Use of Cis- and Trans-Ribozymes to Remove 5′ and 3′ Heterogeneities From Milligrams of In Vitro Transcribed RNA

In vitro transcription with phage T7 RNA polymerase is the method of choice for obtaining multi-milligram quantities of RNA for structural studies. However, run-off transcription with this enzyme results in molecules that are heterogeneous at their 3'-, and depending on template sequence, 5...

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Bibliographic Details
Published in:Nucleic acids research 1996-03, Vol.24 (5), p.977-978
Main Authors: Ferré-D'Amaré, Adrian R., Doudna, Jennifer A.
Format: Article
Language:English
Subjects:
Base Sequence
Molecular Sequence Data
phage T7
RNA - isolation & purification
RNA, Catalytic - chemistry
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Summary:In vitro transcription with phage T7 RNA polymerase is the method of choice for obtaining multi-milligram quantities of RNA for structural studies. However, run-off transcription with this enzyme results in molecules that are heterogeneous at their 3'-, and depending on template sequence, 5'-termini. For transcripts longer than similar to 50 nucleotides (nt), these impurities cannot be removed by preparative purification techniques. Use of cis-delta, or trans-VS ribozymes allows preparation of homogeneous RNA with any 3'-terminal sequence. If present, 5' heterogeneity can be overcome with a cis-hammerhead ribozyme. During the course of a structural investigation of Group II self-splicing introns, we sought to prepare a 70 nt RNA molecule comprising domains V and VI (d56) of the ai5 gamma intron.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/24.5.977