Abstract
Tagging RNAs with fluorogenic aptamers has enabled imaging of transcripts in living cells, revealing novel aspects of RNA metabolism and dynamics. While a diverse set of fluorogenic aptamers has been developed, a new generation of aptamers uses the ring-opening of spirocyclic rhodamine dyes to achieve robust performance in live mammalian cells. These fluorophores have two chemical states: a cell-permeable spirocyclic state and a fluorescent zwitterionic state. The SpyRho555 dye is mostly non-fluorescent in solution and becomes fluorescent in complex with the RhoBAST aptamer. To understand the basis for RhoBAST-SpyRho555 fluorogenicity, we have determined crystal structures of RhoBAST in complex with 5-carboxytetramethylrhodamine and a SpyRho555 analogue, MaP555. RhoBAST is organized by a perfect four-way junction that positions two loops to form the dye-binding pocket. The core of the ligand resides between a tri-adenine floor and a single guanine base, largely driven by π-stacking interactions. Importantly, the unpaired guanine interacts with the 3-position group of MaP555 to stabilize the open conformation, supported by mutagenesis data, and may play an active role in promoting the open conformation of the dye. This work has implications for the development of new fluorogenic aptamers with improved properties using structure-guided design approaches.