Bovine prion protein aptamer

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Timeline

1997

The first aptamer for PrP was selected in 1997 using the SELEX method directed against recombinant Syrian hamster full-length prion protein (rPrP23–231)[1]

2008

RNA aptamers against a bovine bPrP were obtained by means of an in vitro selection method from RNA pools containing a 55-nt randomized region[2]

2009

High-resolution structure of an RNA aptamer (R12) against isolated domains of the bovine PrP by NMR was first reported and two lysine clusters of bPrP have been identified as binding sites for R12[3]

2013

NMR measurements provide the first high-resolution 3D-structure of the complex formed with N-terminal PrP peptides (P1 and P16) and the R12 aptamer. What's more, R12 reduced the PrPSc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent[4]

2014

The hybrid method and the 3D-RISM theory are employed to demonstrate that the driving force for the binding between R12 and P16 (a PrP peptide) is a robust gain of water entropy, and the energy decrease driven by attractive interactions between R12 and P16 is compensated by the energetic dehydration effect after binding or vice-versa[5]

2019

An anti-prion RNA aptamer, R12, inhibits the interaction of PrP with Aβto prevent Alzheimer's disease[6]

Description

In 2008, Nishikawa et al. employed the SELEX method to isolate aptamers and identified four consecutive GGA triplet repeats (GGA4) in the major RNA aptamers obtained. Subsequently, in 2013, Mashima et al. utilised NMR spectroscopy to determine the first high-resolution three-dimensional structure of the complex formed by the N-terminal PrP peptides (P1 and P16) and the R12 aptamer[3,4].



SELEX

In 2008, Nishikawa et al. used the SELEX method to isolate aptamers and identified four consecutive GGA triplet repeats (GGA4) in the major RNA aptamers obtained. They carried out SELEX using 97-nt RNA pool that has 55-nt randomized sequences. To increase the selection stringency, they applied the following selection pressures: (1) decreased protein concentration and reaction time, and increased washing volumes; (2) increased tRNA concentration as a non-specific competitor; and (3) increased concentration of anti-mPrP RNA aptamer16 as a specific competitor[3].

Detailed information are accessible on SELEX page.



Structure

2D representation

R12 aptamer forms an intramolecular parallel quadruplex, which consists of G:G:G:G tetrads and G(:A):G:G(:A):G hexad planes. Two quadruplexes assemble into a dimer through intermolecular hexad–hexad stacking. Here we utilized Ribodraw to complete the figure based on the 3D structure information[3].

5'-GGAGGAGGAGGA-3'

drawing

3D visualisation

Mashima et al. utilised NMR spectroscopy to determine the first high-resolution three-dimensional structure of the complex formed between the N-terminal PrP peptides (P1 and P16) and the R12 aptamer. There are the similar structures between 2RQJ，2RSK and 2RU7, so only the 2RU7 with last literature is chosen[4].

Additional available structures that have been solved and detailed information are accessible on Structures page.

(Clicking the "Settings/Controls info" to turn Spin off)      

drawing PDBe Molstar




Binding pocket

Left: A surface representation of the aptamer’s binding pocket, generated from PDB ID: 2RU7 at a resolution of 2.8 Å. The bovine prion protein is depicted with vacuum electrostatics, where blue represents positive charges and red represents negative charges. Right: Hydrogen bonds at the binding sites of the aptamer bound to the bovine prion protein.

drawing drawing


Ligand information

SELEX ligand

Radioisotope labeling of RNA by in vitro transcription was carried out using α-32P-ATP. Refolded 32P labeled aptamer (10 nM) was mixed with varying concentrations of bPrP, or its derivatives, to a total volume of 25 µl in reaction buffer [20 mM Tris-HCl (pH 7.5), 100 mM NaCl or 10 mM KCl]. After 20 min incubation, the mixture was passed through a nitrocellulose filter and washed with 500 µl of the reaction buffer. The amount of bound RNA was measured with BAS 2500, and binding activities were calculated as the percentage of input RNA retained on the filter in the protein-RNA complex. We determined the equilibrium dissociation constant (Kd) using GraphPad PRISM using non-linear regression curve fitting[2].

Name Sequence Ligand Affinity
apt #1 5′-CAAUCCAUUCAUCUCUCGAAUGAGGAAGUAGCCCAAGAGGAGGAGGAGGAUGAGC-3′ bovine prion protein (bPrP) 82 nM
apt #6 5′-ACCUUCUGUUCAUCGUGACCAACCCAAUAGAUUGUGAUAAAGGAGGAGGAGGA-3′ bovine prion protein (bPrP) 166 nM

Structure ligand

The major prion protein (PrP) is encoded in the human body by the PRNP gene also known as CD230 (cluster of differentiation 230).Expression of the protein is most predominant in the nervous system but occurs in many other tissues throughout the body. The protein can exist in multiple isoforms: the normal PrPC form, and the protease-resistant form designated PrPRes such as the disease-causing PrPSc (scrapie) and an isoform located in mitochondria.-----From Wiki

UniProt ID: uniquely identifies protein sequences in the UniProt database, a resource for protein information.

Pfam: a widely recognised database of protein families and domains.

GenBank: maintained by NCBI(National Center for Biotechnology Information), is a database of nucleotide sequences from various organisms, vital for genetic and molecular biology research.

Mass: an intrinsic property of a body.

Uniprot ID Pfam Mass Protein sequence PDB ID GenBank
P10279 IPR000817 28.61 KDa
GGPYLQ ...... MVKSHIGSWILVLFVAMWSDVGLCKKRPKPGGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGGWGQGGTHGQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGSDYEDRYYRENMHRYPNQVYYRPVDQYSNQNNFVHDCVNITVKEHTVTTTTKGENFTETDIKMMERVVEQMCITQYQRESQAYYQRGASVILFSSPPVILLISFLIFLIVG
1DX0 281427
drawing

Similar compound

We used the Dail server website to compare the structural similarities of ligand proteins, and selected the previous information with high similarity for presentation.

Dail server website: a network service for comparing protein structures in 3D. Dali compares them against those in the Protein Data Bank (PDB).

Z-score: a standard score that is converted from an original score. The list of neighbours is sorted by Z-score. Similarities with a Z-score lower than 2 are spurious.

RMSD: (Root Mean Square Deviation) is used to measure the degree to which atoms deviate from the alignment position.

PDB: PDB ID+ chain name.

PDB Z-score RMSD Description
1DX0-A 22.5 0 Original chain
2KUN-A 12 2.7 major prion protein
1I17-A 6.1 3.5 prion-like protein
6THH-C 5.2 2.8 sirv2 acrid1 (gp02) anti-crispr protein
7BU0-A 5 5 uncharacterized protein
2VWA-A 5 3.1 putative uncharacterized protein pf13_0012
6WB9-6 4.9 3.4 endoplasmic reticulum membrane protein complex su
2KHM-A 4.8 3.6 fibroin-3
3WFW-A 4.8 2.9 hemoglobin-like flavoprotein fused to roadblock/l
8CR1-C 4.7 2.5 atpase asna1


References

[1] An intramolecular quadruplex of (GGA)4 triplet repeat DNA with a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad, and its dimeric interaction.
Matsugami, A., Ouhashi, K., Kanagawa, M., Liu, H., Kanagawa, S., Uesugi, S., & Katahira, M.
Journal of Molecular Biology, 313(2):255-69. (2001)
[2] Detection of structural changes of RNA aptamer containing GGA repeats under the ionic condition using the microchip electrophoresis.
Nishikawa, F., Murakami, K., Noda, K., Yokoyama, T., & Nishikawa, S.
Nucleic Acids Symposium Series,(51):397-8. (2007)
[3] Anti-bovine Prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its β isoform with high affinity.
Murakami, K., Nishikawa, F., Noda, K., Yokoyama, T., & Nishikawa S.
Prion, 2(2):73-80. (2008)
[4] Structural analysis of r(GGA)4 found in RNA aptamer for bovine prion protein.
Matsugami, A., Mashima, T., Nishikawa, F., Murakami, K., & Nishikawa, S.
Nucleic Acids Symposium Series, (52):179-80. (2008)
[5] Unique quadruplex structure and interaction of an RNA aptamer against bovine prion protein.
Mashima, T., Matsugami, A., Nishikawa, F., Nishikawa, S., & Katahira, M.
Nucleic Acids Research, 37(18):6249-58. (2009)
[6] Anti-prion activity of an RNA aptamer and its structural basis.
Mashima, T., Nishikawa, F., Kamatari, YO., Fujiwara, H., Saimura, M., Nagata, T., Kodaki, T., Nishikawa, S., Kuwata, K., & Katahira, M.
Nucleic Acids Research, 41(2):1355-62. (2012)
[7] Cross-talk between prion protein and quadruplex-forming nucleic acids: a dynamic complex formation.
Cavaliere, P., Pagano, B., Granata, V., Prigent, S., Rezaei, H., Giancola, C., & Zagari, A.
Nucleic Acids Research, 41(1):327-39. (2013)
[8] Binding of an RNA aptamer and a partial peptide of a prion protein: crucial importance of water entropy in molecular recognition.
Hayashi, T., Oshima, H., Mashima, T., Nagata, T., Katahira, M., & Kinoshita, M.
Nucleic Acids Research, 42(11):6861-75. (2014)
[9] The anti-prion RNA aptamer R12 disrupts the Alzheimer's disease-related complex between prion and amyloid β.
Iida, M., Mashima, T., Yamaoki, Y., So, M., Nagata, T., & Katahira, M.
FEBS Journal, 286(12):2355-2365. (2019)
[10] Development and structural determination of an anti-PrPC aptamer that blocks pathological conformational conversion of prion protein.
Mashima, T., Lee, JH., Kamatari, YO., Hayashi, T., Nagata, T., Nishikawa, F., Nishikawa, S., Kinoshita, M., Kuwata, K., & Katahira, M.
Scientific Reports, 10(1):4934. (2020)