Squash aptamer

Jun 30, 2024 • Zhizhong Lu, Ying Ao

Timeline

Description

In 2022, Dey, S. K. et al. evolved a naturally occurring well-folded adenine riboswitch into a fluorogenic aptamer-Squash，Squash exhibits markedly improved in-cell folding and highly efficient metabolite-dependent folding when fused to a S-adenosylmethionine (SAM)-binding aptamer. The same year, Truong, L. et al. reported the 2.7 Å-resolution co-crystal structure of fluorophore-bound Squash. Squash did not harbor a G-quadruplex, sandwiching its fluorophore between a base triple and a non-canonical base quadruple in a largely apolar pocket^[1,2].

SELEX

To evolve the adenine aptamer into a fluorogenic aptamer, and selected for aptamers that bind to agarose-immobilized DFHBI. By the seventh round of SELEX, the pool exhibited fluorescence upon incubation with DFHBI-1T. At this point, the library was cloned into a bacterial expression vector, transformed into E. coli, and fluorescent aptamers were identified by sorting cells based on DFHBI-1T-induced fluorescence. After recovery of plasmids from the brightest cells, they found three library members which induced fluorescence activation of DFHBI-1T. For directed evolution, they synthesized a DNA library of 9–1 aptamer mutants such that each nucleotide in the ligand-binding pocket has a controlled probability of being converted into one of the other three nucleotides^[1].
Detailed information are accessible on SELEX page.

Structure

2D representation

Here we used ribodraw to complete the figure, through the 3D structure information^[2].

5'-GGGAAGAUACAAGGUGAGCCCAAUAAUAUGGUUUGGGUUAGGAUAGGAAGUAGAGCCUUAAACUCUCUAAGCGGUAUCUUCCC-3'

3D visualisation

Determined the co-crystal structures of Squash bound to DFHBI-1T and DFHO. Our structures reveal that the fluorescence turn-on aptamer retains the overall fold of the adenine riboswitch, and that the fluorophores are recognized in a binding site lacking a G-quadruplex.This shows Squash-DFHBI-1T co-crystal structure in here. The PDB ID is 7KVU^[2].
Additional available structures that have been solved and detailed information are accessible on Structures page.

Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 7KVU by X-ray crystallography. DFHBI-1T(shown in sticks) is labeled in yellow. Right: The hydrogen bonds of binding sites of the aptamer bound with DFHBI-1T^[2].

Ligand information

SELEX ligand

Dissociation constants (Kd) for the RNA-fluorophore complexes were determined. The RNA aptamer at a fixed concentration (50 nM) was titrated with increasing fluorophore concentration and the resulting increase in fluorescence was recorded. For each fluorophore concentration, a background signal for fluorophore only solution was also measured separately and subtracted from the measured RNA-fluorophore signal. Data was fitted to a single site saturation model using nonlinear regression analysis in Sigmaplot software.

Structure ligand

DFHBI-1T is a membrane-permeable RNA aptamers-activated fluorescence probe (ex/em=472 nm/507 nm). DFHBI-1T binds to RNA aptamers (Spinach, iSpinach, and Broccoli) and causes specific fluorescence and lower background fluorescence. DFHBI-1T is used to image RNA in live cells.-----From MedChemExpress

Name	PubChem CID	Molecular Formula	MW	CAS	Solubility	MedChemExpress
DFHBI-1T	101889712	C13H9F5N2O2	320.21 g/mol	1539318-36-9	100 mg/ml(in DMSO)	HY-110251

Similar compound

We used the PubChem database to screen compounds that were more similar to DFHBI-1T, and selected the most similar compounds for display. For those without CAS numbers, we will supplement them with Pubchem CID.

Named	CAS	Pubchem CID	Structure
DFHBI	1241390-29-3	70808995
SCHEMBL14662970	NA	71240545
3,5-difluoro-4-hydroxybenzylidene 1-trifluoroethyl-imidazolinone	NA	129847459
(z)-4-(2-(difluoroboryl)-4-hydroxybenzylidene)-1,2-dimethyl-1h-imidazol-5(4h)-one	NA	132599529
DFAME	1420815-55-9	71240548
DFHBI-2T	1539318-40-5	129080921

References

[1] Repurposing an adenine riboswitch into a fluorogenic imaging and sensing tag.
Dey, S. K., Filonov, G. S., Olarerin-George, A. O., Jackson, B. T., Finley, L. W. S., & Jaffrey, S. R.
Nature chemical biology, 18(2), 180–190. (2022)
[2] The fluorescent aptamer Squash extensively repurposes the adenine riboswitch fold.
Truong, L., Kooshapur, H., Dey, S. K., Li, X., Tjandra, N., Jaffrey, S. R., & Ferré-D'Amaré, A. R.
Nature chemical biology, 18(2), 191–198. (2022)
[3] A universal orthogonal imaging platform for living-cell RNA detection using fluorogenic RNA aptamers.
Yin, P., Ge, M., Xie, S., Zhang, L., Kuang, S., & Nie, Z.
Chemical science, 14(48), 14131–14139. (2023)