There is a need to develop and expand approaches to regulate and alter the function of RNA using light. Although there are many optogenetic tools involving light-responsive proteins, RNA tools remain underdeveloped. The highly useful nature of RNA makes them valuable tools in regulating intracellular functions. For example, riboswitches are RNAs that mediate gene expression in archaea, plants, and fungi and play significant roles in feedback mechanisms. Other RNAs, like ribozymes, have enzymatic activity and can self-cleave in the presence of adequate concentrations of its metabolite. The functionality of RNA has grasped the attention of synthetic biologists in recent years, leading to new discoveries and the start of RNA optogenetic tools.

In this thesis, I report the first example of a riboswitch that can reversibly regulate translation in response to a sub-millisecond pulse of light in vivo. This is the first optogenetic tool that is a light-regulated RNA switch that responds to a reversible photoactive ligand to control gene expression, giving great spatial and temporal resolution. This work also investigates two more potential photoriboswitches, significantly expanding the field of optogenetic RNA tools.