Demir, T. D.; Ram-Mohan, N.; Yang, S.

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 for the first time the existence of the 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 B-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 B-DNA counterpart post nuclease treatment compared to untreated, offering more resistance to the NETs. 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. NETs are likely endogenous antigens for anti-Z-DNA antibodies, and the Z-DNA offers a protein-free explanation for the persistence of NETs observed in DNase-rich vasculature.