Synthesis of RNA by In Vitro Transcription

Abstract

In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in μg to mg quantities. It is based on the engineering of a template that includes a bacteriophage promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In vitro transcripts are used in analytical techniques (e.g. hybridization analysis), structural studies (for NMR and X-ray crystallography), in biochemical and genetic studies (e.g. as antisense reagents), and as functional molecules (ribozymes and aptamers).

Rapid Production of Novel Pre-MicroRNA Agent hsa-mir-27b in Escherichia coli Using Recombinant RNA Technology for Functional Studies in Mammalian Cells

“…These synthetic RNA agents all consist of unnatural modifications, and it is unknown how chemical modifications may alter the biologic activity of miRNAs despite the mimics exhibiting a longer halflife. In vitro transcription (Beckert and Masquida, 2011) may produce RNA agents in variable lengths, whereas a large-scale production requires more but inexpensive RNA polymerases. Recently, tRNA (Ponchon and Dardel, 2007;Ponchon et al, 2009;Nelissen et al, 2012) and ribosomal RNA (Liu et al, 2010) have been used as scaffolds for successful production of recombinant RNAs in Escherichia coli (E. coli) for structural analyses.…”

New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coli

“…Advantages and drawbacks of different sources of RNA biopharmaceuticals Systematic studies of the structure and function of RNA and the establishment of RNA-based therapeutics usually require large quantities of the target RNA with adequate integrity, purity and biological activity. 2 The required RNA for biomolecular applications has mostly been obtained by two distinct methods 11 : the longer oligoribonucleotides by in vitro transcription from DNA templates using T7 RNA polymerase 12 whereas short oligoribonucleotides are preferentially obtained via chemical synthesis by phosphoamidite chemistry. 13 Despite recent improvements, these sources of agents for RNA research remain costly, laborious and have their limitations with respect to sequence requirements, variations in yield, non-templated nucleotide additions and/or the maximum length of the oligonucleotide.…”

Bioengineered non-coding RNA agent (BERA) in action

“…Currently, RNA agents are commonly produced through chemical synthesis or in vitro transcription with recombinant T7 RNA polymerase. 9 The major limitation of those RNA agents is the addition of excessive artificial modifications and/or the lack of necessary posttranscriptional modifications occurring in natural RNAs, which may lead to different folding properties, biological activities, and safety profiles. Another approach to introduce target ncRNAs into mammalian cells is the use of DNA materials such as viral or non-viral vector-based ncRNA expression plasmids.…”