RNA Labeling with Fluorophore.

Fluorescent labeling of RNA has become an essential technique for visualizing RNA molecules and interrogating their functions in vitro and in vivo. This chapter describes a detailed protocol for synthesizing fluorophore-labeled enhancer RNAs (eRNAs) through in vitro transcription with dye-modified nucleotides such as Cy3-UTP. The method allows efficient incorporation of fluorophores into RNA transcripts while maintaining their structural and functional integrity. Labeled RNAs can be used in a variety of downstream applications, including biochemical assays of RNA-protein interactions and phase separation studies. As demonstrated in prior studies, fluorophore-labeled eRNAs are sufficient to drive condensate formation with RNA-binding proteins such as YTHDC1, enabling direct visualization of RNA-mediated compartmentalization processes. The chapter also provides guidance on template preparation, RNA purification, and fluorescence validation, along with critical notes on optimizing labeling conditions and minimizing degradation. This method offers a robust and adaptable approach for producing fluorescent RNAs applicable to studies of RNA localization, nuclear architecture, and post-transcriptional gene regulation.