ZBP1 specifically binds pre-formed Z-RNA, limiting its potential pool of nucleic acid ligands compared to ADAR1.

ZBP1 senses foreign nucleic acids in the unusual, left-handed Z-conformation via binding through its Zα1 and Zα2 domains and initiates cell death pathways that enable viral clearance. dsDNA and dsRNA can adopt Z-conformations; however, both typically require chemical modifications or protein binding to induce Z-form conversion and stabilization. ZBP1 has been previously shown to bind and convert B-DNA to the Z-conformation and was assumed to be able to convert A-RNA as well, despite the lack of experimental validation. Here, we use NMR and other biophysical and biochemical experiments to characterize the Z-DNA and Z-RNA binding properties of ZBP1's Zα1 and Zα2 domains. While ZBP1's Zα domains are able to convert and stabilize unmodified dsDNA in the Z-conformation, they are incapable of flipping unmodified (CpG)n repeat dsRNA. ZBP1's Zα domains require dsRNAs with Z-promoting chemical modifications to be able to bind and stabilize the Z-conformation. These results contrast with ADAR1's Zα domain, which can bind and flip both dsDNA and dsRNA into the Z-conformation, potentially indicating finely tuned competition between ADAR1 and ZBP1 for pro-survival and pro-death outcomes, respectively. This work highlights the functional variability of Zα domains and narrows down potential physiological substrates of ZBP1 in infection and disease.