Reagents and instruments
Bovine serum albumin (BSA), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich, Inc. (Saint Louis, MO). Integrin αIIbβ3 was purchased from Enzyme Research Laboratories, Inc. (South Bend, IN). Integrin αVβ3 and Integrin αVβ6 were purchased from R&D Systems (Minneapolis, MN). The single-stranded DNA (ssDNA) library and all PCR primers were synthesized and purified by Integrated DNA Technologies (Coralville, IA). HotStart Master Mix and water for PCR were purchased from Qiagen (Hilden, Germany). The magnetic beads, including Dynabeads MyOne C1 streptavidin-coated beads and M-270 carboxylic acid-coated beads, were purchased from Invitrogen (Carlsbad, CA). Fluorescence measurements were performed in black 96-well microplates (Microfluor 2, Thermo Scientific, Waltham, MA) using a microplate reader (Tecan, Männedorf, Switzerland), and surface plasmon resonance (SPR) measurements were performed on a Biacore 3000 instrument (GE Healthcare, Waukesha, WI). Real-time PCR equipment (IQ5, Bio-Rad, Hercules, CA) was used to measure the DNA concentrations.
2′-F RNA MAI-SELEX: target and library preparation
Both M-270 and MyOne C1 beads were used to immobilize integrin proteins. M270 beads were activated with EDC and NHS, after which the proteins were immobilized according to the manufacturer’s procedure. Prior to immobilization onto MyOne C1 beads, the protein was first biotinylated using the sulfo-NHS-LC-biotin reagent (Thermo Scientific) and then incubated with MyOne C1 beads, following the manufacturer’s procedure. The immobilized proteins were quantified using the LavaPep Quantification Kit (Gel Company, San Francisco, CA). Each element of the ssDNA random library was composed of 50 randomized nucleotides flanked by 5′ and 3′ primer sites (5′-TAATACGACTCACTATAGGGAGGACGATGCGG-[50N]-CAGACGACTCGCCCGA-3′). The ssDNA library was amplified by PCR, and then transcribed to generate 2′-F RNA using a DuraScribe T7 Transcription Kit (EPICENTRE Biotechnologies, Madison, WI). We then digested the DNA template in the reaction using Turbo DNAse I (Ambion, Austin, TX), and purified the RNA library by urea-PAGE gel followed by electro-elution. The eluate was desalted and concentrated by ethanol precipitation, dissolved in PBS and quantified with a NanoDrop spectrophotometer (Wilmington, DE).
MAI-SELEX: affinity module
We used commercially available Dynabeads and a DynaMag magnet (Invitrogen) for MAI-SELEX in order to demonstrate the broad applicability of the method. Protein-coated magnetic beads were washed two times with 500 μl of PBSMT buffer (PBS supplemented with 2.5 mM MgCl2 and 0.01% Tween-20) before each selection. Integrin αVβ3-coated M270 beads were used in the affinity module: 100 nM in the first three rounds, followed by 8 nM for rounds 4 and 5. At the start of SELEX, ~50 pmole random DNA library was transcribed into ~500 pmole of RNA library to be used in the first round. The 2′-F RNA library was folded by denaturing at 95 °C for 5 min and snap-cooling on ice for 5 min. We used an initial library concentration of 5 μM in round 1, and then reduced the concentration to 1 μM in rounds 2 and 3, and to 370 nM in rounds 4 and 5. Integrin αVβ3-coated beads were incubated with the library in 100 μl selection buffer (PBS supplemented with 2.5 mM MgCl2, 0.1% BSA and 100 μM yeast tRNA) for 2 hours at room temperature. After incubation, the beads were magnetically trapped in order to remove the supernatant. The trapped beads were washed three times with 500 μl wash buffer (PBS supplemented with 2.5 mM MgCl2 and 0.1% BSA); the total duration of washing was 15 min for round 1 and 1 hour for subsequent rounds. Target-bound aptamers were eluted from beads by heating at 95 °C for 5 min, and then reverse-transcribed to generate cDNA using the ThermoScript RT-PCR System (Invitrogen). We monitored the progress of the selection by subjecting 1 μl cDNA to quantitative PCR (qPCR). Everything else being equal, the decrease in threshold cycle (Ct) value from one round to the next indicates the increase in the proportion of target-binding sequences. The qPCR result is also valuable in predicting the proper number of PCR cycles to use for full-scale library amplification. After reverse transcription and PCR amplification of the total aptamer eluate, we transcribed the PCR product back to 2′-F-modified RNA and purified the RNA pool for the next round of selection, following the procedure described above.
MAI-SELEX: specificity module
MyOne C1 beads were used in the specificity module, to avoid accumulating sequences that bind to M270 beads. 100 nM aptamer pool RNA was incubated with 5 μM integrin αIIbβ3-coated beads in 30 μl selection buffer for 2 hours, after which the integrin αIIbβ3-coated beads were trapped and the supernatant was transferred to another tube to incubate with 20 nM integrin αVβ3-coated beads in 30 μl selection buffer for 2 hours. The integrin αIIbβ3-coated beads were then washed with wash buffer for 1 hour. Finally, the integrin αVβ3-coated beads were also trapped and washed with wash buffer for 1 hour. The aptamers were eluted from both sets of beads and amplified following the same procedure as described in the affinity module.
Cloning and sequencing of aptamer pools
The αV and β3 pools were reverse transcribed and amplified by PCR, and then cloned into E. coli using the TOPO TA Cloning Kit (Invitrogen). 25 colonies from each pool 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 the αV pool and three from the β3 pool were selected for further affinity measurements.
RNA aptamers were treated with Antarctic phosphatase (New England Biolabs, Ipswich, MA) and then labeled at the 5′ end with radioactive γ-32P ATP using T4 polynucleotide kinase (New England Biolabs). We then incubated 0, 10, 25, 100, 400, and 500 nM of protein with 1 nM radiolabeled RNA in 20 μl reactions, and separated protein-bound RNA from unbound RNA by passing the solution through a 0.45 μm pore-size Millipore (Billerica, MA) mixed cellulose ester filter membrane. This membrane has strong affinity for protein but not RNA, such that protein and protein-bound RNA will be retained while unbound RNA will flow through. We then quickly washed the membrane twice with 1 ml PBS and measured the amount of protein-bound RNA by measuring remaining scintillation counts on the membrane. For Kd measurement, at least six different concentrations of protein were used in the filter-binding assay, and the scintillation count values were plotted against protein concentrations to generate a binding curve. The curve was then fitted using the GraphPad Prism software (La Jolla, CA).
Surface Plasmon Resonance Measurements
We biotinylated the αV-1 and β3-1 aptamers at the 5′ end by adding a mixture of 5′-biotin-G-monophosphate (Trilink, San Diego, CA) and GTP (molar ratio 3:2) to the in vitro transcription reaction. Biotinylated aptamers were then purified as described above, and immobilized by flowing 10 nM aptamer solution at 20 μl/min onto the surface of the Biacore SA chip (GE Healthcare). Next, we obtain a series of SPR sensorgrams using the Kinetics Wizard software: at each cycle of the kinetic measurement, we applied varying concentrations of integrin protein for association (0/25/50/100 nM for αV-1 measurement, where duplicate tests were performed at the 25 nM and 50 nM concentration points; 0/10/50/100/200/500 nM for β3-1 measurement) at 20 μl/min for 2 mins, stopped the protein injection, and allowed 5 mins for dissociation. We then regenerated the sensor surface by injecting 10 mM glycine pH 3.0 at 30 μl/min for 30 s. A flow cell without immobilized aptamers was used as reference.
Enzyme-linked oligonucleotide assay (ELONA)16
Individual aptamers were biotinylated at the 5′ end as described above. For ELONA, microtiter plate wells were coated with integrin proteins by adding 50 μl protein solution (at 25 nM unless noted otherwise) and incubating at 4 °C for 37 hours. All subsequent steps were performed at room temperature. After incubation, we washed the plate once with 200 μl PBS supplemented with 0.05% Tween-20 (PBST buffer) and then blocked each well with 100 μl 1% BSA in PBST for 1 hour. We then washed the plate with 200 μl PBST three times, added 100 μl of biotinylated aptamers (at 10 nM unless otherwise noted) dissolved in PBST supplemented with 0.1% BSA (PBSTB buffer), and incubated for 1 hour. Next, we washed the plate three more times with 200 μl PBST, and added streptavidin-conjugated horseradish peroxidase (HRP) dissolved in 100 μl PBSTB buffer at 1:500 dilution. After 30 min incubation, we washed the plate five times and added the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) substrate. This substrate becomes oxidized by HRP to produce a blue-green color, which we measured with a Tecan plate reader at 405 nm wavelength (OD405). The ELONA test in serum was performed in a similar manner, except that the biotinylated aptamers were dissolved in 100 μl undiluted fetal bovine serum (FBS) instead of PBSTB buffer.