Quality Control of mRNA Vaccines by Synthetic Ribonucleases: Analysis of the Poly‐A‐Tail

• Published in: Chembiochem : a European journal of chemical biology 2024-07, Vol.25 (13), p.e202400347-n/a
• Main Authors: Zellmann, Felix, Schmauk, Nina, Murmann, Nina, Böhm, Madeleine, Schwenger, Alexander, Göbel, Michael W.
• Format: Article
• Language: English
• Subjects: 5’ cap; artificial ribonucleases; Catalysts; Chromatography, High Pressure Liquid; Mass Spectrometry; Mass spectroscopy; Metal ions; mRNA; mRNA vaccine; mRNA Vaccines; Poly A - chemistry; poly-A tail; Quality Control; Ribonuclease H; Ribonucleases - chemistry; Ribonucleases - metabolism; RNA cleavage; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sample preparation; Vaccines
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.202400347

The effectivity and safety of mRNA vaccines critically depends on the presence of correct 5’ caps and poly‐A tails. Due to the high molecular mass of full‐size mRNAs, however, the direct analysis by mass spectrometry is hardly possible. Here we describe the use of synthetic ribonucleases to cleave off 5’ and 3’ terminal fragments which can be further analyzed by HPLC or by LC–MS. Compared to existing methods (e. g. RNase H), the new approach uses robust catalysts, is free of sequence limitations, avoids metal ions and combines fast sample preparation with high precision of the cut. Oligonucleotide conjugates of tris(2‐aminobenzimidazoles) are known as potent and sequence‐specific RNA cleavers. Here we apply such “chemical scissors” as analytical tools to evaluate critical quality attributes of mRNA vaccines, like the poly‐A tail or the 5’ terminus. To assure continuous access to tris(2‐aminobenzimidazole) conjugates, an optimized synthetic procedure using microwave irradiation has been developed.