Arginine aptamer

Apr 9, 2024 • Yuhang Luo, LinFei Li

Timeline

Description

In 1994, Michael Famulok employed in vitro selection techniques to isolate aptamers with high-affinity binding sites for L-Citrulline. Subsequently, following the selection for L-citrulline, one of the citrulline-binding sequences (Clone 16) was used to generate a mutated pool for further selection. The capability to shift binding specificity from L-citrulline to L-arginine was demonstrated. In 1996, Michael Famulok et al. elucidated the structure of the aptamer complexed with L-Citrulline and L-arginine using multidimensional nuclear magnetic resonance spectroscopy and molecular dynamics calculations^[1].

SELEX

The SELEX began with an initial RNA pool consisting of approximately 10¹⁵ different RNA molecules. This pool was subjected to in vitro selection targeting specific binding to L-citrulline. RNA molecules that demonstrated affinity for L-citrulline were isolated using an epoxy-activated Sepharose 6B agarose column, to which L-citrulline was coupled. After seven cycles, the selected RNA pool was cloned and sequenced. Sequencing of 21 aptamers from this pool revealed 19 different sequences, with two sequences appearing twice, indicating a convergence towards certain sequence motifs that have higher affinity for L-citrulline. Following the selection for L-citrulline, one of the citrulline-binding sequences (Clone 16) was used to generate a mutated pool for further selection. The pool was mutated at a rate of 30% per base position to introduce diversity and potential for adaptation to new targets. This mutated pool was then used in subsequent selection cycles aimed at isolating RNA aptamers with binding specificity for L-arginine. After four cycles, 22 sequences derived from the L-arginine pool were analyzed. Of these, 11 sequences were found to potentially fold into a motif slightly different from the L-citrulline-binding motif but were highly specific to L-arginine^[1].
Detailed information are accessible on SELEX page.

Structure

2D representation

Here we used ribodraw to complete the figure, through the 3D structure information. Here's another aptamer: ag.06 aptamer that was isolated in 1996^[1,5].

A:5'-AGAAGGAGCGU-3'
B:5'-ACAGGUAGGUCGCU-3'

3D visualisation

The solution structures of the L-citrulline and L-arginine RNA aptamers complexed with their respective ligands were determined by Michael Famulok et al. through multidimensional nuclear magnetic resonance (NMR) spectroscopy. The PDB ID of this structure is 1KOC^[4].
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: 1KOC by NMR. Arginine (shown in sticks) is labeled in yellow. Right: The hydrogen bonds of binding sites of the aptamer bound with Arginine^[4].

Ligand information

SELEX ligand

Michael Famulok determined the dissociation constants Kd for L-citrulline RNA conjugates and L-arginine RNA conjugates in solution.The methods used for this purpose are isocratic elution chromatography and equilibrium gel filtration, which allow for a comprehensive assessment of the stability and affinity of the interactions under a variety of experimental conditions. These methods allow a robust assessment of the binding affinity dynamics of RNA with L-citrulline and L-arginine in different environments. The affinity of Citrulline RNA aptamer for L-Citrulline is 68 μM, and the rest demonstrates the affinity of Arginine RNA aptamer^[1].

Structure ligand

L-arginine is an L-alpha-amino acid that is the L-isomer of arginine. It has a role as a nutraceutical, a biomarker, a micronutrient, an Escherichia coli metabolite and a mouse metabolite. It is a glutamine family amino acid, a proteinogenic amino acid, an arginine and a L-alpha-amino acid. It is a conjugate base of a L-argininium(1+). It is a conjugate acid of a L-argininate. It is an enantiomer of a D-arginine.-----from ChEBI

PubChem CID	Molecular Formula	MW	CAS	Solubility	Drugbank ID
6322	C6H14N4O2	174.20 g/mol	74-79-3	50 mg/mL	DB00125

Similar compound

We screened the compounds with great similarity to Citrulline and Arginine 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
ZINC000003873047	L-Thiocitrulline	156719-37-8	2733514
ZINC000001886324	n5-Iminoethyl-l-ornithine	36889-13-1	107984
ZINC000003869452	Canavanin	543-38-4	439202
ZINC000035881877	5-Azidonorvaline	156463-09-1	53662155

References

[1] Molecular Recognition of Amino Acids by RNA-Aptamers: An L-Citrulline Binding RNA Motif and Its Evolution into an L-Arginine Binder.
Michael Famulok
Journal of the American Chemical Society 1994 116 (5), 1698-1706 (1994)
[2] Structural probing and damage selection of citrulline- and arginine-specific RNA aptamers identify base positions required for binding.
P Burgstaller, M Kochoyan, M Famulok
Nucleic acids research. 1995 Dec 11;23(23):4769-76. (1995)
[3] RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity.
A Geiger, P Burgstaller, H von der Eltz, A Roeder, M Famulok
Nucleic acids research. 1996 Mar 15;24(6):1029-36. (1996)
[4] Structural basis of ligand discrimination by two related RNA aptamers resolved by NMR spectroscopy.
Y Yang, M Kochoyan, P Burgstaller, E Westhof, M Famulok
Science. 1996 May 31;272(5266):1343-7. (1996)
[5] Mirror-design of L-oligonucleotide ligands binding to L-arginine.
A Nolte, S Klussmann, R Bald, V A Erdmann, J P Fürste
Nature biotechnology. 1996 Sep;14(9):1116-9. (1996)
[6] Chiral stationary phase based on a biostable L-RNA aptamer.
Agnès Brumbt, Corinne Ravelet, Catherine Grosset, Anne Ravel, Annick Villet, Eric Peyrin
Analytical chemistry. 2005 Apr 1;77(7):1993-8. (2005)