Bidirectional Direct Sequencing of Noncanonical RNA by Two-Dimensional Analysis of Mass Chromatograms.
Mass spectrometry (MS) is a powerful technique for characterizing noncanonical nucleobases and other chemical modifications in small RNAs, yielding rich chemical information that is complementary to high-throughput indirect sequencing. However, mass spectra are often prohibitively complex when fragment ions are analyzed following either solution phase hydrolysis or gas phase fragmentation. For all but the simplest cases, ions arising from multiple fragmentation events, alternative fragmentation pathways, and diverse salt adducts frequently obscure desired single-cut fragment ions. Here we show that it is possible to take advantage of predictable regularities in liquid chromatographic (LC) separation of optimized RNA digests to greatly simplify the interpretation of complex MS data. A two-dimensional analysis of extracted compound chromatograms permits straightforward and robust de novo sequencing, using a novel Monte Carlo algorithm that automatically generates bidirectional paired-end reads, pinpointing the position of modified nucleotides in a sequence. We demonstrate that these advances permit routine LC-MS sequencing of RNAs containing noncanonical nucleotides, and we furthermore examine the applicability of this approach to the study of oligonucleotides containing artificial modifications as well as those commonly observed in post-transcriptionally modified RNAs.
Howard Hughes Medical Institute , Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|Department of Genetics, Harvard Medical School , Boston, Massachusetts 02115, United States.|Åbo Akademi University , Department of Biosciences, Artillerigatan 6, FI-20520 Åbo, Finland.
Anders
A
Björkbom
Howard Hughes Medical Institute , Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|Department of Genetics, Harvard Medical School , Boston, Massachusetts 02115, United States.
Victor S
VS
Lelyveld
Howard Hughes Medical Institute , Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.
Shenglong
S
Zhang
Howard Hughes Medical Institute , Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|Department of Chemistry and Chemical Biology, Harvard University , Cambridge, Massachusetts 02138, United States.
Weicheng
W
Zhang
Howard Hughes Medical Institute , Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|Department of Chemistry and Chemical Biology, Harvard University , Cambridge, Massachusetts 02138, United States.
Chun Pong
CP
Tam
Howard Hughes Medical Institute , Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.
J Craig
JC
Blain
Howard Hughes Medical Institute , Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, United States.|Department of Chemistry and Chemical Biology, Harvard University , Cambridge, Massachusetts 02138, United States.|Department of Genetics, Harvard Medical School , Boston, Massachusetts 02115, United States.
Jack W
JW
Szostak
D001483:Base Sequence; D013058:Mass Spectrometry; D012313:RNA / Q000737:chemistry*; D017423:Sequence Analysis, RNA / Q000379:methods*
D012313:RNA