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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 ASEP 0570750 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title trans-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, ORCIDSource Title Bioconjugate Chemistry. - : American Chemical Society - ISSN 1043-1802
Roč. 34, č. 4 (2023), s. 772-780Number of pages 9 s. Language eng - English Country US - United States Keywords triphosphates OECD category Organic chemistry R&D Projects GX20-00885X GA ČR - Czech Science Foundation (CSF) EF16_019/0000729 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Method of publishing Open access Institutional support UOCHB-X - RVO:61388963 UT WOS 000958185200001 DOI 10.1021/acs.bioconjchem.3c00064 Annotation A 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. Workplace Institute of Organic Chemistry and Biochemistry Contact asep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Jana Procházková, Tel.: 220 183 418 Year of Publishing 2024 Electronic address https://doi.org/10.1021/acs.bioconjchem.3c00064
Number of the records: 1