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Crystal Structure and Fluorescence Properties of the iSpinach Aptamer in Complex With DFHBI

Crystal Structure and Fluorescence Properties of the iSpinach Aptamer in Complex With DFHBI

Pablo Fernandez-Millan, Alexis Autour, Eric Ennifar, Eric Westhof, Michael Ryckelynck

RNA. 2017 Dec;23(12):1788-1795.

PMID: 28939697

Abstract:

Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach, comprising five mutations for which the exact role has not yet been deciphered. In this work, we cocrystallized a reengineered version of iSpinach in complex with the DFHBI and solved the X-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach sheds some light on the possible function of the different beneficial mutations carried by iSpinach.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP1241390293	DFHBI		1241390-29-3	Price

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