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. 2024 May 23;29(11):2461.
doi: 10.3390/molecules29112461.

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription

Wei He  1   2 , Xinya Zhang  2 , Yangxiaoyu Zou  2 , Ji Li  2 , Chong Wang  2 , Yucai He  3 , Qiuheng Jin  2 , Jianren Ye  1
Affiliations

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription

Wei He et al. Molecules. .

Abstract

mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5'-cap and a 3'-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.

Keywords: T7 RNA polymerase; fragmented mRNA; mRNA integrity; mutation; synthesis; transcription.

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Conflict of interest statement

Wei He, Xinya Zhang, Yangxiaoyu Zou, Ji Li, Chong Wang and Qiuheng Jin were employed by Vazyme Biotech Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Figures

Figure 1
Figure 1
Schematic showing one theory of metal-based RNA degradation.
Figure 2
Figure 2
Schematic showing the generation of mRNA fragments.
Figure 3
Figure 3
Interactions among the site chains of amino acids K389, K172, N171, Q754, and R756 from subdomain H (purple), the specificity loop (green), and the thumb domain (red) with mRNA products (orange).
Figure 4
Figure 4
Analysis of mRNA products: (a) capillary electrophoresis analysis; (b) comparison of mRNA integrity and proportion of fragmented mRNA generated when using uridine (U) and m1ψ as substrates (the experiments were replicated ≥4 times; error bars represent ±SD; * p < 0.05, *** p < 0.001).
Figure 4
Figure 4
Analysis of mRNA products: (a) capillary electrophoresis analysis; (b) comparison of mRNA integrity and proportion of fragmented mRNA generated when using uridine (U) and m1ψ as substrates (the experiments were replicated ≥4 times; error bars represent ±SD; * p < 0.05, *** p < 0.001).
Figure 5
Figure 5
Structural analysis of (a) T7 RNAP WT and (b) T7RNAP K389A.
Figure 5
Figure 5
Structural analysis of (a) T7 RNAP WT and (b) T7RNAP K389A.
Figure 6
Figure 6
Effect of T7 RNAP substitutions on (a) mRNA integrity (%) and (b) fragmented mRNA (%) (the experiments were replicated ≥4 times; error bars represent ±SD; ** p  <  0.01, *** p  <  0.001, **** p  <  0.0001).
Figure 7
Figure 7
Comparison of mRNA integrity (%) (histograms) and yield of mRNA products (lines) catalyzed by various substitutions (the experiments were replicated ≥4 times; error bars represent ±SD).
Figure 8
Figure 8
Effect of optimized DNA template on (a) capillary electrophoresis analysis of mRNA products; (b) mRNA integrity; and (c) proportion of fragmented mRNA (The experiments were replicated ≥4 times; error bars represent ±SD; * p  <  0.05, ** p  <  0.01, *** p  <  0.001, **** p  <  0.0001).
Figure 9
Figure 9
Effect of different organic acids on mRNA degradation: (a) citrate reduced the Mg2+-based degradation efficiency of mRNA; (b) mRNA integrity (%) (histograms) and the yield of mRNA products (lines) (the experiments were replicated ≥3 times; error bars represent ±SD).
Figure 9
Figure 9
Effect of different organic acids on mRNA degradation: (a) citrate reduced the Mg2+-based degradation efficiency of mRNA; (b) mRNA integrity (%) (histograms) and the yield of mRNA products (lines) (the experiments were replicated ≥3 times; error bars represent ±SD).
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