Ion Mobility Gas-Phase Separation Enhances Top-Down Mass Spectrometry of Heavily Modified Guide RNA

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Gene editing technologies, including CRISPR, are advancing into clinical applications.
• Characterizing complex therapeutic molecules like guide RNAs (gRNAs) is crucial for drug safety and efficacy.
• Conventional sequencing methods may not fully identify all chemical modifications in synthetic oligonucleotides.

Purpose of the Study:

• To develop and validate an advanced analytical method for comprehensive characterization of large, chemically modified synthetic guide RNAs (gRNAs).
• To improve the identification of sequence and chemical modifications in CRISPR-gRNAs using mass spectrometry coupled with ion mobility.
• To achieve high sequence coverage and precise localization of modifications in a 100-mer gRNA.

Main Methods:

• Utilized top-down mass spectrometry combined with ion mobility spectrometry.
• Performed gas-phase separation of fragment ions to enhance characterization.
• Applied de novo sequencing and modification localization techniques.

Main Results:

• Achieved a 95% sequence coverage for a heavily modified 100-mer gRNA.
• Significantly increased fragment identifications, particularly in modified regions and the spacer sequence.
• Enabled simultaneous de novo sequencing and modification mapping at both 5' and 3' ends.

Conclusions:

• Top-down mass spectrometry with ion mobility provides near-complete sequence and chemical modification confirmation for complex synthetic gRNAs.
• This enhanced characterization method is vital for the quality control of CRISPR-based gene editing therapeutics.
• The developed approach facilitates deeper understanding and validation of oligonucleotide therapeutics.