Structural Characterization of Normal and Modified Oligonucleotides by Matrix-assisted Laser Desorption Fourier...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Molecular Biology

Background:

• Oligonucleotide structural characterization is crucial for understanding their biological functions.
• Accurate sequencing and isomer differentiation are essential for studying normal and modified oligonucleotides.
• Mass spectrometry offers powerful tools for molecular analysis.

Purpose of the Study:

• To investigate the utility of matrix-assisted UV laser desorption Fourier transform mass spectrometry (MALDI-FTMS) for detailed structural characterization of oligonucleotides.
• To determine the role of the nicotinic acid matrix in generating characteristic ions for oligonucleotide analysis.
• To explore fragmentation pathways for sequence determination and isomer differentiation.

Main Methods:

• Matrix-assisted UV laser desorption Fourier transform mass spectrometry (266 nm, nicotinic acid matrix).
• Analysis of negative ion spectra, including (M - H) (-) ions and fragment ions.
• Collision-induced dissociation (CID) experiments.

Main Results:

• The nicotinic acid matrix was essential for producing (M - H) (-) ions for oligonucleotide dimers to hexamers.
• Phosphate ester bond cleavage was the primary fragmentation pathway, with charge retention on the 3' end.
• Fragmentation patterns allowed for isomeric differentiation (e.g., d(S'-CGCG-3') vs. d(S'-CCGG-3')).
• CID confirmed preferential nucleotide loss from the 5' end.
• Oligonucleotide modifications (e.g., methyl, ethyl groups) were identifiable.

Conclusions:

• MALDI-FTMS with a nicotinic acid matrix is a robust method for detailed oligonucleotide structural characterization.
• The technique effectively determines oligonucleotide sequence, differentiates isomers, and identifies modifications.
• Understanding fragmentation patterns provides insights into oligonucleotide structure and stability.