Ribonucleic Acid Sequence Characterization by Negative Electron Transfer Dissociation Mass Spectrometry.

Area of Science:

• Analytical Chemistry
• Biochemistry
• Molecular Biology

Background:

• Modified oligonucleotides, such as small interfering RNAs (siRNA) and microRNAs, are crucial in therapeutic drug development.
• Mass spectrometry (MS) is a powerful tool for characterizing complex nucleic acid structures.
• Negative electron transfer dissociation (NETD) is a fragmentation technique for sequencing oligonucleotide anions, preserving modifications.

Purpose of the Study:

• To demonstrate the implementation of NETD on a standard quadrupole-Orbitrap-linear ion trap mass spectrometer.
• To characterize unmodified and modified ribonucleic acids using NETD.
• To present the first application of activated-ion NETD (AI-NETD) for nucleic acid analysis.

Main Methods:

• Utilized a quadrupole-Orbitrap-linear ion trap mass spectrometer with a glow discharge source.
• Employed radical fluoranthene reagent cations for NETD.
• Applied both NETD and AI-NETD to analyze a 6-mer ribonucleic acid with 2'-O-methyl modifications and a 21-mer luciferase antisense siRNA.

Main Results:

• Achieved 100% sequence coverage for both the modified 6-mer and the 21-mer siRNA using AI-NETD.
• NETD and AI-NETD provided complete sequence coverage while minimizing base-loss and internal fragments compared to collision-based methods.
• Successfully implemented NETD on a widely available mass spectrometry platform.

Conclusions:

• NETD and AI-NETD are highly effective and versatile methods for sequencing modified oligonucleotides, including therapeutic siRNAs.
• These techniques offer superior sequence coverage and modification preservation compared to traditional methods.
• The successful implementation on standard MS instruments broadens accessibility for nucleic acid characterization.