icRNA: in vivo circular RNAs for efficient expression and control of genes and polyproteins

Potent, stable and controllable gene expression is a long-standing goal of synthetic biology. Thanks to Watson-Crick base pairing, RNA-based controllers have the potential to be more programmable and predictable, and to impose less metabolic burden to host cells compared to protein-based controllers. In microbes, however, RNA-based systems typically offer limited dynamic range and require high-levels of RNA expression due to the short half-lives of linear RNAs. This project aims to develop an RNA-based controller with a high dynamic range and low host burden by using in vivo circularisation and linearisation of mRNA. Circular RNAs (cRNAs) lack 5' and 3' ends, which eliminates end-dependent degradation by exoribonucleases and several endoribonucleases, causing cRNAs to exhibit far longer half-lives compared to linear mRNAs. However, due to a lack of efficient methods for in-vivo cRNA production, the use of cRNAs has been limited so far.

This project will focus on the development of a novel in-vivo cRNA expression system in E. coli (and potentially in S. cerevisiae, too), adapting the Tornado RNA circularisation system to circularise long mRNAs. The main task of the project will be to rationally engineer the sequence of the ribozymes and transcripts to optimise cleavage and ligation. To further increase the dynamic range of the circuit, we will implement an RBS-repairing strategy where the RBS is initially split in two parts placed at the ends of the linear mRNA and gets repaired only when the RNA is circularised. In parallel with the experimental work, computational models accurately describing RNA circularisation and linearisation kinetics and circular mRNA translation dynamics will be built to understand and predict the dynamic behaviour of these RNA-circularisation systems.

This cRNA control system will be used as a platform for controlling gene expression with applications in production of materials, therapeutics, and potentially vaccines. Additionally, by removing the stop codon from the coding sequence in a circular mRNA, rolling circle translation can be achieved, which can be used for the efficient production of valuable multi-unit repeat proteins with useful properties for the production of novel biomaterials.