B.2 aptamer

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Timeline

2002

Antonino Biroccio et al. used SELEX procedure to find small RNA molecules that are specific and high-affinity ligands of nonstructural 5B (NS5B) polymerase[1]

2003

Nam Viet Vo et al. showed Most (60%) of the aptamers they selected carry the conserved YGUAGR hexamer ((Y = pyrimidine, R = purine) at the 5' end of the 40-nt randomized region, and 74% of the aptamers end in (A/C)U at the 3' end[2]

2011

Changhyun Roh et al. demonstrated a streptavidin-biotin conjugation method, namely, the RNA aptamer sensor system that can quantify viral protein with detection level of 700 pg mL(-1) using a biotinylated RNA oligonucleotide on an Octet optical biosensor[3]

2013

Chang Ho Lee et al. identified specific and avid RNA aptamers consisting of 2'-hydroxyl- or 2'-fluoropyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication[4]

2015

Chang Ho Lee et al. modified a 29 nucleotide-long 2'-F pyrimidine modified RNA aptamer through conjugation of cholesterol for in vivo availability[5]

2023

CRISPR-Cas9 was used to insert an expression cassette encoding an RNA aptamer targeting HCV NS5B replicase as an anti-HCV agent into adeno-associated virus integration site 1 (AAVS1), leading to the stable expression of aptamer RNA in the developed cell line[6]

Description

 In 2002, Antonino Biroccio and colleagues isolated RNA aptamers that bind specifically to the HCV NS5B polymerase (HCV NS5BΔC55 protein). It is worth mentioning that the full-length purified enzyme has a very poor catalytic activity. Deletions of the C-terminal membrane localization signal have allowed the production of proteins with enhanced solubility and activity. Therefore, the NS5B protein lacking of the C-terminal 55 amino acids was used in this study[1].

SELEX

In 2002, Antonino Biroccio and colleagues selected a structurally constrained combinatorial RNA library by using the SELEX procedure to isolate high affinity RNA ligands for the HCV NS5B polymerase (HCV NS5BΔC55 protein). The library contained 35-nt random sequences in two segments of 25 and 10 nt, divided by a constant core sequence of 10 nt. This was in turn flanked by 18-nt constant regions at the 5' and 3' ends, respectively[1].
Detailed information are accessible on SELEX page.



Structure

B.2 was the aptamer sequence mainly studied in the article, which had a high affinity with HCV NS5BΔC55 protein. The 2D structure of the figure is based on the article by ribodraw tool to draw[1].

5'-GGGAUGCUUCGGCAUCCCCGAAGCCGCUAUGGACCAGUGGCGCGGCUUCGGCCCGACGGAGUGGUACCGCUUCGGCGGUACGUAAGCUUGGG-3'

 drawing


Ligand information

SELEX ligand

HCV NS5B polymerase (HCV NS5B protein) performs primer-template recognition and RNA synthesis during viral replication. Initiates RNA transcription/replication at a flavin adenine dinucleotide (FAD), resulting in a 5'- FAD cap on viral RNAs. In this way, recognition of viral 5' RNA by host pattern recognition receptors can be bypassed, thereby evading activation of antiviral pathways.-----From Uniprot

Name Uniprot ID Pfam MW Amino acids sequences PDB Gene ID
HCV NS5B polymerase (HCV NS5B protein) O92972 PF00998 62.16 kDa SMSYTWTGALITPCAAEESKLPINPLSNSLLRHHNMVYATTSRSASLRQKKVTFDRLQVLDDHYRDVLKEMKAKASTVKAKLLSIEEACKLTPPHSAKSKFGYGAKDVRNLSSRAVNHIRSVWEDLLEDTETPIDTTIMAKSEVFCVQPRKPARLIVFPDLGVRVCEKMALYDVVSTLPQAVMGSSYGFQYSPKQRVEFLVNTWKSKKCPMGFSYDTRCFDSTVTESDIRVEESIYQCCDLAPEARQAIRSLTERLYIGGPLTNSKGQNCGYRRCRASGVLTTSCGNTLTCYLKATAACRAAKLQDCTMLVNGDDLVVICESAGTQEDAAALRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDASGKRVYYLTRDPTTPLARAAWETARHTPINSWLGNIIMYAPTLWARMILMTHFFSILLAQEQLEKALDCQIYGACYSIEPLDLPQIIERLHGLSAFTLHSYSPGEINRVASCLRKLGVPPLRTWRHRARSVRAKLLSQGGRAATCGRYLFNWAVRTKLKLTPIPAASQLDLSGWFVAGYSGGDIYHS 3MWV AAC15722.1

Some isolated sequences bind to the affinity of the protein.

Name Sequence Ligand Affinity
B.2 GGGAUGCUUCGGCAUCCCCGAAGCCGCUAUGGACCAGUGGCGCGGCUUCGGCCCGACGGAGUGGUACCGCUUCGGCGGUACGUAAGCUUGGG HCV NS5BΔC55 protein 1.5 ± 0.2 nM
A.2 GGGAUGCUUCGGCAUCCCAGUCGAUGCGUAUCGCAGACUAUGUGGCUUCGGCCGUUGGAGUUGGUACCGCUUCGGCGGUACGUAAGCUUGGG HCV NS5BΔC55 protein 16 ± 1.6 nM
B.3 GGGAUGCUUCGGCAUCCCGCUCUGGGCCGAAUAUGGACCACGUGGCUUCGGCCGCCAGCUCGUGUACCGCUUCGGCGGUACGUAAGCUUGGG HCV NS5BΔC55 protein 10 ± 1.6 nM
drawing


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
3GNW-B 58.9 0.4 RNA-directed RNA polymerase
5Y6R-A 25.2 3.4 Genome polyprotein
4K6M-A 21.2 3.1 Polyprotein
8WU3-B 20.7 3.3 RNA polymerase
1SH0-B 19.9 4.1 RNA polymerase
2IJD-1 19.6 3.8 Picornain 3C, RNA-directed RNApolymerase
5I61-A 17.6 3.4 Potential RNA-dependent RNA polymerase
8GWE-A 16.1 4.0 RNA-directed RNA polymerase
8IIC-B 15.8 4.5 Polymerase polyprotein
8FM9-M 15.4 4.1 RNA-directed RNA polymerase


References

[1] Selection of RNA aptamers that are specific and high-affinity ligands of the hepatitis C virus RNA-dependent RNA polymerase.
Biroccio, A., Hamm, J., Incitti, I., De Francesco, R., & Tomei, L.
Journal of virology, 76(8), 3688–3696. (2002)
[2] Identification of RNA ligands that bind hepatitis C virus polymerase selectively and inhibit its RNA synthesis from the natural viral RNA templates.
Vo, N. V., Oh, J. W., & Lai, M. M.
Virology, 307(2), 301–316. (2003)
[3] Label free inhibitor screening of hepatitis C virus (HCV) NS5B viral protein using RNA oligonucleotide.
Roh, C., Kim, S. E., & Jo, S. K. 
Sensors (Basel, Switzerland), 11(7), 6685–6696. (2011)
[4] Inhibition of hepatitis C virus (HCV) replication by specific RNA aptamers against HCV NS5B RNA replicase.
Lee, C. H., Lee, Y. J., Kim, J. H., Lim, J. H., Kim, J. H., Han, W., Lee, S. H., Noh, G. J., & Lee, S. W.
Journal of virology, 87(12), 7064–7074. (2013)
[5] Pharmacokinetics of a Cholesterol-conjugated Aptamer Against the Hepatitis C Virus (HCV) NS5B Protein.
Lee, C. H., Lee, S. H., Kim, J. H., Noh, Y. H., Noh, G. J., & Lee, S. W.
Molecular therapy. Nucleic acids, 4(10), e254. (2015)
[6] Generation of hepatitis C virus-resistant liver cells by genome editing-mediated stable expression of RNA aptamer.
Kim, T. H., & Lee, S. W.
Molecular therapy. Methods & clinical development, 31, 101151. (2023)