Published online 2015 Mar 27. doi: 10.1093/nar/gkv145
PMCID: PMC4446411
Abstract
Ribosomal
ribonucleic acid (RNA), transfer RNA and other biological or synthetic
RNA polymers can contain nucleotides that have been modified by the
addition of chemical groups. Traditional Sanger sequencing methods
cannot establish the chemical nature and sequence of these
modified-nucleotide containing oligomers. Mass spectrometry (MS) has
become the conventional approach for determining the nucleotide
composition, modification status and sequence of modified RNAs. Modified
RNAs are analyzed by MS using collision-induced dissociation tandem
mass spectrometry (CID MS/MS), which produces a complex dataset of
oligomeric fragments that must be interpreted to identify and place
modified nucleosides within the RNA sequence. Here we report the
development of RoboOligo, an interactive software program for the robust
analysis of data generated by CID MS/MS of RNA oligomers. There are
three main functions of RoboOligo: (i) automated de novo
sequencing via the local search paradigm. (ii) Manual sequencing with
real-time spectrum labeling and cumulative intensity scoring. (iii) A
hybrid approach, coined ‘variable sequencing’, which combines the user
intuition of manual sequencing with the high-throughput sampling of
automated de novo sequencing.
