Synthesis of 2'-Fluoro RNA by Syn5 RNA polymerase.
Synthesis of 2'-Fluoro RNA by Syn5 RNA polymerase.
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DOI：
10.1093/nar/gkv367
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发表时间：
2015-08-18
影响因子：
14.9
通讯作者：
Richardson CC
中科院分区：
文献类型：
--
作者：
Zhu B;Hernandez A;Tan M;Wollenhaupt J;Tabor S;Richardson CC
用2‘-氟取代了RNA中的2’-羟基，大大提高了RNA的稳定性。RNA的稳定性是涉及RNA的RNA研究和应用中的一个主要问题。我们报道了海洋噬菌体Syn5的RNA聚合酶对2‘-氟代dNMPs在转录延伸过程中的掺入具有固有的低分辨能力。高浓度的镁和锰离子的存在进一步降低了这种辨别力，而不会降低掺入效率。我们构建了一个用苯丙氨酸(Y564F)取代酪氨酸564的Syn5 RNA聚合酶，进一步降低了在RNA合成过程中对2‘-氟-dNTPs的识别能力。DNA模板中影响RNA产量和Syn5 RNA聚合酶掺入2‘-氟-dNMPs的序列元件已被确定。
The substitution of 2′-fluoro for 2′-hydroxyl moieties in RNA substantially improves the stability of RNA. RNA stability is a major issue in RNA research and applications involving RNA. We report that the RNA polymerase from the marine cyanophage Syn5 has an intrinsic low discrimination against the incorporation of 2′-fluoro dNMPs during transcription elongation. The presence of both magnesium and manganese ions at high concentrations further reduce this discrimination without decreasing the efficiency of incorporation. We have constructed a Syn5 RNA polymerase in which tyrosine 564 is replaced with phenylalanine (Y564F) that further decreases the discrimination against 2′-fluoro-dNTPs during RNA synthesis. Sequence elements in DNA templates that affect the yield of RNA and incorporation of 2′-fluoro-dNMPs by Syn5 RNA polymerase have been identified.