Milligan J F, Groebe D R, Witherell G W, Uhlenbeck O C
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Nucleic Acids Res. 1987 Nov 11;15(21):8783-98. doi: 10.1093/nar/15.21.8783.
A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the -17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3' terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.
本文描述了一种使用T7 RNA聚合酶和含有T7启动子的合成DNA模板来合成特定长度和序列的小RNA的方法。仅在-17至+1启动子区域碱基配对的部分单链模板在转录中与线性质粒DNA一样活跃。径流转录物在一个独特的、可预测的位置起始，但在3'末端可能多一个或少一个核苷酸。除了全长产物外，反应还产生大量2至6个核苷酸范围内的较小寡核糖核苷酸，这些似乎是流产起始事件的结果。启动子+1至+6区域的变体转录效率降低，但增加了可合成的RNA种类。转录反应条件已得到优化，以允许合成毫克量的长度从12至35个核苷酸的几乎任何RNA。
