Discovering Design Principles to Re-Engineer Functional RNA Elements

Abstract

Functional RNA elements play critical roles in biological processes, including refolding to regulate protein synthesis, responding to cofactors or self-splicing. The activation of these widespread elements is typically triggered by biological partners such as small molecules, proteins or ions. The induction of RNA function makes these elements attractive therapeutic targets and potentially highly sensitive biosensors, where a designed response could be engineered for a known input. Despite the potential importance of controlling and exploiting RNA's molecular response, the tightly coupled balance of RNA energetics can challenge our ability to tune it. Here we use microfluidic mixing technology to measure non-equilibrium, single-molecule kinetics for a set of small molecule sensing riboswitch mutants. This benchmark set of constructs elucidates how differing regions and bases interact to alter RNA functionality, suggesting themes that are potentially transferable to similarly structured riboswitches. We use these principles to design a new set of RNA mutants which exhibit significant variation in small-molecule sensing.