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Review
. 2023 Sep 11:10:1229246.
doi: 10.3389/fmolb.2023.1229246. eCollection 2023.

Real-time monitoring strategies for optimization of in vitro transcription and quality control of RNA

Kyung Hyun Lee  1 , Jaehwi Song  1 , Seongcheol Kim  1 , Seung Ryul Han  1 , Seong-Wook Lee  1   2
Affiliations
Review

Real-time monitoring strategies for optimization of in vitro transcription and quality control of RNA

Kyung Hyun Lee et al. Front Mol Biosci. .

Abstract

RNA-based therapeutics and vaccines are opening up new avenues for modern medicine. To produce these useful RNA-based reagents, in vitro transcription (IVT) is an important reaction that primarily determines the yield and quality of the product. Therefore, IVT condition should be well optimized to achieve high yield and purity of transcribed RNAs. To this end, real-time monitoring of RNA production during IVT, which allows for fine tuning of the condition, would be required. Currently, light-up RNA aptamer and fluorescent dye pairs are considered as useful strategies to monitor IVT in real time. Fluorophore-labeled antisense probe-based methods can also be used for real-time IVT monitoring. In addition, a high-performance liquid chromatography (HPLC)-based method that can monitor IVT reagent consumption has been developed as a powerful tool to monitor IVT reaction in near real-time. This mini-review briefly introduces some strategies and examples for real-time IVT monitoring and discusses pros and cons of IVT monitoring methods.

Keywords: IVT; RNA; in vitro transcription; real-time monitoring; transcription.

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Conflict of interest statement

KHL, JS, SK, and SRH are employee of Rznomics Inc. S-WL is CEO of Rznomics Inc.

Figures

FIGURE 1
FIGURE 1
(A–C) Light-up RNA aptamer and fluorescence dye pair for real-time IVT monitoring. (A) An example of light-up RNA aptamer (Spinach) and fluorescence dye (DFHBI) pair. DFHBI dye is nonfluorescent due to subtle movement. However, its nonfluorescent form can switch to a fluorescent form when it binds to a specific RNA aptamer such as Spinach. (B) DNA template containing promoter and a universal fluorescence module (UFM) consisting of a highly active hammerhead ribozyme (HHR) and Spinach RNA aptamer can be used to monitor the synthesis of RNA of interest (ROI) during IVT in the presence of DFHBI dye. (C) FRET between water-soluble conjugated polymer (PFP, blue line) that binds to RNA by nonspecific electrostatic interaction as a FRET donor and RNA aptamer/DFHBI complex as a FRET acceptor. (D–G) Fluorophore-labeled antisense probe-based strategies for real-time IVT monitoring. (D) Fluorophore-labeled antisense probe for fluorescence turn-on. (E) FRET-based strategy. (F) Molecular beacon-based strategy. (G) Strategy using fluorophore-labeled and quencher-labeled probes.
FIGURE 2
FIGURE 2
Schematic representation of at-line HPLC-based IVT optimization workflow, which allows fed-batch approach. Representative chromatograms are shown in peak analysis part. Elution peaks corresponding to anti-reverse cap analog (ARCA), NTPs, and mRNA are indicated in chromatograms.
See this image and copyright information in PMC

References

    1. Akoopie A., Müller U. F. (2018). Cotranscriptional 3′-end processing of T7 RNA polymerase transcripts by a smaller HDV ribozyme. J. Mol. Evol. 86 (7), 425–430. 10.1007/s00239-018-9861-9 - DOI - PubMed
    1. Autour A., Westhof E., Ryckelynck M. (2016). iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications. Nucleic Acids Res. 44 (6), 2491–2500. 10.1093/nar/gkw083 - DOI - PMC - PubMed
    1. Babendure J. R., Adams S. R., Tsien R. Y. (2003). Aptamers switch on fluorescence of triphenylmethane dyes. J. Am. Chem. Soc. 125 (48), 14716–14717. 10.1021/ja037994o - DOI - PubMed
    1. Brauns E. B., Dyer R. B. (2005). Time-resolved infrared spectroscopy of RNA folding. Biophys. J. 89 (5), 3523–3530. 10.1529/biophysj.105.061531 - DOI - PMC - PubMed
    1. Cao G. J., Sarkar N. (1992). Identification of the gene for an Escherichia coli poly(A) polymerase. Proc. Natl. Acad. Sci. U. S. A. 89 (21), 10380–10384. 10.1073/pnas.89.21.10380 - DOI - PMC - PubMed