Synthesis of mixed ribo/deoxyribopolynucleotides by mutant T7 RNA polymerase

FEBS Letters

Grishchenko, O.I¹  Tunitskaya, V.L¹  Gudima, S.O¹  Kostyuk, D.A¹  Memelova, L.V¹  Kochetkov, S.N¹ 

[1]Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov st., 32, 117984 Moscow, Russia

关键词: T7 RNA polymerase;    dNTP utilization;    Mixed ribo/deoxypolynucleotides;   

DOI  :  10.1016/S0014-5793(98)01393-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown