Large-Scale in Vitro Transcription, RNA Purification and Chemical Probing Analysis

Kanwal, Fariha; Chen, Ting; Zhang, Yunlong; Yunlon, Zhang; Simair, Altaf Ahmed; Cai, Rujie; Zaidi, Najam us Sahar Sadaf; Guo, Xinhang; Wei, Xiao-Long; Siegel, Geoffrey; Lu, Changrui

doi:10.1159/000492512

Cited by 30 publications

(22 citation statements)

References 52 publications

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“…We tested the effect of lowering the magnesium concentration on the formation of dsRNA by-products. As expected, overall RNA yield was reduced rather than just that of the by-products (data not shown) (Suh et al 1992;Zaychikov et al 1997;Kanwal et al 2018).…”

Section: A B C Dsupporting

confidence: 76%

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

Asahara

Tzertzinis

et al. 2020

RNA

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The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in vitro-transcribed RNAs, predominantly using phage RNA polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity in vivo that is often undesirable. Previous studies have identified double-stranded RNA (dsRNA), a major by-product of the in vitro transcription (IVT) process, as a trigger of cellular immune responses. Here we describe the characterization of a high-temperature IVT process using thermostable T7 RNAPs to synthesize functional mRNAs that demonstrate reduced immunogenicity without the need for a post-synthesis purification step. We identify features that drive the production of two kinds of dsRNA by-products-one arising from 3 ′ ′ ′ ′ ′ extension of the runoff product and one formed by the production of antisense RNAs-and demonstrate that at a high temperature, T7 RNAP has reduced 3 ′ ′ ′ ′ ′-extension of the runoff product. We show that template-encoded poly(A) tailing does not affect 3 ′ ′ ′ ′ ′-extension but reduces the formation of the antisense RNA by-products. Combining high-temperature IVT with template-encoded poly(A) tailing prevents the formation of both kinds of dsRNA by-products generating functional mRNAs with reduced immunogenicity.

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Section: A B C Dsupporting

confidence: 76%

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

Asahara

Tzertzinis

et al. 2020

RNA

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show abstract

“…We tested the effect of lowering the magnesium concentration on formation of dsRNA byproducts. As expected, overall RNA yield was reduced rather than just that of the byproducts (data not shown) [34][35][36].…”

Section: High-temperature Ivt Does Not Affect Formation Of Antisense supporting

confidence: 77%

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

Asahara

Tzertzinis

et al. 2019

Preprint

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The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in vitro-transcribed mRNAs, predominantly using phage RNA polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity in vivo, that is often undesirable. Previous studies have identified double-stranded RNA (dsRNA), a major by-product of the in vitro transcription (IVT) process, as a trigger of cellular immune responses. Here we describe the characterization of a high-temperature IVT process using thermostable T7 RNAPs to synthesize functional mRNAs that demonstrate reduced immunogenicity without the need for a post-synthesis purification step. We identify features that drive the production of two kinds of dsRNA by-products-one arising from 3' extension of the run-off product and one formed by the production of antisense RNAs-and demonstrate that at a high temperature, T7 RNAP has reduced 3'-self extension of the run-off product. We show that template-encoded poly-A tailing does not affect 3'-self extension but reduces the formation of the antisense RNA by-products and that combining high-temperature IVT with templateencoded poly-A tailing prevents formation of both kinds of by-products.

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“…Concerning factors influencing the yield of RNA transcription, we noted that the most important were DNA template concentration (50-100 μg/mL) and reaction incubation time (12-16 h). Although the adjusting concentration of magnesium ions and ribonucleotides often improves the yield of transcription, we did not observe significant differences in the amount of synthesized RNA (data not shown) [142].…”

Section: Discussionmentioning

confidence: 58%

Overview of Methods for Large-Scale RNA Synthesis

et al. 2022

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In recent years, it has become clear that RNA molecules are involved in almost all vital cellular processes and pathogenesis of human disorders. The functional diversity of RNA comes from its structural richness. Although composed of only four nucleotides, RNA molecules present a plethora of secondary and tertiary structures critical for intra and intermolecular contacts with other RNAs and ligands (proteins, small metabolites, etc.). In order to fully understand RNA function it is necessary to define its spatial structure. Crystallography, nuclear magnetic resonance and cryogenic electron microscopy have demonstrated considerable success in determining the structures of biologically important RNA molecules. However, these powerful methods require large amounts of sample. Despite their limitations, chemical synthesis and in vitro transcription are usually employed to obtain milligram quantities of RNA for structural studies, delivering simple and effective methods for large-scale production of homogenous samples. The aim of this paper is to provide an overview of methods for large-scale RNA synthesis with emphasis on chemical synthesis and in vitro transcription. We also present our own results of testing the efficiency of these approaches in order to adapt the material acquisition strategy depending on the desired RNA construct.

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Large-Scale in Vitro Transcription, RNA Purification and Chemical Probing Analysis

Cited by 30 publications

References 52 publications

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

Overview of Methods for Large-Scale RNA Synthesis

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