DNA Oligonucleotide 3'-Phosphorylation by a DNA Enzyme.
DNA Oligonucleotide 3'-Phosphorylation by a DNA Enzyme.
复制标题
DOI：
10.1021/acs.biochem.6b00151
复制
发表时间：
2016-05-10
期刊：
影响因子：
2.9
通讯作者：
Silverman SK
中科院分区：
文献类型：
--
作者：
Camden AJ;Walsh SM;Suk SH;Silverman SK
T4多核苷酸激酶广泛用于DNA和RNA寡核苷酸末端的5′-磷酸化，但没有天然蛋白酶能够3′-磷酸化。在这里，我们报告的脱氧核酶（DNA酶）的体外选择能够DNA寡核苷酸3′-磷酸化，使用5′-三磷酸化的RNA转录本（pppRNA）作为磷酰基供体。选择的基础是在每轮选择期间，通过2-甲基咪唑活化3 ′-磷酸（形成3′-MeImp）并随后与5′-氨基DNA寡核苷酸进行夹板连接来捕获3′-磷酸化的DNA底物末端。竞争性的和先前的DNA催化反应是DNA磷酸二酯水解或去糖基化，每种反应也会导致3′-磷酸，但在DNA底物内的不同核苷酸位置。从N40随机库中获得的一种寡核苷酸3′-激酶脱氧核酶命名为3′-Kin 1，它能3′-磷酸化几乎所有3′-末端具有序列基序5′-NKR-3′的DNA寡核苷酸底物，其中N表示任何寡核苷酸序列，K = T或G，R = A或G。这些结果确立了体外筛选鉴定3′-磷酸化DNA寡核苷酸的DNA酶的可行性。
T4 polynucleotide kinase is widely used for 5′-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3′-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3′-phosphorylation, using a 5′-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3′-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3′-phosphate (forming 3′-MeImp) and subsequent splint ligation with a 5′-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3′-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3′-kinase deoxyribozyme, obtained from an N40 random pool and named 3′Kin1, can 3′-phosphorylate nearly any DNA oligonucleotide substrate for which the 3′-terminus has the sequence motif 5′-NKR-3′, where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3′-phosphorylate DNA oligonucleotides.