Truncated and "double-headed" derivatives of TO1-B dye for Mango-based imaging systems with increased brightness and selectivity and large Stocks shift

Fluorogenic dyes that light up in complexes with genetically encodable RNA aptamers are increasingly used for intracellular RNA imaging. The growing toolbox of dyes and aptamers allows simultaneous or sequential monitoring of different RNAs. However, few orthogonal aptamer-dye pairs meet all the requirements of multiplex imaging in terms of brightness, contrast, etc., thus necessitating their optimization and further diversification. Here, we report new fluorogenic ligands for the Mango II aptamer, one of the most stable and widely used RNA tags. They are based on the cognate Mango ligand TO1-B, which is a thiazole orange (TO) derivative with a biotinylated tetraethylene glycol (TEG) residue attached via an amide linker, and its analogue with an isosteric triazolyl linker (TO1-triazolyl-TEG-biotin). Structural data suggested that the biotin residue might be dispensable for interactions with Mango, so we replaced it with a second TO residue or the alternative push-pull system, namely the dimethylaminophenyl (DMAP) group linked to the benzothiazolyl (BzT) group via the ethenyl linker. The resulting "double-headed" symmetric and asymmetric dyes showed reduced fluorescence in the free state due to intramolecular TO/TO or TO/DMAP-BzT interactions. The asymmetric dye exhibited intramolecular FRET in complex with Mango, resulting in a remarkably large (130 nm) Stocks shift, which may be advantageous for multiplex imaging. The main limitation of the "double-headed" dyes was their low brightness. To improve brightness, we further optimized the truncated (biotin-free) TO1-triazolyl-TEG dye by introducing a hydroxy or a methoxy group into the methylqunolinium (MQ) fragment of its push-pull system. The brightness of the methoxy-MQ derivative was increased by 40% compared to the reported TO1-triazolyl-TEG-biotin. Importantly, the methoxy-MQ derivative also showed increased selectivity for Mango over other noncanonical nucleic acids structures, making it a promising alternative to known TO-based dyes for high-contrast RNA imaging.