Biotin aptamer

May 8, 2024 • Zhizhong Lu, Ying Ao

Timeline

Description

In 1998, Szostak, J employed in vitro selection techniques to isolate aptamers with affinity binding sites for biotin. In 2000, Nix, J. C. determined the 1.3 Å crystal structure of the aptamer complexed with biotin. Biotin is bound at the interface between the pseudoknot's stacked helices in a pocket defined almost entirely by base-paired nucleotides. The structure provides general insight into the mechanisms by which RNA function is mediated by divalent metals^[1,3].

SELEX

A randomized RNA library, consisting of approximately 10¹⁴ molecules, was synthesized via in vitro transcription from a DNA template and resuspended in a selection buffer comprising 0.1 M KCl, 5 mM MgCl2, and 10 mM Na-HEPES at pH 7.4. Following equilibration at ambient temperature, the RNA pool was applied to a 500 μL biotin-agarose affinity column to selectively bind biotin-interacting RNAs. Non-specific binders were eliminated through a series of 15 washes with 500 μL of the selection buffer. The biotinylated RNAs were eluted using 5 volumes of 500 μL elution buffer, which was the selection buffer supplemented with 5 mM D-biotin at pH 7.4. The affinity-purified RNAs were then pooled, concentrated, and subjected to enzymatic amplification. The resulting enriched RNA pool underwent gel purification and was cycled through additional rounds of selection and amplification. Notably, biotin-binding RNA species were first identified in the fourth selection round, and by the seventh round, these RNAs constituted over 50% of the total sequences present in the pool^[1].
Detailed information are accessible on SELEX page.

Structure

2D representation

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

5'-GGACCGUCAGAGGACACGGUUAAAAAGUCCUCU-3'

3D visualisation

Nix, J. et al. determined the structure of the biotin RNA aptamer using X-ray crystallography. The aptamer structure reveals how a highly complementary binding pocket for an unusual ligand can be built by an RNA using mostly Watson-Crick base-paired nucleotides and a collection of bound solvent molecules. The PDB ID of this structure is 1F27^[3].
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: 1F27 by X-ray crystallography. Biotin (shown in sticks) is labeled in yellow. Right: The hydrogen bonds of binding sites of the aptamer bound with Biotin.

Ligand information

SELEX ligand

The affinity of the aptamer for biotin was determined by measuring the fraction of RNA bound to biotin agarose at a range of immobilized ligand concentrations. The concentration of biotin available for binding was determined by saturating biotin agarose with excess labeled aptamer and measuring the amount of RNA specifically eluted following washing^[1].

Structure ligand

Biotin is one of the vitamin B groups, also known as vitamin H, vitamin B7, coenzyme R (Coenzyme R) and so on. Biotin is abundant in liver, kidney, yeast and milk, and it is an important factor for organisms to fix carbon dioxide. It is easy to combine with protein Avidin in egg white. Eating a large amount of raw protein can hinder the absorption of biotin and lead to biotin deficiency, such as depilation, weight loss and dermatitis. Because its deficiency rarely occurs, it is often called biotin directly.-----From Wiki

Name	PubChem CID	Molecular Formula	MW	CAS	Solubility	Drugbank ID
Biotin	171548	C10H16N2O3S	244.31 g/mol	58-85-5	220 mg/L (at 25 °C)	DB00121

Similar compound

We screened the compounds with great similarity to biotin 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	Named	CAS	Pubchem CID	Structure
ZINC4095742	Biotin Amide	6929-42-6	439597
ZINC3870809	2-Iminobiotin	NA	46936649
ZINC13543600	Lydimycin	NA	70685126
ZINC2169827	vitamin H	NA	6560210
ZINC4321698	Biotin Sulfone	40720-05-6	83863
ZINC4096827	Biocytin	576-19-2	83814
ZINC5192424	Bisnorbiotin	16968-98-2	86492
ZINC4521221	Biotin-X	72040-64-3	446905

References

[1] Functional requirements for specific ligand recognition by a biotin-binding RNA pseudoknot.
Wilson, C., Nix, J., & Szostak, J.
Biochemistry, 37(41), 14410–14419. (1998)
[2] Preliminary crystallographic characterization of an in vitro evolved biotin-binding RNA pseudoknot.
Nix, J. C., Newhoff, A. R., & Wilson, C.
Acta crystallographica. Section D, Biological crystallography, 55(Pt 1), 323–325. (1999)
[3] The 1.3 A crystal structure of a biotin-binding pseudoknot and the basis for RNA molecular recognition.
Nix, J., Sussman, D., & Wilson, C.
Journal of molecular biology, 296(5), 1235–1244. (2000)
[4] A replaceable liposomal aptamer for the ultrasensitive and rapid detection of biotin.
Sung, T. C., Chen, W. Y., Shah, P., & Chen, C. S.
Scientific reports, 6, 21369. (2016)
[5] Pull-down of Biotinylated RNA and Associated Proteins.
Uppala, J. K., Ghosh, C., Sabat, G., & Dey, M.
Bio-protocol, 12(4), e4331. (2022)