Selective Chemical Labeling Strategy for Oligonucleotides Determination: A First Application to Full-Range Profiling of Transfer RNA Modifications

To date, the extremely high polarity and poor signal
intensity
of macromolecular nucleic acids are greatly impeding the progress
of mass spectrometry technology in the quality control of nucleic
acid drugs and the characterization of DNA oxidation and RNA modifications.
We recently described a general N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) labeling method for
oligonucleotide determination and applied it to the full-range profiling
of tRNA in vitro and in vivo studies for the first time. The primary
advantages of this method include strong retention, no observable
byproducts, predictable and easily interpreted MS2 data,
and the circumvention of instrument harmful reagents that were necessary
in previous methods. Selective labeling of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide to the terminal phosphate groups
of oligonucleotides endows it broadly applicable for DNA/RNA profiling.
Moreover, the improvement of sequence coverage was achieved in yeast
tRNAphe(GAA) analysis owing to this method’s good
detection capability of 1–12 nucleotides in length. We also
extended this strategy to determine the abundance of modified bases
and discover new modifications via digesting RNA into single-nucleotide
products, promoting the comprehensive mapping of RNA. The easy availability
of derivatization reagent and the simple, rapid one-step reaction
render it easy to operate for researchers. When applied in characterizing
tRNAs in HepG2 cells and rats with nonalcoholic fatty liver disease,
a fragment of U[m1G][m2G], specific for tRNAAsn(QUU) in cells, was significantly upregulated, indicating
a possible clue to nonalcoholic fatty liver disease pathogenesis.