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trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging

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    SYSNO ASEP0570750
    Document TypeJ - Journal Article
    R&D Document TypeJournal Article
    Subsidiary JČlánek ve WOS
    Titletrans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging
    Author(s) Spampinato, Ambra (UOCHB-X)
    Kužmová, Erika (UOCHB-X) ORCID
    Pohl, Radek (UOCHB-X) RID, ORCID
    Sýkorová, Veronika (UOCHB-X) ORCID, RID
    Vrábel, Milan (UOCHB-X) RID, ORCID
    Kraus, Tomáš (UOCHB-X) RID, ORCID
    Hocek, Michal (UOCHB-X) RID, ORCID
    Source TitleBioconjugate Chemistry. - : American Chemical Society - ISSN 1043-1802
    Roč. 34, č. 4 (2023), s. 772-780
    Number of pages9 s.
    Languageeng - English
    CountryUS - United States
    Keywordstriphosphates
    OECD categoryOrganic chemistry
    R&D ProjectsGX20-00885X GA ČR - Czech Science Foundation (CSF)
    EF16_019/0000729 GA MŠMT - Ministry of Education, Youth and Sports (MEYS)
    Method of publishingOpen access
    Institutional supportUOCHB-X - RVO:61388963
    UT WOS000958185200001
    DOI10.1021/acs.bioconjchem.3c00064
    AnnotationA series of 2′-deoxyribonucleoside triphosphates (dNTPs) bearing 2- or 4-linked trans-cyclooctene (TCO) or bicyclononyne (BCN) tethered through a shorter propargylcarbamate or longer triethyleneglycol-based spacer were designed and synthesized. They were found to be good substrates for KOD XL DNA polymerase for primer extension enzymatic synthesis of modified oligonucleotides. We systematically tested and compared the reactivity of TCO- and BCN-modified nucleotides and DNA with several fluorophore-containing tetrazines in inverse electron-demand Diels–Alder (IEDDA) click reactions to show that the longer linker is crucial for efficient labeling. The modified dNTPs were transported into live cells using the synthetic transporter SNTT1, incubated for 1 h, and then treated with tetrazine conjugates. The PEG3-linked 4TCO and BCN nucleotides showed efficient incorporation into genomic DNA and good reactivity in the IEDDA click reaction with tetrazines to allow staining of DNA and imaging of DNA synthesis in live cells within time periods as short as 15 min. The BCN-linked nucleotide in combination with TAMRA-linked (TAMRA = carboxytetramethylrhodamine) tetrazine was also efficiently used for staining of DNA for flow cytometry. This methodology is a new approach for in cellulo metabolic labeling and imaging of DNA synthesis which is shorter, operationally simple, and overcomes several problems of previously used methods.
    WorkplaceInstitute of Organic Chemistry and Biochemistry
    Contactasep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Jana Procházková, Tel.: 220 183 418
    Year of Publishing2024
    Electronic addresshttps://doi.org/10.1021/acs.bioconjchem.3c00064
Number of the records: 1  

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