Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1

Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1

G Model VIRMET 12095 1–8 ARTICLE IN PRESS Journal of Virological Methods xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Jour...

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G Model VIRMET 12095 1–8

ARTICLE IN PRESS Journal of Virological Methods xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet

Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1

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Ronghui Wang a , Jingjing Zhao b , Tieshan Jiang c , Young M. Kwon c , Huaguang Lu d , Peirong Jiao e , Ming Liao e , Yanbin Li a,b,c,∗

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Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR 72701, USA c Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA d Animal Diagnostic Laboratory, Pennsylvania State University, State College, PA 16802, USA e College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China

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a b s t r a c t 11

Article history: Received 6 August 2012 Received in revised form 10 December 2012 Accepted 11 March 2013 Available online xxx

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Aptamers are artificial oligonucleotides (DNA or RNA) that can bind to a broad range of targets. In diagnostic and detection assays, aptamers represent an alternative to antibodies as recognition agents. The objective of this study was to select and characterize DNA aptamers that can specifically bind to avian influenza virus (AIV) H5N1 based on Systematic Evolution of Ligands by EXponential enrichment (SELEX) and surface plasmon resonance (SPR). The selection was started with an ssDNA (single-stranded DNA) library of 1014 molecules randomized at central 74 nt. For the first four selection cycles, purified hemagglutinin (HA) from AIV H5N1 was used as the target protein, and starting from the fifth cycle, entire H5N1 virus was applied in order to improve the specificity. After 13 rounds of selection, DNA aptamers that bind to the H5N1 were isolated and three aptamer sequences were characterized further by sequencing and affinity binding. Dot blot analysis was employed for monitoring the SELEX process and conducting the preliminary tests on the affinity and specificity of aptamers. With the increasing number of selection cycles, a steady increase in the color density was observed, indicating that the aptamers with good binding affinity to the target were enriched. The best aptamer candidate had a dissociation constant (KD ) of 4.65 nM as determined by SPR, showing a strong binding between the HA and the selected aptamer. The specificity was determined by testing non-target AIV H5N2, H5N3, H5N9, H9N2 and H7N2. Negligible cross-reactivity confirmed the high specificity of selected aptamers. The developed aptamer was then applied for detection of AIV H5N1 in spiked poultry swab samples. The obtained aptamers could open up possibilities for the development of aptamer-based medical diagnostics and detection assays for AIV H5N1. (The H5N1 used in this study was inactivated virus.) © 2013 Published by Elsevier B.V.

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Keywords: Avian influenza H5N1 DNA aptamer SELEX Detection

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1. Introduction

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The avian influenza virus (AIV) H5N1, discovered in the late 1990s, is a long filamentous or spherical virus (Shortridge et al., 1998) with a segmented genome that belongs to type A in the family Orthomyxoviridae. It has been reported in more than 60 countries for animal cases and in 15 countries for human cases with 607 people infected and 358 deaths since 2003 (WHO, 2012). It has drawn global attentions due to the potential pandemic threat for public health and enormous economic losses. Rapid detection and identification of AIV is crucial for public health protection and

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∗ Corresponding author at: Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA. Tel.: +1 4795752881/4795752424; fax: +1 4795752846. E-mail address: [email protected] (Y. Li).

controlling the outbreaks. In addition to viral isolation, RT-PCR and ELISA are used commonly for AIV detection with bio-recognition ligands of nucleic acid probes and antibodies, respectively. Currently, diagnostic techniques for in-field detection of AI virus are based on rapid diagnostic test kits and/or strip tests (Cho et al., in press; Dusek et al., 2011). At present, some direct antigen detection assays are available commercially for detection of AIV, such as Directizen EZ Flu A and B (Becton Dickinson, Sparks, MD), Binax Now Influenza A/B antigen kit (Binax, Portland, ME), and Humasis Influenza A/B antigen test (Humasis, Anyang, ROK). These assays use antibodies as bio-recognition ligands for virus detection. In recent years, aptamers have been also investigated as an alternative of bio-recognition ligands. Aptamers are artificial nucleic acid ligands that can bind to a wide range of target molecules (Tuerk and Gold, 1990; Jayasena, 1999; Ellington and Szostak, 1990; Huizenga and Szostak, 1995; Zhou et al., 2010; Nadal et al., 2012; Duan et al., 2012), ranging from large molecules such as protein

0166-0934/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jviromet.2013.03.006

Please cite this article in press as: Wang, R., et al., Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.03.006

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to simple organic small molecules like ATP, dyes, amino acids or simple small cations, with high affinity and specificity. They are generated by an in vitro selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment) which was first reported in 1990 (Ellington and Szostak, 1990; Robertson and Joyce, 1990; Tuerk and Gold, 1990). Aptamers show a very high affinity for their targets, with dissociation constants typically in the micromolar to low picomolar range, comparable to those monoclonal antibodies and sometimes even better (Jenison et al., 1994). In addition, as bio-recognition ligands, aptamers possess numerous advantages, including small size, rapid and reproducible synthesis, simple and controllable modification to fulfill different diagnostic and therapeutic purposes, slow degradation kinetics, nontoxicity, and a lack of immunogenicity (Duan et al., 2012; Famulok et al., 2007). Aptamers have been successfully employed in strip tests as substitutes (Wang et al., 2011a,b; Xu et al., 2009) and aptamerbased assays show great potentiality for in-field applications. A number of recent reviews have positioned aptamers to make a significant impact in many areas (Binning et al., 2012; Syed and Pervaiz, 2010). So far, several high-affinity DNA and RNA aptamers have been successfully selected against viral proteins or whole virus, such as human immunodeficiency virus HIV glycoprotein 120 (gp120) (Zhou et al., 2009), human hepatitis B virus polymerase (P protein) (Feng et al., 2011), hepatitis C virus envelope glycoprotein E2 (Chen et al., 2009), and whole human cytomegalo virus and RSV (Konopka et al., 2000). A number of aptamers have been isolated against influenza A HA that inhibit viral infectivity (Dhar and Datta, 2009; Jeon et al., 2004; Gopinath et al., 2006a; Misono and Kumar, 2005; Cheng et al., 2008; Park et al., 2011). Jeon et al. (2004) have selected a DNA aptamer that prevents influenza infection by efficiently blocking the receptor binding region of the viral HA. Kumar’s group also reported an RNA aptamer that discriminates the closely related HA derived from the H3N2 strain of influenza A virus and inhibits membrane fusion (Gopinath et al., 2006a). They also developed an efficient RNA aptamer against human influenza B virus hemagglutinin (Gopinath et al., 2006b). In the studies by Cheng et al. (2008) and Park et al. (2011), DNA and RNA aptamers targeted to HA1 proteins of influenza virus H5 subtypes were selected, respectively. Their studies focused on potent inhibition of influenza virus, and therefore, their selected aptamers may be promising candidates for treatment and prophylaxis of influenza virus infection. The objective of this study was to select and characterize DNA aptamers that can specifically bind to AIV H5N1, and use the selected aptamer as a bio-recognition ligand in the development of aptamer-based detection methods for specific detection of AIV H5N1 for in-field or on-site application. Thirteen selection cycles were performed to obtain the DNA aptamers with high affinity and specificity. The best aptamer candidate was evaluated and characterized using dot blot assay and SPR (surface plasmon resonance). The results presented in this report provide the first-step in the development of a simple, rapid, robust, sensitive and cost-effective detection method based on aptamers, offering a possible viable alternative to current methods for AIV H5N1 detection.

activity and tertiary structure. The inactivated AIV H5N1 strain (A/Chicken/Scotland/59) (GenBank accession number X07826.1) was obtained from USDA-APHIS National Veterinary Services Laboratories (NVSL, Ames, IA), and an inactivated recent Asian H5N1 field strain (A/Duck/Guangdong/383/2008) was obtained from College of Veterinary Medicine, South China Agricultural University, China. Related tests on the Asian H5N1 stain (A/Duck/Guangdong/383/2008) were conducted in the Avian Research Laboratory at College of Veterinary Medicine, South China Agricultural University, China. The non-target influenza viruses used in evaluation tests were the inactivated AIV subtypes of A/H5N2/PA/chicken/85, A/H5N3/WileyLab/87, A/H5N9/WileyLab/85, A/H1N1/WileyLab/87, A/H2N2/PA/chicken/ 1117-6/04, A/H7N2/PA/chicken/3779-2/97, A/H9N2/WileyLab/87, which were prepared at Wiley Laboratory at Pennsylvania State University (University Park, PA). Because of biosecurity requirement in handling live AIV strains and the biosafety limitation of our laboratory condition (biosafety level 2), only inactivated AIV strains were used in this study. The pGEM-T easy vector was obtained from Promega (Falls Church, VA).

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2.2. DNA library and primers

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The oligonucleotide template was synthesized as a singlestranded 115 bases with the following sequence: 5 -CCG GAA TTC CTA ATA CGA CTC – (N)74 – TAT TGA AAA CGC GGC CGC GG – 3 where the central 74 nucleotides represent random oligonucleotides based on equal incorporation of A, T, G and C at each position. The dsDNAs were obtained by PCR amplification using Forward 5 -CCG GAA TTC CTA ATA CGA CTC-3 and Reverse 5 CCG CGG CCG CGT TTT CAA TA-3 primers. Biotinylated forward primer with 5 biotinylation was for Dot blot hybridization. Reverse primer with 5 phosphorylation was used to obtain PCR products for lambda exonuclease digestion to produce ssDNA. Both the library and primers were synthesized by Integrated DNA Technologies (Coralville, IA).

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2.3. In vitro selection procedure

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Aptamer candidates against target AIV H5N1 were selected using SELEX technique (Tuerk and Gold, 1990). The detailed protocol for SELEX procedure is described as follows. To exclude filter-binding ssDNA sequences from the pool, the ssDNAs were passed three times prior to the selection cycle through a pre-wetted nitrocellulose acetate membrane (0.45 ␮m HAWP filter, Millipore, MA, USA) in a filter holder (“pop-top”, a diameter of 13 mm, Millipore). For the first cycle of selection, 35.5 ␮l (1 ␮g/␮l) DNAs were added in 114.5 ␮l binding buffer solution. The molecules of first selection cycle are:

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35.5 ␮l × 1 ␮g/␮l = 1 nmol = 6.02 × 1014 35441.4 g/mol (DNA Oligo Molecular weight : 35, 441.4 g/mol).

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(1)

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2. Materials and methods

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2.1. Target protein, virus and plasmid

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The full-length glycosylated recombinant HA protein of AIV subtype H5N1 (A/Vietnam/1203/04) with a concentration of 524 ␮g/ml was purchased from Protein Science Corporation (Meriden, CT, USA). The protein was produced in insect cells using the baculovirus expression vector system and purified to >90% purity under conditions that preserve its biological

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This randomized pool contained approximately 1014 (14 orders of magnitude) units DNA species under experimental conditions. To initiate in vitro selection, ssDNA library was denatured at 95 ◦ C for 10 min and was allowed to cool down at room temperature for 30 min. Denatured DNAs were incubated with the target protein HA/virus for 1 h and 45 min at room temperature in a binding buffer (50 mM Tris–HCl, 25 mM NaCl, 5 mM MgCl2 , 10 mM DTT (dithiothreitol), pH 7.5). This reaction mixture was filtered over a HAWP filter (Millipore, MA) and washed three times with the binding buffer. ssDNAs that were retained with HA/virus on the filter were eluted twice with elution buffer (0.4 M sodium acetate,

Please cite this article in press as: Wang, R., et al., Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.03.006

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R. Wang et al. / Journal of Virological Methods xxx (2013) xxx–xxx Table 1 Concentrations of DNA and HA protein/AIV H5N1 used in selection cycles. Cycle HA protein (M) 1 2 3 4 H5N1virus (l) 5 6 7 8 9 10 11 12 13

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ssDNA pool (␮M) 6.7 5.0 5.0 5.0

0.5 0.25 0.125 0.063 30 25 20 15 10 5 2 1 0.5

1.7 1.7 1.1 1.1 1.1 0.9 0.9 0.8 0.8

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5 mM EDTA, 7 M urea; pH 5.5) at for over the course of 5 min. Afterwards the eluted DNA was diluted with an equal volume of deionized water (dH2 O) and was precipitated (0.12 mg glycogen, equal volume of 7.5 M ammonium acetate and 1 ml of 100% ethanol) and incubated for 1 h at −80 ◦ C. After centrifugation at 9447 × g at 4 ◦ C for 1 h, supernatant was discarded and the pellet was washed twice with 75% ethanol solution and resuspended in dH2 O. Selected ssDNAs were amplified by PCR (Mastercycler Gradient, Eppendorf) and used for the next round of selection. Subsequent selection cycles were the same as the earlier ones with the exception that the stringency of selection was increased to promote competition between binding species. Therefore, as the cycles progress, the molar ration of HA/virus to DNA increased (Table 1). 2.4. PCR amplification and conversion of oligonucleotides into ssDNA

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The recovered ssDNA pools were used as a template in the subsequent PCR reaction using a thermocycler (Mastercycler Gradient, Eppendorf). The reaction was carried out in a total volume of 50 ␮l of ThermoPol reaction buffer (20 mM Tris–HCl, pH 8.8, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 ) containing 0.4 ␮M of each primer (forward and reverse), 200 ␮M dNTPs, and 2.5 U of Taq polymerase. The amplification conditions were as follows: initial denaturation at 95 ◦ C for 5 min and final extension at 72 ◦ C for 10 min; and 30 cycles of denaturation at 94 ◦ C for 45 s; annealing at 64 ◦ C for 45 s and extension at 72 ◦ C for 45 s. Amplified products were analyzed on non-denaturing 6% Tris–borate EDTA (TBE) – polyacrylamide gels (Invitrogen, Carlsbad, CA) at 200 V for 20 min after binding with SYBR Green 1 (Invitrogen, Carlsbad, CA). The PCR products were converted into ssDNA by incubation in a total volume of 50 ␮l of lambda exonuclease reaction buffer with 10 U of lambda exonuclease (New England Biolabs, Ipswich, MA) for 1 h at 37 ◦ C, followed by 10 min at 75 ◦ C to inactivate the enzyme. Digested products were precipitated as above (resuspension of pellet in binding buffer not in water) and used for the next round of SELEX. The concentration of ssDNA was measured using NanoDrop 1000 Spectrophotometer (ThermoScientific, Wilmington, DE).

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2.5. Cloning and sequencing

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After 13 rounds of selection cycle, the PCR product pool was purified using Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany) and then was cloned into the pGEM-T easy vectors according to the manufacturer’s manual (Promega, Madison, WI). Twenty colonies were randomly picked. Plasmid DNA was extracted using the QIAprep Miniprep kit (Qiagen, Hilden, Germany). Ampicillin and IPTG were purchased from Calbiochem

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(San Diego, CA), and X-Gal (5-bromo-4-chloro-3-indolyl-␤-Dgalactoside) and S.O.C medium were purchased from Invitrogen (Invitrogen, Carlsbad, CA). The plasmids were analyzed by NanoDrop 1000 Spectrophotometer (ThermoScientific, Wilmington, DE) and then were sent for sequencing. Sequencing of plasmid DNA of the selected transformants was done by automated DNA sequencing using ABI 3130xl analyzer BigDye 3.1 diemistry (ABI 7300 Sequence Detector, Foster City, CA). Secondary structures of sequenced aptamers were predicted by web-based UNAFold software in OligoAnalyzer 3.1 program from IDT (Integrated DNA Technologies, Coralville, IA), which was based on free energy minimization algorithm. Sequences were aligned using ClustalX v.1.81 (Thompson et al., 1997) and pattern analysis was performed using ABI software (Applied Biosystems, Foster City, CA).

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2.6. Dot blot analysis with aptamers and antibodies

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Dot blot assay (Vivekananda and Kiel, 2006) was used for a rapid analysis of the affinity of aptamers from selected SELEX rounds. Biotinylated forward primer and phosphorylated reverse primer were used to obtain dsDNA which were further digested by lambda exonuclease to obtain biotinylated ssDNA for Dot blot analysis. Target AIV H5N1 (5 ␮l) with different titers were spotted onto nitrocellulose membrane (BA85 Protran, 0.45 ␮m, Whatman, USA) and allowed to air dry. Then these samples were followed by blocking with blocking buffer (12.5 g casein; 4.5 g NaCl; 605 mg Tris; 100 mg Thimerosal) for 30 min and incubated with biotinylated aptamers (2.2 ␮g/ml) from selected cycles for 45 min at room temperature. After washing three times with 1× KPL (KPL, Gaithersburg, MD, USA) washing solution (0.002 M imidazole, 0.02% Tween 20, 0.5 mM EDTA, 160 mM NaCl), the membrane was reacted with streptavidin-conjugated alkaline phosphatase (1:500 dilution) for 30 min. Excess enzyme was removed by three subsequent washes with 1× KPL washing solution. Finally, the membrane was coated in BCIP/NBT (5-bromo-4-chloro-3-indoxyl-phosphate and nitrobluetetrazolium) (KPL, Gaithersburg, MD) substrate in the dark for color development. PBS buffer was served as a negative control and monoclonal antibody (mAb) specifically against AIV H5 subtypes (provided by Animal Diagnostic Laboratory at Pennsylvania State University) with a concentration of 4.4 ␮g/ml was employed in the dot blot analysis to replace the aptamer as a comparison.

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2.7. Surface plasmon resonance

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BIAcore 3000 SPR instrument with CM5 chips (BIAcore, Piscataway, NJ) were used in all experiments that were conducted at 25 ◦ C with a flow rate of 10 ␮l/min. The running buffer used for the experiments was HEPES and 10 mM NaOH (10 ␮l) was used for regeneration to remove the bound target protein. Selected aptamers were immobilized onto the surface of SPR chips using biotin-streptavidin method. The SPR chip was first coated with streptavidin, and then biotin-labeled aptamers with a concentration of ∼1 ␮M was injected for 10 min (10 ␮l/min, 25 ◦ C) and followed by injecting biotin (10 ␮M) for 5 min and ethanolamine (1 M, pH 8.5) 7 min as blocking reagents. The channel with streptavidin coating and ethanolamine blocking, but without aptamer immobilization, was used as a reference channel. For binding assay, HA proteins with different concentrations (2, 5, 10, 20 and 40 ␮g/ml) were injected onto the sensor chip and the affinity binding was monitored for 2 min followed by washing with running buffer. After measuring SPR signals, the association and dissociation rate constants (Ka and Kd ), and equilibrium dissociation constants (KD ) were calculated by a 1:1 [Langmuir] fitting model using the data analysis software (BIA evaluation software).

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Further experiments were conducted to study the binding affinity between the selected aptamers and the H5N1 virus with different titers of 0.064, 0.128, 0.32 and 0.64 HAU. Then the specificity was investigated by testing non-target AIV H5N2, H5N9, H9N2, H7N2 and H2N2 with a titer of 0.64 HAU.

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2.8. Swab sample preparation

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Two tracheal swabs were taken from two healthy chickens and then placed in per tube containing 3 ml of Viral Transport Media (VTM). The VTM included 500 ml of Minimum Essential Media (MEM), 7.5 ml of HEPES buffer (1 M), 10 ml of Gentamicin (10 mg/ml), 2.5 ml of Kanamycin (10 mg/ml), 5 ml of Antibiotic/Antimycotic (Pen-Strep-Amp), and 5 ml of Horse Serum (heat inactivated at 56 ◦ C for 30 min). Four tubes of tracheal swab samples were mixed together to prepare the uniform swab sample for further tests. First, each tube of VTM solution with two swabs was mixed sufficiently using a vortex mixer. After mixing, each swab was pressed against the tube wall several times to squeeze the solution in swabs and then discarded. Four tubes of the solution were then put into one tube and mixed sufficiently. Finally, the solution was filtered using syringe filter (0.45 ␮m) and spiked with AI virus for further use. Virus titers of inactivated AIVs were provided by USDA-APHIS National Veterinary Services Laboratories, Animal Diagnostic Laboratory at Pennsylvania State University and the College of Veterinary Medicine at South China Agricultural University in China. Swab solution without spiking was used as a control.

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2.9. Chemicals

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All chemicals used in this study unless specified were purchased from Sigma–Aldrich (St. Louis, MI).

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3. Results

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3.1. Selection of H5N1-biniding aptamers by SELEX

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In the first selection cycle, about 1014 DNA random sequences were incubated with target protein. A commercially available fulllength glycosylated recombinant hemagglutinin (HA) protein of influenza A virus H5N1 (A/Vietnam/1203/04) was used as target protein. Started from the fifth cycle, entire inactivated H5N1 virus was applied in order to improve the specificity. As the cycles progressed, the molar ratio of target HA/virus to DNA was increased (Table 1) to promote competition between binding species. Following incubation, the bound oligonucleotides were separated from unbound oligonucleotides via nitrocellulose filtration. Then, the target-bound aptamers were eluted from the filters, and amplified by PCR. All DNAs were quantified using spectrophotometer and analyzed with PAGE (polyacrylamide gel electrophoresis) gel. The observation of a 115-bp band on the gel after each round of selection and PCR amplification suggested that the target HA/virus were able to bind to a pool of aptamer sequences. Single-stranded DNA (ssDNA) templates are required during the SELEX and the preparation of ssDNA is an essential and important step. In this study, the lambda exonuclease digestion method was used for generation of ssDNA. Lambda exonuclease is a highly processive 5 –3 exodeoxyribonuclease that selectively digest the 5 -phosphorylated strand of dsDNA. Therefore, a 5 -phosphorylated group was introduced into one strand of dsDNA by performing PCR where only the reverse primer was 5 -phosphorylated. This phosphorylated strand was removed by digestion with lambda exonuclease while the other strand was retained. After lambda exonuclease digestion, phenol/chloroform exaction and subsequent ethanol precipitation were performed to eliminate the extra lambda exonuclease from the aptamer pool. Finally, the generated

Fig. 1. Dot blot analysis of affinity binding between aptamer pools from the 4th, 8th and 10th selection cycles and target AIV H5N1, and the cross-reactivity with non-target AIV H5N2 and H7N2.

ssDNA was used as the input for the next selection cycle. A total of thirteen repeated separation-amplification cycles were completed in order to yield high affinity and specificity DNA aptamers.

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3.2. Binding affinity and specificity of aptamer pools following selection cycle

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After two or three rounds of selection, the aptamer pools were checked for binding affinity and specificity to the target AIV H5N1 using dot blot assay for a rapid assessment. In this format, target AIV H5N1 was spotted onto nitrocellulose membrane and air dried at room temperature. PBS buffer without target virus was used as a control. To test the binding specificity of the aptamer pools, parallel dot blot assay was performed using non-target AIV subtypes H5N2 and H7N2. The results of dot blot assay for the aptamer pool at 4th, 8th and 10th cycles are presented in Fig. 2. For the 4th cycle aptamer pool, the target AIV H5N1 with a titer of 25.6 HAU developed a clear dot; however, the titer of 12.8 HAU only displayed a faint dot. For the aptamer pools of the 8th and 10th cycles, the stronger dots were observed for the H5N1 even with the titer of 12.8 HAU, and the bigger size of dots was observed at the 10th cycle than that of the 8th cycle. The dot blot results (Fig. 1) clearly showed that with the increase of SELEX cycles, the selected aptamer pools displayed stronger binding to the target AIV H5N1 with the increase of both dot size and intensity. No cross-reaction was observed with nontarget AIV subtypes H5N2 and H7N2, indicating good specificity toward the target virus.

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3.3. Cloning and sequence analysis

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Twenty clones were sequenced, since there were some repeat sequences and wrong matches, from which three unique sequences were obtained as the follows: (1) 5 -GAC GGG TAA CGT ATG TTT TAC ATT ACG AAA TTT AGA GCA CCC TTA CAG CGA GAC TCG TTG ACC TGT AGC AGT G-3 , (2) 5 -GTG TGC ATG GAT AGC ACG TAA CGG TGT AGT AGA TAC GTG CGG GTA GGA AGA AAG GGA AAT AGT TGT CCT GTT G-3 , and (3) 5 -GGC CGA ATT GGT TCG TCG AGC GAG TCA CAC CAA CAA TGC TGC GAT AGA AAC TTC GTA CGA GCT TTC TTA CGC TG-3 . Among these three sequences, only sequence (3) has 74-nucleotide sequence while the other two have 73-nucleotide sequences. The one base was probably missing during the PCR amplification process. According to the mutagenic PCR theory (Cadwell and Joyce, 1994), the error rate of Taq polymerase is high in the range 0.1 × 10−4 to 2 × 10−4 per nucleotide per pass of the polymerase. Secondary structures of the sequenced aptamers (as shown in Fig. 2) were predicted by web-based UNAFold software

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Please cite this article in press as: Wang, R., et al., Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.03.006

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Fig. 2. The predicted secondary structures of the three aptamers: (a) aptamer sequence (1); (b) aptamer sequence (2); and (c) aptamer sequence (3).

Please cite this article in press as: Wang, R., et al., Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.03.006

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Table 2 Dot blot analysis for the comparison study between aptamer sequence (2) and H5N1 monoclonal antibody (anti-H5 mAb): (a) Detection of target H5N1 in the titers ranging from 0.0128 to 128 HAU; and (b) specificity test using non-target AIV subtypes. AIV H5N1 titer (HAU)

(a) Anti-H5 mAb Aptamer sequence (2)

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12.8

1.28

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0.0128

++ ++

++ ++

+ +

− −

− −

Specificity test

(b) Anti-H5 mAb Aptamer sequence (2)

H5N1

H5N2

H5N3

H5N9

H7N2

++ ++

++ −

+ −

++ −

− −

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in OligoAnalyzer 3.1 program from IDT (Integrated DNA Technologies), which was based on free energy minimization algorithm. The G value for the three structures was −5.03, −7.03 and −6.24 kcal/mole, respectively; and the GC contents for the three sequences were 45.2%, 47.9% and 51.4%, respectively. The secondary structure of sequence (1) had one external loop and three hairpin loops. The structure of sequence (2) contained one external loop and two hairpin loops. Structure of sequence (3) had one external loop, one interior loop, one bulge loop and two hairpin loops. These secondary structures of the nucleotides such as hairpin loops and bulge loops played an important role of contacting and binding the target (Zhou et al., 2010). The binding between aptamers and their targets relies on aptamers’ secondary structures and three-dimensional (3D) structures (Zhou et al., 2010; Song et al., 2008), hydrogen bonding, electrostatic and hydrophobic interactions rather than Watson–Crick base pairing (Citartan et al., 2012).

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3.4. Evaluation and characterization of DNA aptamers

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The preliminary evaluation of the obtained three aptamers was carried out using dot blot assay to check the binding-interaction between the selected aptamers and the target AIV H5N1. Non-target AIV subtypes H5N3, H5N9, H7N2, H2N2 and H9N2 with a titer of 25.6 HAU were also tested to check any crossreactivity. The results showed that there is no binding between the selected three aptamers and the non-target AIV subtypes, indicating good specificity of the aptamers. Among the three aptamers, aptamer sequence (2) showed the best binding affinity to the target H5N1 virus, displaying the largest dot size and the highest intensity, which was employed for the comparison study with H5N1 monoclonal antibody and then further used for characterization with surface plasma resonance. Table 2 shows the results of dot blot using both H5N1 monoclonal antibody (anti-H5 mAb) and aptamer sequence (2) for the target AIV H5N1. Table 2(a) indicates that both anti-H5 mAb and aptamer sequence (2) could detect H5N1 with dot blot, and the detection limit was observed to be 1.28 HAU for both ligands. The specificity of the aptamer for detection of H5N1 was also compared with anti-H5 mAb by testing other non-target AIV subtypes including H5N2, H5N3, H5N9 and H7N2, and the results are presented in Table 2(b). It was found that the developed aptamer (2) had greater specificity to H5N1, and other AIV subtypes (H5N2, H5N3, H5N9, and H7N2) did not bind with it. The anti-H5 mAb displayed visible dots for all the tested H5 subtypes (H5N1, H5N2, H5N3, and H5N9) and no cross-reactivity was observed for other non-H5. subtype (H7N2). The result indicated that the selected aptamer was specific against AIV H5N1.

Fig. 3. An overlay of the SPR signals from the interaction between HA protein and the selected aptamer.

Surface plasma resonance (SPR) is one of the most sensitive techniques particularly suitable for study the surface binding reaction of molecules. In this study, SPR was used to measure the affinity of the aptamer sequence (2) against recombinant HA protein of the subtype H5N1 and AIV H5N1 virus. Immobilization of the selected aptamer on SPR chip surface was carried out using the biotin-streptavidin method, which resulted in increasing of 997 RU (resonance units) SPR signal, indicating success of the aptamer immobilization. Then, HA proteins with different concentrations (2, 5, 10, 20 and 40 ␮g/ml) were injected onto the sensor chip and the affinity binding was monitored. The analytical signal, recorded in resonance units (RUs), was computed as the difference between the aptamer and corresponding reference channel. We evaluated the affinity binding of HA protein to the selected aptamer by injecting HA with the concentrations from low to high over the aptamer and reference surfaces. In this way, the portion of the SPR signal that was attributed to specific affinity binding could be calculated by subtracting the non-specific binding and bulk refractive index effects detected on the reference surface from the total SPR signal measured on the aptamer surface. An overlay of the SPR sensor signals from the interactions between HA and the selected aptamer is shown in Fig. 3. As expected, increasing HA concentration resulted in an increase of the SPR signal. The calculated Ka (association rate constants) and Kd (dissociation rate constants) was 4.69 × 104 (M−1 s−1 ) and 2.18 × 10−4 (s−1 ), respectively, and the KD (dissociation constants) was 4.65 nM, indicating strong binding between the HA protein and the selected aptamer. Further experiments were conducted to study the binding between the selected aptamer and the H5N1 virus. A linear relationship was found in the virus titer range from 0.064 to 0.64 HAU and the corresponding equation was described as: y = 208.39x + 2.23 (R2 = 0.99), where y is SPR signal in RU and x is virus titer in HAU. The result showed that the selected aptamer could be used as a recognition ligand to detect target AIV H5N1.

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3.5. Detection of AIV H5N1in poultry swab samples using the developed aptamer

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Dot blot assay was applied further to the detection of tracheal swab samples experimentally spiked with inactivated AIV H5N1 viruses (strain A/Chicken/Scotland/59 and A/Duck/Guangdong/ 383/2008), and other subtypes (A/H5N2/PA/chicken/85, A/ H5N3/WileyLab/87, A/H5N9/WileyLab/85, A/H1N1/WileyLab/87, A/H2N2/PA/chicken/1117-6/04, A/H7N2/PA/chicken/3779-2/97,

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Fig. 4. Dot blot assay for detection of tracheal swab samples experimentally spiked with AIV: (a) AIV H5N1 strain A/Chicken/Scotland/59 with the titer range from 0.0128 HAU to 64 HAU; (b) AIV H5N1 strain A/Duck/Guangdong/383/2008 with the titer range from 1 HAU to 64 HAU; (c) specificity test with other AIV subtypes (H5N9, H7N2, H2N2, H9N2 and H1N1).

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and A/H9N2/WileyLab/87) using the developed aptamer as recognition agent. AIV H5N1 strain A/Duck/Guangdong/383/2008 belongs to clade 2.3.2, which have been circulating widely in China since 2008 and may cause a new wave of cross-continental spreading from Asia to Europe (Sun et al., 2011). As shown in Fig. 4, the results indicated that the developed aptamer was able to recognize target AIV H5N1 (both strains A/Chicken/Scotland/59 and A/Duck/Guangdong/383/2008) in chicken swab samples and no interference was observed from other AIV subtypes, such as H5N9, H7N2, H2N2, H9N2 and H1N1.

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The development of aptamers that display good binding affinity and high specificity against AIV H5N1 suggests that the selected aptamer has great potential to be a viable alternative for detection and screening of AIV H5N1 for in-field or on-site application. Since aptamers can be chemically synthesized in an easy and economical manner, manufacturing and assay costs of aptamers are expected to be significantly lower than the antibody-based immunoassays currently used for rapid AIV detection. It is estimated that the production cost of aptamers could be about 10–50 times less than antibodies (Low et al., 2009). In addition, the results from this study indicated that the selected aptamer had great specificity for AIV H5N1, whereas the anti-H5 monoclonal antibody showed binding-interaction not only for H5N1, but also

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for other H5 AIV subtypes. The aptamer-based assay would likely be less prone to false positive results in AIV H5N1 detection. Moreover, the thermal stability of DNA aptamers and their tolerance of harsh chemical, physical and biological conditions, as compared to antibodies, add attractive advantages for the development of aptamer-based assay for AIV detection, specifically for in-field use. Aptamers can also readily be labeled with reporter and quencher moieties to form molecular beacons and functional groups for immobilization on microarrays, facilitating the construction of a biosensor which represents great potential for the development of aptamer-based detection technologies. The selection of these sensitive and specific aptamers is the first step in developing a simply, rapid, robust, cost-effective and reliable detection method. In summary, DNA aptamers that target specifically AIV H5N1 were generated using SELEX technology in the study. Results showed that the selected aptamer displayed strong binding affinity and high specificity against target AIV H5N1. Future research will focus on the development of a new detection technology using the selected aptamer as a bio-recognition ligand for in-field or on-site detection of AIV H5N1.

Uncited reference Zhou et al. (2011).

Please cite this article in press as: Wang, R., et al., Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.03.006

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Acknowledgments

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This research was supported in part by National Science Foundation (NSF/STTR #0932661) and Arkansas Biosciences Institute (ABI).

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