Systematic reconstruction of binding and stability landscapes of the fluorogenic aptamer spinach

Abstract

Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new generation of in vitro and in vivo sensors for bioanalytics. With the resolved crystal structure of the RNA/small molecule complex, the engineering map becomes available, but comprehensive information regarding the thermodynamic profile of the molecule is missing. Here, we reconstructed the full thermodynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a systematic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermodynamic profile with single base resolution was used to engineer an improved fluorogenic spinach generation via a directed rather than evolutional approach.

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

“…Therefore, even though additional experiments will be required to conclude on the exact contribution of U17, our data suggest that this residue could act by increasing the folding stability of the DFHBI-binding pocket. This mutation was also found to be the only one tolerated in the G-quadruplex region and its introduction in the original Spinach aptamer was also found to increase the fluorescence of the complex (Ketterer et al 2015). Moreover, Broccoli, another DFHBI-binding aptamer (Filonov et al 2014) proposed to share the same core structure as Spinach (Ageely et al 2016), also possesses a U at the same position, further supporting the key role of this residue.…”

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

“…A 98‐nt FLAP, termed Spinach (Figure A, 3) was selected for DFHBI and showed roughly 50 % of the fluorescent brightness of eGFP when bound to the fluorogen (Figure and Table ) . It was successfully tagged to 5S ribosomal RNA (rRNA), which could be visualized in the presence of DFHBI in HEK293T cells in real time (Figure B) as granules or accumulated in the nucleus . Further development of the FLAP to Spinach2 (96 nt) in 2013 using systematic mutagenesis led to increased thermal stability in living cells .…”

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

“…[35,41,42] Af undamental issue with in vitro SELEX is the artificial environment during the selection process resulting in aptamers that often show divergent properties in the cellular environment (e.g.,d ecreased fluorescence due to incorrect folding). [46] In addition, FLAPs are expressed on an aptamer scaffold (e.g., tRNA Lys 3 ,F 30, Figure 1) for efficient transcription and folding. [46] In addition, FLAPs are expressed on an aptamer scaffold (e.g., tRNA Lys 3 ,F 30, Figure 1) for efficient transcription and folding.…”

“…[36,61] It was successfully tagged to 5S ribosomal RNA( rRNA), which could be visualized in the presence of DFHBI in HEK293T cells in real time (Figure 1B)a sg ranules or accumulated in the nucleus. [36,46] Further development of the FLAP to Spinach2 (96 nt) in 2013 using systematic mutagenesis led to increased thermal stability in living cells. [44] Spinach2 was used to localize CGG RNArepeats in COS-7 cells in the presence of DFHBI.…”

“…[43][44][45] Therefore,f urther optimization by directed evolution or rational design based on the crystal structure,i fa vailable,i su sually necessary. [46] In addition, FLAPs are expressed on an aptamer scaffold (e.g., tRNA Lys 3 ,F 30, Figure 1) for efficient transcription and folding. [47,48] More recently,strategies have been developed to select aptamers under more cell-like conditions [45] to minimize alternative non-fluorescent RNAc onformations and improve folding of the fluorescent aptamer in the cell and for fluorescence brightness [49][50][51] rather than solely fluorogen affinity.…”