Ivanov S A, Welz R, Gottikh M B, Müller S
Mol Biol (Mosk). 2004 Sep-Oct;38(5):798-803.
Transcription of RNA molecules from synthetic DNA templates with T7 RNA polymerase is a common procedure for the preparation of long RNA molecules. However, enzymatic synthesis does not allow for site-specific incorporation of modified nucleotides. RNA synthesis by chemical methods on the other side can satisfy this purpose, but it is limited to RNA fragments of about 80 nucleotides at maximum. We aimed to combine both chemical and enzymatic procedures to synthesise RNA molecules by RNA primed transcription with T7 RNA polymerase. To this end we have chemically synthesised a fluorescein labelled RNA primer and studied elongation of this primer by T7 RNA polymerase on a single-stranded DNA template. We show that the enzyme is capable of extending the primer to the full-length product. The 34-mer RNA that has been synthesised by RNA primed transcription served as substrate for a twin ribozyme and was successfully cleaved in the expected manner.
用T7 RNA聚合酶从合成DNA模板转录RNA分子是制备长RNA分子的常见方法。然而，酶促合成不允许位点特异性掺入修饰核苷酸。另一方面，通过化学方法进行RNA合成可以满足这一目的，但它最多限于约80个核苷酸的RNA片段。我们旨在结合化学和酶促方法，通过用T7 RNA聚合酶进行RNA引发转录来合成RNA分子。为此，我们化学合成了一种荧光素标记的RNA引物，并研究了T7 RNA聚合酶在单链DNA模板上对该引物的延伸。我们表明该酶能够将引物延伸至全长产物。通过RNA引发转录合成的34聚体RNA用作双核糖酶的底物，并以预期方式成功切割。
