Extracellular nucleic acids (eNA) are ubiquitous components of bacterial biofilms and play central roles in maintaining biofilm architecture, contributing to antimicrobial resistance, and facilitating immune evasion. Historically, eNA in biofilms have been conceptualised mainly as extracellular DNA in the canonical B‑form. This narrower view has shaped both experimental approaches and interpretations of how nucleic acids contribute to biofilm biology.
Recent work emphasises that eNA are not a monolithic entity but a heterogeneous mixture of nucleic acid species and structures. Recognising the diversity of eNA is important for accurate imaging, functional studies, and for developing strategies to disrupt biofilms therapeutically.
The biofilm matrix contains multiple non‑canonical nucleic acid conformations beyond B‑DNA. These include left‑handed Z‑DNA, G‑quadruplexes, i‑motifs, triplex structures, and extracellular RNA. Such structures coexist within the matrix and can differ substantially in chemical and physical properties from canonical double‑stranded B‑DNA.
The discovery of these non‑canonical forms challenges prior assumptions about matrix composition and necessitates updated imaging and analytical strategies that can detect a wider range of nucleic acid architectures.
Non‑canonical nucleic acid structures display functional properties that set them apart from B‑DNA. Key emergent features reported include:
Resistance to degradation by mammalian DNase I, which has implications for interpreting results of nuclease treatments commonly used to probe matrix composition.
The ability to form catalytically active DNAzymes with peroxidase activity, indicating potential enzymatic contributions from eNA to biofilm chemistry.
Participation as conduits for extracellular electron transfer, suggesting a role for eNA in biofilm electrochemistry and redox biology.
These properties underscore the biological significance of structural diversity among eNA and suggest that overlooking non‑canonical forms may miss important functional aspects of biofilms.
Imaging approaches that rely on fluorescent DNA‑binding dyes are widely used to visualise eNA in biofilms, but many of these dyes have pronounced structural biases. Examples cited include propidium iodide and TOTO™‑1, which are commonly applied in biofilm imaging workflows.
Such dyes can preferentially bind canonical DNA structures or particular conformations, thereby rendering non‑canonical structures effectively invisible to fluorescence microscopy. This selective detection has direct consequences for experimental interpretation: dyes that fail to label certain eNA structures can lead to underestimates of total eNA, misinterpretation of nuclease efficacy, and incomplete pictures of matrix architecture.
Because the PubMed abstract does not provide quantitative comparisons or a complete list of dye‑structure affinities, readers should consult the full text for detailed performance characteristics and recommended dye combinations.
To overcome the structural selectivity of generic DNA‑binding dyes, structure‑specific immunolabelling has been used to detect non‑canonical nucleic acid conformations. Commercial monoclonal antibodies that recognise specific structures are highlighted as validation tools in imaging workflows. Examples named include Z22 (for Z‑DNA), BG4 (for G‑quadruplexes), 1H6, iMab (for i‑motifs), Jel466, and J2 (presumably for double‑stranded RNA), as reported in the abstract.
These antibodies permit targeted detection of distinct nucleic acid architectures and can corroborate dye‑based imaging results. The abstract notes them as part of a recommended validation strategy but does not provide binding affinities, recommended protocols, or cross‑reactivity data; those details are available in the full article and original antibody documentation.
The review provides practical guidance aimed at achieving broader and more accurate visualisation of eNA in biofilms. Key recommendations reported in the abstract include:
Integrate multiple detection methods rather than relying on a single dye or assay.
Combine far‑red fluorescent dyes with TOTO™‑1 to improve structural coverage across different eNA forms.
Validate dye‑based observations with commercial monoclonal antibodies that recognise specific non‑canonical motifs (for example, Z22, BG4, 1H6, iMab, Jel466, and J2).
Use functional assays alongside imaging to test for enzymatic activities (such as DNAzyme peroxidase activity) or nucleic‑acid–dependent functions (for example, electron transfer), thereby linking structure to function.
The abstract does not enumerate precise staining protocols, concentrations, imaging settings, or validation criteria; investigators should consult the full text and manufacturer guidance before implementing these recommendations.
Understanding the structural diversity of eNA and choosing appropriate detection tools are framed as essential steps for rational design of structure‑specific nuclease therapeutics aimed at biofilm infections. Because non‑canonical eNA can resist mammalian DNase I and possess unique biochemical activities, therapeutics that target only canonical B‑DNA may be ineffective against the full complement of matrix nucleic acids.
The review argues that comprehensively characterising eNA structure in situ — using combined dye and antibody strategies plus functional assays — will inform the development of nucleases or other agents engineered to recognise and degrade the specific conformations present in pathogenic biofilms.
The summary here is derived from the PubMed abstract and article metadata. Specific experimental details, quantitative comparisons of imaging reagents, antibody validation data, and protocol recommendations are not reported in the abstract and require review of the full text. For implementation in laboratory or clinical research, consult the complete article (Essays Biochem. 2026;70(3):347–360, doi: 10.1042/EBC20250031) and primary sources for antibodies and dyes.
Accurate detection of the full spectrum of eNA structures is a prerequisite for mechanistic research into biofilm resilience and for development of targeted anti‑biofilm interventions.