Selective Chemical Labeling Strategy for Oligonucleotides Determination: A First Application to Full-Range Profiling...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Molecular Biology

Background:

• High polarity and low signal intensity of nucleic acids hinder mass spectrometry (MS) applications.
• Existing MS methods for nucleic acids often require harsh reagents and lack efficiency.

Purpose of the Study:

• To introduce a novel labeling method for improved oligonucleotide determination using MS.
• To enhance the quality control of nucleic acid drugs and characterization of DNA/RNA modifications.
• To apply the method for comprehensive RNA profiling and discovery of disease-specific biomarkers.

Main Methods:

• Development and application of a general N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) labeling strategy for oligonucleotides.
• Utilizing selective labeling of terminal phosphate groups for broad DNA/RNA profiling.
• Employing RNA digestion into single-nucleotide products for comprehensive RNA mapping and modification analysis.

Main Results:

• The MTBSTFA method provides strong retention, no byproducts, and interpretable MS2 data for oligonucleotide analysis.
• Achieved improved sequence coverage in yeast tRNA analysis, detecting fragments from 1-12 nucleotides.
• Identified a significantly upregulated fragment (U[m1G][m2G]) specific to tRNAAsn(QUU) in HepG2 cells and rats with nonalcoholic fatty liver disease.

Conclusions:

• The MTBSTFA labeling method offers a robust, efficient, and broadly applicable approach for nucleic acid profiling and modification analysis via MS.
• This technique facilitates the comprehensive mapping of RNA and the discovery of potential disease biomarkers.
• The method's simplicity and reagent availability make it accessible for researchers in various fields.