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) la-beling 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-trifluor-oacetamide 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.

Automated summary

Despite the challenges posed by macromolecular nucleic acids, a recent labeling method using N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) has shown promise in the profiling and characterization of DNA and RNA. This method offers advantages such as strong retention, predictable MS2 data, and avoidance of harmful reagents. It has been successfully applied to the analysis of tRNA, including the detection of modified bases and the discovery of new modifications. The ease of access and simplicity of this method make it a valuable tool for researchers.