A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs
A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs
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DOI：
10.1093/nar/gnf138
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发表时间：
2002-12-15
影响因子：
14.9
通讯作者：
Sousa, R
中科院分区：
文献类型：
--
作者：
Padilla, R;Sousa, R
Tyr639突变为Phe的T7 RNA聚合酶很容易利用2'-脱氧，2'-NH2和2'-F ntp作为底物，广泛用于合成各种应用的修饰RNA。该突变体不容易利用具有较大2'-取代基的NTPs，也不容易将NTPs与其他位置的修饰结合。在Y639F背景中引入第二个突变（H784A）显著提高了2'-取代基（2'-OMe和2'-N-3）的NTPs的利用率，也可能提高了2'-位置以外修饰的NTPs的利用率。因此，Y639F/H784A双突变体可能对将各种非规范nmp整合到RNA中特别有用。
A T7 RNA polymerase in which Tyr639 is mutated to Phe readily utilizes 2'-deoxy, 2'-NH2 and 2'-F NTPs as substrates and has been widely used to synthesize modified RNAs for a variety of applications. This mutant does not readily utilize NTPs with bulkier 2'-substituents, nor does it facilitate incorporation of NTPs with modifications at other positions. Introduction of a second mutation (H784A) into the Y639F background markedly enhances utilization of NTPs with bulky 2'-substituents (2'-OMe and 2'-N-3), and may also enhance use of NTPs with modifications at other than the 2'-position. The Y639F/H784A double mutant may therefore be exceptionally useful for incorporation of a variety of non-canonical NMPs into RNA.