Analysis of mRNA Capping and Tailing

In recent years, in vitro synthesis of mRNA molecules is a widely used laboratory procedure that is critical to mRNA biopharmaceuticals as well as related research. Apart from the production of high-quality, scalable mRNA to meet a wide range of clients' requirements, Creative Biogene also provides the services to analyze mRNA capping and tailing efficiency. To ensure the quality, safety, and efficacy of mRNA, the assessment of mRNA at the biophysical and biochemical level is essential, including the capping and tailing efficiency. Creative Biogene aims to establish a streamlined service platform to evaluate mRNA's quality, advancing the drugs into preclinical testing.

Capping and tailing- proper quality control parameters of mRNAs

A 7-methylguanylate cap structure linked to the RNA through a reverse 5' to 5' triphosphate linkage is an evolutionarily conserved modification as well as a hallmark of eukaryotic mRNA. The 5' m7G cap serves as a unique molecular module that not only protects the mature mRNA from degradation, but also affects the translation of mRNA, involving regulation of pre-mRNA splicing, nuclear export, and cap-dependent protein synthesis. Therefore, due to the importance of a cap structure in the synthesis of functional mRNA, as a proper quality control parameter, the quantitative assessment of capped RNAs is necessary.

For a functional mRNA to obtain efficient translation in eukaryotic cells, besides the basic requirements of a 5' m7G cap, a poly(A) tail at the 3' end is also required. The addition of the poly(A) tail, also known as polyadenylation, confers stability to the mRNA, mediates the export of the mRNA, and takes part in the formation of a translation-competent ribonucleoprotein (RNP). Since mRNA-based drugs are emerging as a promising therapeutic alternative, the heterogeneity assessment of mRNA tailing is important for obtaining a consistent and well defined product.

Fig1. A typical mRNA construct. (Versteeg, L., et al. 2019)

The methods used to analyze mRNA capping and tailing

Since the mRNA cap structure and poly(A) tail play major roles in cell biology, numerous approaches have been developed to measure them, liquid chromatography-mass spectrometry (LC-MS), RNaseH/Oligo(dT) northern blot analysis and next-generation sequencing protocols. Here, we've listed some of the methods.

Our strategy for mRNA capping and tailing assessment

For IVT mRNA produced for therapeutic purposes, the assessment of capping efficiency and the heterogeneity of poly(A) tails in mRNA is necessary, which profoundly impacts the quality of the products. We provide comprehensive strategies and assessment services according to our customers' experimental goals and required resolution. Among them, the LC-MS-based method is simple and rapid for directly measuring mRNA capping efficiency and poly(A) tail length. First, the magnetic oligo dT beads, a common technique for mRNA isolation, is employed. Then, the dissociated fragments are analyzed by LC-MS.

Based on the advanced instruments and a team of skillful scientists, Creative Biogene is confident to offer accurate and rapid assessment methods to characterize IVT mRNA, including the mRNA capping efficiency and tailing heterogeneity. If you need to evaluate the mRNA quality, please contact us or directly send us an inquiry. We are looking forward to cooperating with you!