{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T04:26:42Z","timestamp":1787632002431,"version":"build-2736575974"},"reference-count":33,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003443","name":"The Ministry of Education and Science of the Russian Federation","doi-asserted-by":"crossref","award":["075-15-2025-473"],"award-info":[{"award-number":["075-15-2025-473"]}],"id":[{"id":"10.13039\/501100003443","id-type":"DOI","asserted-by":"crossref"}]},{"award":["075-15-2025-473"],"award-info":[{"award-number":["075-15-2025-473"]}],"id":[{"id":"https:\/\/ror.org\/00ghqgy32","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Methyl-dependent restriction endonucleases are promising tools for analyzing eukaryotic DNA methylation patterns. However, quantitative assessment of their substrate specificity requires the determination of the kinetic parameters of enzymatic reactions. Here, we present a method for determining initial reaction rates based on fluorescent probes and real-time monitoring of changes in fluorescence intensity. Initial rates of methyl-dependent GlaI and BlsI restriction endonucleases were determined as the slope of the linear part of the kinetic curves, after which the Michaelis\u2013Menten constants (KM) and reaction rates (Vmax) were calculated using nonlinear regression. For both enzymes, KM values were determined for the first time, indicating a high affinity of the methyl-dependent restriction endonucleases for methylated sites. KM values for fully methylated duplexes were in the range of (4.4\u20137.0)\u00b7102 nM for GlaI and 2.4\u201355 nM for BlsI. KM values were significantly lower for the hemimethylated duplexes: (1.9\u20138.0)\u00b7102 nM for GlaI and (0.7\u201315.0)\u00b7102 nM for BlsI; and even lower for unmethylated duplexes: (27\u201346)\u00b7102 nM for GlaI and (0.28\u201323)\u00b7102 nM for BlsI. Maximum reaction rates varied within relatively narrow limits: Vmax values were in range (4.5\u201319.5)\u00b710\u22124 nM\/s for GlaI and (1.4\u201318)\u00b710\u22124 nM\/s for BlsI, respectively. Vmax values were depended weakly on the degree of methylation compared to the KM. The proposed fluorescence method was applied to determine the kinetic parameters of methyl-dependent restriction endonucleases for the first time. It may serve as a simpler and more environmentally friendly alternative to traditional electrophoretic approaches that use radioactive labels.<\/jats:p>","DOI":"10.3390\/biom16081217","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T16:05:19Z","timestamp":1787241919000},"page":"1217","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Fluorescent Analysis of KM and Vmax Values for Methyl-Dependent Restriction Endonucleases"],"prefix":"10.3390","volume":"16","author":[{"given":"Vladislava","family":"Martyshova","sequence":"first","affiliation":[{"name":"Faculty of Natural Sciences, School of Advanced Engineering Studies, Novosibirsk State University, Lyapunova St., 1, 630090 Novosibirsk, Russia"},{"name":"Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Lavrentieva Ave., 8, 630090 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0882-8171","authenticated-orcid":false,"given":"Sergey","family":"Sedykh","sequence":"additional","affiliation":[{"name":"Faculty of Natural Sciences, School of Advanced Engineering Studies, Novosibirsk State University, Lyapunova St., 1, 630090 Novosibirsk, Russia"},{"name":"Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Lavrentieva Ave., 8, 630090 Novosibirsk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1038\/s41580-018-0016-z","article-title":"Dynamics and Function of DNA Methylation in Plants","volume":"19","author":"Zhang","year":"2018","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1016\/j.pbi.2011.03.004","article-title":"Epigenetic Contribution to Stress Adaptation in Plants","volume":"14","author":"Mirouze","year":"2011","journal-title":"Curr. Opin. Plant Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1186\/s13148-022-01382-9","article-title":"Gene Body Methylation in Cancer: Molecular Mechanisms and Clinical Applications","volume":"14","author":"Wang","year":"2022","journal-title":"Clin. Epigenet."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1007\/978-981-15-3449-2_1","article-title":"Epigenetics in Health and Disease","volume":"1253","author":"Zhang","year":"2020","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1186\/s13072-024-00548-4","article-title":"The Role of Ribosomal DNA Methylation in Embryonic Development, Aging and Diseases","volume":"17","author":"Yang","year":"2024","journal-title":"Epigenet. Chromatin"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1111\/nph.16529","article-title":"DNA Methylation in Plants: Mechanisms and Tools for Targeted Manipulation","volume":"227","year":"2020","journal-title":"New Phytol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"514","DOI":"10.1038\/s41584-020-0470-9","article-title":"Clinical Value of DNA Methylation Markers in Autoimmune Rheumatic Diseases","volume":"16","author":"Ballestar","year":"2020","journal-title":"Nat. Rev. Rheumatol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1010","DOI":"10.1101\/gad.2037511","article-title":"CpG Islands and the Regulation of Transcription","volume":"25","author":"Deaton","year":"2011","journal-title":"Genes Dev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1038\/npp.2012.112","article-title":"DNA Methylation and Its Basic Function","volume":"38","author":"Moore","year":"2013","journal-title":"Neuropsychopharmacology"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Lynch-Sutherland, C., Stockwell, P., Chatterjee, A., Eccles, M.R., and Macaulay, E. (2026). Epigenetic Deregulation of Transposable Elements Links Developmental Processes and Tumorigenesis. Int. J. Mol. Sci., 27.","DOI":"10.3390\/ijms27062690"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"9939","DOI":"10.1038\/s41467-025-66033-y","article-title":"Ultra-Mild Bisulfite Outperforms Existing Methods for 5-Methylcytosine Detection with Low Input DNA","volume":"16","author":"Dai","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1007\/978-1-61779-316-5_2","article-title":"DNA Methylation Detection: Bisulfite Genomic Sequencing Analysis","volume":"791","author":"Li","year":"2011","journal-title":"Methods Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1186\/s13059-021-02510-z","article-title":"DNA Methylation-Calling Tools for Oxford Nanopore Sequencing: A Survey and Human Epigenome-Wide Evaluation","volume":"22","author":"Liu","year":"2021","journal-title":"Genome Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"23664","DOI":"10.17161\/sjm.v2i2.23664","article-title":"Impact of Nanopore Flow Cell Types on DNA Methylation Detection","volume":"2","author":"Shi","year":"2025","journal-title":"Serican J. Med."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Pagano, L., Lagrotteria, D., Facconi, A., Saraceno, C., Longobardi, A., Bellini, S., Ingannato, A., Bagnoli, S., Ducci, T., and Mingrino, A. (2025). Evaluation of Illumina and Oxford Nanopore Sequencing for the Study of DNA Methylation in Alzheimer\u2019s Disease and Frontotemporal Dementia. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26094198"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1348","DOI":"10.1038\/s41587-021-01108-x","article-title":"Nanopore Sequencing Technology, Bioinformatics and Applications","volume":"39","author":"Wang","year":"2021","journal-title":"Nat. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1805","DOI":"10.1093\/nar\/gkg274","article-title":"A Nomenclature for Restriction Enzymes, DNA Methyltransferases, Homing Endonucleases and Their Genes","volume":"31","author":"Roberts","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"434","DOI":"10.1128\/mr.57.2.434-450.1993","article-title":"Biology of DNA Restriction","volume":"57","author":"Bickle","year":"1993","journal-title":"Microbiol. Rev."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Tarasova, G.V., Nayakshina, T.N., and Degtyarev, S.K.H. (2008). Substrate Specificity of New Methyl-Directed DNA Endonuclease GlaI. BMC Mol. Biol., 9.","DOI":"10.1186\/1471-2199-9-7"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1038\/s41467-022-34828-y","article-title":"Comparative Analysis of Genome-Scale, Base-Resolution DNA Methylation Profiles across 580 Animal Species","volume":"14","author":"Klughammer","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"568","DOI":"10.18632\/aging.100792","article-title":"Stochastic Epigenetic Mutations (DNA Methylation) Increase Exponentially in Human Aging and Correlate with X Chromosome Inactivation Skewing in Females","volume":"7","author":"Gentilini","year":"2015","journal-title":"Aging"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Chen, C.-Y., Wu, J.-J., Lin, Y.-J., Hsu, C.-H., Hu, J.-M., Chang, P.-K., Sun, C.-A., Yang, T., Su, J.-Q., and Chou, Y.-C. (2022). Significance of Hypermethylation of Tumor-Suppressor Genes PTGER4 and ZNF43 at CpG Sites in the Prognosis of Colorectal Cancer. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms231810225"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"17018","DOI":"10.1038\/s41598-017-17072-z","article-title":"Beyond the Michaelis-Menten Equation: Accurate and Efficient Estimation of Enzyme Kinetic Parameters","volume":"7","author":"Choi","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_24","first-page":"551","article-title":"Application of GLAD-PCR Assay for Study on DNA Methylation in Regulatory Regions of Some Tumor-Suppressor Genes in Lung Cancer","volume":"22","author":"Smetannikova","year":"2019","journal-title":"Zhongguo Fei Ai Za Zhi"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2089","DOI":"10.1093\/nar\/30.9.2089","article-title":"Effect of Primary and Secondary Structure of Oligodeoxyribonucleotides on the Fluorescent Properties of Conjugated Dyes","volume":"30","author":"Nazarenko","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3155","DOI":"10.1093\/nar\/22.15.3155","article-title":"Real Time Kinetics of Restriction Endonuclease Cleavage Monitored by Fluorescence Resonance Energy Transfer","volume":"22","author":"Ghosh","year":"1994","journal-title":"Nucleic Acids Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.bbrc.2009.09.125","article-title":"Expression of Small RNAs in Rhizobiales and Protection of a Small RNA and Its Degradation Products by Hfq in Sinorhizobium Meliloti","volume":"390","author":"Voss","year":"2009","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1203","DOI":"10.1093\/nar\/gkr809","article-title":"Illuminating the Reaction Pathway of the FokI Restriction Endonuclease by Fluorescence Resonance Energy Transfer","volume":"40","author":"Pernstich","year":"2012","journal-title":"Nucleic Acids Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1038\/nbt896","article-title":"FRET Imaging","volume":"21","author":"Jovin","year":"2003","journal-title":"Nat. Biotechnol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Medintz, I., and Hildebrandt, N. (2013). FRET\u2013F\u00f6rster Resonance Energy Transfer, Wiley.","DOI":"10.1002\/9783527656028"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Lakowicz, J.R. (2006). Principles of Fluorescence Spectroscopy, Springer US.","DOI":"10.1007\/978-0-387-46312-4"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1515","DOI":"10.1021\/cr960386p","article-title":"Signaling Recognition Events with Fluorescent Sensors and Switches","volume":"97","author":"Gunaratne","year":"1997","journal-title":"Chem. Rev."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3159","DOI":"10.1093\/nar\/9.13.3159","article-title":"Reaction Kinetics of Some Important Site-Specific Endonucleases","volume":"9","author":"Hinsch","year":"1981","journal-title":"Nucleic Acids Res."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/8\/1217\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T04:13:25Z","timestamp":1787631205000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/8\/1217"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,20]]},"references-count":33,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["biom16081217"],"URL":"https:\/\/doi.org\/10.3390\/biom16081217","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,20]]}}}