{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T11:03:36Z","timestamp":1785323016799,"version":"3.55.0"},"posted":{"date-parts":[[2026,7,25]]},"group-title":"Biophysics","reference-count":48,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,25]],"date-time":"2026-07-25T00:00:00Z","timestamp":1784937600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,25]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Left-handed (Z-form) double-stranded nucleic acid conformers (Z-DNA and Z-RNA, collectively called Z-NA) are molecular patterns recognized by ADAR1 and ZBP1, which are sensor proteins involved in innate immunity. Monoclonal antibodies raised against Z-DNA, such as Z22 and Z-D11, are employed as probes for the study of Z-NAs, but their substrate specificity and functional equivalence remain unclear. Here, we used biochemical, biophysical, and structural approaches to compare the binding modes and target specificies of Z22 and Z-D11, using Z-prone CG-rich dsDNA, dsRNA, and DNA\u2013RNA hybrids as substrates. Both antibodies failed to form stable complexes with short CG-rich dsRNA under conditions compatible with antibody stability, which suggests that they are unable to induce A-to-Z transitions in dsRNA. In contrast, both antibodies robustly complexed with dsDNA and DNA\u2013RNA hybrid substrates. Cryo-EM structural analysis confirmed that both antibodies interact with Z-NA through a conserved interaction network, and demonstrated that complexes between the antibodies and DNA-RNA hybrids adopted distinct higher-order organizations. Z22 retained binding to both Z-DNA and Z-RNA segments, whereas Z-D11 displayed substrate-dependent organization and was restricted to Z-DNA segments Molecular modelling provided mechanistic explanation for the differences in Z-RNA binding ability between the two antibodies, which was confirmed\n                  <jats:italic>in situ<\/jats:italic>\n                  with ADAR1-depleted, IAV- infected and JTE607-treated cells. We also demonstrate that Z-D11, in contrast to previously described Z22, is unable to induce B-to-Z-transitions in short d(CG)\n                  <jats:sub>6<\/jats:sub>\n                  oligos under physiological conditions. Together, these results show that anti Z-NA monoclonal antibodies are not functionally interchangeable: whereas Z22 recognizes pre-formed Z-RNA, Z-DNA- RNA hybrids, and Z-DNA, Z-D11 recognizes Z-DNA, and DNA segments in Z-DNA-RNA hybrids. These differences are dictated by substrate composition, local geometry, and conformational accessibility, which also likely impact their ability to induce NA structural transitions. These findings have important implications for interpreting antibody-based detection of left-handed nucleic acids in biological systems.\n                <\/jats:p>","DOI":"10.64898\/2026.07.22.740205","type":"posted-content","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T05:31:49Z","timestamp":1785130309000},"source":"Crossref","is-referenced-by-count":0,"title":["Anti-Z-NA antibodies can distinguish accessible Z-form states across DNA, RNA, and DNA\u2013RNA substrates"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9391-9339","authenticated-orcid":false,"given":"Danielle","family":"Chin","sequence":"first","affiliation":[{"name":"Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"},{"name":"Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2806-3161","authenticated-orcid":false,"given":"Yongbo","family":"Luo","sequence":"additional","affiliation":[{"name":"Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"},{"name":"Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7058-835X","authenticated-orcid":false,"given":"Zengyting","family":"He","sequence":"additional","affiliation":[{"name":"Center for Immunology, Fox Chase Cancer Center, Philadelphia, PA 19111, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4193-7022","authenticated-orcid":false,"given":"Chaoran","family":"Yin","sequence":"additional","affiliation":[{"name":"Center for Immunology, Fox Chase Cancer Center, Philadelphia, PA 19111, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5422-0733","authenticated-orcid":false,"given":"Riley M.","family":"Williams","sequence":"additional","affiliation":[{"name":"Center for Immunology, Fox Chase Cancer Center, Philadelphia, PA 19111, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5447-738X","authenticated-orcid":false,"given":"Peter","family":"Dr\u00f6ge","sequence":"additional","affiliation":[{"name":"LambdaGentherapeutics Pte. Ltd., 1557 Keppel Road, Singapore 089066"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2084-1803","authenticated-orcid":false,"given":"Siddharth","family":"Balachandran","sequence":"additional","affiliation":[{"name":"Center for Immunology, Fox Chase Cancer Center, Philadelphia, PA 19111, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7637-7275","authenticated-orcid":false,"given":"Dahai","family":"Luo","sequence":"additional","affiliation":[{"name":"Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"},{"name":"Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921"},{"name":"National Centre for Infectious Diseases, 16 Jln Tan Tock Seng, Singapore 308442"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026072903301112000_2026.07.22.740205v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/282680a0"},{"key":"2026072903301112000_2026.07.22.740205v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/311584a0"},{"key":"2026072903301112000_2026.07.22.740205v1.3","doi-asserted-by":"crossref","unstructured":"Krall, J.B. , et al., Structure and Formation of Z-DNA and Z-RNA. 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