Method development for the detection and quantification of biological and synthetic RNA cap structures by LC-MS/MS

Abstract

5’ caps on messenger RNA (mRNA) play a crucial role in regulating mRNA function and processing. Apart from the canonical cap 0 structure, comprising a 7-methylguanosine (m7G) linked to the first transcribed nucleotide by a reverse 5’-5’-triphosphate bridge, additional methylations, such as 2’O-methylations of the penultimate nucleotide and additional N6-methylation of adenosine contribute to the complex cap epitranscriptome. Based on the critical role of 5’ caps in mRNA function, this study developed a sensitive and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) approach to detect and quantify these structures, while also providing the capability to simultaneously analyze modified nucleosides. The comprehensive method development of chromatographic and mass spectrometric parameters considerably impacted signal intensity and peak separation, pushing detection limits into the attomole to femtomole range. Examination of eukaryotic RNA revealed distinct cap methylation patterns for cell and tissue samples. Investigating the knockout of the cap-modifying enzyme CMTR1 provided insights into its substrate specificity and contributed to the understanding of RNA cap methylation processes. To enhance mass spectrometry-based quantification, an accessible approach for isotope labeling of cap dinucleotides was developed. The method aimed to achieve deuteration at the C-8 position of purines through incubation in deuterium oxide at elevated temperatures. Studies with N7-unmethylated cap dinucleotides revealed that temperatures of at least 85 °C and extended incubation times were necessary for near-complete deuteration. However, m7G exhibited rapid exchange at the C-8 position in contact with water, precluding stable isotope labeling of m7G-containing cap structures. In view of the increasing relevance of capped synthetic mRNA for therapeutic applications, another part of this study assessed a novel LC-MS-based method for determining capping efficiencies. Application to co- and post-transcriptionally capped in vitro transcribed (IVT) RNA revealed challenges in IVT purification and quantification accuracy, highlighting the need to optimize IVT processes and purification techniques for reliable evaluation of capping reactions.