Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI

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.

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

“…Fernandez-Millan and colleagues were able to minimize the length and improve the folding and stability,t hereby its spectral characteristics.The new aptamer was called iSpinach, and its structure could be solved without cleavage,s equence insertions or auxiliary protein binders ( Figure 4D). [49,64]…”

“…[68] Fernandez et al used this loop to optimize intramolecular contacts of the loop nucleotides,t hereby strengthening the apical region. [64] Affinity maturation of the Fab3-6 crystallization chaperon resulted in the highest resolution structure up to now (1.64 ,P DB ID: 6b14). [64] Affinity maturation of the Fab3-6 crystallization chaperon resulted in the highest resolution structure up to now (1.64 ,P DB ID: 6b14).…”

“…Schematic overview of FLAP. [49,64] In an effort to make the selection of FLAPs more efficient, Jaffrey and co-workers integrated fluorescence enhancement into their SELEX process and used fluorescence-activated cell sorting (FACS) with E. coli cells. This leads to ad rastic increase in fluorescence.…”

“…A) Secondary-structure prediction by mfold using default settings for RNA. [64] Dotted line indicates loop used for separation of the two strands during construct design. B-D) Spinach X-ray crystal structure variants.…”

“…[62,63] Another variant, termed iSpinach (69 nt), was optimized by Ryckelynck and coworkers in 2016 using random mutagenesis with microfluidic-assisted compartmentalization for in vitro applications. [49,64] In an effort to make the selection of FLAPs more efficient, Jaffrey and co-workers integrated fluorescence enhancement into their SELEX process and used fluorescence-activated cell sorting (FACS) with E. coli cells. [45] This selection method resulted in Broccoli (49 nt) in 2014, which showed superior properties for applications in cells (no requirement for high-magnesium medium or at RNAs caffold) and around 84 %o fe GFP brightness with DFHBI-1T ( Figure 3a nd Table 1).…”