Dental biofilms contain DNase I-resistant Z-DNA and G-quadruplexes

eDNA is a major component of the extracellular matrix of bacterial biofilms, and recent studies have shown that biofilms from many pathogens contain both Z-DNA and G-quadruplex (G4) structures in addition to the canonical B-DNA double helix. These secondary DNA structures provide new emergent functions to the biofilm, most notably by making biofilms more resilient by protecting them from nucleases. In dental biofilms, it is largely unknown what conformation eDNA adopts, and the aim of this study was to determine if dental biofilms contain non-canonical secondary DNA structures. In this study, we visualised B-DNA, G4, and Z-DNA in dental biofilms from 10 healthy subjects and from 10 caries-active subjects using fluorescence immunolabeling and confocal microscopy. eDNA formed large structures surrounding clusters of microorganisms that contained B-DNA, G4, and Z-DNA in the majority of the samples. We also identified microorganisms outside of these large eDNA structures that contained smaller G4 and Z-DNA structures associated to cell surfaces. G4 and Z-DNA are resistant to degradation by the commonly used mammalian DNase I. We verified this resistance in dental biofilms, and we suggest that these structures present a novel target for improved enzyme formulations for controlling oral biofilms and, more broadly, any biofilm that contains G4 and Z-DNA.