Team:Peking R/Project/RNAToolkit

Overview

In recent years, RNA devices have emerged as powerful tools to regulate gene expression in vivo, and particularly, ligand-responsive riboswitches/ribozymes enable us to manipulate translation strength of specific genes upon different concentrations of ligands. Ligand-responsive riboswitches/ribozymes regulate the translation rate of downstream gene by changing conformations, cleaving or splicing upon external addition of ligand. Compared with transcriptional and post-translational regulation, riboswitches/ribozymes function through allostery of RNA structure, which requires little or no assistance from proteins, so the regulation mechanism is relatively simpler and their functions are more decoupled from native biological activities.

In order to fulfill the goal of establishing an extensible and versatile platform for softcoding of genetic program, our team reached out to a great extent to search for available ligand-responsive riboswitches/ribozymes that meet our criteria and selected them as our genetic rheostats.

Candidates for genetic rheostats must meet two basic criteria: firstly, they should possess a relatively plain dose-response curve, which would allow for precise translation strength modulation within a wide range of ligand concentration; secondly, ligands they recognize should be genetically and biochemically orthogonal to the host cells, in our case, E.coli cells, as much as possible.

Two candidates emerged as promising genetic rheostats that satisfied our requirements: thiamine pyrophosphate (TPP)-responsive hammerhead ribozymes and theophylline-responsive riboswitches. By altering the upstream promoter and downstream coding sequence of the genetic rheostats, we demonstrated that their performance was independent from sequence context, which proved that our genetic rheostats are modular.

To further extend the range of application of our genetic rheostats, we created a ribozyme that functions with a different mechanism, which has an extreme low basal level. We substituted the aptamer domain of c-di-GMP group I intron to theophylline-responsive aptamer, thus invented a group I intron that senses theophylline to perform splicing function.

Moreover, we introduced a general method to evolve hammerhead ribozyme that senses a new ligand. By coupling an adenine aptamer with hammerhead ribozyme and randomizing nucleotides in the linker domain, we evolved new hammerhead ribozymes through dual selection, whose self-cleavage could be regulated by adenine.

In summary, our project harvested two RNA devices and performed detailed characterization; we also provided new design principles for rational or semi-rational design of riboswitches/ribozymes.