We designed and synthesized a series of 2'-deoxyribonucleoside triphosphates (dNTPs) bearing various lipid moieties. Fatty acid- and cholesterol-modified dNTPs proved to be substrates for KOD XL DNA polymerase in primer extension reactions. They were also mutually compatible for simultaneous multiple incorporations into the DNA strand. The methodology of enzymatic synthesis opened a pathway to diverse structurally unique lipid-ON probes containing one or more lipid units. We studied interactions of such probes with the plasma membranes of live cells. Employing a rational design, we found a series of lipid-ONs with enhanced membrane anchoring efficiency. The in-membrane stability of multiply modified ONs was superior to that of commonly studied ON analogues, in which a single cholesterol molecule is typically tethered to the thread end. Notably, some of the probes were detected at the cell surface even after 24 h upon removal of the probe solution. Such an effect was general to several studied cell lines.

• Publikační typ
• časopisecké články MeSH

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.

• Publikační typ
• časopisecké články MeSH

Thymidine triphosphate bearing benzylidene-tetrahydroxanthylium near-IR fluorophore linked to the 5-methyl group via triazole was synthesized through the CuAAC reaction and was used for polymerase synthesis of labelled DNA probes. The fluorophore lights up upon incorporation to DNA (up to 348-times) presumably due to interactions in major groove and the fluorescence further increases in the single-stranded oligonucleotide. The labelled dsDNA senses binding of small molecules and proteins by a strong decrease of fluorescence. The nucleotide was used as a light-up building block in real-time PCR for detection of SARS-CoV-2 virus.

• Klíčová slova
• DNA, fluorescence, nucleotides, real-time PCR,
• MeSH
• COVID-19 * MeSH
• DNA sondy MeSH
• lidé MeSH
• nukleotidy MeSH
• replikace DNA * MeSH
• SARS-CoV-2 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA sondy MeSH
• nukleotidy MeSH

We report proof of principle biomimetic switching of transcription in vitro through non-natural chemical reactions in the major groove of DNA templates. Photocaged DNA templates containing nitrobenzyl-protected 5-hydroxymethyluracil or - cytosine permitted no transcription with E. coli RNA polymerase (OFF state). Their irradiation with 400 nm light resulted in DNA templates containing hydroxymethylpyrimidines, which switched transcription ON with a higher yield (250-350%) compared to non-modified DNA. Phosphorylation of templates containing 5-hydroxymethyluracil (but not 5-hydroxymethylcytosine) then turned transcription OFF again. It is the first step towards artificial bioorthogonal chemical epigenetics.

• Publikační typ
• časopisecké články MeSH