Screening and structural engineering of lariat-capping ribozymes for use as an alternative mRNA 5`-capping system

Abstract

Aim. Identification of native and structurally-engineered variants of LCRs with faster processing kinetics and increased lariat capping, and their use for mRNA translation in cells. Conclusions. The capless translation system constructed from functionally-coupled lariat capping ribozyme and viral IRES significantly increased overall protein production compared to IRES-only configuration, while still inferior to co-translational capping with ARCA.Structural engineering of DiLCR stems by modulation of their thermodynamic stability, allowed us to control lariat cap/cleavage products ratio and design variants with near-quantitative capping achievable in vitro, leading to increased protein accumulation in the cell-based translation assay.Alternative LCR-IRES reporter combinations demonstrate high dependence of functional activity on sequence context possibly due to the mutual folding/interaction environment interference.