A Flexible, High-Throughput System for Studying Live mRNA Translation with HiBiT Technology
Ascanelli C. et al. (2025). A Flexible, High-throughput System for Studying Live mRNA Translation with HiBiT Technology. Nuc. Acids Res. 53, 11 gkaf496. DOI: 10.1093.nar/gkaf
Researchers at the University of Cambridge developed and validated a high-throughput assay system to measure messenger RNA (mRNA) translation in real time, designed to support the optimization of mRNA-based therapeutics. Rather than relying on reporter proteins such as green fluorescent protein (GFP) or luciferase, the authors fused the small 11-amino-acid HiBiT peptide tag directly to proteins of interest (POIs), enabling quantification of translation without the confounding effects of reporter-specific expression dynamics. The system was validated in two contexts: an in vitro rabbit reticulocyte lysate (RRL) assay using the Promega Flexi Rabbit Reticulocyte Lysate Kit and LgBiT/Furimazine detection reagents, and a live-cell kinetic assay in HEK293 cells constitutively expressing LgBiT, using the Nano-Glo® Endurazine Live Cell Substrate to enable continuous luminescence monitoring over 18 hours.
Using this platform, the authors systematically evaluated how individual mRNA components affect translation output. Removal of the 5' cap reduced luminescence by more than 80% in live cells, and a structured 5' UTR hairpin reduced translation area under the curve (AUC) by approximately 55–58% compared to unmodified controls. Poly(A) tails of only three adenosines nearly abolished expression (86–90% reduction in AUC vs. 120A constructs), while tails of 40 adenosines or more supported efficient translation. Nucleotide modification effects were shown to be POI-dependent: pseudouridine substitution improved Myc-HiBiT translation AUC by approximately 60% over 18 hours, while eGFP-HiBiT showed minimal sensitivity to the same modifications. The assay also distinguished protein stability differences — the Myc T58A phosphodegron mutant showed a 67% increase in signal over wild-type Myc at 4 hours, an effect confirmed by proteasome inhibition with MG132.
The study further demonstrated that codon optimization strategies produce POI-specific outcomes: a codon adaptation index (CAI)-optimized Myc sequence generated an 84% increase in 18-hour signal compared to wild-type, while minimum free energy (MFE) optimization reduced translation by 65% despite improving mRNA stability. Across multiple cell types including A549 cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs), translation rankings between POIs were broadly consistent, supporting the reproducibility of the approach. The authors conclude that the HiBiT-based pipeline provides a sensitive, flexible, and scalable tool for screening mRNA design parameters against therapeutically relevant proteins, offering advantages over reporter-based methods that may not reflect the dynamics of the actual POI.Keywords: HiBiT, LgBiT, NanoLuc, mRNA therapeutics, mRNA translation, rabbit reticulocyte lysate, live cell assay, codon optimization, poly(A) tail, nucleotide modification, protein turnover, bioluminescence
