OLIGORIBONUCLEOTIDE SYNTHESIS USING T7 RNA-POLYMERASE AND SYNTHETIC DNA TEMPLATES
OLIGORIBONUCLEOTIDE SYNTHESIS USING T7 RNA-POLYMERASE AND SYNTHETIC DNA TEMPLATES
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DOI：
10.1093/nar/15.21.8783
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发表时间：
1987-11-11
影响因子：
14.9
通讯作者：
UHLENBECK, OC
中科院分区：
文献类型：
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作者：
MILLIGAN, JF;GROEBE, DR;UHLENBECK, OC
描述了一种使用T7RNA聚合酶和含有T7启动子的合成DNA的模板来合成限定长度和序列的小RNA的方法。仅在−17~+1启动子区域碱基配对的部分单链模板在转录中的活性与线性质粒DNA相同。径流转录本起始于一个独特的、可预测的位置，但在3‘端可能有或多或少的一个核苷酸。除了全长产物外，反应还产生大量较小的寡核苷酸，范围从2到6个核苷酸，似乎是流产引发事件的结果。启动子+1到+6区域的变异被转录，效率降低，但增加了可以制造的RNA的种类。转录反应条件已经优化，允许合成毫克量的几乎任何长度从12到35个核苷酸的RNA。
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.