Bidirectional Direct Sequencing of Noncanonical RNA by Two-Dimensional Analysis of Mass Chromatograms

Anders Björkbom, VS Lelyveld, SL Zhang, WC Zhang, CP Tam, JC Blain, JW Szostak

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    17 Citations (Scopus)


    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 LCMS 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.
    Original languageUndefined/Unknown
    Pages (from-to)14430–14438
    Number of pages9
    JournalJournal of the American Chemical Society
    Issue number45
    Publication statusPublished - 2015
    MoE publication typeA1 Journal article-refereed

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