Multifaceted mRNA analysis using programmed RNA cleavage by Mucilaginibacter paludis Argonaute

Argonaute (Ago) endonucleases are ubiquitous across all domains of life and, like Cas nucleases, can be programmed by guide oligonucleotides to target and cleave specific nucleic acid sequences. However, unlike Cas enzymes, Agos are not restricted by sequence requirements such as protospacer adjacent motifs (PAMs) enhancing their utility as molecular tools. This study demonstrates diverse applications of the DNA-guided RNA-endonuclease Mucilaginibacter paludis Argonaute (MpaAgo) for in vitro mRNA processing, e.g., 3’ end polishing, and for mRNA analysis in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS). Applications of the latter include the comprehensive assessment of 5’ capping efficiency, 3’ poly(A) tail length and composition, one-pot multiplexed cleavage for RNA sequencing, modification detection, and fingerprinting.