Increasing Oligonucleotide Sequencing Information and Throughput with Ion Mobility Spectrometry-Mass Spectrometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Synthetic oligonucleotides, including antisense oligonucleotides and small interfering RNA, are crucial for therapeutic gene expression control.
• Accurate sequence verification of these nucleic acid polymers is essential for their therapeutic application, relying on Watson-Crick base pairing.
• Developing high-quality synthetic oligonucleotides necessitates comprehensive analytical workflows for rigorous evaluation.

Purpose of the Study:

• To develop and apply a novel analytical platform for efficient and accurate synthetic oligonucleotide sequence confirmation.
• To improve the speed and robustness of sequence verification methods for therapeutic nucleic acids.
• To address the challenges in spectral interpretation and sequence determination for complex oligonucleotide data.

Main Methods:

• A platform coupling liquid chromatography, ion mobility spectrometry, collision-induced dissociation, and mass spectrometry (LC-IMS-CID-MS) was employed.
• Ion mobility spectrometry (IMS) was utilized to separate and simultaneously analyze multiple charge states of oligonucleotides.
• All-ion fragmentation was implemented for comprehensive sequence coverage across different charge states with fewer injections.

Main Results:

• The LC-IMS-CID-MS platform successfully facilitated oligonucleotide sequence confirmation.
• Sequence coverage was found to be inversely proportional to oligonucleotide length, ranging from 40% to 80% for 20-40 residue molecules.
• An analysis workflow using PNNL preprocessor and Agilent's BioConfirm software was developed to simplify data interpretation.

Conclusions:

• The developed LC-IMS-CID-MS platform offers a robust method for synthetic oligonucleotide sequence verification.
• The platform demonstrates efficiency in analyzing multiple charge states and providing sequence information.
• Further optimization may be needed for achieving higher sequence coverage for longer oligonucleotide therapeutics.