{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T16:00:20Z","timestamp":1790006420358,"version":"4.0.1"},"posted":{"date-parts":[[2026,9,17]]},"group-title":"Genomics","reference-count":41,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institute of Health","award":["R01 HG006827"],"award-info":[{"award-number":["R01 HG006827"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,17]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  5-Hydroxymethylcytosine (5hmC) is a relatively stable chemical mark formed by oxidation of 5-methylcytosine in DNA, while its RNA counterparts, 5-hydroxymethylcytidine (hm\n                  <jats:sup>5<\/jats:sup>\n                  C) and 2\u201d-\n                  <jats:italic>O<\/jats:italic>\n                  -methyl-5-hydroxymethylcytidine (hm\n                  <jats:sup>5<\/jats:sup>\n                  C\n                  <jats:italic>m<\/jats:italic>\n                  ), have recently emerged as potential regulators of post-transcriptional processes. Existing 5hmC enrichment methods rely on antibodies or enzymatic\n                  <jats:italic>\u03b2<\/jats:italic>\n                  -glucosyltransferase labeling, are largely DNA-specific, and are not readily applicable to RNA. Here we report bisulfite-assisted labeling by thiol sequencing (BALT-seq), a one-step chemoselective strategy that installs a biotin-thioether handle directly onto hydroxymethylcytosine in DNA or RNA under mild acidic bisulfite conditions, without using enzymes. Mechanistic studies show that thiol capture of the bisulfite-activated 5hmC intermediate competes with canonical cytosine-5-methylenesulfonate formation, yielding a stable adduct that DNA polymerases bypass efficiently and that resists APOBEC-mediated deamination. In mouse embryonic stem cells, BALT-seq produces genome-wide 5hmC profiles highly concordant with 5hmC-Seal, TAB-seq, and oxBS-seq, recovers expected gene-body and chromatin-state enrichment, and extends to low-input cell-free DNA. On RNA, BALT-seq enriches hm\n                  <jats:sup>5<\/jats:sup>\n                  C\/hm\n                  <jats:sup>5<\/jats:sup>\n                  C\n                  <jats:italic>m<\/jats:italic>\n                  -containing transcripts more effectively than antibody-based hMeRIP and reveals structured modification landscapes across tRNA species and LINE\/LTR repeat families. BALT-seq thus establishes a unified, enzyme-free chemical platform for hydroxymethylcytosine profiling across DNA, cell-free DNA, and RNA.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Abstract Figure<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"751533v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.14.751533","type":"posted-content","created":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T20:50:16Z","timestamp":1789678216000},"source":"Crossref","is-referenced-by-count":0,"title":["A One-Step Chemoselective Strategy for Hydroxymethylcytosine Sequencing in DNA and RNA"],"prefix":"10.64898","author":[{"given":"Jiahao","family":"Li","sequence":"first","affiliation":[{"name":"Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA"},{"name":"Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pei-Hong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA"},{"name":"Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanchu Arvin","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA"},{"name":"Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuhao","family":"Zhong","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA"},{"name":"Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiding","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA"},{"name":"Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruitu","family":"Lyu","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA"},{"name":"Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenyou","family":"Zhu","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA"},{"name":"Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing","family":"Dai","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA"},{"name":"Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chuan","family":"He","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA"},{"name":"Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, 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