Researchers developed RNA aptamers that bind fluorophores similar to those in green fluorescent protein (GFP), creating a range of fluorescent RNA-fluorophore complexes spanning the visible spectrum. These complexes, such as Spinach, emit green fluorescence comparable to fluorescent proteins and are highly resistant to photobleaching. Spinach is a RNA mimic of GFP that can be used to fluorescently tag RNA in living cells, offering a new method for studying RNA biology and applications. The fluorophore in GFP is formed from three residues in the nascent protein, which undergo an autocatalytic intramolecular cyclization. The resulting fluorophore, 4-hydroxybenzlidene imidazolinone (HBI), is encased within the protein, enabling its fluorescence. Chemically synthesized HBI is nonfluorescent, but upon refolding, the fluorescence of GFP is recovered. The folded GFP protein forms specific contacts with the fluorophore that prevent intramolecular motions, making fluorescence the major pathway for energy dissipation. To create RNA-fluorophore complexes with GFP-like properties, researchers used SELEX to identify RNA sequences that bind and activate the fluorescence of GFP-like fluorophores. They identified an RNA aptamer, 13-2, which significantly enhances the fluorescence of DMHBI. Further studies showed that RNA sequences can be tailored to produce a range of fluorescence properties, including cyan, greenish-yellow, and yellow. To generate RNA-fluorophore complexes with EGFP-like properties, researchers designed a new HBI derivative, DFHBI, which is exclusively in the phenolate form. An RNA aptamer, 24-2, was identified that binds DFHBI exclusively in the phenolate form, resulting in a significantly enhanced quantum yield. This RNA-fluorophore complex, termed Spinach, exhibits green fluorescence and is highly resistant to photobleaching. Spinach was used to fluorescently tag RNA in living cells, demonstrating its utility for studying RNA dynamics. When fused to a small noncoding RNA, 5S, Spinach was detected in living cells and showed dynamic localization in response to cellular stress. Spinach was also used to monitor RNA trafficking and dynamics in living cells, revealing its potential for applications in RNA biology and imaging.Researchers developed RNA aptamers that bind fluorophores similar to those in green fluorescent protein (GFP), creating a range of fluorescent RNA-fluorophore complexes spanning the visible spectrum. These complexes, such as Spinach, emit green fluorescence comparable to fluorescent proteins and are highly resistant to photobleaching. Spinach is a RNA mimic of GFP that can be used to fluorescently tag RNA in living cells, offering a new method for studying RNA biology and applications. The fluorophore in GFP is formed from three residues in the nascent protein, which undergo an autocatalytic intramolecular cyclization. The resulting fluorophore, 4-hydroxybenzlidene imidazolinone (HBI), is encased within the protein, enabling its fluorescence. Chemically synthesized HBI is nonfluorescent, but upon refolding, the fluorescence of GFP is recovered. The folded GFP protein forms specific contacts with the fluorophore that prevent intramolecular motions, making fluorescence the major pathway for energy dissipation. To create RNA-fluorophore complexes with GFP-like properties, researchers used SELEX to identify RNA sequences that bind and activate the fluorescence of GFP-like fluorophores. They identified an RNA aptamer, 13-2, which significantly enhances the fluorescence of DMHBI. Further studies showed that RNA sequences can be tailored to produce a range of fluorescence properties, including cyan, greenish-yellow, and yellow. To generate RNA-fluorophore complexes with EGFP-like properties, researchers designed a new HBI derivative, DFHBI, which is exclusively in the phenolate form. An RNA aptamer, 24-2, was identified that binds DFHBI exclusively in the phenolate form, resulting in a significantly enhanced quantum yield. This RNA-fluorophore complex, termed Spinach, exhibits green fluorescence and is highly resistant to photobleaching. Spinach was used to fluorescently tag RNA in living cells, demonstrating its utility for studying RNA dynamics. When fused to a small noncoding RNA, 5S, Spinach was detected in living cells and showed dynamic localization in response to cellular stress. Spinach was also used to monitor RNA trafficking and dynamics in living cells, revealing its potential for applications in RNA biology and imaging.