Introduction

Messenger RNA (mRNA) technology has revolutionized the field of genetic research and therapeutic development. Central to this advancement is the use of plasmids for mRNA production. An essential feature of these mRNA molecules is the polyadenylate (polyA) tail, a sequence of adenine nucleotides at the 3′ end, playing a pivotal role in mRNA stability and efficiency. This essay explores the necessity of the polyA tail, current methods for its synthesis, and its incorporation in plasmid design.

The Necessity of the PolyA Tail in mRNA

The polyA tail is more than a simple nucleotide stretch; it is a crucial element for mRNA functionality in eukaryotic cells. Firstly, it enhances mRNA stability, protecting the mRNA from rapid degradation. In the cellular milieu, RNAs are vulnerable to exonucleases, and the polyA tail acts as a buffer against this degradation. Secondly, it plays a regulatory role in nuclear export. Only mRNAs with adequate polyA tails are efficiently transported from the nucleus to the cytoplasm. Furthermore, the polyA tail is integral to translation initiation. It interacts with poly(A) binding proteins, which, in concert with the 5′ cap structure, facilitate the recruitment of ribosomes. Additionally, the length of the polyA tail can influence mRNA half-life, with longer tails generally enhancing stability.

Current Methods for mRNA PolyA Tail Synthesis

Currently, several methods are employed for the synthesis of mRNA polyA tails. Enzymatic polyadenylation, where a poly(A) polymerase adds a polyA tail to the 3′ end of RNA, is a common technique. In vitro transcription (IVT) is another prevalent method, where the polyA sequence is encoded within the DNA template, allowing the tail to be transcribed directly as part of the mRNA. Chemical synthesis, though less common, is emerging as a method to add polyA tails, offering more control over tail length. Each method has its advantages and limitations. For instance, enzymatic polyadenylation allows precise control over tail length but can be less efficient than IVT in certain contexts.

Commen Issue with polyA containing Plasmid: instability!

The core issue with PolyA tails in plasmids is their inherent instability in bacterial systems. This instability can lead to alterations in the plasmid’s structure and function, impeding its use in mRNA production. The recombination events that occur as a result of this instability are particularly problematic, as they can result in the loss or modification of the intended genetic sequences, rendering the plasmids ineffective for their intended use.

Increased Stability Through PolyA Tail Segmentation

Addressing this challenge, recent advancements have focused on the segmentation of PolyA tails. This innovative approach involves splitting the conventional stretch of approximately 120 adenosines into shorter segments. These segments are separated by non-adenosine nucleotide spacers, a strategic design choice that has shown to significantly mitigate the issue of instability. The segmented PolyA tails have demonstrated a marked reduction in recombination events within E. coli, thereby maintaining the plasmid’s integrity and enhancing its functionality for mRNA production.

There are several polyA segmentation options:

1. Two segments of 60 adenosines. As tested by Trepotec(2019), the most effective pattern emerged as the one consisting of two segments of 60 adenosines each, separated by a Guanine nucleotide spacer, denoted as poly(A)2 × 60_G. This particular segmentation not only minimized recombination rates to below 20% but also preserved the mRNA’s stability and expression efficiency. This finding is particularly significant as it offers a viable and efficient solution to one of the major challenges in plasmid-based mRNA production.
2. 30A+GCATATGACT+70A, Pfizer-BioNTech Vaccine. This segmentation pattern proved to be effective on human by BNT162b2 Vaccine.