Beetroot aptamer

Apr 30, 2024 • Bo Fu, Jiali Wang

Timeline

Description

In 2022, Jiahui Wu, et al. developed Beetroot aptamer, which enables to bind DFAME and forms an RNA-fluorophore complex. In 2023, Luiz F. M. Passalacqua et al. analyzed the structure of the Beetroot-ThT complex through crystallization, diffraction data collection and NMR spectroscopy^[1,2].

SELEX

About ~10¹⁴ random RNA sequences was used in the systematic evolution of ligands by exponential enrichment approach to generated aptamers that bind DFHO. DFHO with an aminohexyl linker was synthesized so that this fluorophore could be attached to solid support. After eight rounds of SELEX, a single 119 nucleotides-long aptamer (aptamer 6-1) that induced the fluorescence of DFHO was identified^[1].
Detailed information are accessible on SELEX page.

Structure

2D representation

Here we used ribodraw to complete the figure, through the 3D structure information^[1].

5'-GUUAGGCAGAGGUGGGUGGUGUGGAGGAGUAUCUGUC-3'

3D visualisation

In 2023, Luiz F. M. Passalacqua et al. analyzed the structure of the Beetroot-DFAME complex through crystallization, diffraction data collection, structure determination and refinement. The atomic coordinates and structure factor amplitudes have been deposited with Protein Data bank under accession codes 8EYU (Beetroot-DFAME), 8EYV (Beetroot-DFHO), 8EYW (Beetroot-ThT), and 8F0N (Beetroot A16U, U38G-DFHO)^[3].
Additional available structures that have been solved and detailed information are accessible on Structures page. In 2023, Luiz F. M. Passalacqua et al. analyzed the structure of the Beetroot-DFAME complex through crystallization, diffraction data collection, structure determination and refinement. The atomic coordinates and structure factor amplitudes have been deposited with Protein Data bank under accession codes 8EYU (Beetroot-DFAME), 8EYV (Beetroot-DFHO), 8EYW (Beetroot-ThT), and 8F0N (Beetroot A16U, U38G-DFHO)^[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: 8EYW by X-ray crystallography. Thioflavin T(ThT)(shown in sticks) is labeled in yellow. Right: The hydrogen bonds of binding sites of the aptamer bound with ThT or other nucleotides surround small molecules.

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 8EYU by X-ray crystallography. DFAME (shown in sticks) is labeled in yellow. Right: The hydrogen bonds of binding sites of the aptamer bound with DFAME or other nucleotides surround small molecules.

Ligand information

SELEX ligand

K_d is calculated by both ITC and measuring the increase in fluorescence as a function of increasing fluorophore concentration in the presence of a fixed concentration (50 nM) of RNA aptamer^[2].

The K_d was determined by and measuring the increase in fluorescence as a function of increasing RNA aptamer concentration in the presence of a fixed concentration of fluorophore^[1].

Structure ligand

ThT are fluorescent dyes used for histology staining and biophysical studies of protein aggregation and investigate amyloid formation. They are also used in biophysical studies of the electrophysiology of bacteria.-----From Wikipedia

PubChem CID	Molecular Formula	MW	CAS	Solubility	Drugbank ID
16953	C17H19ClN2S	318.9 g/mol	2390-54-7	16.67 mg/mL in DMSO; 5 mg/mL in H2O	HY-D0218

DFAME is a red fluorophore(Ex=508 nm, Em=641 nm). Beetroot and Corn are dimeric fluorogenic RNA aptamers that can bind to DFAME to form Beetroot-DFAME (Kd=460 nM) and Corn-DFAME (Kd= 3600 nM). Beetroot-DFAME (Kd=460 nM) and Corn-DFAME can be used to form RNA assemblies in living cells. Creating RNA assemblies can be used for the study of RNA Nanostructures. DNA/RNA Nanostructures would be useful in cell and gene therapy (CGT) research.-----From MedChemExpress

PubChem CID	Molecular Formula	MW	CAS	Solubility	Drugbank ID
71240548	C15H12F2N2O4	322.26 g/mol	1420815-55-9	100 mg/mL (DMSO)	HY-W073524

Similar compound

We screened the compounds with great similarity to ThT by using the ZINC database and showed some of the compounds' structure diagrams. For some CAS numbers not available, we will supplement them with Pubchem CID.

Source ligand	Named	CAS	Pubchem CID	Structure
ThT	p-Dimethylaminostyrylbenzothiazole	1628-58-6	5378081
ThT	DHPT	92-36-4	7087
ThT	4-(1,3-benzothiazol-2-yl)aniline	6278-73-5	234475
DFAME	DFHBI	1241390-29-3	70808995
DFAME	DFHBI 1T	1539318-36-9	101889712
DFAME	SCHEMBL14662970	NA	71240545

References

[1] Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex.
Song, W., Filonov, G. S., Kim, H., Hirsch, M., Li, X., Moon, J. D., & Jaffrey, S. R.
Nature chemical biology, 13(11), 1187–1194. (2017)
[2] Binding between G quadruplexes at the homodimer interface of the corn RNA aptamer strongly activates thioflavin T fluorescence.
Sjekloća, L., & Ferré-D'Amaré, A. R.
Cell chemical biology, 26(8), 1159–1168.e4. (2019)
[3] Self-assembly of intracellular multivalent RNA complexes using dimeric Corn and Beetroot Aptamers.
Wu, J., Svensen, N., Song, W., Kim, H., Zhang, S., Li, X., & Jaffrey, S. R.
Journal of the American Chemical Society, 144(12), 5471–5477. (2022)
[4] Co-crystal structures of the fluorogenic aptamer Beetroot show that close homology may not predict similar RNA architecture.
Passalacqua, L. F. M., Starich, M. R., Link, K. A., Wu, J., Knutson, J. R., Tjandra, N., Jaffrey, S. R., & Ferré-D'Amaré, A. R.
Nature communications, 14(1), 2969. (2023)