Ion Mobility Gas-Phase Separation Enhances Top-Down Mass Spectrometry of Heavily Modified Guide RNA

As gene editing technologies enter the clinic, state-of-the-art characterization methods have been developed in parallel to assess the components of these paradigm-shifting medicines. One such component, the guide RNA (gRNA) element of CRISPR-based drugs, is a large synthetic heavily modified oligonucleotide that programs for the desired gene edit. Conventional oligonucleotide sequencing technologies can inform gRNA composition, but these methods may not completely capture the chemical modifications that are introduced during synthesis. Circumventing these challenges, mass spectrometry has demonstrated use in oligonucleotide analyses and has been combined here with ion mobility to deepen its characterization power. The use of ion mobility enabled us to perform gas-phase separation of the fragment ions produced by top-down mass spectrometry, yielding a significant increase in fragment identifications for a highly modified 100-mer gRNA by uncovering high-confidence assignments for heavily modified regions and for the important spacer region. Furthermore, the high-confidence fragment assignments empowered simultaneous de novo sequencing and chemical modification localization for the 5'-end spacer region as well as for 15 nucleotides on the heavily modified 3'-end. Overall, a total sequence coverage of 95% was achieved for the heavily modified 100-mer, ushering near complete sequence and chemical modification confirmation by top-down mass spectrometry.