RNA synthesis by T7 RNA polymerase supported primer extensionстатья
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Дата последнего поиска статьи во внешних источниках: 18 июля 2013 г.
Аннотация: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. On the other hand, RNA synthesis by chemical methods can be used for this purpose, but it is limited to RNA fragments of about 80 nucleotides at maximum. We aimed at combining both chemical and enzymatic procedures to synthesise RNA molecules by RNA primed transcription with T7 RNA polymerase. To this end, we have chemically synthesized a fluorescein-labelled RNA primer and studied elongation of this primer by T7 RNA polymerase on a single-stranded DNA template. We have shown that the enzyme is capable of extending the primer to the full-length product. The 34-mer RNA that has been synthesized by RNA primed transcription served as substrate for a twin ribozyme and was successfully cleaved in the expected manner.