A flexible, high-throughput system for studying live mRNA translation with HiBiT technology

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• HiBiT is a small peptide tag that forms a functional luciferase with LgBiT.
• HiBiT technology is established for protein turnover studies.
• Quantifying mRNA translation dynamics is crucial for understanding gene expression.

Purpose of the Study:

• To adapt HiBiT technology for temporal mRNA translation quantification in vitro and in cellulo.
• To investigate the impact of mRNA features (cap, 5'UTR, nucleotides, CDS, poly(A) tail) on translation.
• To establish protein-specific optimal mRNA compositions for therapeutic development.

Main Methods:

• Utilized HiBiT-LgBiT complementation assay for luciferase activity measurement.
• Performed in vitro translation assays in rabbit reticulocyte lysate.
• Conducted cellulo assays in HEK293 cells expressing LgBiT.
• Varied mRNA elements including cap, 5'UTR, coding sequence, and poly(A) length.

Main Results:

• Developed a novel assay to monitor mRNA translation in real-time.
• Demonstrated the ability to detect subtle differences in mRNA elements affecting translation efficiency.
• Established that optimal mRNA composition is protein-dependent, not universal.
• Showcased HiBiT's utility for screening mRNA constructs for therapeutic applications.

Conclusions:

• HiBiT technology is a versatile tool for real-time mRNA translation monitoring in diverse systems.
• Protein-specific mRNA optimization is critical for efficient translation, especially for therapeutic applications.
• This method provides a favorable approach for developing next-generation mRNA-based therapeutics.