Single-Molecule RNA Imaging in Live Cells with an Avidity-Based Fluorescent Light-Up Aptamer biRhoBAST

Observing individual RNA molecules provides valuable insights into their regulation, interactions with other cellular components, organization, and functions. Although fluorescent light-up aptamers (FLAPs) have recently shown promise for RNA imaging, their wider applications have been mostly hindered by poor brightness and photostability. We recently developed an avidity-based FLAP known as biRhoBAST that allows for single-molecule RNA imaging in live or fixed cells and tracking individual mRNA molecules in living cells due to its excellent photostability and high brightness. Here, we present step-by-step detailed protocols starting from cloning biRhoBAST repeats into the target RNA sequence, to imaging dynamics of single mRNA molecules. Additionally, we address the validation of single-molecule imaging experiments through single-molecule fluorescence in situ hybridization (smFISH) and colocalization studies.