A flexible, high-throughput system for studying mRNA translation kinetics in vitro and in cellulo with HiBit technology

Abstract HiBit is an engineered luciferase’s 11 amino acid component that can be introduced as a tag at either terminus of a protein of interest. When the LgBit component and a substrate are present, HiBit and LgBit dimerise forming a functional luciferase. The HiBit technology has been extensively used for high-throughput protein turnover studies in cells. Here, we have adapted the use of the HiBit technology to quantify mRNA translation temporally in vitro in the rabbit reticulocyte system and in cellulo in HEK293 cells constitutively expressing LgBit. The assay system can detect differences in Cap, 5’UTR, modified nucleotide composition, coding sequence optimisation and poly(A) length. Importantly, using these assays we established the optimal mRNA composition varied depending on the encoded protein of interest, highlighting the importance of screening methods tailored to the protein of interest, and not reliant on reporter proteins. Our findings demonstrated that HiBit can be easily and readily adapted to monitor mRNA translation and offers a novel and highly favourable method for the development of mRNA-based therapeutics. Graphical abstract