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Antimicrobial Agents and Chemotherapy, September 2006, p. 3019-3027, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.01603-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

High-Affinity Aptamers to Subtype 3a Hepatitis C Virus Polymerase Display Genotypic Specificity

Louisa A. Jones,1,2 Leighton E. Clancy,1,2 William D. Rawlinson,1,2,3 and Peter A. White2*

Virology Division, Department of Microbiology, SEALS, Prince of Wales Hospital, Randwick, Sydney, NSW 2031, Australia,1 School of Biotechnology and Biomolecular Sciences, Faculty of Science,2 School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia3

Received 6 December 2005/ Returned for modification 13 April 2006/ Accepted 4 July 2006


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ABSTRACT
 
Research into antiviral agents directed at hepatitis C virus (HCV) proteins is commonly based and tested on a single genotype, namely, genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than genotype 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach used in this study that allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNA-dependent RNA polymerase (RdRp) (nonstructural protein 5B [NS5B]) of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 as the partitioning system. Two aptamers, r10/43 and r10/47, were chosen for further studies on the basis of their abilities to bind the HCV RdRp and inhibit polymerase activity. The affinities (equilibrium dissociation constants) of these aptamers for the HCV subtype 3a polymerase were estimated to be 1.3 ± 0.3 nM (r10/43) and 23.5 ± 6.7 nM (r10/47). The inhibition constants of r10/43 and r10/47 were estimated to be 1.4 ± 2.4 nM and 6.0 ± 2.3 nM, respectively. Inhibition of HCV 3a polymerase was specific for r10/47, while r10/43 also demonstrated some inhibitory effect on norovirus and {phi}6 polymerase activity. Neither r10/43 nor r10/47 was able to inhibit the RdRp activity of HCV genotype 1a and 1b polymerases. This study is the first description of an inhibitor specific to the HCV subtype 3a polymerase.


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INTRODUCTION
 
Hepatitis C virus (HCV) is estimated to infect 3% of the world's population, approximately 170 million people (55). HCV was discovered in 1989 as the major causative agent of non-A, non-B hepatitis in blood transfusion recipients (10, 38). Chronic hepatitis, which results from infection with HCV in 50 to 80% of cases (19), is a major cause of liver disease and liver cancer and is the leading cause of liver transplantation in the United States and Australia (9, 40, 48). There is no vaccine for HCV, and current treatments are limited by variable response rates and a high incidence of side effects (21, 26, 41). Therefore, HCV is a significant human viral pathogen that is responsible for a large worldwide disease burden.

HCV is a member of the family Flaviviridae and is an enveloped virus with a single-stranded RNA genome approximately 9.5 kb in length that contains a single open reading frame. The genome is replicated via a negative-stranded intermediate by the nonstructural protein 5B (NS5B), which is the viral RNA-dependent RNA polymerase (RdRp). HCV is classified into six major genotypes (50), whose genomes vary by approximately 30% at the nucleotide level. HCV genotypes also demonstrate differential geographical distribution (55), with genotype 1 the most prevalent worldwide, genotype 5 predominant in South Africa, genotype 4 predominant in the Middle East, while genotype 6 is commonly found in Southeast Asia. HCV genotype 3 infects approximately 33 million people worldwide or 20% of the estimated number of HCV infections (30). In Australia alone, subtype 3a is estimated to account for 33.5% of the infections with HCV (42, 53).

Treatment of HCV currently involves pegylated interferon alone or in combination with ribavirin. This treatment is poorly tolerated, with a high incidence of side effects (20) and variable sustained response rates. Only 50 to 80% of treated patients resolve the infection (21, 26, 41). The range reflects treatment outcomes, which vary according to the infecting genotype. Genotypes 2 and 3 tend to respond better to treatment than genotypes 1 and 4 do (27, 46). The search for novel HCV antiviral agents has therefore become an intense area of research.

Proteins that are essential for the replication of the virus, such as the HCV protease (NS3), and the RNA polymerase (NS5B), are current targets for antiviral agents, paralleling the evolution of human immunodeficiency virus treatments. Treatments directed at the HCV polymerase, in particular, have less chance of adversely affecting the patient, as no corresponding enzyme exists in humans. NS5B was identified as a putative RdRp by discovery of the Gly-Asp-Asp (GDD) motif (36), which is essential for polymerase activity and common to many RdRps characterized to date. The crystal structure of the HCV RdRp has recently been solved, revealing a completely enclosed active site, and the canonical right-handed-like structure with fingers, palm, and thumb subdomains (1, 7, 39).

The systematic evolution of ligands by exponential enrichment (SELEX) is one approach that has been used in the search for novel antiviral agents. SELEX involves sequential rounds of selection and amplification from a vast pool of nucleic acids (~1014 molecules) for ligands that bind with high affinity to target molecules. HCV targets have so far included the internal ribosome entry site (IRES) (32-35), the NS3 protease (22, 23, 37), and the HCV subtype 1a and 1b polymerases (3, 4, 52).

The HCV polymerase varies between different HCV genotypes by approximately 25% at the amino acid level. Despite this variation, the majority of research has been performed solely on genotype 1, in particular, subtype 1b. One study has tested a non-nucleoside analogue inhibitor, HCV-371, against multiple HCV genotypes (28). In this study, subtype 1b polymerases had similar susceptibilities to HCV-371 (50% inhibitory concentration [IC50], 0.3 to 0.5 µM); however, subtypes 1a and 3 and genotype 4 polymerases were 16-, 6-, and 59-fold less susceptible, respectively (28). Variation in the properties of the polymerase may have major implications on the rational design of antiviral agents that are effective against all genotypes and not just genotype 1. The ability of new drugs to be effective against multiple genotypes will be an important consideration in future developments.

Advances in therapeutic options for the treatment of HCV infection are of paramount importance. Inhibitory aptamers, directed at key enzymatic targets could be used as novel antiviral agents or useful tools in research. Hence, the aims of this study were to isolate aptamers to the HCV subtype 3a polymerase by SELEX and to test selected aptamers for their ability to inhibit HCV subtype 3a polymerase activity. The inhibitory effects of these aptamers on the polymerases of HCV subtypes 1a and 1b and other RNA polymerases were also investigated.


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MATERIALS AND METHODS
 
Plasmids. The RdRp-encoding region from the HCV subtype 3a polymerase CB strain (GenBank. accession number AF046866), subtype 1a strain H77 (GenBank accession number AF009606), and subtype 1b strain JK1 (GenBank accession number X61596 [57]) excluding the C-terminal 21 amino acids was amplified by PCR using primers incorporating BamHI and EcoRI restriction sites. The HCV subtype 3a, 1a, and 1b polymerase cDNA was derived from the respective plasmids: pVRL17 (kind gift from Dharma Shukla, Biomolecular Research Institute, Melbourne, Australia), p90/HCV-Fl H77 (kind gift from Charles Rice, Centre for the Study of Hepatitis C, The Rockefeller University, New York, New York), and pGENKS-HCVNS5Bt (kind gift from Seishi Murakami, Cancer Research Institute, Kanazawa University, Kanazawa, Japan). PCR products were cloned into the bacterial expression vector pTrcHis2C (Invitrogen Corporation, Carlsbad, California) to create pVRL69 (subtype 3a), pVRL118 (subtype 1a), and pVRL75 (subtype 1b). Plasmids were transformed into Escherichia coli BL21 cells by heat shock for expression.

Recombinant polymerase expression. For expression of HCV polymerases, bacteria were grown in overnight cultures in Luria-Bertani (LB) broth with 50 µg/ml ampicillin (Sigma) at 37°C. Overnight cultures (5 ml) were diluted in 500 ml LB with ampicillin (50 µg/ml), and protein expression was induced after 3 to 4 h of shaking incubation at 37°C by the addition of 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). Cultures were incubated for a further 16 h at 22°C. Cells were collected by centrifugation, washed with phosphate-buffered saline, and resuspended in binding buffer (50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 10 mM imidazole, 10 mM ß-mercaptoethanol, 10% glycerol, 0.2% n-octyl glucoside, 1:200 [vol/vol] protease inhibitor cocktail [Calbiochem, San Diego, California]). Cells were lysed on ice by six 10-s bursts of sonication, and the insoluble fraction was removed by centrifugation at 10,000 x g for 30 min. The soluble fraction was loaded onto a Hi-Trap column (Amersham Biosciences, Buckinghamshire, United Kingdom) charged with Ni2+ and washed with 5 column volumes of binding buffer. The histidine-tagged recombinant protein was eluted by using binding buffer containing increasing amounts of imidazole from 50 to 500 mM. The purity of recombinant RdRp was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its identity was confirmed by mass spectrometry and Western blotting using an anti-six-histidine probe. Recombinant RdRp was buffer exchanged by dialysis using Slide-A-Lyser dialysis cassettes (Pierce, Rockford, Illinois) into storage buffer (20 mM Tris, pH 7.0, 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol, and 300 mM NaCl) and stored at –80°C.

Recombinant norovirus (strain norovirus Mc17/03/TH) RdRp, genogroup II, genotype 7 was a kind gift from Rowena Bull, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia. Bacteriophage {phi}6 RdRp was a kind gift from D. H. Bamford, University of Helsinki, Finland.

Single-stranded DNA (ssDNA) library. The random oligonucleotide library (hep128) (Proligo/Sigma-Aldrich) consisted of two fixed regions and a 36-bp randomized region. hep128 was based on previously published libraries for SELEX (18) and had a total length of 83 nucleotides (nt) (5'-GGGAGACAAGAATAAACGCTCAA-N36-TTCGACAGGAGGCTCACAACAGGC-3'). Primers hep129 (5'-GGGAGACAAGAATAAACGCTCAA-3') and hep130 (5'-GCCTGTTGTGAGCCTCCTGTCGAA-3') were designed to anneal to the two fixed regions of hep128, and hep130b was identical to hep130 with the addition of a 5'-phosphate group.

SELEX rounds. The Biacore 2000 (Biacore International AB, Uppsala, Sweden) was used to perform 10 rounds of selection to isolate aptamers that bind to the HCV subtype 3a polymerase. Operations on the Biacore 2000 were controlled using Biacore control software version 3.0. HEPES-buffered saline containing 1 mM CaCl2, 2.7 mM KCl, 1 mM MgCl2, and 0.005% surfactant P-20 (HBS-CKM) and regeneration buffer (3 M NaCl, 10 mM EDTA) were used for the fluid phase of the Biacore system. Recombinant RdRp (10,000 response units [RU]) was immobilized to a CM5 sensor chip (Biacore International) by amine coupling at pH 4.5.

The starting library consisted of 6.3 x 1014 molecules, or 1 nmol of ssDNA, at a concentration of 5 µM. Prior to each round, ssDNA was heated to 95°C and snap-cooled on ice. ssDNA was then allowed to equilibrate to room temperature prior to use to allow formation of secondary structures (47). The ssDNA was passed through flow cells 1 and 2 at 3 µl/min in three separate injections, where flow cell 1 was a blank flow cell activated and deactivated, and flow cell 2 contained immobilized RdRp. For the first two injections, the flowthrough of unbound DNA was recovered and included in the next injection to increase the chance of selecting all possible binding species. Binding species were eluted from flow cell 2 only, using 100 µl of regeneration solution containing 3 M NaCl and 10 mM EDTA at a flow rate of 100 µl/min.

The HCV polymerase was expressed with a six-histidine tag fused to the C terminus of the protein. To ensure irrelevant aptamers that bound to the histidine-tagged portion of the immobilized protein were not retained in the selection process, a prescreening step was included prior to round 2. The library was injected over a histidine-tagged protein (chloramphenicol acetyltransferase), and the flowthrough (without elution) was recovered as starting material for round 2. For round 2, ssDNA was diluted in HBS-CKM to a concentration of 0.5 µM, and 210 µl passed over flow cells 1 and 2 at 3 µl/min without recovery of flowthrough fractions. Weakly bound species were allowed to dissociate for 2 min prior to elution. Round 3 was performed as per round 2, with dissociation time increased to 3 min. Rounds 4 to 10 were performed with the dissociation time increased for each round to up to 60 min in round 10.

PCR and generation of ssDNA. To generate the single-stranded DNA library after each round of SELEX, selected oligonucleotides were amplified by PCR, and one strand of the double-stranded DNA product was digested.

The following PCR conditions were used: 93°C for 3 min; then 20 cycles of 93°C for 30 s, 55°C for 10 s, and 72°C for 60 s; and a final extension of 72°C for 7 min. PCR mixtures contained 2 µM of each primer (hep129 and hep130b), 4 U of Taq DNA polymerase (Promega, Madison, Wisconsin), 7.5 mM MgCl2, 1 mM deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) (Promega), 1x Taq DNA polymerase buffer (Promega), and ~20 pmol template DNA per 50-µl reaction mixture (five reactions per round were used). PCR products were ethanol precipitated with the inclusion of 20 µg of glycogen and resuspended in 200 µl of water. A fraction of the double-stranded DNA (30 µl of 200 µl) after each round of selection was stored for further analysis. Double-stranded PCR products were converted into ssDNA using a technique not previously applied to SELEX. In brief, the PCR product was incubated with {lambda} exonuclease (New England Biolabs, Massachusetts), which preferentially degrades DNA that is 5' phosphorylated (strands containing hep130b), leaving ssDNA. Gel purification of ssDNA was performed after amplification following rounds 3, 5, and 7 to remove nonspecific products. ssDNA was purified by phenol chloroform extraction and ethanol precipitation and resuspended in 20 µl of water. The quality of the ssDNA was assessed by nondenaturing polyacrylamide gel electrophoresis and SYBR green II staining, and the quantity of recovered DNA was measured by densitometry and confirmed by spectrometry at 260 nm. ssDNA aptamers were diluted in 125 µl HBS-CKM in preparation for the next round. An average of 82.8 ± 36.3 pmol of ssDNA was used for each round of selection (rounds 2 to 10).

SPR analysis. The increase in affinity of the library from selected rounds (rounds 0, 2, 3, 6, 8, and 10) was assessed by surface plasmon resonance (SPR) analysis, performed on a Biacore 2000 (Biacore, Sweden). Evolved aptamers (60 µl at a concentration of 125 nM in HBS-CKM) were injected at a flow rate of 30 µl/min over 5,000 RU of immobilized RdRp, and the binding response was measured. The screening of selected aptamers for the "best binders" was performed in a similar manner.

Cloning and sequencing. After rounds 2, 4, 8, and 10, a fraction (approximately 1% of the total pool) of selected aptamers were amplified by PCR and cloned into pGEM-T Easy (Promega). Cloned inserts were amplified by PCR using the T7 promoter primer (5'-TATTTAGGTGACACTATA-3') and the SP6 promoter primer (5'-TAATACGACTCACTATAGGG-3'). PCR products were precipitated using polyethylene glycol (PEG) reagent (26.7% PEG 8000, 0.6 M sodium acetate, 6.5 M magnesium chloride) and sequenced on an ABI 3730 DNA analyzer (Applied Biosystems) using dye terminator chemistry.

Aptamer motif analysis. Sequence data were analyzed using programs provided in WebANGIS by the Australian National Genomic Information Service (http://www.angis.org.au). Aptamers were first organized into groups, determined by the presence of sequence relatedness. Motifs were defined as at least five consecutive identical nucleotide positions. Manipulation of groups was then performed in GeneDoc Multiple Sequence Alignment Editor and Shading Utility (43), where shading was performed to indicate levels of conservation as follows: black, 100% conservation; dark gray, 80% conservation; light gray, 60% conservation; and white, less than 60% conservation.

Structures of aptamers were predicted using MFOLD (59), available at http://www.bioinfo.rpi.edu/applications/mfold/ using a salt correction algorithm and temperature correction for 25°C. Potential G-quartet structures were identified by comparison with previously published G-quartet consensus sequences (56) and the thrombin aptamer G-quartet consensus sequence. The thrombin aptamer consensus sequence is d(GGtTGGN2-5GGtTGG), where uppercase letters are invariant bases, while lowercase letters indicate the most likely base found at this position (6).

Polymerase assays. Polymerase assays were performed at 25°C in six replicates. Each 20-µl reaction mixture contained 20 mM Tris, pH 7.0, 1 mM dithiothreitol, 1 mM EDTA, 25 mM NaCl, 150 nM poly(C) RNA, 4 mM sodium glutamate, 0.2 U/µl RNasin (Promega), 0.04 µCi/µl [8-3H]GTP (Amersham), 1.8 mM MnCl2, 250 ng polymerase (HCV, norovirus, or bacteriophage {phi}6 polymerase) and in some experiments, aptamer at various concentrations. For standard assays, all reagents were added with the exception of [8-3H]GTP, which was added last to initiate the reaction. For assays with aptamers (at 20 nM, 100 nM, or 500 nM), all reagents were added with the exception of the [8-3H]GTP and the polymerase. The polymerase was then added to ensure that the aptamer and poly(C) RNA had equal time to interact with and compete for the polymerase. [8-3H]GTP was then added last to initiate the reaction. Polymerase assays were incubated for 2 h and stopped by adding 5 µl of 0.5 M EDTA and 20 µg glycogen.

Determination of inhibition constants. Kinetic and inhibition constants were determined using various concentrations of poly(C) RNA (substrate) and aptamer. For each of the substrate concentrations (3.75 to 120 nM), aptamer concentration was also varied from 0 to 100 nM. Assays were performed in triplicate over 10 min, and reactions were stopped at 5 and 10 min to determine the velocity of the reaction. RNA products were precipitated with 20% trichloroacetic acid for 1 h on ice. RNA products were then harvested using a Filtermate harvester (Packard Biosciences, Shelton, Connecticut), fitted with a GF/C 96-well Unifilter microplate (Falcon, Franklin Lakes, New Jersey). Filter plates were dried, and 25 µl of Microscint scintillation fluid (Packard Biosciences) added to each well. The filter plate was counted in a Packard liquid scintillation counter (Packard Biosciences), and results were plotted using Graphpad Prism version 4.02 for Windows (Graphpad Software, San Diego, California).

To determine kinetic inhibition constants, the aptamers were first classed as competitive or noncompetitive inhibitors with respect to the poly(C) template RNA. In order to "diagnose" the type of inhibition, a reciprocal plot method, described by Dixon (17) was employed. The reciprocal of velocity (1/v) is taken and plotted against the concentration of inhibitor i at different concentrations of substrate s. Diagnosis of the type of inhibition is possible by observing the arrangement of the best-fit lines, representing the plots at the different concentrations of substrate (12). Determination of inhibition constants was then carried out according to the most appropriate method for the type of inhibition. Inhibition constants for competitive inhibitors were estimated with nonlinear regression using Graphpad Prism 4.02 software. Michaelis-Menten mechanics was assumed. For uncompetitive or mixed inhibitors, data were transformed by multiplication of 1/v by a factor of [s] for each inhibitor concentration. A plot of [s]/v against i then gave the value of Ki as the negative x value of the intersection of the plots (13).

Affinity estimation for selected aptamers. Equilibrium dissociation constant (KD) values were estimated using a novel real-time quantitative PCR method. Aptamers were heated to 95°C for 2 min, snap cooled, and then allowed to equilibrate to room temperature to allow formation of secondary structures prior to incubation with the polymerase (47). Binding reaction mixtures were incubated for 30 min at room temperature in 30-µl reaction mixtures containing 5 nM aptamer, RdRp from 1 to 640 nM, and nonspecific competitor tRNA at 50 nM in HBS-CKM.

The polymerase and bound aptamers were removed using Strataclean resin (Stratagene Corporation, La Jolla, California), which binds protein but not nucleic acids. Strataclean resin was resuspended, and 5.5 µl was added to each reaction mixture. Reaction mixtures were vortexed, incubated for 1 min at room temperature and transferred to the spin cup of a 40-µm membrane cutoff spin column (Pall Life Sciences, East Hills, New York). Spin columns were centrifuged for 1 min at 2,000 x g, and the spin column and resin were rinsed with 30 µl of HBS-CKM and recentrifuged. DNA was ethanol precipitated, resuspended in 50 µl water, and used as template for real-time quantitation. All reactions were performed in triplicate.

In order to determine that binding was specific to the polymerase, aptamer was incubated with 640 nM of an unrelated protein (bovine serum albumin). To exclude the possibility that aptamer was binding to the resin, reaction mixtures without protein were included in every assay.

Unbound DNA was quantified using a MyiQ real-time PCR detection system (Bio-Rad Laboratories, Hercules, California), in 20-µl reaction mixtures with 1x iQ SYBR green supermix (Bio-Rad), and 0.5 µM each of primer hep129 and hep130. Reaction temperatures were as follows: 95°C for 3 min; and 50 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The threshold value was calculated automatically using the maximum correlation coefficient approach by the MyiQ optical system software, version 1.0 (Bio-Rad). The threshold cycle is defined as the number of cycles for fluorescence emission to exceed the calculated threshold value. The threshold cycles for the standard curve (aptamer concentrations ranging from 10–1 to 10–8 µM) were plotted against the starting quantity, and the unknowns were calculated by comparison to the standard curve. These unknown values represented the amount of unbound DNA in the binding reaction mixtures. Bound aptamer concentrations were calculated by subtracting the amount of unbound aptamer from the starting quantity of aptamer. Bound aptamer concentrations were plotted against polymerase concentration using the one-site binding equation on Graphpad Prism software, which established a value for KD.


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RESULTS
 
Selection of aptamers. A library of approximately 6 x 1014 molecules was screened for ligands that bind to the HCV 3a polymerase using the Biacore 2000. The pool of DNA was prescreened by passing the pool of DNA over a control (blank) flow cell to remove oligonucleotides that bound to the carboxy-methylated dextran surface of the sensor chip prior to the first round of selection. Prior to the second round, the pool was precleared by passing the enriched library over a flow cell immobilized with chloramphenicol acetyltransferase fused to a six-histidine tag in order to remove sequences that bind specifically to the histidine tag. For each of the selections, a low flow rate of 3 µl/min was used to ensure maximum interaction of the oligonucleotide pool with the surface of the sensor chip. Rounds 3 to 10 were performed as rounds 1 and 2 were but with increasing amounts of dissociation time.

Aptamer evolution. SPR analysis was used to measure the increase in polymerase affinity of ssDNA aptamer populations from rounds 0, 2, 3, 6, 8, and 10. Pooled DNA from individual rounds (125 nM) was passed over immobilized RdRp, and the binding was assessed by measuring the response units. Affinity of the pool of DNA increased from round 0 to round 10 demonstrated by an increase in the affinity of the pool of DNA from 14.7 RU for round 0, 49.9 RU in round 6, 77.5 in round 8, and 197.4 RU in round 10.

Aptamer analysis. Aptamers were clustered into groups of closely related sequences or sequences that had common motifs (Fig. 1). Round 2 and 4 aptamers did not have common sequences or common motifs with each other or with aptamers from round 8 or 10. However, one cloned sequence identified in round 8 (r8c/5) was also found in round 10 (r10/47), and the sequences of aptamers found in round 8 had common motifs with sequences of aptamers found in round 10 (Fig. 1B). Two cloned sequences were found twice (r8c/5 and r10/47) and four times (r10/24, r10/46, r10/66, and r10/79) each (Fig. 1A and B). The length of the random region was found to be 36 nucleotides for 39 of 48 aptamers sequenced. The lengths of the random regions were 38 nt for one aptamer, 37 nt for three aptamers, 35 nt for two aptamers, 34 nt for two aptamers, and 31 nt for one aptamer (data not shown).


Figure 1
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  		FIG. 1. Alignment of the 36-bp random region of selected aptamer sequences containing aptamers that were used in further studies. Clusters A and B contain identical sequences and closely related variants. Cluster B feature common motifs, particularly at the 5' end. Cluster C contains two conserved motifs 5 nt in length at the 3' end. Shading indicates conservation as follows: black, 100% conservation; dark gray, 80% conservation; and light gray, 60% conservation. The length of the random sequence is indicated after the colon to the right of the sequence, and the names of the sequences indicate the round and clone number (e.g., r10/43 is the aptamer sequence from clone 43 of round 10). The four aptamers used for further analysis are underlined.

SPR analysis was used to screen 31 of 48 aptamers in order to find the "best binders." Injection of aptamers at a concentration of 125 nM allowed comparison of the binding responses to the HCV polymerase by measurement of the relative response. Aptamers with the top four binding responses to the HCV RdRp were selected for further work (data not shown). These aptamers were r8c/10, r10/43, r10/46, and r10/47 (sequences underlined in Fig. 1).

Polymerase assays. Aptamers were tested to determine whether they had an inhibitory effect on polymerase activity in vitro. The four selected aptamers and two control aptamers (one unselected round 0 aptamer [r0/9] and an aptamer selected using an unrelated target [c10-14]) were included in the assays at three different concentrations, 20 nM, 100 nM, and 500 nM. The selected aptamers (r8c/10, r10/43, r10/46, and r10/47) demonstrated at least fourfold-greater inhibition of the HCV subtype 3a polymerase than the control aptamers did at the minimum concentration of aptamer tested. The control aptamers r0/9 and c10-14 showed the least inhibitory activity against the HCV 3a polymerase at all three concentrations. At 20 nM, the four best aptamers (as assessed by SPR analysis) were all able to significantly inhibit the polymerase activity of the HCV genotype 3a RdRp compared to the two control aptamers (P < 0.05). Values were 67.8% inhibition for aptamer r10/43, 67.1% for r10/47, 49.8% for r10/46, and 48.9% for r8c/10. In comparison, control aptamers showed mean values of 14.8% inhibition of polymerase activity for r0/9 and 13.7% for c10-14. At 100 nM, aptamer r10/43 inhibited polymerase activity by almost 80%, while r8c/10 inhibited activity by 76.2%, r10/46 inhibited by 62.0%, and r10/47 inhibited by 70.6%. This level of inhibition for all four aptamers was significantly higher (P < 0.003) than that seen with control aptamer c10-14 (21.3% inhibition of RdRp activity). At 500 nM, these four aptamers display from 80.7 to 95.0% inhibitory activity. Two aptamers, r10/43 and r10/47, that showed the greatest level of inhibition at the lowest concentration, were analyzed further to determine the Ki (inhibition constant), polymerase specificity, and KD (dissociation constant).

Inhibition constants of aptamers r10/43 and r10/47. Inhibition constants of r10/43 and r10/47 were determined by measuring the initial velocity of polymerase assays at six substrate [poly(C) RNA] concentrations ranging from 3.75 to 120 nM and at five aptamer concentrations (from 0 to 100 nM) for each given substrate concentration.

Aptamer r10/43 inhibited the HCV genotype 3a polymerase in a competitive manner with respect to the poly(C) RNA template, since the values of V did not change, while the apparent Km (the Michaelis constant) increased in the presence of the inhibitor. Therefore, Ki was determined by fitting the curves using nonlinear regression according to Michaelis-Menten mechanics. Aptamer r10/43 was estimated to have a Ki of 1.4 ± 2.8 nM by this method.

The Dixon plot of 1/v against aptamer concentration for r10/47 indicated that this aptamer was not a competitive inhibitor with respect to substrate RNA (data not shown). The data was converted by multiplication of 1/v by the values for [s]. The resulting Cornish-Bowden plot (13) revealed that the type of inhibition was mixed with respect to the template RNA (data not shown). The Ki for aptamer r10/47 was estimated to be 6.0 ± 2.3 nM.

To test whether inhibition by aptamer r10/43 and r10/47 was specific to the HCV subtype 3a polymerase, RdRps from other sources were used and included HCV subtype 1a and 1b RdRps, bacteriophage {phi}6 RdRp, and norovirus RdRp. Bacteriophage {phi}6 polymerase was chosen, as it is structurally the most closely related to the HCV polymerase (8), while norovirus polymerase is an unrelated RdRp.

At 100 nM, aptamers r10/43 and r10/47 did not inhibit the activity of the HCV subtype 1a and 1b polymerases to the same extent as the HCV subtype 3a polymerase (Fig. 2). Aptamers r10/43 and r10/47 (100 nM) inhibited subtype 1a polymerase activity by 10.3% and 10.9%, respectively. Inhibition of 1b polymerase activity was slightly higher than the inhibition of 1a polymerase activity, with 24.2% and 12.6% inhibition for r10/43 and r10/47, respectively. However, this was not significantly higher than the inhibition of 1a and 1b polymerase activity by the control aptamer r0/9 (17.1% and 20.9%, respectively). This is compared with an inhibition of the 3a polymerase activity of 84.6% for aptamer r10/43 and 70.6% for aptamer r10/47 at the same concentration (100 nM) (Fig. 2).


Figure 2
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  		FIG. 2. Inhibition of non-HCV subtype 3a polymerases by selected aptamers. Specificity of inhibition by selected aptamers to HCV subtype 3a RdRp, r10/43, and r10/47 at 100 nM was tested with HCV subtype 1a and 1b polymerases. The corresponding results for inhibition of the HCV subtype 3a polymerase by these aptamers at 100 nM are shown for comparison. To test whether inhibition of the 3a polymerase by aptamers r10/43 and r10/47 was specific to the HCV polymerase, aptamers were also tested at 500 nM against two non-HCV polymerases, bacteriophage {phi}6 and norovirus (NoV) RdRps. A control aptamer, r0/9, was also included in assays for each polymerase. Asterisks indicate that the value is significantly different from the value for the control aptamer r0/9 in the corresponding assay, with P values of P = 0.01 to 0.05 (*) and P = 0.001 to 0.01(**). P values were calculated using a two-tailed, paired t test with 95% confidence intervals. Data shown are the means of three replicates, and error bars represent the standard errors of the means.

Aptamer r10/43 showed significantly higher inhibition for both non-HCV polymerases than r0/9 and r10/47 did (P = 0.01 to 0.05). r10/43 inhibited bacteriophage {phi}6 polymerase activity by 24.7% and the norovirus RdRp by 32.7% (Fig. 2). Aptamer r10/47 showed no inhibition of the {phi}6 polymerase and 7.7% inhibition of the norovirus polymerase activity (Fig. 2). Minimal inhibition of the {phi}6 polymerase (6.8%) and no inhibition of the norovirus polymerase was seen with the control aptamer r0/9 (Fig. 2).

Dissociation constants of r10/43 and r10/47. Apparent dissociation constants were calculated by incubating various concentrations of polymerase with a fixed concentration of aptamer, with subsequent quantitation of the unbound fraction of DNA by real-time PCR (Fig. 3). The affinity of aptamer r10/43 was the highest, with a KD value of 1.3 ± 0.3 nM, while the KD value for r10/47 was 23.5 ± 6.7 nM. The KD value of control aptamer r0/9 was 258.2 nM (Fig. 3).


Figure 3
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  		FIG. 3. Estimated dissociation constants for selected aptamers. Aptamers are represented as follows: {blacktriangleup}, r0/9; {blacksquare}, r10/43; and {blacktriangledown}, r10/47. The percentage of aptamer bound is plotted against the concentration of polymerase. Estimated values for KD are also shown. Results are expressed as the means of three replicates, and error bars represent the standard errors of the means.

Structural analysis of aptamers r10/43 and r10/47. Structural analysis of r10/43 and r10/47 was performed by using MFOLD to predict the most likely minimum energy structures (Fig. 4). The structural predictions included the conserved regions. r10/43 was predicted to form a single large stem-loop structure, with an internal bulge (Fig. 4A). Aptamer r10/47 also appeared to have a stem-loop/bulge structure with two stem-loops (Fig. 4B).


Figure 4
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  		FIG. 4. Most likely predicted structures of aptamers. (A) r10/43. (B) r10/47 as predicted by MFOLD (59). (C) Potential G-quartet structure formed by aptamer r10/43. The direction of the sequence is indicated by arrows, and the three loops of the G-quartet structure are numbered.

Presence of G quartets. Since many of the aptamers were rich in G residues, the presence of G quartets, a commonly found stabilizing feature in ssDNA was investigated. G quartets are made up of four guanine bases in a square planar array, which stack, stabilizing the overall molecule (24, 58). Upon inspection, it was predicted that aptamer r10/43 could form a G-quartet-like structure (Fig. 4C). Although r10/47 is rich in G residues, it does not contain a sequence that fits the G-quartet consensus.


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DISCUSSION
 
ssDNA aptamers to the HCV subtype 3a polymerase were isolated by SELEX by using a 83-bp library and a 36-bp random region. Both RNA aptamers to the HCV subtype 1b polymerase (4, 52) and DNA aptamers to the HCV subtype 1a polymerase (3) have recently been described. However, aptamers to the subtype 3a polymerase have not been previously reported, and no genotype-specific potential antiviral agent has been developed to date. Subtype 3a is estimated to account for 20% of HCV infections worldwide (30). This is a significant proportion of total infections, yet published research into the polymerase inhibitors as potential antiviral agents remains focused on genotype 1.

The present study demonstrated that it is possible to isolate high-affinity DNA aptamers to the HCV RNA polymerase, despite the strict discrimination for RNA over DNA templates (2, 15, 57). Biroccio and colleagues demonstrated that the affinity of the best of their isolated RNA aptamers against HCV subtype 1b polymerase was 1.5 ± 0.2 nM (4), which is very similar to the value of 1.3 ± 0.3 nM obtained for r10/43. This value is comparable to the values for the aptamers developed by Vo and colleagues, who estimated the affinity of their RNA aptamers to the subtype 1b polymerase to be between 1 and 5 nM (52). The only comparable ssDNA aptamer, 27v, developed by Bellecave and colleagues against the subtype 1a polymerase was shown to have a KD of 98.3 ± 2 nM (3), an affinity that was at least 75-fold lower than of the aptamers developed in the present study.

The aptamers developed in this study bore no sequence homology to the RNA aptamers developed to HCV genotype 1 polymerase that were recently published (4, 52). RNA and DNA aptamers directed at the same target have not been reported to yield homologous sequences (25). Furthermore, there is no evidence to suggest that a high-affinity RNA aptamer will retain the same properties when converted to DNA sequence or vice versa (25). Due to practical constraints, only a fraction (1/107) of the possible 5 x 1021 unique sequences were sampled. Therefore, it is likely that sequences containing motifs found in previously reported aptamers were not present in the library and were not sampled in this particular selection.

The structures of RNA aptamers to the HCV subtype 1b polymerase (4, 52) as well as the predicted structure of aptamers from the current study displayed a predominance of stem-loop structures with internal bulges (Fig. 4A and B). This is in contrast to the study by Bellecave and colleagues (3), who reported the presence of hairpin structures in their DNA aptamers developed against the subtype 1a HCV polymerase. Step-loops with internal bulges are found at the 3' end of the X region of HCV RNA, a known binding site of the HCV RdRp (5, 29). The structural resemblance of the RNA aptamers and the aptamers from the present study to the X-region RNA may indicate that they were preferentially selected for structural similarity to this region, despite the lack of primary sequence homology.

The sequences of several aptamers in this study contain motifs reminiscent of the common motif of a guanine quartet (G quartet). DNA aptamers that have been found to form G quartets include those developed against thrombin (6), human immunodeficiency virus type 1 RNase H (16), and hematoporphyrin (45). Hence, although computer-generated structural predictions show a minimum energy conformation, it is possible that other structures are adopted by the aptamer. These structures need to be confirmed by methods such as chemical probing with nucleases, nuclear magnetic resonance technology, or X-ray crystallography (reviewed in reference 56).

Polymerase assays revealed that the selected aptamers (r10/43 and r10/47) were able to inhibit polymerase activity at a low concentration (20 nM). The lower KD value for r10/43 (1.3 nM) for the HCV subtype 3a polymerase compared to r10/47 (23.5 nM) correlated with a lower Ki value of 1.4 nM for r10/43 compared to 23.5 nM for r10/47. This suggests that the more tightly an aptamer binds, the greater the extent of interference with polymerase activity. The observation that aptamers that bind more tightly to their polymerase target have enhanced inhibitory properties has been previously reported (52). Both the KD and Ki values for aptamer r10/43 fall in the low nanomolar range, and comparisons to other antiviral agents can be made. The inhibitory activity of a number of nonnucleoside inhibitors and nucleoside analogues has been determined using in vitro polymerase assays containing recombinant HCV RdRp. While IC50 values for the nonnucleoside inhibitors are generally around 100 nM, those of nucleoside analogues fall in the low micromolar range (reviewed in references 30 and 49), a concentration 1,000-fold higher than that of the best aptamer described in the current study (r10/43).

Although the control aptamers displayed the least inhibitory activity against the polymerase, some inhibition was nonetheless observed (Fig. 2). The low-level inhibition of polymerase activity and binding (KD = 258 nM) by the control aptamers is probably due to polymerase's natural affinity for RNA and DNA as nucleic acid-binding proteins. Recently, Cramer et al. (14) reported that the HCV genotype 1b RdRp has a KD for single-stranded and partially double-stranded RNA of 250 nM, similar to the value we obtained in the current study for the control ssDNA aptamers.

Aptamers r10/43 and r10/47 had inhibitory activity against the HCV subtype 3a polymerase but had differing mechanisms of action. r10/43 inhibited the polymerase in a competitive manner with respect to the template poly(C) RNA, while r10/47 inhibited in a mixed-competitive manner. This suggests that the aptamers bind at different sites on the polymerase, and it is more likely that r10/43 binds to or near the template binding region, preventing template access to the polymerase. The possibility that r10/43 binds elsewhere on the polymerase and causes a change in conformation that affects the use of poly(C) RNA as a template in a competitive manner exists and has not been excluded. For aptamer r10/47, mixed competition with respect to the template can be explained mechanistically by the binding of the aptamer to both the free enzyme and to the enzyme-template complex (12). This indicates that r10/47 may bind to a region that does not change greatly in conformation upon binding of the template RNA. Aptamers against the HCV polymerase have been reported as having both competitive (aptamer R20-43 [52] and 27v [3]) and uncompetitive (aptamer B.2 [4]) interactions with the polymerase.

The inhibition of polymerase activity observed for r10/47 appeared to be specific for the HCV subtype 3a polymerase. At 500 nM, aptamers r10/43 and r10/47 were able to almost completely inhibit the 3a polymerase activity. This effect was not observed using the norovirus and bacteriophage {phi}6 polymerases (Fig. 2). However, some inhibition of the norovirus and {phi}6 polymerases by aptamer r10/43 was observed. It is possible that this observation is due to the competitive nature of the interaction of r10/43 with the polymerase with respect to the poly(C) template RNA. r10/43 may conceivably bind to some common feature of the template binding region of polymerases in general.

Cross-genotypic testing for aptamer specificity has not previously been reported for aptamers against the HCV polymerase. Aptamers selected in this study did not inhibit the subtype 1a or 1b polymerase activity above background levels (control aptamers) or to the same extent as that of the 3a polymerase (Fig. 2). This is not surprising, given that the level of amino acid variation between genotype 1 and 3 polymerases is approximately 25% (44) and that aptamers typically have very high specificity for their targets (11).

This is the first description of inhibitors directed at the HCV subtype 3a polymerase. Furthermore, it is the first HCV aptamer study to compare the inhibitory effect against the enzymatic function of the target protein in a cross-genotypic manner. The results of this study, which suggest that aptamers are not effective cross-genotypically, have implications for the future design of antiviral agents. The aptamers in this study have estimated affinity and inhibition constants among the best reported for inhibitory aptamers of the HCV enzymes (polymerase and protease). With their promise of high specificity, low toxicity, and their ability to be chemically synthesized in large quantities (unlike antibodies), aptamers could lead a revolution in infectious disease and cancer therapeutics. However, the field of therapeutic nucleic acids is in its infancy, and many hurdles need to be overcome before their clinical use becomes more widespread (51). ssDNA aptamers have advantages over RNA aptamers in terms of cost of production and stability; however, fundamental challenges on their delivery remain. The liver has a number of amenable attributes as a target organ for nucleic acid-based therapy, although studies have demonstrated that the nucleic acids tend to accumulate in nonparenchymal liver cells, rather than hepatocytes (31). Other important considerations include how to improve the bioavailability of the aptamer. The clearance rate of the aptamer can be reduced by attaching the aptamer to a moiety of large molecular size, which decreases the rate at which it is removed from the body. Examples that have been used in the past include PEG or other hydrophobic groups (reviewed in reference 54). Although clinical use may be some distance away, the HCV aptamers described here will provide a useful, readily available, inexpensive research tool with which to study HCV replication.


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ACKNOWLEDGMENTS
 
This study was funded by an Australian Research Council linkage grant (LP 0210513). We thank BTF PLC Ltd. for support of this project. P.A.W.'s visit to the University of Oxford was supported by a Menzies Australian Bicentennial fellowship. L.A.J. and L.E.C. were supported by Australian postgraduate awards.

We thank Yong Pan for technical assistance. We also thank William James (Sir William Dunn School of Pathology, University of Oxford, United Kingdom) for advice on SELEX methodology and accommodating P.A.W. in his laboratory.


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FOOTNOTES
 
* Corresponding author. Mailing address: School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, NSW 2052, Australia. Phone: 61 9385 3780. Fax: 61 9385 1483. E-mail: p.white{at}unsw.edu.au. Back


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Antimicrobial Agents and Chemotherapy, September 2006, p. 3019-3027, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.01603-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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