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.