Developing Orthogonal Fluorescent RNAs for Photoactive Dual‐Color Imaging of RNAs in Live Cells

Fluorogenic RNA aptamers have revolutionized the visualization of RNAs within complex cellular processes. A representative category of them employs the derivatives of green fluorescent protein chromophore, 4-hydroxybenzlidene imidazolinone (HBI), as chromophores. However, the structural homogeneity of their chromophoric backbones causes severe cross-reactivity with other homologous chromophores. This limitation impairs their multiplexing capabilities, which are essential for the simultaneous visualization of multiple RNA species in live cells. Herein, we rationally designed a series of red-shifted chromophores and employed SELEX-independent engineering to develop a novel fluorogenic RNA aptamer, mSquash. mSquash displays specific and intense fluorescence upon binding with our red-shifted chromophore DFHBFPD (Ex/Em=501/624 nm). The mSquash/DFHBFPD allows orthogonal imaging of selected RNA targets alongside the established Broccoli/DFHBI-1T (Ex/Em=472/501 nm), facilitating multiplexed live cell imaging of various targets. Moreover, we expanded the application of fluorescent RNA to photoactive imaging by constructing two genetically encoded photoactivatable fluorescent RNAs for the first time. This innovative approach allows photoactivatable control of fluorescent RNAs via specific light wavelengths (365 nm and 450 nm), enabling spatiotemporal dual-color imaging of RNAs in live cells. Our findings represent a significant advancement in fluorescent RNA-based orthogonal imaging and spatiotemporal analysis of RNAs.