Z-DNA Hunter tool for straightforward detection of Z-DNA forming regions and a case study in Drosophila.

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.