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G9-II aptamer
Timeline
An aptamer called 10G-1, which binds specifically to the NS3 protease, was selected for the first time by in vitro genetic-selection strategy[1]
Keigo Machida et al. isolated aptamers that inhibited the activity of NS3 protease and helicase[2]
Kotaro Fukuda et al. isolated RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide ΔNS3[3]
The binding sites of NS3 protease domain and aptamer were analyzed by alanine scanning mutagenesis[4]
Fumiko Nishikawa et al. constructed a G9 aptamer expression system in cultured cells, using the cytomegarovirus enhancer + chicken beta-actin globin (CAG) promoter[5]
Kotaro Fukuda et al. designed a new aptamer called NEO-III-14U to inhibit both protease and helicase activity[6]
Robert Vaughan et al. showed that RNAs can bind directly to the active site cleft of the NS3 protease domain (NS3P) and inhibit proteolysis of peptide substrates. RNAs that are less apt to form intramolecular structures have a stronger inhibitory activity than RNAs with more stable base paired regions[7]
Description
In 2000, Kotaro Fukuda and colleagues isolated RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide ΔNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX)[3].SELEX
In 2000, Kotaro Fukuda and colleagues designed a selecting method based on existing research. The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of ΔNS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9-I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC[3].
Detailed information are accessible on SELEX page.
Structure
G9-II was the aptamer sequence mainly studied in the article, which had a high affinity with ΔNS3. The 2D structure of the figure is based on the article by ribodraw tool to draw[3].5'-GGGAGAAUUCCGACCAGAAGUGCUCUUAGAAUGGGACUAAGACACGGGACCCUUUCCUCUCUCCUUCCUCUUCU-3'
Ligand information
SELEX ligand
Hepatitis C virus NS3 protein is a serine protease which has a trypsin-like fold. The non-structural (NS) protein NS3 is one of the NS proteins involved in replication of the HCV genome. The action of NS3 protease (NS3P), which resides in the N-terminal one-third of the NS3 protein, then yields all remaining non-structural proteins.-----From Pfam
| Name | Uniprot ID | Pfam | MW | Amino acids sequences | PDB | Gene ID |
|---|---|---|---|---|---|---|
| HCV NS3 protease (ΔNS3) | B2Y2M9 | PF02907 | 19.15 kDa | APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRS | 3KF2 | ABY67662.1 |
Some isolated sequences bind to the affinity of the protein.
| Name | Sequence | Ligand | Affinity |
|---|---|---|---|
| G9-II | GGGAGAAUUCCGACCAGAAGUGCUCUUAGAAUGGGACUAAGACACGGGACCCUUUCCUCUCUCCUUCCUCUUCU | HCV NS3 protease (ΔNS3) | 6.3 nM |
| G9-I | GGGAGAAUUCCGACCAGAAGCUUCGGGAUUUGAGGGUAGAAUGGGACUACCUUUCCUCUCUCCUUCCUCUUCU | HCV NS3 protease (ΔNS3) | 11.6 nM |
| G9-III | GGGAGAAUUCCGACCAGAAGUACGACACGAUUGGGACGUGUCUAUGGGACCCUUUCCUCUCUCCUUCCUCUUCU | HCV NS3 protease (ΔNS3) | 8.9 nM |
Similar compound
We used the Dail server website to compare the structural similarities of ligand proteins, and chose the top 10 in terms of similarity for presentation. The Dali server is a network service for comparing protein structures in 3D. Dali compares them against those in the Protein Data Bank (PDB). Z-score is 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) value is used to measure the degree to which atoms deviate from the alignment position.
| PDB | Z-socre | RMSD | Description |
|---|---|---|---|
| 1CU1-A | 20.7 | 1.0 | Protein (protease/helicase NS3) |
| 2W5E-A | 15.3 | 2.2 | Putative sering protease |
| 3K6Y-A | 14.7 | 2.7 | Possible membrane-associated sering protease |
| 2R3Y-A | 14.4 | 2.8 | Protease degs |
| 6Z05-A | 14.2 | 2.8 | Dego family sering endoprotease |
| 4INK-A | 13.4 | 2.8 | Sering protease spld |
| 5LKL-A | 13.3 | 3.0 | Genome polyprotein |
| 1SGP-E | 13.0 | 3.0 | Streptomyces griseus proteinase B |
| 7SJY-A | 12.8 | 3.1 | Anti-sigma-I factor RSGI9 |
| 8W20-C | 12.7 | 3.0 | Secreted protein |
References
[1] Selection of RNA aptamers that bind specifically to the NS3 protease of hepatitis C virus.Urvil, P. T., Kakiuchi, N., Zhou, D. M., Shimotohno, K., Kumar, P. K., & Nishikawa, S.
European journal of biochemistry, 248(1), 130–138. (1997)
[2] Isolation of RNA aptamers specific to the NS3 protein of hepatitis C virus from a pool of completely random RNA.
Kumar, P. K., Machida, K., Urvil, P. T., Kakiuchi, N., Vishnuvardhan, D., Shimotohno, K., Taira, K., & Nishikawa, S.
Virology, 237(2), 270–282. (1997)
[3] Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease.
Fukuda, K., Vishnuvardhan, D., Sekiya, S., Hwang, J., Kakiuchi, N., Taira, K., Shimotohno, K., Kumar, P. K., & Nishikawa, S.
European journal of biochemistry, 267(12), 3685–3694. (2000)
[4] The RNA aptamer-binding site of hepatitis C virus NS3 protease.
Hwang, J., Fauzi, H., Fukuda, K., Sekiya, S., Kakiuchi, N., Shimotohno, K., Taira, K., Kusakabe, I., & Nishikawa, S.
Biochemical and biophysical research communications, 279(2), 557–562. (2000)
[5] Inhibition of HCV NS3 protease by RNA aptamers in cells.
Nishikawa, F., Kakiuchi, N., Funaji, K., Fukuda, K., Sekiya, S., & Nishikawa, S.
Nucleic acids research, 31(7), 1935–1943. (2003)
[6] An RNA ligand inhibits hepatitis C virus NS3 protease and helicase activities.
Fukuda, K., Umehara, T., Sekiya, S., Kunio, K., Hasegawa, T., & Nishikawa, S.
Biochemical and biophysical research communications, 325(3), 670–675. (2004)
[7] RNA binding by the NS3 protease of the hepatitis C virus.
Vaughan, R., Li, Y., Fan, B., Ranjith-Kumar, C. T., & Kao, C. C.
Virus research, 169(1), 80–90. (2012)