pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences

Abstract

Increasing demand for large-scale synthesis of in vitro transcribed (IVT) mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3′ polyadenosine (poly(A)) tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A) tail lengths to ~120 base pairs (bp). Here, we have developed a novel method for generation of extended poly(A) tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A) tracts up to ~500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A) tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A) tracts and 3′ termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s).

Biomedical applications of mRNA nanomedicine

“…In another study, IVT mRNA with a poly(A) length of 100 nucleotides allowed for efficient protein expression [42]. A linear plasmid vector system, pEVL, was recently explored as the template for generating IVT mRNA with poly(A) tails of up to approximately 500 bases and with a defined length [43]. Besides the tail extension, incorporation of adenosine analogs, such as 3’-deoxyadenosine or 8-azaadenosine, provided better mRNA protection from 3’-exonuclease degradation, which could also be used to increase protein expression [44].…”

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

“…Ensuring an optimal length of the poly(A) tail is vital for efficient translation and shortening of these sequences leads to decay of mRNA (reviewed in [3,4]). Multiple A residues may be added to synthetic RNA using a poly(A) polymerase from Escherichia coli (EPAP) [5] or during transcription from an encoding template [6,7]. When using EPAP to add A residues to the 3 end of mRNA, achieving consistency of the tail lengths may be difficult [6,7].…”

“…Multiple A residues may be added to synthetic RNA using a poly(A) polymerase from Escherichia coli (EPAP) [5] or during transcription from an encoding template [6,7]. When using EPAP to add A residues to the 3 end of mRNA, achieving consistency of the tail lengths may be difficult [6,7]. A complication of using poly(A:T)-containing circular plasmids is that the homopolymeric A:T base pairs in these vectors are unstable and prone to shortening during replication in bacteria [6,8].…”

“…When using EPAP to add A residues to the 3 end of mRNA, achieving consistency of the tail lengths may be difficult [6,7]. A complication of using poly(A:T)-containing circular plasmids is that the homopolymeric A:T base pairs in these vectors are unstable and prone to shortening during replication in bacteria [6,8]. Use of a linear plasmid vector was recently employed to increase stability of longer poly(A:T) tracts [6].…”

Metabolic stability and persistence of expression of mRNA for nonviral gene delivery

“…Encoding this length of poly(A) tail into a circular plasmid, therefore, is an unreliable method for generating a poly(A) tail, so the poly(A) tracts in these mRNAs are usually shorter (typically up to 120 bp). One group improved this problem by using a linear plasmid with terminal hairpin loops (termed the pEVL vector), resulting in stability in bacteria of over 500 bp of a homopolymeric A-tract 64 .…”