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Their function critically depends on their structural stability and nonspecific interactions in cells. However, the behavior of exogenously delivered G4s in living cells remains poorly understood. Here, using 19F NMR, we monitored three representative G4 sequences, HT, hVEGFP, and TBA, in intact Xenopus laevis oocytes, HeLa cells, and derived lysates. We found that G4 structural stability, interactions, and NMR signal detectability are cell-type-dependent. In oocytes, all three G4s maintain detectable folded signals, with TBA showing slight time-dependent degradation. In HeLa cells, folded signals are absent for all three G4s due to intracellular interactions. HT and TBA undergo degradation in the cytoplasm rather than in the nucleus, whereas hVEGFP uniquely remains protected from degradation owing to both its higher thermal stability and protective interactions. These findings highlight the non-negligible effect of the cellular environment and the necessity of studying G4s in their native cellular context. Furthermore, we demonstrated that cell lysates can serve as a practical platform for reflecting G4 stability in intact cells. Collectively, this work deepens our understanding of G4 behavior in cells and provides a basis for improvement of G4-based agents.<\/jats:p>","DOI":"10.1021\/jacsau.6c01086","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T03:17:40Z","timestamp":1788319060000},"page":"5410-5419","source":"Crossref","is-referenced-by-count":0,"title":["Assessing G-Quadruplex\nStability and Nonspecific\nInteractions in Living Cells Using 19F NMR"],"prefix":"10.1021","volume":"6","author":[{"given":"Qiong","family":"Wu","sequence":"first","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenwen","family":"Zheng","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chen","family":"Wang","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiajing","family":"Zhao","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]},{"name":"Xi\u2019an Modern Chemistry Research Institute , , ,","place":["Xi\u2019an, P.R. China, 710065"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoli","family":"Liu","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]},{"name":"Graduate University of Chinese Academy of Sciences , , ,","place":["Beijing, P.R. China, 100049"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaofei","family":"Chai","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]},{"name":"Graduate University of Chinese Academy of Sciences , , ,","place":["Beijing, P.R. China, 100049"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing","family":"Liu","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinbo","family":"Yu","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9478-6056","authenticated-orcid":true,"given":"Yansheng","family":"Ye","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]},{"name":"Graduate University of Chinese Academy of Sciences , , ,","place":["Beijing, P.R. China, 100049"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guohua","family":"Xu","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]},{"name":"Graduate University of Chinese Academy of Sciences , , ,","place":["Beijing, P.R. China, 100049"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maili","family":"Liu","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]},{"name":"Graduate University of Chinese Academy of Sciences , , ,","place":["Beijing, P.R. China, 100049"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5798-1722","authenticated-orcid":true,"given":"Conggang","family":"Li","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , , ,","place":["Wuhan, P.R. China, 430071"]},{"name":"Graduate University of Chinese Academy of Sciences , , ,","place":["Beijing, P.R. 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Drug Del."},{"key":"2026092808091206300_cit14","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1038\/425027a","article-title":"Join the crowd","volume":"425","author":"Ellis","year":"2003","journal-title":"Nature"},{"key":"2026092808091206300_cit15","doi-asserted-by":"publisher","first-page":"114","DOI":"10.1016\/S0959-440X(00)00172-X","article-title":"Macromolecular\ncrowding: an important but neglected aspect of the intracellular environment","volume":"11","author":"Ellis","year":"2001","journal-title":"Curr. Opin. Struc. Biol."},{"key":"2026092808091206300_cit16","doi-asserted-by":"publisher","first-page":"12444","DOI":"10.1073\/pnas.231479198","article-title":"A human nuclease\nspecific for G4\nDNA","volume":"98","author":"Sun","year":"2001","journal-title":"Proc. Natl. Acad. Sci. 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Magn.\nReson."},{"key":"2026092808091206300_cit23","doi-asserted-by":"publisher","first-page":"171","DOI":"10.1146\/annurev-biophys-051013-023136","article-title":"Live cell NMR","volume":"43","author":"Freedberg","year":"2014","journal-title":"Annu. Rev. Biophys."},{"key":"2026092808091206300_cit24","doi-asserted-by":"publisher","first-page":"9497","DOI":"10.1021\/acs.chemrev.1c00937","article-title":"In-cell\nstructural biology by NMR: the benefits of the atomic scale","volume":"122","author":"Theillet","year":"2022","journal-title":"Chem. Rev."},{"key":"2026092808091206300_cit25","doi-asserted-by":"publisher","first-page":"676","DOI":"10.1038\/s41592-019-0525-5","article-title":"Atomic-level\nin-cell protein NMR","volume":"16","author":"Singh","year":"2019","journal-title":"Nat. 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Soc."},{"key":"2026092808091206300_cit35","doi-asserted-by":"publisher","first-page":"14317","DOI":"10.1021\/bi011751w","article-title":"In-cell NMR spectroscopy","volume":"40","author":"Serber","year":"2001","journal-title":"Biochemistry"},{"key":"2026092808091206300_cit36","doi-asserted-by":"publisher","first-page":"171","DOI":"10.1146\/annurev-biophys-051013-023136","article-title":"Live cell NMR","volume":"43","author":"Freedberg","year":"2014","journal-title":"Annu. Rev. Biophys."},{"key":"2026092808091206300_cit37","doi-asserted-by":"publisher","first-page":"5501","DOI":"10.1093\/nar\/gkx109","article-title":"Characterization\nof human telomere\nRNA G-quadruplex structures in vitro and in living cells using 19F NMR spectroscopy","volume":"45","author":"Bao","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"2026092808091206300_cit38","doi-asserted-by":"publisher","first-page":"1992","DOI":"10.1038\/s41467-024-46221-y","article-title":"In-cell NMR suggests that DNA i-motif levels\nare strongly depleted in living human cells","volume":"15","author":"Vi\u0301s\u030ckova\u0301","year":"2024","journal-title":"Nat. Commun."},{"key":"2026092808091206300_cit39","doi-asserted-by":"publisher","first-page":"6042","DOI":"10.3390\/ijms22116042","article-title":"Towards profiling of the G-quadruplex targeting drugs\nin the living human cells using NMR spectroscopy","volume":"22","author":"Krafcik","year":"2021","journal-title":"Int. J. Mol. Sci."},{"key":"2026092808091206300_cit40","doi-asserted-by":"publisher","first-page":"6547","DOI":"10.1039\/D0CC02053B","article-title":"Telomeric DNA-RNA-hybrid\nG-quadruplex exists in environmental conditions\nof HeLa cells","volume":"56","author":"Bao","year":"2020","journal-title":"Chem. Commun."},{"key":"2026092808091206300_cit41","doi-asserted-by":"publisher","first-page":"407","DOI":"10.1007\/978-1-4939-9666-7_26","article-title":"19F NMR spectroscopy for\nthe analysis of DNA G-quadruplex structures using 19F-labeled\nnucleobase","volume":"2035","author":"Ishizuka","year":"2019","journal-title":"Methods Mol. Biol."},{"key":"2026092808091206300_cit42","doi-asserted-by":"publisher","first-page":"4940","DOI":"10.1093\/nar\/gkz276","article-title":"Hybrid-type\nand two-tetrad antiparallel\ntelomere DNA G-quadruplex structures in living human cells","volume":"47","author":"Bao","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"2026092808091206300_cit43","doi-asserted-by":"publisher","first-page":"12622","DOI":"10.1021\/jacs.8b08436","article-title":"A dual-app nucleoside probe provides\nstructural insights into the human telomeric overhang in live cells","volume":"140","author":"Manna","year":"2018","journal-title":"J. Am. Chem. Soc."},{"key":"2026092808091206300_cit44","doi-asserted-by":"publisher","first-page":"652","DOI":"10.1038\/nprot.2017.156","article-title":"Investigation of higher-order RNA G-quadruplex structures in vitro\nand in living cells by 19F NMR spectroscopy","volume":"13","author":"Bao","year":"2018","journal-title":"Nat. Protoc."},{"key":"2026092808091206300_cit45","doi-asserted-by":"publisher","first-page":"3314","DOI":"10.1039\/C4SC03853C","article-title":"G-quadruplex DNA\nand ligand interaction in living cells using NMR spectroscopy","volume":"6","author":"Salgado","year":"2015","journal-title":"Chem. Sci."},{"key":"2026092808091206300_cit46","doi-asserted-by":"publisher","first-page":"9963","DOI":"10.1021\/ja062791w","article-title":"Structure\nof the human telomere in K+ solution: an intramolecular\n(3 + 1) G-quadruplex scaffold","volume":"128","author":"Luu","year":"2006","journal-title":"J. Am. Chem.\nSoc."},{"key":"2026092808091206300_cit47","doi-asserted-by":"publisher","first-page":"10584","DOI":"10.1093\/nar\/gkt784","article-title":"Solution\nstructure of the major G-quadruplex formed in the human VEGF promoter\nin K+: insights into loop interactions of the parallel\nG-quadruplexes","volume":"41","author":"Agrawal","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"2026092808091206300_cit48","doi-asserted-by":"publisher","first-page":"3745","DOI":"10.1073\/pnas.90.8.3745","article-title":"Thrombin-binding\nDNA aptamer forms a unimolecular quadruplex structure in solution","volume":"90","author":"Macaya","year":"1993","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026092808091206300_cit49","doi-asserted-by":"publisher","first-page":"17651","DOI":"10.1016\/S0021-9258(17)46749-4","article-title":"The structure\nof alpha-thrombin inhibited by a 15-mer single-stranded DNA aptamer","volume":"268","author":"Padmanabhan","year":"1993","journal-title":"J. Biol. Chem."},{"key":"2026092808091206300_cit50","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1016\/S0005-2736(99)00135-2","article-title":"Ion currents\nof Xenopus laevis oocytes: state of the art","volume":"1421","author":"Weber","year":"1999","journal-title":"Biochim. Biophys. Acta-Biomembranes"},{"key":"2026092808091206300_cit51","doi-asserted-by":"publisher","first-page":"5328","DOI":"10.1002\/anie.201500261","article-title":"Direct observation\nof Ca2+-induced calmodulin conformational transitions in\nintact xenopus laevis oocytes by 19F NMR spectroscopy","volume":"54","author":"Ye","year":"2015","journal-title":"Angew. Chem., Int. 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