Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI.
Fiche publication
Date publication
décembre 2017
Journal
RNA (New York, N.Y.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr ENNIFAR Eric, Dr RYCKELYNCK Mickael
Tous les auteurs :
Fernandez-Millan P, Autour A, Ennifar E, Westhof E, Ryckelynck M
Lien Pubmed
Résumé
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.
Mots clés
Aptamers, Nucleotide, chemistry, Base Sequence, Benzyl Compounds, chemistry, Biocatalysis, Fluorescent Dyes, chemistry, High-Throughput Screening Assays, methods, Humans, Imidazolines, chemistry, Nucleic Acid Conformation
Référence
RNA. 2017 12;23(12):1788-1795