Protected 5-(hydroxymethyl) uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA
Protected 5-(hydroxymethyl) uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA
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DOI：
10.1039/c8ob00160j
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发表时间：
2018-03-07
影响因子：
3.2
通讯作者：
Hocek, Michal
中科院分区：
文献类型：
--
作者：
Bohacova, Sona;Ludvikova, Lucie;Hocek, Michal
制备并测试了含有受光裂解基团（2-硝基苄基、6-硝基胡椒基或 9-蒽甲基）保护的 5-（羟甲基）尿嘧啶的核苷、核苷酸和 2'-脱氧核糖核苷三磷酸 (dNTP)，作为光笼寡核苷酸聚合酶合成的构建模块 和DNA。在模型核苷单磷酸上详细研究了光脱保护（光释放）反应，并测定了它们的光反应量子产率。然后测试光笼 dNTP 并将其用作引物延伸或 PCR 中 DNA 聚合酶的底物。制备了限制性内切酶识别序列中含有光笼或游离5-羟甲基U的DNA探针，用于研究不同波长的紫外或可见光对笼中DNA的光释放。硝基胡椒基保护的核苷酸被发现是一种优异的构建模块，因为相应的 dNTP 是 DNA 聚合酶的良好底物，并且保护基团可通过紫外线或可见光（高达 425 nm）的照射有效裂解。
Nucleosides, nucleotides and 2'-deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxy-methyl) uracil protected with photocleavable groups (2-nitrobenzyl-, 6-nitropiperonyl or 9-anthrylmethyl) were prepared and tested as building blocks for the polymerase synthesis of photocaged oligonucleotides and DNA. Photodeprotection (photorelease) reactions were studied in detail on model nucleoside mono-phosphates and their photoreaction quantum yields were determined. Photocaged dNTPs were then tested and used as substrates for DNA polymerases in primer extension or PCR. DNA probes containing photocaged or free 5-hydroxymethylU in the recognition sequence of restriction endonucleases were prepared and used for the study of photorelease of caged DNA by UV or visible light at different wavelengths. The nitropiperonyl-protected nucleotide was found to be a superior building block because the corresponding dNTP is a good substrate for DNA polymerases, and the protecting group is efficiently cleavable by irradiation by UV or visible light (up to 425 nm).