iSpinach: a fluorogenic RNA aptamer optimized forin vitroapplications

Abstract

Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme.

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

“…Therefore, we suggest that GoG has a positive impact during this transition by holding the K2 in position via the water W1 and helping to stabilize the G-quartet core. This observation is consistent with the fact that, in our original screen, the two G's were always found together (Autour et al 2016). This G pair was also recently found to increase the fluorescence of Broccoli, but at the expense of an increased dependency on magnesium (Ageely et al 2016).…”

“…4, top left). This confirms our initial suggestion (Autour et al 2016) that the evolutionary process selected the mutation U39C only to increase the stability of the helix without modifying its structure. Since iSpinach and Spinach-P shared the same sequence from G28-C39 to the first layer of G-quartet, both molecules superposed well over this region.…”

“…Nevertheless, our model now allows us to propose that these mutations could, at least in part, act by forming a new electrostatic interaction (U17) as well as ensuring a tighter coordination of the second potassium ion of the G-quartet via a water molecule (G14 and G56). The net effect of these different mutations is to compact and stabilize the structure of the aptamer, especially that of the Gquadruplex core and of the DFHBI binding pocket, making the iSpinach/DFHBI complex far less salt sensitive and more thermostable than the parental molecule as previously observed (Autour et al 2016). This improved folding capacity is also supported by the fact that we could obtain diffracting crystals with minimal modification of the sequence.…”

“…The remainder of the DFHBI-binding pocket consists of a top layer made of a base-triple while the side of the dye establishes interactions with nucleobases. Despite the tight accommodation of the dye and the improved properties of Spinach 2, we recently found that the folding properties of the aptamer were still suboptimal (Autour et al 2016). To further optimize Spinach RNA, we generated mutant libraries and screened them for brighter and better folding mutants using our recently introduced microfluidic-assisted in vitro compartmentalization pipeline (Ryckelynck et al 2015).…”

“…To further optimize Spinach RNA, we generated mutant libraries and screened them for brighter and better folding mutants using our recently introduced microfluidic-assisted in vitro compartmentalization pipeline (Ryckelynck et al 2015). During this process we used selection pressures (i.e., warming, complete absence of K + and use of Na + instead) aimed at challenging RNA structural stability, especially that of the G-quadruplex core (Autour et al 2016). Sequence analysis of the most fluorescent mutants allowed us to identify several beneficial mutations, of which the five displaying the highest effect were combined into the same aptamer.…”