Bright and Stable Cyan Fluorescent RNA Enables Multicolor RNA Imaging in Live Escherichia coli

Abstract Fluorescent RNAs (FRs), which are RNA aptamers that bind and activate their cognate small fluorogenic dyes, have provided a particularly useful approach for imaging RNAs in live cells. Although the color palette of FRs is greatly expanded, a bright and stable cyan FR with good biocompatibility and biorthogonality with currently available FRs remains desirable but is not yet developed. Herein, the development of Myosotis is described, an RNA aptamer that emits bright cyan fluorescence upon binding a novel GFP chromophore‐like fluorophore called DBT. Myosotis has a nanomolar affinity for DBT and shows a weak dependence on magnesium for folding. Further studies reveal that the Myosotis‐DBT complex has a long fluorescence lifetime, good photostability, and enhance cellular brightness. It is further shown that Myosotis‐DBT is biorthogonal to Pepper and Clivia FRs, allowing multiplex fluorescence imaging of RNA in live bacteria. Myosotis can also use to image mRNA in live bacteria, revealing potential coupling between mRNA translation and stability. It is believed that this cyan FR will be a useful tool for studying the functionality and mechanism of RNA underlying diverse biological processes.