Thiophene-linked tetramethylbodipy-labeled nucleotide for viscosity-sensitive oligonucleotide probes of hybridization and protein-DNA interactions.,Organic & Biomolecular Chemistry

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Thiophene-linked tetramethylbodipy-labeled nucleotide for viscosity-sensitive oligonucleotide probes of hybridization and protein-DNA interactions.
Organic & Biomolecular Chemistry ( IF 2.9 ) Pub Date : 2020-01-10 , DOI: 10.1039/c9ob02634g
Pedro Güixens-Gallardo ₁ , Jana Humpolickova ₂ , Sebastian Paul Miclea ₂ , Radek Pohl ₂ , Tomáš Kraus ₂ , Piotr Jurkiewicz ₃ , Martin Hof ₃ , Michal Hocek ₁

Affiliation

Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.

中文翻译：

噻吩连接的四甲基二联体标记的核苷酸，用于粘度敏感的寡核苷酸探针的杂交和蛋白质-DNA相互作用。

设计并合成了胞嘧啶2'-脱氧核糖核苷dCTBdp及其三磷酸（dCTBdpTP），其在位置5处带有四甲基化的噻吩-二联荧光团。绿色荧光核苷dCTBdp显示出荧光寿命与粘度的完美关系。修饰的三磷酸dCTBdpTP是几种DNA聚合酶的底物，并用于通过引物延伸的体外酶促合成标记寡核苷酸（ON）或DNA。标记的单链ONs与DNA或RNA的互补链杂交时，平均荧光寿命显着降低，并且对错配也敏感。标记的dsDNA感知到蛋白结合（p53），从而延长了其荧光寿命。

更新日期：2020-02-13

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