A One-Step Chemoselective Strategy for Hydroxymethylcytosine Sequencing in DNA and RNA
- 1University of Chicago
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.
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BibTeX
@misc{li2026onestep,
title = {{A One-Step Chemoselective Strategy for Hydroxymethylcytosine Sequencing in DNA and RNA}},
author = {J. Li and P.-H. Zhang and A. Y. Wang and Y. Zhong and Y. Wang and R. Lyu and C. Zhu and Q. Dai and C. He},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.14.751533},
url = {https://www.biorxiv.org/content/10.64898/2026.09.14.751533v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - A One-Step Chemoselective Strategy for Hydroxymethylcytosine Sequencing in DNA and RNA
AU - J. Li
AU - P.-H. Zhang
AU - A. Y. Wang
AU - Y. Zhong
AU - Y. Wang
AU - R. Lyu
AU - C. Zhu
AU - Q. Dai
AU - C. He
PY - 2026
DA - 2026/09/14
PB - bioRxiv
DO - 10.64898/2026.09.14.751533
UR - https://www.biorxiv.org/content/10.64898/2026.09.14.751533v1
AB - 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.
ER -