Z-DNA is an Intrinsic Structural Component of Neutrophil Extracellular Traps
Neutrophil extracellular traps (NETs), with their networked extracellular DNA (eDNA), are key effectors of innate immunity which when ineffectively cleared can lead to immunopathologies. Here, we demonstrate the existence of intrinsic, host-derived left-handed noncanonical Z-DNA in NETs eDNA. Immunofluorescence staining of NETs formed in response to diverse NETosis pathways resulted in extensive filamentous Z-DNA tracing the non-Z-DNA backbone irrespective of the stimulus suggesting converging mechanisms. Circular dichroism validated the existence of Z-DNA in NETs. Temporal analysis revealed Z-DNA formation through a staged process sourced from early mitochondrial DNA release engaging with ZBP1 and transitioning to nuclear-derived as NETs mature, increasing through nucleation of new domains, revealing a heterogeneous and dynamically evolving B-to-Z-DNA landscape. The intrinsic Z-DNA showed greater relative retention than its non-Z-DNA counterpart following nuclease treatment. Modulation of DNA structure using chloroquine induced Z-to-B transition and restored susceptibility to nuclease-mediated degradation thereby establishing conformation as a tunable determinant of NET architecture. Critically, Z-DNA-enriched NETs induce significantly greater IFN- from PBMCs compared to non-Z-DNA fractions, linking NETs Z-DNA to innate immune activation. NETs may also represent endogenous antigens for anti-Z-DNA antibodies, and Z-DNA may contribute as an additional intrinsic structural determinant of NET persistence observed in DNase-rich vasculature.