Christian E Loo, Jared B Parker, Aleksia Barka, Ruiyao Zhu, Matthew R Schnell, Rosella G Cuomo, Heqiao Zhu, Laura Liu, Ashley Chen, Jasleen Gill, Wanding Zhou, Rahul Kohli. Unnatural Cytosine Analogs Potentiate a Customizable, Enzymatic Method for Integrated Epigenetic and Four-Base Genetic Sequencing. J Am Chem Soc. 2026 Mar 11. doi: 10.1021/jacs.5c18450. Epub ahead of print. PMID: 41812666.

Abstract

The interplay of genetic and epigenetic information shapes cell identity, development, and disease. However, standard methods for profiling DNA modifications (e.g., bisulfite sequencing) rely on selective C-to-T conversions, hindering the simultaneous examination of both genetic and epigenetic information. Here, we introduce Integrated Sequencing, which provides high-fidelity mapping of DNA modifications while preserving the native four-base genetic code in single DNA molecules. Integrated-Seq leverages the synthesis of a tethered copy strand with unnatural cytosine analogs that resist enzymatic conversion, combined with a novel DNA deaminase-helicase fusion that drives selective C-to-T conversion of natural cytosines in the original template strand. We demonstrate that Integrated-Seq is compatible with customizable enzymatic readouts to parse 5-methylcytosine and 5-hydroxymethylcytosine, and that preservation of the original four-base genetic code markedly improved enrichment, facilitating analysis of targeted genomic regions. Integrated-Seq thus provides a platform for simultaneous genetic and epigenetic analyses, paving the way for deep insights into fundamental biology and next-generation diagnostics.