Direct LC-HRMS/MS characterisation and RT-qPCR detection for potential mRNA-based doping agent

Area of Science:

• Biochemistry
• Analytical Chemistry
• Sports Science

Background:

• Messenger ribonucleic acid (mRNA) therapeutics show promise for protein expression.
• Potential misuse of mRNA for gene doping in sports presents an emerging risk.
• Detection of modified mRNA, like erythropoietin (EPO) mRNA, is crucial for anti-doping efforts.

Purpose of the Study:

• To verify the identity and chemical composition of an online-available human EPO mRNA product.
• To develop a sensitive detection assay for equine anti-doping.
• To establish a framework for identifying novel mRNA-based doping agents.

Main Methods:

• Mass spectrometry (MS)-based bottom-up workflow with RNase 4 digestion and LC-MS/MS analysis.
• Automated sequence mapping against an mRNA database for identity verification.
• Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay development for equine plasma.

Main Results:

• Successful verification of mRNA sequence and 5-methoxyuridine base modification using MS.
• Achieved mean sequence coverage above 74% for product identity confirmation.
• Validated RT-qPCR assay with a limit of detection at 1250 copies/mL of EPO mRNA in equine plasma.

Conclusions:

• The combined MS and RT-qPCR approach effectively verifies mRNA identity and composition.
• The developed assay provides a reliable method for detecting EPO mRNA in equine anti-doping.
• This framework can be extended to monitor other emerging mRNA-based doping agents in sports.