{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T07:05:50Z","timestamp":1790665550784,"version":"4.1.0"},"reference-count":31,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>Site-specific 5-hydroxymethylcytosine (5hmC) has been identified as a potential biomarker involving diagnosis, prognosis, and tumor immune microenvironment across multiple cancers. However, directly distinguishing trace levels of 5hmC from its highly abundant cytosine\/5-methylcytosine (C\/5mC) analogs remains a technical challenge. Herein, we present a 5hmC selective ligation-rolling circle amplification (5hmC-RCA) that enables the direct, high-specificity discrimination of 5hmC from C\/5mC. The high specificity of 5hmC-RCA depends on the high selectivity of HiFi Taq DNA Ligase, which exhibits robust tolerance for mismatched 5hmC\/A base pairs, thereby efficiently ligating the associated nicking site. While the mismatched 5mC\/A or C\/A pairs fail to be recognized by HiFi Taq DNA Ligase. As a proof of principle, the 5hmC-RCA assay was evaluated for its performance using Septin9 gene, which considered as key biomarkers in the prognosis and diagnosis of CRC, as the target, demonstrating a detection sensitivity of approximately 0.5%. Notably, the assay exhibited no cross-reactivity with unmodified target even at a high background concentration of 5,000 copies per reaction. By coupling HiFi Taq DNA Ligase-mediated high selective ligation with RCA, sensitive and selective quantification of site-specific 5hmC is achieved. Due to the simplicity of 5hmC-RCA, it also has potential for use in cfDNA originating from the tumor microenvironment and immune cells.<\/jats:p>","DOI":"10.3389\/fimmu.2026.1960016","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T06:34:00Z","timestamp":1790663640000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Selective rolling circle amplification detection of site-specific DNA 5-hydroxymethylcytosine"],"prefix":"10.3389","volume":"17","author":[{"given":"Ningning","family":"Dong","sequence":"first","affiliation":[{"name":"Department of Oncology, The First Affiliated Hospital of Zhengzhou University","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing","family":"Hong","sequence":"additional","affiliation":[{"name":"Department of Oncology, The First Affiliated Hospital of Zhengzhou University","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Sui","sequence":"additional","affiliation":[{"name":"Department of Oncology, The First Affiliated Hospital of Zhengzhou University","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Yan","sequence":"additional","affiliation":[{"name":"Department of Oncology, The First Affiliated Hospital of Zhengzhou University","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaping","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of Oncology, The First Affiliated Hospital of Zhengzhou University","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zehua","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Oncology, The First Affiliated Hospital of Zhengzhou University","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yao","family":"Luo","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Sichuan Medical Laboratory Clinical Medicine Research Center, West China Hospital, Sichuan University","place":["Chengdu, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanru","family":"Qin","sequence":"additional","affiliation":[{"name":"Department of Oncology, The First Affiliated Hospital of Zhengzhou University","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,29]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.9b11393","article-title":"Differentiated visualization of single-cell 5-hydroxymethylpyrimidines with microfluidic hydrogel encoding","volume":"142","author":"Chen","year":"2020","journal-title":"J Am Chem 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