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Improvement of fluorogenic RNA aptamers by directed evolution

Fluorogenic RNAs aptamers are molecules able to specifically interact with and activate the fluorescence of small fluorogenic compounds. By analogy with the fluorescent proteins (e.g. GFP), one can anticipate that this new class of genetic tags will open exciting perspectives in biology provided bright enough molecules can be isolated. Several fluorogenic aptamers have been described, Spinach and Mango being the best characterized. Both molecules were isolated by SELEX, so they were primarily selected for their capacity to interact with their cognate fluorogenic dye rather than for their capacity to turn fluorescence on. Using, our ultrahigh-throughput directed evolution pipeline we recently improved the fluorogenic properties and affinities of both Spinach and Mango molecules and we developed respectively iSpinach and Mango II, III and IV. These news variants offer an increased sensitivity for gene expression monitoring by live-cell imaging experiments.

Crystal structure of iSpinach
Crystal structure of iSpinach and its expression in vivo in the presence of its fluorogenic co-factor (DFHBI-1T)

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