Randomized single-stranded DNA library, aptamers, and primers.
The single-stranded DNA library (WAP40) consisting of randomized 40-mer DNA sequence flanked by constant primer-binding regions, primers (sense strand [WP18] and antisense strand [WP20]), and aptamers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Sequences of primers and DNA library are shown in .
Sequences of primers and DNA library used in this study
Recombinant His6-tagged H3 hemagglutinin protein.
Recombinant His6-tagged H3 hemagglutinin protein from an H3N2 strain A/swine/Minnesota/SG-00235/2007 (swH3) was cloned and expressed in a baculovirus system in our laboratory. To verify that the recombinant swH3 protein was a functional protein, we performed a hemagglutination (HA) test parallel with the corresponding swH3 virus, and the results were similar. The recombinant swH3 protein was used as a template for the selection and characterization of the DNA aptamers. To test the specificity of aptamers, the reference recombinant HA proteins in different subtypes of IAVs obtained from Biodefense and Emerging Infections Research (BEI) resources were used. Detailed descriptions of the recombinant HA proteins used in the present study are provided in .
Detailed descriptions of the recombinant HA proteins used in this study
Preparation of recombinant swH3 protein and Ni-NTA magnetic agarose bead matrix.
Approximately 100 μl of Ni-NTA magnetic agarose beads (Qiagen, Hilden, Germany) was separated from solution by using magnetic apparatus. The beads were washed three times with 500 μl of 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 0.005% Tween 20 (pH 8.0) (washing buffer) and then conjugated with recombinant swH3 protein in 500 μl of 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole (pH 8.0) (binding buffer) at 4°C with gentle shaking. After 48 h of incubation, protein-bead matrix was washed three times with 500 μl of washing buffer. After washing, the protein-bead matrix was stored at 4°C and used within a day. In subsequent iterations of SELEX, the recombinant swH3 protein was gradually reduced from 30 to 0.4 μg.
Negative selection was performed in every iteration of SELEX to remove any nonspecific candidates that bind to the Ni-NTA magnetic agarose beads. The aptamer library was denatured at 95°C for 10 min and then incubated at room temperature for 10 min prior use. Three sets of ~100 μl of Ni-NTA beads were prepared by separating them from solution by using a magnetic separation apparatus and three washes with 500-μl portions of washing buffer. The denatured aptamer library was added to Ni-NTA beads in 500 μl of 50 mM NaH2PO4–50 mM NaCl–20 mM imidazole pH 8.0 (interaction buffer), followed by incubation at room temperature for 30 min in a rotisserie shaker. After the third negative selection, the unbound library was subjected to positive SELEX against H3.
The aptamer library was conjugated with protein-bead matrix in 500 μl of interaction buffer, followed by incubation at room temperature using a rotisserie shaker. After 1 h of incubation, the unbound library or poor binders were removed by 11 wash steps with 1× phosphate-buffered saline (PBS) containing 0.05% Tween 20 (1× PBST). Protein-bound aptamers were recovered with 100 μl of nuclease-free water and then subjected to PCR. PCR was performed to amplify all protein-bound aptamer candidates. Briefly, 3 μl of protein-bound aptamers were mixed with 5 pmol of forward primer (WP18) and biotinylated reverse primer (Bio-WP20), 2× HotStarTaq DNA polymerase (Qiagen), and nuclease-free water (final volume, 50 μl). PCR was performed using the following conditions: 95°C for 15 min, followed by 15 cycles of 95°C for 30 s, 63°C for 30 s, and 72°C for 30 s, and finally 72°C for 7 min. Amplicons from PCR were then used as a template for asymmetric-touchdown PCR to enrich for the sense strand to be applied back in the next round of SELEX. Briefly, 2 μl of amplicons was mixed with 30 pmol of WP18 and 1.2 pmol of Bio-WP20 (forward primer/reverse primer ratio of 25:1). Touchdown PCR conditions used were 95°C 15 min, followed by 9 cycles of 95°C for 15 s, 72°C for 15 s (gradually decreasing by 1°C each cycle), and 72°C for 15 s, followed by 11 cycles of 95°C for 15 s, 63°C for 15 s, and 72°C for 15 s, and a final extension at 72°C for 3 min. Approximately 400 μl of amplicons from asymmetric-touchdown PCR was purified by MiniElute PCR purification kit (Qiagen) and then eluted with 60 μl of nuclease-free water. Subsequent rounds of SELEX were performed only with the sense strand aptamers. To remove the antisense strand, PCR amplicons were purified by heat denaturing and quick chilling on ice and then mixture with Dynal M280 streptavidin super-paramagnetic beads (Invitrogen/Dynal AS, Oslo, Norway) for 15 min. The SELEX process was repeated 15 times. The binding affinity and the specificity of aptamer candidates were investigated by chemiluminescent electromobility shift analysis (LightShift chemiluminescent EMSA kit; Pierce, Rockford, IL) before cloning and sequencing.
Counter-SELEX to remove any His tag binding candidates.
During the last two rounds of SELEX (i.e., rounds 14 and 15), His6-tagged recombinant nucleoprotein (rNP) from an H1N1 virus, A/swine/Minnesota/07002083/2007 (swH1), was used for counter-SELEX to remove any nonspecific candidates that bound to the His6-tagged portion of the protein. A protein-bead matrix was prepared according to the same procedures used for the recombinant swH3 protein. The aptamer pool was incubated with rNP at room temperature for 1 h, and then the supernatant was collected using a magnetic apparatus. The supernatant containing aptamer candidates was then submitted to positive SELEX.
Cloning and sequencing of the aptamer candidates.
PCR amplicons containing the enriched aptamer pools of rounds 8 and 15 were purified by using a MiniElute PCR purification kit (Qiagen). The purified amplicons were cloned into TA vector (pCR2.1-TOPO) and then transformed into chemically competent TOP10 Escherichia coli
cell by heat shock at 42°C according to the manufacturer's recommendations (TOPO TA cloning kit; Invitrogen, Carlsbad, CA). Transformed E. coli
cells were plated on Luria-Bertani agar containing ampicillin (50 μg/ml) and X-Gal (5-bromo-4-chloro-3-indolyl-β-d
-galactopyranoside). White colonies of E. coli
cells were chosen and amplified by PCR with M13 primers. PCR amplicons were then submitted to the Biomedical Genomics Center at the University of Minnesota (BMGC; St. Paul, MN) for sequencing using the Sanger sequencing method (ABI Prism 3730xl DNA analyzer). The DNA sequences were analyzed, and a phylogenetic tree was prepared to identify redundancy in the selected pool using MEGA4 (29
). Selected aptamer sequences were further analyzed for secondary structure prediction using the Mfold web server (30
Selected aptamer sequences were synthesized and labeled with biotin (5′), while amplicons containing enriched aptamer pool were amplified by PCR with 5′-biotin-labeled forward primer (Bio-WP18). Aptamer-protein binding assay was analyzed by using a LightShift chemiluminescent electrophoretic mobility shift assay (EMSA) kit (Pierce) in accordance with the manufacturer's instructions with a few modifications. Briefly, selected aptamers (20 fmol) were added to recombinant swH3 protein in 1× binding buffer and nuclease-free water (final volume, 20 μl), followed by incubation at room temperature for 30 min. The samples were loaded onto an 8% native polyacrylamide gel (Bio-Rad, Hercules, CA) in 0.5× Tris-borate-EDTA buffer. Electrophoresis was performed at 80 V for 60 min, and then the samples were electrotransferred to a positively charged nylon membrane (Biodyne B; 0.45-μm pore size; Biodyne, Pensacola, FL). The membrane was processed and developed with a chemiluminescent nucleic acid detection module (Pierce) in accordance with the manufacturer's instructions. Reactions on the membrane were then visualized and imaged with the LabWorks 4.5 imaging system (UVP Products, Upland, CA).
HA inhibition test.
HA inhibition (HI) was performed to prove that the selected aptamers recognized the active site of the HA protein of swH3 IAV and could inhibit the viral infectivity. Turkey red blood cells (RBC) were diluted to 0.5% in PBS. The HA test was performed in a microtiter 96-well plate with 50-μl samples containing virus and 50 μl of 0.5% RBC. The samples were incubated at room temperature for 30 to 45 min, and the agglutination of RBC was inspected. HI was performed with 500 pmol of each aptamer added to the virus before the addition of the RBC, followed by incubation at room temperature for 30 min.
DNase I footprinting assay.
A DNase I footprinting assay was performed to identify the binding site(s) of the selected aptamer (31
). The recombinant swH3 protein (4 to 6 μg) was mixed with 1 pmol of HA68 labeled with 6-carboxyfluorescein (FAM) at the 5′ end in 1× binding buffer (LightShift; Pierce), nuclease-free water was added to a final volume 50 μl, followed by incubation at room temperature for 1 h. After incubation, the samples were treated with 0.2 U of DNase I (amplification-grade; Invitrogen), followed by incubation at 37°C for 5 min. To inactivate the DNase I, 2 mM EDTA was added to each sample, followed by incubation at 70°C for 10 min. Recombinant protein from an H1N1 human influenza virus (A/Solomon Islands/3/06 [human H1]) was used as a negative control and treated under identical conditions. A negative control (without protein) was applied using nuclease-free water in place of protein. After the reaction, the samples were purified by using a MiniElute PCR purification kit (Qiagen) and eluted with 14 μl of nuclease-free water. Approximately 12 μl of purified samples were submitted to the BMGC for fragment analysis on a 3130XL genetic analyzer. The binding region of aptamer was analyzed by Peak Scanner software (v.1.0; Applied Biosystems, Foster City, CA) with Liz500 as an internal size standard.
Specificity of each aptamer against recombinant HA proteins of different subtypes of IAVs.
The aptamer dot blot assay was modified and performed to test the specificity of each aptamer by using recombinant HA proteins in different subtypes of IAVs from the BEI resource: H1, H2, H3, H5, H6, H7, and H9 (). Each protein was diluted with nuclease free-water to a concentration of 50 ng/μl, and 2 μl each was blotted onto nitrocellulose membrane (Protran; Whatman/Pierce). The membrane was blocked with 5% fish gelatin (Sigma, St. Louis, MO) in 1× PBST at 4°C. After 12 h of incubation, the membrane was incubated with 5′ digoxigenin (DIG)-labeled aptamer at the final concentration 100 nM in 1% fish gelatin at room temperature with gentle shaking for 1 h and then washed five times with 1× PBST in 5-min increments. The membrane was incubated with peroxidase-conjugated IgG fraction monoclonal mouse anti-DIG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) in 1:12,500 dilutions in 1% fish gelatin in 1× PBST at room temperature for 30 min. The membrane was then washed once with 1× PBS and developed with luminal enhancer and stable peroxide solution in a 1:1 dilution (LightShift; Thermo) at room temperature for 5 min. The membrane was visualized and imaged by LabWorks 4.5 software (UVP Products).
Binding affinity of selected aptamers.
The recombinant swH3 protein was prepared at a concentration 25 ng/μl by dilution with 50 mM Tris–250 mM NaCl (pH 8.2) (protein buffer). Nitrocellulose membranes (Protran) were blotted with 2 μl of the protein (50 ng) in triplicate and dried at room temperature for 1 h. The membranes were blocked with 5% fish gelatin in 1× PBST at 4°C. After 12 h of incubation, the membranes were incubated with different concentrations of 5′ Bio-aptamer in 2-fold dilutions (1 to 1,024 nM) in 1% fish gelatin in 1× PBST at room temperature for 1 h with gentle shaking. The membranes were washed with 1× PBST for 5 min with gentle shaking. After five washes, the membranes were incubated with NeutrAvidin protein and horseradish peroxidase (Thermo) conjugated in 1:12,500 dilutions in 1% fish gelatin in PBST at room temperature for 30 min. The membranes were washed once with 1× PBS and developed with luminol enhancer and stable peroxide solution in 1:1 dilution (LightShift) at room temperature for 5 min. The membranes were visualized and imaged by LabWorks 4.5 software. The chemiluminescence intensity was calculated by using ImageJ 1.45s software. The dissociation constant (Kd) was calculated based on a nonlinear regression equation.
Influenza live virus detection by aptamer dot blot assay.
All processes involving work with live viruses were performed in a class II biosafety cabinet. IAV isolates from North American swine belonging to phylogenetic lineages of H3 were obtained from the University of Minnesota Veterinary Diagnostic Laboratory (UMVDL) as blind samples in minimal essential medium (MEM). AIV H3N2 in allantoic fluid was used as the representative for H3 cluster I (A/mallard/South Dakota/SE128/2007) (33
). Detailed descriptions of the reference viruses used in the present study are given in and . Samples containing live viruses were centrifuged at full speed (13,000 rpm) for 5 min prior use. Then, 2 μl of each culture (supernatant) was blotted onto the nitrocellulose membranes in triplicate (Protran). The membranes were dried at room temperature for 1 h and blocked with 5% fish gelatin (Sigma) in 1× PBST at 4°C. After 12 h of incubation, denatured sheared salmon sperm DNA (Ambion, Austin, TX) was added to the membranes with blocking buffer at a concentration of 10 ng/μl, followed by incubation at room temperature for 20 min. Membrane was incubated with DIG-aptamers (100 nM) in 1% fish gelatin at room temperature for 1 h and then washed five times with 1× PBST. The washing and developing steps were performed as described above.
Fig 1 Phylogenetic clusters of IAV H3 subtypes used for aptamer specificity testing. The relative phylogenetic positions of HA sequences of archived IAVs from North American swine (blue) used for aptamer dot blot assay are shown. Isolates were obtained from (more ...)
Detail descriptions of reference influenza live viruses used in this study and results of each aptamer detected by aptamer dot blot assay