Characterization of Z-DNA dynamics across the tree of life

Abstract Z-DNA/Z-RNA is an alternative left-handed nucleic acid conformation with established and emerging roles in gene regulation, immunity, and genome instability. However, its occurrence dynamics and lineage specificity across the tree of life have not yet been fully characterized. Utilizing the recently developed and improved Z-DNA searching tool, ZSeeker, we analyzed 281,139 complete organismal genomes, including multiple Telomere-to-Telomere genome assemblies, and generated genome-wide Z-nucleic acid maps, examined their topography, and compared them to dinucleotide-preserving controls. Cellular genomes featured pervasive Z-DNA enrichment relative to expectation, with enrichments of ∼1.5 and ∼1.7-fold in Bacteria and Archaea and ∼3-fold in Eukaryota. In contrast, Viruses exhibited large differences between lineages, with modest enrichment in several DNA viral groups and pronounced depletion across RNA clades, most notably Influenza A/B strains. We built a LASSO regression model trained on non-Influenza viruses (cross-validated R² ≈ 0.73), which identified GC content, genome type, and host type as the leading predictors for Z-nucleic acid density, yet it significantly over-predicted Z-RNA density in Influenza A/B. More than 99% of assemblies exceeded the +2 SD threshold, and a “typical Influenza” genome was predicted at 2.76 bp/kb compared to ∼0.016 bp/kb observed (a ∼170-fold overestimation based on chance alone). Together, these results reveal domain- and lineage-specific regimes: cellular genomes are enriched for Z-DNA consistent with regulatory roles, whereas influenza viruses appear to have undergone strong, lineage-specific depletion of Z-RNA-forming sequences, likely reflecting evolutionary pressure tied to host sensing pathways. Competing Interest Statement The authors have declared no competing interest.