Z-DNA is a left-handed DNA conformation linked to gene regulation, chromatin dynamics, and immunity. Despite its importance, genome-wide prediction of Z-DNA forming sequences (ZFS) remains limited by the absence of fast and accessible tools. Here, we present Z-DNA Hunter, a user-friendly web server for genome-scale ZFS prediction utility. The algorithm employs a pattern-based approach optimized for canonical motifs such as (GC)n and (CA)n repeats, with adjustable parameters for detection stringency. Compared with existing methods, Z-DNA Hunter achieves similar or higher accuracy while reducing runtime from hours to seconds, making large-scale analyses feasible. To demonstrate its application, we analyzed the Drosophila melanogaster genome and uncovered a pronounced enrichment of long ZFS on the X chromosome, contrasting with their near absence on the satellite repeat- and transposable element-rich Y chromosome. These findings illustrate both the scalability of Z-DNA Hunter and its potential to reveal biologically meaningful patterns of non-B-DNA. The tool provides direct visualization and export options (e.g. BedGraph for UCSC Genome Browser) and is freely available at https://bioinformatics.ibp.cz/#/analyse/zdna.

Department of Biology and Ecology University of Ostrava 710 00 Ostrava Czech Republic

Department of Informatics Mendel University in Brno Zemědělská 1 613 00 Brno Czech Republic

Faculty of Chemistry Brno University of Technology Purkyňova 118 612 00 Brno Czech Republic

Institute of Biophysics of the Czech Academy of Sciences Královopolská 135 612 00 Brno Czech Republic

The Brainstem Group Institute for Cell Biology and Neurobiology Center for Anatomy Charité University Hospital 10117 Berlin Germany

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