This study describes the development of RNA aptamers that bind fluorophores, creating a palette of RNA-fluorophore complexes spanning the visible spectrum. The researchers focused on generating RNA sequences that can activate the fluorescence of GFP-like fluorophores, such as 4-hydroxybenzilene imidazolinone (HBI). Through systematic evolution of ligands by exponential enrichment (SELEX), they identified RNA aptamers that bind HBI and enhance its fluorescence. These aptamers, including 13-2, 24-2, and others, exhibit different spectral properties, allowing for a range of colors from cyan to orange-red. The study also developed a new HBI derivative, DFHB1, which is exclusively in the phenolate form at pH 7.4, leading to the discovery of an RNA aptamer, 24-2, that preferentially binds this form. The 24-2-DFHB1 complex, named Spinach, shows enhanced quantum yield and resistance to photobleaching, making it suitable for live cell imaging. Spinach was successfully used to tag and track the dynamics of 5S RNA in living mammalian cells, demonstrating its potential for studying RNA biology and applications.This study describes the development of RNA aptamers that bind fluorophores, creating a palette of RNA-fluorophore complexes spanning the visible spectrum. The researchers focused on generating RNA sequences that can activate the fluorescence of GFP-like fluorophores, such as 4-hydroxybenzilene imidazolinone (HBI). Through systematic evolution of ligands by exponential enrichment (SELEX), they identified RNA aptamers that bind HBI and enhance its fluorescence. These aptamers, including 13-2, 24-2, and others, exhibit different spectral properties, allowing for a range of colors from cyan to orange-red. The study also developed a new HBI derivative, DFHB1, which is exclusively in the phenolate form at pH 7.4, leading to the discovery of an RNA aptamer, 24-2, that preferentially binds this form. The 24-2-DFHB1 complex, named Spinach, shows enhanced quantum yield and resistance to photobleaching, making it suitable for live cell imaging. Spinach was successfully used to tag and track the dynamics of 5S RNA in living mammalian cells, demonstrating its potential for studying RNA biology and applications.