Abstract

RNAs exhibit complex dynamics in cells, including expression, splicing, localization, translation and degradation, and these processes are highly coordinated and tightly regulated both spatially and temporally. To better understand the biological function of diverse RNAs, approaches that allow monitoring of RNA with high spatiotemporal resolution are essential. Fluorescent RNAs (FRs), fluorescent protein–like entities consisting of RNA aptamers and their cognate fluorogenic dyes, have emerged as a promising approach for imaging RNA dynamics in live cells. We recently reported the development of several high-performance FRs, named Pepper, Clivia and Okra, that show advantageous properties, including high cellular brightness and photostability, low ion dependence and/or large Stokes shifts, and have been used to image diverse RNA species in live cells. In this protocol, we provide easy, efficient and generalizable strategies for using FRs to visualize different RNA species in bacteria and mammalian cells by expressing the RNA of interest tagged with one or more copies of the aptamer. We also provide a detailed procedure for multiplexed RNA imaging using orthogonal FRs and the steps to perform super-resolution live imaging of RNAs. The protocol typically takes 5–7 d, including cloning, transfection of mammalian cells or transformation of bacteria, live imaging and results analysis. This protocol is applicable to the real-time monitoring of the localization and dynamics of RNAs of interest in live cells.