Streptavidin was purchased from Roche Applied Science (Penzberg, Germany). Human α-thrombin was purchased from Hematologic Technologies Inc. (Essex Junction, VT). Bovine serum albumin (BSA), N-hydroxy-succinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich (Saint Louis, MO) and used without further purification. Agarose was purchased from Fisher Scientific (Pittsburgh, PA). GoTaq Hot Start Colorless Master Mix and PCR water were purchased from Promega (Madison, WI). Dynabeads MyOne C1 SA-coated beads and M-270 carboxylic acid-coated beads were purchased from Invitrogen (Carlsbad, CA). Lambda exonuclease enzyme (5000 U/mL) and 10× lambda exonuclease reaction buffer were purchased from New England BioLabs (Ipswich, MA). The random DNA library was synthesized with hand-mixing by Integrated DNA Technologies (Coralville, IA) and purified via PAGE. Each single-stranded DNA (ssDNA) library component consists of a central 60-mer random region flanked by two 20-mer PCR primer sequences. Unlabeled, phosphorylated and FAM-labeled PCR primers (see Supporting Information, Table S1
) were synthesized by Biosearch Technologies (Novato, CA) and purified via HPLC. The binding and washing buffer used for SA aptamer selection consisted of 20 mM Tris, pH 7.6, 150 mM NaCl, 2 mM MgCl2
, and 0.02% Tween20 (v/v).
We performed PCR amplification with a 100 μL reaction mixture containing 50 μL of GoTaq Hot Start Master Mix, 4 μL of 100 μM forward primer, 4 μL of 100 μM phosphorylated reverse primer, 4 μL of 1 μM random ssDNA stock and 38 μL of nuclease-free water. The PCR program consisted of 5 cycles at 95 °C for 5 min, 51 °C for 1 min and 72 °C for 20 min, followed by an additional 4 cycles of 95 °C for 30 sec, 58 °C for 1 min and 72 °C for 1 min, and finally a 14-min extension at 72 °C. PCR products were concentrated by ethanol precipitation and resolved on a 2% agarose gel. 100-base pair double-stranded DNAs (dsDNAs) were extracted and purified using a QIAquick gel extraction kit (Qiagen, Venlo, Netherlands) following the manufacturer's protocol. Purified dsDNAs were subsequently digested with lambda exonuclease using the method described below, and the resulting ssDNA pool was further purified by phenol/chloroform extraction and ethanol precipitation. The total PCR reaction volume was adjusted to yield 1 nmole of amplified ssDNA pool.
We performed a pilot PCR to optimize the number of cycles for amplifying the eluted sample during selection. A PCR mixture containing 50 μL of GoTaq Hot Start Master Mix, 0.25 μL of 100 μM forward primer, 0.25 μL of 100 μM phosphorylated reverse primer and 39.5 μL of nuclease-free water was prepared, and then combined with 10 μL of the eluted beads from the MMS chip for a final volume of 100 μL. GoTaq Hot Start polymerase was activated prior to PCR by heating the reaction mixture to 95 °C for 2 min, followed by 20 cycles of a rapid three-step PCR (30 sec at 95 °C, 30 sec at 56 °C, 30 sec at 72 °C). During the extension step of each cycle, 6 μL of PCR mixture were collected and resolved on a 10% PAGE-TBE (1× TBE: 89 mM Tris borate, 2 mM Na2EDTA, pH 8.3) gel to determine the optimal PCR amplification cycle number with minimal by-products. The collected aptamer pools from each round were PCR amplified at this optimized cycle number.
Characterization of Washing Efficiency
0.5 μL of MyOne C1 SA-beads were incubated with 50 pmol of ssDNA library in 50 μL of SA binding buffer for 2 hours followed by a 30-min wash using one of three different methods: MMS chip washing, volume dilution challenge and combined washing. For continuous washing in the MMS chip, the flow rate was 50 mL/hr, and for the dilution challenge the volume was diluted 100-fold. The combined method consisted of a 25-min dilution challenge and 5-min additional MMS chip washing. After washing, beads were eluted into 600 μL of fresh buffer. To compare washing efficiencies, real-time PCR (RT-PCR) was performed, and the amounts of ssDNA bound to SA were determined by monitoring threshold cycles (CT
). The reaction solutions were prepared with 10 μL iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), 8.8 μL PCR water, 0.1 μL each of 0.1 mM forward and reverse primer and 1 μL eluted bead solution. CT
values for each washing method were monitored using the iQ5
RT-PCR Detection System (Bio-Rad Laboratories).
Purified dsDNA solution was mixed with a specific volume of lambda exonuclease enzyme (e.g., 1 μg of dsDNA with 1 μL of lambda exonuclease stock), and the mixture was adjusted to yield 1× lambda exonuclease reaction buffer conditions using 10× concentrated buffer stock. This mixture was immediately incubated at 37 °C for 2 hrs followed by an additional 10-min incubation at 75 °C to stop the enzymatic reaction. After digestion, ssDNA was purified with phenol/chloroform extraction and ethanol precipitation and completely dried out before use.
Affinity and Specificity Measurements
The average dissociation constant of each ssDNA pool was measured via a fluorescence binding assay. FAM-labeled ssDNA pools were diluted to several different concentrations (from 0 to 100 nM) in 140 μL of binding buffer; these dilutions were heated at 95 °C for 10 min, immediately cooled on ice for 10 min and then incubated for another 10 min at room temperature (RT). Subsequently, these various heat-treated ssDNA solutions were incubated with target-coated beads at RT for 1 hour in a total volume of 150 μL. We measured the Kd for pools from each round, including the amplified library, using 0.1 μL of Dynabeads MyOne C1 SA-coated beads (7×109 beads/mL, 3.01×105 SA molecules/bead, 1.0 μm in diameter). The specificity of the synthesized aptamer sequences was tested with M-270 carboxylic acid-coated beads (2×109 beads/ml, 2.8 μm in diameter) because these beads yielded closer matching of surface densities of SA, BSA and thrombin. The molecules were immobilized on the beads through the EDC-NHS coupling process using the manufacturer's protocol, and quantified with the NanoOrange Protein Quantitation Kit (Invitrogen). We used 0.3 μL of SA (3.42×105 molecules/bead), 0.4 μL of BSA (2.66×105 molecules/bead), and 0.3 μL of thrombin (3.22×105 molecules/bead) and 0.3 μL of tris coated beads. After incubating the ssDNA solutions with target-coated beads, the magnetic beads were washed three times with binding buffer and magnetic separation was used to eliminate unbound ssDNAs. Bound ssDNAs were then released into 55 μL of binding buffer by heating the aptamer-bound beads at 95 °C for 10 min while shaking (300 rpm). Next, 50 μL of each eluate was transferred to a black 96-well microplate (Microfluor 2, Thermo Scientific, Waltham, MA), and the amount of released ssDNA was determined by fluorescence measurement using a microplate reader (Tecan, San Jose, CA). Finally, dissociation constants (Kd) were calculated from the calibrated curve fitting using the equation Y = Bmax × X/ (Kd + X) where X is the concentration of the ssDNA and Bmax is the fluorescence value at saturation.
Cloning and Sequencing of Selected Aptamers
PCR products were amplified with unlabeled primers at the optimized cycle number, purified using the MiniElute PCR Purification Kit (Qiagen) with the manufacturer's protocol and cloned into Escherichia coli using the TOPO TA Cloning Kit (Invitrogen). 20 colonies each from rounds 2–3 were randomly picked and sequenced at Genewiz Inc. (South Plainfield, NJ). The sequences were then analyzed and aligned using Geneious v5.1 (Biomatters Ltd., New Zealand). Two representative aptamer sequences from each round were amplified with phosphorylated reverse primers and FAM-labeled forward primers for further fluorescence affinity measurements.
Relative Binding Affinity Assay
A PCR mixture containing 10 μL of GoTaq Hot Start Master Mix, 0.2 μL of 100 μM forward primer, 0.2 μL of 100 μM reverse primer and 9.6 μL of nuclease-free water was prepared, and a small amount of each sequenced colony-derived DNA sample was transferred into 20 μL of PCR mixture. Prepared PCR mixtures were amplified as described above for 25 cycles. PCR products were diluted 100-fold with nuclease-free water, and a small quantity of the diluted solution was further amplified for 8 cycles based on the procedure described above, after which FAM-labeled ssDNA pools were generated for each sample via lambda digestion and purified. 150 μL of 50 nM FAM-labeled ssDNA solution from each colony was prepared in SA binding buffer. The remaining steps of binding measurement were performed as described above.