Squash aptamer

Jun 30, 2024 • Zhizhong Lu, Ying Ao

Timeline

Description

In 2022, Jaffrey, S. R. 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, Jaffrey, S. R., & Ferré-D'Amaré, A. R. 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, they 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 had 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. Squash RNA aptamer was named by Jaffrey, S. R^[2].

5'-GGGAAGAUACAAGGUGAGCCCAAUAAUAUGGUUUGGGUUAGGAUAGGAAGUAGAGCCUUAAACUCUCUAAGCGGUAUCUUCCC-3'

3D visualisation

Determined the co-crystal structures of Squash bound to DFHBI-1T and DFHO. The structures reveal that the fluorescence turn-on aptamer retains the overall fold of the adenine riboswitch, and that the fluorophores were recognized in a binding site lacking a G-quadruplex.This showed Squash-DFHBI-1T 2.7 Å resolution co-crystal structure in here. The PDB ID of the structure 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 magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with DFHBI-1T.

Ligand information

SELEX ligand

Dissociation constants (Kd) for the RNA-fluorophore complexes were determined by Jaffrey, S. R. 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^[1].

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

PubChem CID: a unique identifier for substances in the PubChem database.

CAS number: a global registry number for chemical substances.

MedChemExpress: a global supplier of high-quality chemicals for life science research.

Name	PubChem CID	Molecular Formula	Molecular Weight	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.

ZINC ID: a compound identifier used by the ZINC database, one of the largest repositories for virtual screening of drug-like molecules.

PubChem CID: a unique identifier for substances in the PubChem database.

CAS number: a global registry number for chemical substances.

ZINC ID	Name	CAS	Pubchem CID	Structure
ZINC98208407	DFHBI	1241390-29-3	70808995
ZINC146958962	SCHEMBL14662970	NA	71240545
NA	3,5-difluoro-4-hydroxybenzylidene 1-trifluoroethyl-imidazolinone	NA	129847459
NA	(z)-4-(2-(difluoroboryl)-4-hydroxybenzylidene)-1,2-dimethyl-1h-imidazol-5(4h)-one	NA	132599529
ZINC146958550	DFAME	1420815-55-9	139600407
NA	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)