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Open AccessArticle

One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden
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Academic Editor: Nuno Neng
Molecules 2020, 25(5), 1142; https://doi.org/10.3390/molecules25051142
Received: 24 January 2020 / Revised: 25 February 2020 / Accepted: 1 March 2020 / Published: 4 March 2020
(This article belongs to the Special Issue Modern Sample Preparation Approaches for Separation Science)
There is an increasing demand for efficient and robust production of short RNA molecules in both pharmaceutics and research. A standard method is in vitro transcription by T7 RNA polymerase. This method is sequence-dependent on efficiency and is limited to products longer than ~12 nucleotides. Additionally, the native initiation sequence is required to achieve high yields, putting a strain on sequence variability. Deviations from this sequence can lead to side products, requiring laborious purification, further decreasing yield. We here present transcribing tandem repeats of the target RNA sequence followed by site-specific cleavage to obtain RNA in high purity and yield. This approach makes use of a plasmid DNA template and RNase H-directed cleavage of the transcript. The method is simpler and faster than previous protocols, as it can be performed as one pot synthesis and provides at the same time higher yields of RNA. View Full-Text
Keywords: RNA preparation; in vitro transcription; T7 polymerase; RNase H; tandem repeats; short RNAs RNA preparation; in vitro transcription; T7 polymerase; RNase H; tandem repeats; short RNAs
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MDPI and ACS Style

Feyrer, H.; Munteanu, R.; Baronti, L.; Petzold, K. One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts. Molecules 2020, 25, 1142.

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