Gudima S O, Kostyuk D A, Grishchenko O I, Tunitskaya V L, Memelova L V, Kochetkov S N
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
FEBS Lett. 1998 Nov 20;439(3):302-6. doi: 10.1016/s0014-5793(98)01393-3.
Synthesis of deoxynucleotide-containing RNA-like single-stranded polynucleotides (dcRNAs) using the Y639F, S641A mutant of T7 RNA polymerase (T7 RNAP) was studied. A number of different T7 promoter-containing plasmids were tested as templates for dcRNA synthesis. The dcRNA synthesis efficiency strongly depended on the sequence of the first 8-10 nucleotides immediately downstream of the promoter and increased with the distance of the first incorporated dNMP from the transcription start. The incorporation of dGMP which is obligatory for most T7 promoters in positions +1-+2(3) was practically negligible. Using the constructed plasmid pTZR7G containing seven dG links in the non-coding chain immediately downstream of the promoter, the synthesis of all possible dcRNAs (except dG-containing) was achieved with high yields.
研究了使用T7 RNA聚合酶（T7 RNAP）的Y639F、S641A突变体合成含脱氧核苷酸的RNA样单链多核苷酸（dcRNAs）。测试了许多不同的含T7启动子的质粒作为dcRNA合成的模板。dcRNA的合成效率强烈依赖于启动子下游紧邻的前8 - 10个核苷酸的序列，并随着第一个掺入的dNMP与转录起始位点的距离增加而提高。对于大多数T7启动子而言，在+1 - +2(3)位置掺入dGMP实际上可以忽略不计。使用构建的质粒pTZR7G，其在启动子下游的非编码链中含有七个dG连接，实现了所有可能的dcRNAs（除含dG的以外）的高产率合成。
