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Their abnormalities in structure and composition, such as gene mutations and epigenetic modifications, can trigger multiple diseases. Precise discrimination of bases is critical yet challenging due to their high structural similarity. Herein, we report triphenylamine-based covalent organic framework nanosheets (TPA CONs) as a fluorescent biosensor for nucleobase discrimination and deoxyribonucleic acid (DNA) methylation quantitative profiling, driven by electrostatic potential-mediated base selectivity. Combined experimental and computational studies reveal that the electrostatic potentials of four bases govern their affinity to the negatively charged CONs following an order of thymine (T) &amp;gt; guanine (G) &amp;gt; adenine (A) &amp;gt; cytosine (C). Consequently, T-rich sequences interact strongly with CONs and trigger efficient fluorescence quenching via photoinduced electron transfer, while C-rich sequences restore fluorescence. Leveraging this property, we detected human estrogen receptor \u03b1 (ER\u03b1) gene regional methylation in cultured breast cancer cells with a limit of detection of 2.4% and further extended the platform to human hepatocellular carcinoma (HCC) specimens for eyes absent homologue 2 (EYA2) gene methylation analysis, yielding results highly consistent with gold standard pyrosequencing. This work is the first to exploit COF for direct nucleobase discrimination and quantitative DNA methylation analysis, elucidating the COF-nucleobase interaction mechanism and highlighting their potential for epigenetic detection.<\/jats:p>","DOI":"10.1021\/jacs.5c23031","type":"journal-article","created":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T00:44:17Z","timestamp":1780361057000},"page":"23611-23627","source":"Crossref","is-referenced-by-count":2,"title":["Electrostatic-Driven\nNucleobase Discrimination by\nCovalent Organic Framework Nanosheets for Deoxyribonucleic Acid Methylation\nProfiling"],"prefix":"10.1021","volume":"148","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-3308-5414","authenticated-orcid":true,"given":"Rongjie","family":"Yang","sequence":"first","affiliation":[{"name":"Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences , , ,","place":["Suzhou, China, 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230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lin","family":"Zhou","sequence":"additional","affiliation":[{"name":"Zhejiang University School of Medicine, National Clinical Research Center for Children\u2019s Health , , ,","place":["Hangzhou, China, 310052"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9003-4279","authenticated-orcid":true,"given":"Jia","family":"Yao","sequence":"additional","affiliation":[{"name":"Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences , , ,","place":["Suzhou, China, 215163"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingxue","family":"Li","sequence":"additional","affiliation":[{"name":"Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences , , ,","place":["Suzhou, China, 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