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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

Publikace na Přírodovědecká fakulta |
2023

Tento text není v aktuálním jazyce dostupný. Zobrazuje se verze "en".Abstrakt

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 react i v i t y 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 react i v i t y in the IEDDA click reaction with tetrazines to allow staining of DNA and imaging of DNA synthesis in live cells wit h i n 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.

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