Pros and Cons of In Vitro Methods for Circular RNA Preparation

Lee, Kyung Hyun; Kim, Seong-Cheol; Lee, Seong-Wook

doi:10.3390/ijms232113247

Cited by 17 publications

(12 citation statements)

References 61 publications

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“…In recent years, circRNAs have been implicated in various diseases, including cancer ( Wesselhoeft et al, 2018 ; Li J. et al, 2020 ; Li R. et al, 2020 ; Chen and Shan, 2021 ; Zhu et al, 2021 ; Liu et al, 2022 ), cardiovascular diseases ( Altesha et al, 2019 ; Su and Lv, 2020 ; Tang et al, 2021 ; Ju et al, 2022 ), and neurodegenerative disorders ( Jiao et al, 2023 ). The application of circRNAs has attracted widespread attention among drug researchers, owing to their stable structure and ability to cure complicated diseases ( Zhu et al, 2021 ; Lee et al, 2022 ; Zhao et al, 2022 ). However, circRNA research is still in its early stages, and the synthesis of circRNA as a drug has become a research hotspot.…”

Section: Introductionmentioning

confidence: 99%

“…Unlike mRNA synthesis, linear RNA precursor cyclization is necessary for circRNA preparation in vitro . Several methods have been developed for in vitro ligation: chemical-based methods ( Müller and Appel, 2017 ), enzyme methods ( Abe et al, 2018 ; Kershaw and O'Keefe, 2012 ; Moore and Query, 2000 ), and ribozyme-based methods ( Petkovic and Müller, 2013 ; Obi and Chen, 2021 ; Lee et al, 2022 ). The different methods for linear RNA precursor ligation have both advantages and limitations ( Chen and Lu, 2021 ; Obi and Chen, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

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Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst

He,

Zhang,

Zou

et al. 2024

Front. Bioeng. Biotechnol.

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Introduction:Circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) with transcriptional lengths ranging from hundreds to thousands. circRNAs have attracted attention owing to their stable structure and ability to treat complicated diseases. Our objective was to create a one-step reaction for circRNA synthesis using wild-type T7 RNA polymerase as the catalyst. However, T7 RNA polymerase is thermally unstable, and we streamlined circRNA synthesis via consensus and folding free energy calculations for hotspot selection. Because of the thermal instability, the permuted intron and exon (PIE) method for circRNA synthesis is conducted via tandem catalysis with a transcription reaction at a low temperature and linear RNA precursor cyclization at a high temperature.Methods:To streamline the process, a multisite mutant T7 RNA polymerase (S430P, N433T, S633P, F849I, F880Y, and G788A) with significantly improved thermostability was constructed, and G788A was used.Results:The resulting mutant exhibited stable activity at 45°C for over an hour, enabling the implementation of a one-pot transcription and cyclization reaction. The simplified circRNA production process demonstrated an efficiency comparable to that of the conventional two-step reaction, with a cyclization rate exceeding 95% and reduced production of immunostimulatory dsRNA byproducts.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst

He,

Zhang,

Zou

et al. 2024

Front. Bioeng. Biotechnol.

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“…Currently, there are three methods for circRNA vaccines in in vitro production: chemical-, enzyme-, and ribozyme-based ligations. At the same time, these approaches have shortcomings (reviewed in [ 10 ]). Therefore, new protocols for large-scale production are needed to enable the cost-effective design and production of circRNA vaccines.…”

Section: Introductionmentioning

confidence: 99%

Cap-Independent Circular mRNA Translation Efficiency

et al. 2023

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Recently, the mRNA platform has become the method of choice in vaccine development to find new ways to fight infectious diseases. However, this approach has shortcomings, namely that mRNA vaccines require special storage conditions, which makes them less accessible. This instability is due to the fact that the five-prime and three-prime ends of the mRNA are a substrate for the ubiquitous exoribonucleases. To address the problem, circular mRNAs have been proposed for transgene delivery as they lack these ends. Notably, circular RNAs do not have a capped five-prime end, which makes it impossible to initiate translation canonically. In this review, we summarize the current knowledge on cap-independent translation initiation methods and discuss which approaches might be most effective in developing vaccines and other biotechnological products based on circular mRNAs.

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“…As an example of other RNA modalities, circRNA also can be generated by IVT ( Petkovic and Müller, 2015 ; Lee et al, 2022a ) using group I intron ribozyme-based strategy such as permuted intron-exon method ( Puttaraju and Been, 1992 ). In contrast, chemical- or ligase-based circRNA preparation methods require an additional step after precursor RNA preparation by IVT ( Lee et al, 2022a ).…”

Section: Introductionmentioning

confidence: 99%

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

Lee,

Song,

Kim

et al. 2023

Front. Mol. Biosci.

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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.

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Pros and Cons of In Vitro Methods for Circular RNA Preparation

Cited by 17 publications

References 61 publications

Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst

Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst

Cap-Independent Circular mRNA Translation Efficiency

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

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