Real-time fluorescence imaging of single-molecule endogenous noncoding RNA in living cells

Abstract

Visualizing RNA in living cells is increasingly important to facilitate accumulation of knowledge about the relation between specific RNA dynamics and physiological events. Single-molecule fluorescence imaging of target RNAs is an excellent approach to analyzing intracellular RNA motion, but it requires special techniques for probe design and microscope setup. Herein, we present a principle and protocol of an RNA visualization probe based on an RNA binding protein of the Pumilio homology domain (PUM-HD). We also describe the setup and operation of a microscope, and introduce an application to visualize telomeric repeats-containing RNA with telomeres and a telomere-related protein: hnRNPA1. This imaging technique is applicable to visualization of different RNAs, especially including repetitive sequences, in living cells.