A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells

A key approach to investigating RNA species in live mammalian cells is the ability to label them with fluorescent tags and track their dynamics in the complex cellular environment. The growing appreciation for the diversity of RNAs in nature, especially the roles of small, non-coding RNAs for cell function, calls for development of orthogonal RNA tagging systems. We previously developed Riboglow, a new RNA tagging system that features modular elements and hence the possibility to customize features for each application of choice. Riboglow consists of an RNA tag that is genetically fused to the RNA of interest and a small molecule that binds the RNA tag and elicits a fluorescence light up signal. Here, we present an overview of the Riboglow platform and compare and contrast the system with existing RNA tagging systems. Two step by step protocols for implementation of RNA imaging with Riboglow in live mammalian cells are presented, with special emphasis on guidelines that drive choices for modular elements in the Riboglow platform. Such modular elements include the RNA tag sequence and size, the number of RNA tag repeats per tagged RNA, the fluorescent color of the probe, the identity of the chemical linker in the probe, and the concentration of the probe used in live cells. Together, Riboglow is a new RNA tagging platform that enables robust live cell imaging of RNA dynamics, and this detailed protocol and guidelines for implementation will enable broad usage of Riboglow.