A Convenient Method to Generate and Maintain Poly(A)-Encoding DNA Sequences Required for in Vitro Transcription of mRNA

Abstract

Generating mRNA in vitro to encode therapeutic or cell-modifying proteins is rapidly gaining favor. An important factor that determines efficiency of translation from in vitro transcribed mRNA is the length of the 3 poly(A) sequence. However, reproducibly generating and maintaining templates from circular plasmids to have consistent lengths of the homo poly(A) sequences is challenging. The procedure reported here entails repeated restriction digestion with type IIS enzymes, ligation and circular plasmid propagation. The homopolymeric sequence of approximately 100bp that is generated using the method is approximately equal to the number of 3 A residues found in themRNA ofmammalian cells. Evaluating expression in vivo of a reporter transcript produced using this method showed efficient expression in vivo.

mRNA vaccine in cancer therapy: Current advance and future outlook

“…The simple approach entails repeated asymmetric cleavage with type IIS restriction enzymes, then ligation and propagation to extend the homopolymeric sequence up to approximately 100 bp in circular plasmids, which can serve as a template for mRNA transcription. Moreover, the poly (A) tail of the in vitro transcribed transcript functions in vivo as well 128 (Figure 5B ).…”

Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency

“…Poly(A) tails of approximately 100 nt are ideal for synthesizing mRNA therapies because tail size affects mRNA decay by modulating 3′ exonucleolytic degradation (Schlake et al 2012 ). Arbuthnot et al suggested recurrent restriction digestion with type IIS enzymes, ligation, and propagation to lengthen the homopolymeric sequence up to 100 bp in circular plasmids to overcome the difficulties in cloning (Arbuthnot et al 2019 ). To overcome these constraints, Grier et al advocated using linear plasmids as a template for mRNA synthesis (Grier 2016 ).…”

Current knowledge on the tissue distribution of mRNA nanocarriers for therapeutic protein expression

“…25 Many studies have explored the influence of the poly(A) tail on translational efficiency, reporting optimal lengths ranging between 100 and 240 nucleotides. 21,26,27 This variation has been shown to depend on the specific modifications of the mRNA sequence, as well as the target cell line used. 26…”

“…25 Many studies have explored the influence of the poly(A) tail on translational efficiency, reporting optimal lengths ranging between 100 and 240 nucleotides. 21,26,27 This variation has been shown to depend on the specific modifications of the mRNA sequence, as well as the target cell line used. 26 Another strategy to increase mRNA stability and translational efficiency is the integration of 5′-and 3′-UTRs with optimal length and regulatory elements to influence translational efficiency and stability.…”