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. Beetroot and its variant form heterodimers, serving as the starting point for engineered tags, whose through-space inter-fluorophore interactions can be used to monitor RNA dimerisation^[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. Afterwards, an RNA library containing ∼10¹⁴ random library members was used and DFAME was conjugated to agarose beads using an aminohexyl linker as positive target. After 10 rounds of SELEX, an RNA aptamer that binds DFAME and activates its fluorescence by threefold was identified^[1,3].

Detailed information are accessible on SELEX page.

Structure

2D representation

The co-axial stacking of the two Beetroot protomers results in an eight-tiered quadruplex that spans the dimer interface. Here we used ribodraw to complete the figures, through the 3D structure information^[1,3].

Beetroot aptamer (ThT): 5'-GCGCCGGUUAGGCAGAGGUGGGUGGUGUGGAGGAGUAUCUGUCCGGCGC-3'

Beetroot aptamer (DFAME): 5'-GCGCCGGUUAGGCAGAGGUGGGUGGUGUGGAGGAGUAUCUGUCCGGCGC-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 overall structure of Beetroot bound to different fluorescent molecules closely resembles that of the wild-type Beetroot fluorophore complex. These small molecules bind at both ends of the eight-tiered quadruplex at the Beetroot dimer interface, occupying the same pocket and adopting a nearly planar conformation. 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) with resolutions of 1.95 Å, 2.55 Å, 2.1 Å and 2.85 Å respectively^[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 overall structure of Beetroot bound to different fluorescent molecules closely resembles that of the wild-type Beetroot fluorophore complex. These small molecules bind at both ends of the eight-tiered quadruplex at the Beetroot dimer interface, occupying the same pocket and adopting a nearly planar conformation. 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 (shown in sticks) is labeled in magenta. 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 magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with DFAME or other nucleotides surround small molecules.

Ligand information

SELEX ligand

ThT-Beetroot aptamer complex: 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]. DFAME-Beetroot aptamer complex: 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 (Thioflavin T) 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: a unique identifier for substances in the PubChem database.

CAS number: a global registry number for chemical substances.

MedChemExpress: an entry number for the classification of chemicals by the well-known chemical manufacturing company MedChemExpress.

Name	PubChem CID	Molecular Formula	Molecular Weight	CAS	Solubility	MedChemExpress
ThT	16953	C17H19ClN2S	318.9 g/mol	2390-54-7	16.67 mg/mL in DMSO; 5 mg/mL in H2O	HY-D0218

DFAME (Methyl (Z)-3-(4-(3,5-difluoro-4-hydroxybenzylidene)-1-methyl-5-oxo-4,5-dihydro-1H-imidazol-2-yl)acrylate) 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

Name	PubChem CID	Molecular Formula	Molecular Weight	CAS	Solubility	MedChemExpress
DFAME	71240548	C15H12F2N2O4	322.26 g/mol	1420815-55-9	NA	HY-W073524

Similar compound(s)

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.

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
ZINC38539054	2-[4-(Dimethylamino)phenyl]-6-Hydroxy-3-Methyl-1,3-Benzothiazol-3-Ium	NA	24832019
ZINC56936	N,N-dimethyl-4-(6-methyl-1,3-benzothiazol-2-yl)aniline	10205-62-6	82450
ZINC584593350	N,N-dimethyl-4-(5-methyl-1,3-benzothiazol-2-yl)aniline	NA	125465487
ZINC167917029	4-(1,5-dimethylbenzimidazol-2-yl)-N,N-dimethylaniline	NA	20625541
ZINC553312	N,N-dimethyl-4-(6-methyl-1H-benzimidazol-2-yl)aniline	69570-95-2	950737
ZINC4127888	N,N-dimethyl-4-(6-methyl-1,3-benzoxazol-2-yl)aniline	NA	6486758
ZINC1659652	N,N-dimethyl-4-(5-methyl-1,3-benzoxazol-2-yl)aniline	127753-24-6	401586
ZINC751217940	NA	NA	NA
ZINC1857599770	2-[4-(dimethylamino)phenyl]-1,3-benzothiazol-5-ol	NA	155555168
ZINC218483872	(5E)-5-[(3,5-difluoro-4-hydroxyphenyl)methylidene]-3-methyl-2-(nitrosomethylidene)imidazolidin-4-one	1420815-34-4	154723879
ZINC811701546	(3E)-3-[(3,5-difluoro-4-hydroxyphenyl)methylidene]-1H-indol-2-one	NA	NA
ZINC492568317	NA	NA	NA

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)