Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmaceutical Science

Background:

• Therapeutic RNAs require chemical modifications for optimal drug uptake, stability, and efficacy.
• Phosphorothioate (PS) RNA, featuring sulfur substitution in the phosphate backbone, is a common modification due to synthesis ease and pharmacokinetic advantages.
• Accurate quality assessment of RNA synthesis, including verification of modification incorporation, is critical for therapeutic RNA selectivity and performance.

Purpose of the Study:

• To present a comprehensive analysis of Phosphorothioate (PS) RNA using tandem mass spectrometry (MS/MS).
• To identify and characterize specific MS/MS fragmentation patterns indicative of PS modification incorporation.
• To enhance the diagnostic capabilities for quality control in PS RNA synthesis.

Main Methods:

• Analysis of PS RNA using tandem mass spectrometry (MS/MS).
• Employing activated ion-negative electron transfer dissociation (AI-ETD) MS/MS for PS linkage site diagnosis.
• Utilizing resonant and beam-type collision-based activation methods to study fragmentation differences.

Main Results:

• Activated ion-negative electron transfer dissociation MS/MS effectively diagnoses PS incorporation, yielding diagnostic a- and z-type ions at PS linkage sites.
• PS modification presence leads to more intense sequence and base-loss ions under resonant and beam-type collision-based activation.
• Increased detection of b- and x-type product ions, in addition to standard c- and y-type ions, is observed at sites of PS incorporation.

Conclusions:

• The gas-phase chemical stability imparted by sulfur in PS RNA alters standard RNA dissociation pathways during MS/MS.
• Specific diagnostic ions (a, z, b, x types) should be included in MS/MS analysis for accurate identification of PS RNA.
• This study provides a refined MS/MS approach for comprehensive quality assessment of therapeutic PS RNA.