Messenger RNA (mRNA) serves numerous research and clinical purposes. It is produced through the transcription of a DNA template. In vitro transcription (IVT) of mRNA requires efficient capping strategies to generate biologically active and stable mRNA molecules. In mammalian cells, specific molecular structures are added to one end of an mRNA strand to shield it from degradation and ensure proper translation and function. This process is known as capping. Typically, both in vivo and in various lab procedures, the cap is attached after mRNA synthesis, referred to as post-transcriptional or enzymatic capping. This method necessitates additional enzymatic reactions and purification steps, increasing the complexity and duration of the process.

TriLink Biotechnologies provides a co-transcriptional capping technology using the CleanCap® molecule, which offers a streamlined, one-pot solution that minimizes the number of steps and results in a cap-1 structure crucial for mRNA function. This, CleanCap®-AG Reagent (BCT-38) is a trinucleotide reagent containing the native cap structure and an A and G at the initial two positions of the mRNA.

Conventional capping methods, such as the use of ARCA (anti-reverse cap analogue), sometimes face the problem of capping efficiency and cost. These methods usually result in a cap 0 structure, which can activate pattern recognition receptors and lead to immune responses, reducing the overall efficacy of the mRNA.

The CleanCap®-AG molecule binds to the active site of T7 RNA polymerase when it attaches to the DNA template, stabilizing the site. The AG bases of the CleanCap® molecule form base pairs with the DNA template. T7 RNA polymerase can then extend from the CleanCap® molecule by adding new bases, including various modified NTPs, to produce capped RNA products that closely resemble natural mammalian mRNA.

CleanCap® reagents advantages:

• facilitate efficient manufacturing,
• achieving 95% capping efficiency
• yielding 3x more product than traditional co-transcriptional methods unlike older techniques
• reduce production time
• decrease mRNA immunogenicity
• enhance stability
• create the ideal cap-1 structure for superior in vivo performance.

PLEASE NOTE: CleanCap® Reagent AG requires an AG initiator.

LITERATURE

Development of bis-ANS-based modified fluorescence titration assay for IFIT/RNA studies, Anna Dobieżyńska et al., 2020, ScienceDirect, Vol. 553(3), p. 391-396.

https://doi.org/10.1016/j.bbrc.2020.09.006

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes, Peter J. Chen et al., 2021, Cell, Vol. 184(22), p. 5635-5652.

https://doi.org/10.1016/j.cell.2021.09.018

Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE), Hamideh Parhiz et al., 2022, Journal of Controlled Release, Vol. 344, p. 50-61.

https://doi.org/10.1016/j.jconrel.2021.12.027