Y639F/H784A T7 RNA聚合酶双突变体在使用非标准NTP合成RNA方面表现出优异特性。
A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs.
作者信息
Padilla Robert, Sousa Rui
机构信息
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
出版信息
Nucleic Acids Res. 2002 Dec 15;30(24):e138. doi: 10.1093/nar/gnf138.
Abstract
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'-N3), 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.
摘要
酪氨酸639突变为苯丙氨酸的T7 RNA聚合酶能够轻易地将2'-脱氧、2'-氨基和2'-氟核糖核苷三磷酸用作底物，并且已被广泛用于合成各种应用的修饰RNA。该突变体不易使用具有较大2'-取代基的核糖核苷三磷酸，也不利于掺入在其他位置具有修饰的核糖核苷三磷酸。在Y639F背景中引入第二个突变（H784A）可显著提高对具有较大2'-取代基（2'-甲氧基和2'-叠氮基）的核糖核苷三磷酸的利用率，并且还可能提高对2'-位以外具有修饰的核糖核苷三磷酸的使用。因此，Y639F/H784A双突变体对于将各种非规范的核糖核苷酸掺入RNA可能格外有用。
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