that these motifs are enriched upon treatment with DNase I. Our work shows a novel and robust method for analysis of G4 DNA motifs in complex biological samples using FRET-pair dyes with high affinity toward G4s.