Ribonucleases for Sequencing and Characterization of RNA by LC–MS

The global deployment of mRNA vaccines against SARS-CoV-2 and the projected expansion of therapeutic applications of synthetic mRNA call for robust and high-precision analytical methods to evaluate attributes that are crucial to the safety and efficacy of the mRNA drug substances. Liquid chromatography–mass spectrometry (LC–MS) is one of the few techniques that can provide a direct and high-confidence readout of the identity and incorporation efficiency of the 5′ cap, length of the poly(A) tail, nucleotide sequence, and modification profile of synthetic mRNA molecules. Prior to LC–MS analysis, the RNA molecules are partially digested by specific endoribonucleases into oligonucleotides that are suitable for charge state-dependent fragmentation and mass deconvolution. The most commonly used endoribonuclease for RNA sequence mapping is the guanosine-specific RNase T1. RNase T1 has been employed for analysis of mRNA, rRNA, and tRNA as well as for mRNA poly(A) tail length verification. For mRNA 5′ cap analysis, selective excisions using probe-restrained RNase H or (deoxy)ribozymes are typically required. In this chapter, we will review the application of endoribonucleases for mRNA analysis, with emphasis on a recently characterized endoribonuclease derived from human RNase 4. We will also discuss the latest methods to assess 5′ cap and poly(A) tail incorporation in synthetic mRNA. Finally, we will highlight why more enzymatic tools are needed and how they can contribute to improving the quality of synthetic RNA analysis, and to help understand the biology of RNA modifications in the cell.