{
  "abstract": "5-Hydroxymethylcytosine (5hmC) is a relatively stable chemical mark formed by oxidation of 5-methylcytosine in DNA, while its RNA counterparts, 5-hydroxymethylcytidine (hm5C) and 2'-O-methyl-5-hydroxymethylcytidine (hm5Cm), have recently emerged as potential regulators of post-transcriptional processes. Existing 5hmC enrichment methods rely on anti-bodies or enzymatic {beta}-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 hm5C/hm5Cm-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.",
  "abstract_shown": true,
  "affiliations": [
    "University of Chicago"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 9,
  "authors": [
    {
      "name": "J. Li"
    },
    {
      "name": "P.-H. Zhang"
    },
    {
      "name": "A. Y. Wang"
    },
    {
      "name": "Y. Zhong"
    },
    {
      "name": "Y. Wang"
    },
    {
      "name": "R. Lyu"
    },
    {
      "name": "C. Zhu"
    },
    {
      "name": "Q. Dai"
    },
    {
      "affs": [
        0
      ],
      "name": "C. He"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/10dfa2twcy6nv.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.14.751533",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.14.751533v1.full",
  "id": "10dfa2twcy6nv",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.14.751533v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "genomics"
    ],
    [
      "openalex",
      "1312|Epigenetics and DNA Methylation"
    ]
  ],
  "openalex": "W7213527501",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.14.751533v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-14",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/10dfa2twcy6nv.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:22:55Z",
      "native_id": "10.64898/2026.09.14.751533",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.14.751533v1"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "biorxiv:genomics",
      "subfield": "genetics-genomics"
    }
  ],
  "title": "A One-Step Chemoselective Strategy for Hydroxymethylcytosine Sequencing in DNA and RNA",
  "title_text": "A One-Step Chemoselective Strategy for Hydroxymethylcytosine Sequencing in DNA and RNA",
  "type": "preprint",
  "updated_at": "2026-10-30T12:22:55Z",
  "url": "https://research.draconitic.com/paper/10dfa2twcy6nv",
  "venue": "bioRxiv",
  "versions": []
}