HCV NS5BΔC55 aptamer

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Timeline

2002

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

2003

Lai, M. M. 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

Jo, S. K. 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

Lee, S. W. 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

Lee, S. W. 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, Tomei, L. 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, Tomei, L. 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. Initially, the randomized RNA pool was challenged with streptavidin beads coated with biotinylated NS5BΔC55 protein (NS5BΔC55bio), and the complexed RNA was eluted, amplified (reverse transcription and PCR), and in vitro transcribed by using T7 RNA polymerase in the presence of a labeled nucleotide tracer. The products of transcription were used as an RNA pool for the next cycle of selection. At the fourth selection cycle, the RNA pool was roughly 140-fold enriched in the molecules with high binding affinity for NS5BΔC55bio. As already noticed, in the case of structurally constrained RNA libraries additional selection cycles failed to increase the amount of the selected RNA. Therefore, the PCR DNA from the fourth cycle was cloned, the nucleotide sequences of 15 individual clones were determined, and their structural motifs were analyzed. Based on the presence of common sequence motifs, the clones were grouped in three families[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. The B.2 aptamer was named by Tomei, L. et al. in the article[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

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.

Name Uniprot ID Pfam Mass Protein sequence PDB ID GenBank
HCV NS5B polymerase (HCV NS5B protein) O92972 PF00998 62.16 kDa
...... SMSYTWTGALITPCAAEESKLPINPLSNSLLRHHNMVYATTSRSASLRQKKVTFDRLQVLDDHYRDVLKEMKAKASTVKAKLLSIEEACKLTPPHSAKSKFGYGAKDVRNLSSRAVNHIRSVWEDLLEDTETPIDTTIMAKSEVFCVQPRKPARLIVFPDLGVRVCEKMALYDVVSTLPQAVMGSSYGFQYSPKQRVEFLVNTWKSKKCPMGFSYDTRCFDSTVTESDIRVEESIYQCCDLAPEARQAIRSLTERLYIGGPLTNSKGQNCGYRRCRASGVLTTSCGNTLTCYLKATAACRAAKLQDCTMLVNGDDLVVICESAGTQEDAAALRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDASGKRVYYLTRDPTTPLARAAWETARHTPINSWLGNIIMYAPTLWARMILMTHFFSILLAQEQLEKALDCQIYGACYSIEPLDLPQIIERLHGLSAFTLHSYSPGEINRVASCLRKLGVPPLRTWRHRARSVRAKLLSQGGRAATCGRYLFNWAVRTKLKLTPIPAASQLDLSGWFVAGYSGGDIYHS (Residues 2420-2982)
3MWV AAC15722.1

Some isolated sequences bind to the affinity of the protein[1].

Name Sequence Ligand Affinity
B.2 5'-GGGAUGCUUCGGCAUCCCCGAAGCCGCUAUGGACCAGUGGCGCGGCUUCGGCCCGACGGAGUGGUACCGCUUCGGCGGUACGUAAGCUUGGG-3' HCV NS5BΔC55 protein 1.5 ± 0.2 nM
A.2 5'-GGGAUGCUUCGGCAUCCCAGUCGAUGCGUAUCGCAGACUAUGUGGCUUCGGCCGUUGGAGUUGGUACCGCUUCGGCGGUACGUAAGCUUGGG-3' HCV NS5BΔC55 protein 16 ± 1.6 nM
B.3 5'-GGGAUGCUUCGGCAUCCCGCUCUGGGCCGAAUAUGGACCACGUGGCUUCGGCCGCCAGCUCGUGUACCGCUUCGGCGGUACGUAAGCUUGGG-3' HCV NS5BΔC55 protein 10 ± 1.6 nM
drawing

Similar compound(s)

We used the RCSB PDB website's similar structure search to find the top 10 structures similar to HIV-1 REV PROTEIN (residues T34-R50), and calculated TM-socre values and RMSD values using the TM-align website.

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-socre RMSD (Å) Description
3MWV-A 63.3 0 Genome polyprotein
4J02-A 61.3 0.2 Genome polyprotein
4JY0-A 61.2 0.2 Genome polyprotein
4J10-A 61.1 0.3 Genome polyprotein
4GMC-A 61.0 0.3 NS5B polymerase
4IZ0-A 60.9 0.2 RNA-directed RNA polymerase
2XHU-B 60.7 0.3 RNA-directed RNA polymerase
2XHV-B 60.7 0.2 RNA-directed RNA polymerase
4J04-A 60.5 0.3 Genome polyprotein
3MWW-A 60.4 0.3 Genome polyprotein


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)