Article pubs.acs.org/ac
Monoclonal Surface Display SELEX for Simple, Rapid, Efficient, and Cost-Effective Aptamer Enrichment and Identification Zhi Zhu,† Yanling Song,† Cong Li,† Yuan Zou, Ling Zhu, Yuan An, and Chaoyong James Yang* The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China S Supporting Information *
ABSTRACT: A novel method, monoclonal surface display SELEX (MSD-SELEX), has been designed for simple, rapid, efficient, and cost-effective enrichment and identification of aptamers from a library of monoclonal DNA-displaying beads produced via highly parallel single-molecule emulsion PCR. The approach was successfully applied for the identification of high-affinity aptamers that bind specifically to different types of targets, including cancer biomarker protein EpCAM and small toxin molecule aflatoxin B1. Compared to the conventional sequencing-chemical synthesis-screening work flow, MSDSELEX avoids large-scale DNA sequencing, expensive and time-consuming DNA synthesis, and labor-intensive screening of large populations of candidates, thus offering a new approach for simple, rapid, efficient, and cost-effective aptamer identification for a wide variety of applications.
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and expensive. Therefore, a rapid and efficient aptamer selection approach is of great interest and in high demand. In SELEX, two major steps are involved: the enrichment from a large initial library after many iterative rounds of selection, and the screening of tens to hundreds of aptamer candidates from the enriched library.16 Over the past two decades, significant effort has been devoted to developing innovative methods for rapid and efficient enrichment of aptamers, such as capillary electrophoresis-SELEX,17,18 fluorescence-activated cell sortingSELEX,19,20 and microfluidic-SELEX technologies.21−23 On the other hand, the screening process has remained a rarely explored area. Normally, the enriched library is cloned into plasmids and transfected into bacteria. Then colonies from growing bacteria are picked and sequenced in large quantities to obtain aptamer candidates. After analysis of hundreds to thousands of sequences, the consensus sequences with high repeats are chemically synthesized to screen their binding affinities individually. Such a sequencing-chemical synthesis-screening work flow is time-consuming, labor-intensive, inefficient, and expensive. To address this issue, Krylov et al. reported an elegant non-SELEX method that partitions and screens the individual clones for their affinity to the target by nonequilibrium CE before sequencing.24,25 We recently have developed a highly parallel approach for aptamer screening by partitioning individual aptamer candidates from an enriched library into single agarose droplets for single-molecule PCR.26 The binding
ptamers are single-stranded oligonucleotides that can bind to different types of targets, such as small molecules, proteins, cells, and even tissues, with high affinity and selectivity.1−5 Because of their significant features, including low molecular weight, easy and reproducible synthesis, lack of immunogenicity, low toxicity, and rapid tissue penetration, aptamers are promising affinity ligands for a variety of attractive applications in fundamental research, chemical sensing, clinical diagnosis, targeted drug delivery, and therapy.6−11 Despite great promise and significant effort in aptamer development over the past 20 years, it is astonishing that only a limited number of aptamer−target pairs have been intensively used, mainly for proof-of-principle of novel aptamer assays.12 For example, by far the most frequently used aptamer is the anti-thrombin aptamer, which has been employed in >1000 publications, followed by aptamers targeting adenosine, cocaine, and platelet-derived growth factor, all of which account for over one-third of the total publications on aptamers (Figure S1). This lack of variation severely impedes the development and application of aptamers. Two reasons have been previously proposed to explain this problem. First, the intrinsic property of natural DNA with limited chemical diversity of interaction may lead to the difficulty of selecting aptamers against their targets, especially some proteins.13 This problem has been addressed to some extent by incorporating various artificial nucleobases with functional groups to augment the diversity of a randomized library for improving the success rate of aptamer generation.13−15 Another reason comes from the intrinsic limitations of the conventional aptamer selection technique, known as SELEX (Systematic Evolution of Ligands by EXponential enrichment),1,2 which is time-consuming, labor-intensive, inefficient, © 2014 American Chemical Society
Received: February 19, 2014 Accepted: May 26, 2014 Published: May 27, 2014 5881
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DNA Synthesis and Purification. The oligonucleotide sequences used in this study are listed in Table S1. All DNA sequences were synthesized on a DNA synthesizer (PolyGen Column 12 DNA synthesizer) using standard phosphoramidite chemistry and purified by reversed phase HPLC (Agilent 1100). After detritylation, the DNA were quantified by UV−vis spectrometry and stored at −20 °C for future use. F-Primer Bead Preparation and Characterization. The F-Primer beads are prepared by linking amine modified FPrimer to 34 μm diameter NHS-beads via amine-NHS conjugation chemistry. The amount of F-Primer covalently linked to a bead was quantified by annealing FAM labeled DNA complementary to F-Primer (FAM-FP-cDNA) and then denaturing to obtain the bound FAM-FP-cDNA. The amount of F-Primers on bead was determined by quantifying the annealed FAM-FP-cDNA using a calibration curve and the number of beads. Emulsification, Amplification, and Bead Recovery. The emulsification process creates a heat-stable water-in-oil emulsion. The emulsion oil phase consists of 40% (w/w) DC 5225C Formulation Aid, 30% (w/w) DC 749 Fluid, 30% (w/w) Ar20 Silicone Oil.27 The prepared PCR solution containing PCR reagents as listed in Table S2 was vortexed for 30 s; then 400 μL of emulsion oil was added quickly to the PCR solution, and the mixture was vortexed to create water-in-oil droplets with an average diameter of 50 to 180 μm. The emulsion has been divided into five PCR tubes with 100 μL per tube. After 35 cycles of PCR amplification (30 s at 94 °C, 30 s at 53 °C, 30 s at 72 °C) were performed, the amplified emulsions were combined to a 15 mL tube. Then, 4 mL of acetone was added to break the emulsion. The mixture was vortexed to lower the viscosity of the emulsion. The tube was centrifuged for 4 min at 500 rpm, and the organic phase supernatant was removed. The operation was repeated by successively washing with isopropyl alcohol and ultrapure water. Afterward, the amplified beads were collected and resupsended in 0.2 M NaOH solution to denature and separate the antisense ssDNA from the sense ssDNA on beads. Finally, the library beads were collected and stored in ultrapure water at 4 °C for further use. Protein Expression and Preparation. Human EpCAM plasmid was cloned into a mammalian expression vector pcDNA 3.1/V5-His-TOPO. The recombinant 6 × Histine-tagged EpCAM was transiently expressed in HEK-293T cells. The total cell lysate was incubated with Ni-beads in 0.1 M phosphate buffer with 40 mM imidazole for 3 h at 4 °C, followed by 3 times of washing with PBS buffer (10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4). The Ni-beads functionalized with EpCAM (EpCAM-beads) were kept at 4 °C in PBS buffer until use. SELEX Procedures. For the first round of selection, 5 nmol of the initial ssDNA library was reconstituted thoroughly with 500 μL of binding buffer (5 mM MgCl2 in PBS containing 0.1 mg/mL yeast tRNA to reduce nonspecific binding, pH 7.4), and the resulting solution was heated at 95 °C for 5 min and cooled immediately on ice for 10 min. EpCAM-beads (about 500 pmol protein) were incubated with the initial ssDNA library at 37 °C on a rotary shaker for 30 min for positive selection. After incubation, beads were washed with washing buffer (5 mM MgCl2 in PBS, pH 7.4). The EpCAM-beads collected were added to a PCR cocktail for subsequent amplification by PCR to increase the number of copies of individual sequences. The PCR mixture contained 400 nM each primer, 0.1 mM each dNTP, and 2.5 U Easy-Taq DNA polymerase in a total volume of 50
affinity of individual sequences was evaluated before sequencing and DNA synthesis. However, the non-SELEX method evaluates a group of aptamer candidates, and our previous approach evaluates the individual sequences from an enriched library that contains aptamer candidates having a broad distribution of binding affinity. A time-consuming screening process is still required to identify high-affinity ligands hidden in a large group of less desirable choices. Therefore, new SELEX methods allowing efficient enrichment and identification of high affinity aptamers without cloning, sequencing, chemical synthesis, and screening would greatly accelerate the selection process and speed up the development of aptamers. Toward this end, we have designed a monoclonal surface display SELEX (MSD-SELEX) method for efficient enrichment and identification of aptamers from a library of monoclonal DNA-displaying beads produced via highly parallel singlemolecule emulsion PCR. In MSD-SELEX, the sequences in the random ssDNA library are individually displayed on the microbead surface with millions of repeats. The interaction of DNA with targets is therefore converted to the interaction of beads and targets, which can be directly visualized under the microscope for large target particles such as cells or molecules functionalized on microbeads. Because nonbinding or weak binding beads can be easily removed through a washing process to enrich the library, only beads with high binding affinity sequences are retained. More importantly, due to the one-beadone-sequence nature of the monoclonal beads, the affinity of the aptamer candidate sequences displaying on the surfaces of the enriched beads can be immediately confirmed without the need for sequencing and synthesis. Compared to conventional SELEX methods, the MSD-SELEX approach avoids many rounds of selection, large-scale DNA sequencing, expensive and time-consuming DNA synthesis, and labor-intensive screening, paving a new way for simple, rapid, efficient, and cost-effective molecular evolution of high affinity ligands.
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EXPERIMENTAL SECTION Materials and Reagents. DNA synthesis reagents were purchased from Glen Research (Sterling, VA, USA). Nhydroxysuccinimide (NHS) activated agarose beads, Ni Sepharose beads, and streptavidin-coated sepharose beads were purchased from GE, Healthcare (Beijing, China). DC 5225C Formulation Aid and DC 749 Fluid were obtained from Dow Chemical Co. (Shanghai, China). Ar20 Silicone Oil was purchased from Sigma (St. Louis, MO, USA). Human EpCAM plasmid and mammalian expression vector pcDNA 3.1/V5-HisTOPO were purchased from Invitrogen (Shanghai, China). Cell Lines and Culture. The cell lines of human origin used in this study were purchased from American Type Culture Collection (Manassas, VA, USA), including human gastric cancer Kato III, breast cancer and T47D, human Burkitt’s lymphoma cell line Ramos, and embryonic kidney cell HEK293T. Kato III cells were cultured in modified Iscove’s minimal essential medium (IMEM; Hyclone) with 20% fetal calf serum. T47D and HEK-293T Cells were cultured in the high glucose (4.5 g/L) version of Dulbecco’s Modified Eagle medium (DMEM) (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 100 U/mL penicillin-streptomycin (Hyclone). Ramos cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640; Hyclone) with 10% fetal bovine serum. All cells were maintained at 37 °C in a 5% CO2 atmosphere. 5882
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Figure 1. Working principle of MSD-SELEX technology for aptamer selection and identification. Statistically diluted ssDNA library with forward primer-functionalized beads (F-Primer beads) was compartmentalized into water-in-oil droplets at the single-molecule level. Single copy emulsion droplet PCR was then performed to generate monoclonal beads each displaying millions of identical DNA sequences. A library of monoclonal beads is then incubated with target cells. By removing the unbound beads and collecting bound beads, the positive clones are isolated for subsequent sequence retrieval and aptamer identification.
μL. The thermal cycling conditions of PCR were as follows: 95 °C for 3 min (initial denaturation), eight cycles of 95 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s, followed by a single final extension at 72 °C for 3 min. After denaturing in alkaline conditions (0.2 M NaOH 3 min), the FITC-conjugated sense ssDNA strand was separated from the biotinylated antisense ssDNA strand by streptavidin-coated sepharose beads and used as a new library to perform the second round of selection using the same procedure as described for first selection. Negative selection was introduced after the second round. Before incubation with EpCAM-beads, the DNA library was incubated with Ni-beads for 10 min to remove the sequences that may bind to Ni-beads. The nonbinding supernatant DNA was collected and used in the positive selection by incubation with EpCAM-beads. After five rounds of conventional SELEX, the fifth DNA library was converted to monoclonal bead library by emulsion PCR. Then, the bead library was incubated with EpCAM-positive Kato III cells for MSD-SELEX. After incubation and several washing steps, the beads that did not bound to the adherent Kato III cells were efficiently removed, while the beads forming a complex with Kato III cells were retained in the culture dish and extracted using a capillary tube. Each of these positive clones was amplified with FAM-labeled FPrimers (FAM-F-Primer) by conventional PCR to generate FAM-labeled ssDNA for further confirmation. For AFB1 aptamer SELEX, the AFB1 was immobilized on magnetic beads, and the rest of produces were similar to that of EpCAM. The binding and washing buffer was changed to 20 mM TrisHCl buffer solution (100 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2, pH 8.0). The incubation was performed at room temperature. Flow Cytometry Analysis. To test the clone aptamers or to test the binding ability of selected aptamers, about 1 × 105 target EpCAM-beads or cells were incubated with a final concentration of 200 nM enriched pools or aptamer candidates labeled at the 5′-end with FAM in 200 μL of binding buffer at 37 °C for 30 min. Beads were washed twice with 400 μL of washing buffer and suspended in 200 μL of binding buffer. The fluorescence was determined with a flow cytometer (Becton Dickinson Immunocytometry systems) by counting 10 000 events. The FAM-labeled random sequence was used as a negative control.
To determine the binding affinities of aptamers toward beads or different types of cells, beads or cells were incubated with various concentrations of FAM-labeled aptamers in 200 μL of binding buffer at 37 °C for 30 min in the dark. Beads or cells were washed twice with 600 μL of washing buffer, resuspended in 200 μL of binding buffer, and subjected to flow cytometry analysis. The mean fluorescence intensity of the target−aptamer complex was used to evaluate the binding affinity by subtracting the mean fluorescence intensity of the unselected initial library. Using SigmaPlot software 2.9 (Jandel Scientific), the Kd of the aptamer−cell interaction was obtained by fitting the dependence of fluorescence intensity on the concentration of aptamer to the one-site saturation equation Y = BmaxX/(Kd + X). AFB1-Beads Preparation. The AFB1 beads were prepared according to the synthetic route shown in Figure S3. Carboxymethylhydroxylamine HCl (35 mg) was added to a solution of 7.6 mg of AFB1 in 4 mL of pyridine. The mixture was refluxed at 80 °C overnight. The solution was concentrated under a vacuum, and the residue was chromatographed on a silica column (chloroform/methanol, 10:1, v/v) to separate a fluorescent product AFB1-oxime. To the AFB1-oxime solution in 3 mL of dry dichloromethane, 5.4 mg of N-hydroxysuccinimide (NHS) and 9.6 mg of dicyclohexylcarbodiimide (DCC) were added at room temperature, followed by the addition of 5 mg of 4-dimethylaminopyridine (DMAP). The mixture was stirred overnight and filtered to remove the byproduct. Then, the solvent was evaporated to obtain AFB1-oxime active ester. At the same time, 0.107 g NHS-beads were suspended in 0.9 mL 50 mM K2HPO4 (pH 9.0). Ethanediamine (100 μL) was added into the above solution, stirred overnight to form NH2-beads. Then the excess ethanediamine was removed by washing the beads five times with PBS buffer. At last, the AFB1-oxime active ester dissolved in 0.8 mL of N,N-dimethylformamide (DMF) was mixed with NH2-beads suspended in 0.8 mL of PB buffer (pH 8.0) and stirred overnight. Then the final products AFB1beads were washed 5 times with DMF/PB buffer (1:1, v/v) and PB buffer and stored at 4 °C for future use. Fluorescence Anisotropy Analysis. A fluorescence anisotropy signal was measured on a FluoroMax-4 Spectrofluorometer (Horiba Jobin Yvon, Paris, France) using the Lformat configuration. Excitation was set at 365 nm, and emission 5883
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Figure 2. NHS-activated beads were conjugated with forward primer (F-Primer) by NHS-amine chemistry to generate F-Primer beads (A), which were incubated with FAM-labeled complementary DNA of F-Primer (FAM-FP-cDNA) to verify the conjugation (B). Flow cytometry and fluorescence microscopy (inset) analysis results of blank beads (C) and F-Primer beads (D) with FAM-FP-cDNA.
was collected with 442 nm band-pass filters in relation to the nature of AFB1. All experiments were carried out at room temperature. To determine the binding affinities of aptamers toward AFB1, 200 nM AFB1 was incubated with various concentrations of aptamer in Tris-HCl buffer solution at room temperature for 30 min in the dark prior to the measurement of fluorescence anisotropy. Three anisotropy measurements were taken each time using an integration time of 0.1s, and the resulting anisotropy values were averaged. For the specificity test, 200 nM AFB1 and 10 × analogues or controls for competition were mixed with 1.5 μM aptamer and incubated in a Tris-HCl buffer solution for 30 min at room temperature.
DNA sequencing, expensive and time-consuming DNA synthesis, and labor-intensive screening, paving a new way for simple, rapid, efficient, and cost-effective molecular evolution of high affinity ligands. Preparation and Quantification of Monoclonal Beads. To ensure that each bead displayed sufficient aptamer sequences to form bead/target complexes, we first performed the emulsion droplet PCR and quantified the products on beads. Microbeads were prepared by conjugating forward primers (F-Primer) to 34 μm diameter N-hydroxysuccinimide (NHS) activated agarose beads via NHS-amine chemistry to generate F-Primer beads (Figure 2A). To validate the conjugation, the F-Primer beads were then incubated with FAM-labeled DNA complementary to F-Primers (FAM-FP-cDNA; Figure 2B) and the fluorescence intensity of the resulting beads was characterized by flow cytometry and fluorescence microscopy. As shown in Figure 2C,D, the blank beads before F-Primer functionalization exhibited negligible fluorescence intensity after incubating with FAM-FP-cDNA, while the F-Primer beads showed strong fluorescence intensity, confirming the success of coupling. The amount of F-Primers on each bead was calculated to be around 305 amol/bead by eluting and quantifying the annealed FAMFP-cDNA. Afterward, with the concentration of 1 template/ droplet, the water-in-oil emulsion droplets were generated by agitating the mixture of water, oil, surfactant, beads, and PCR reagents. Then, 35 cycles of emulsion droplet PCR were performed. After bead recovery by washing, the antisense ssDNA was separated from the sense ssDNA on beads by denaturing in NaOH (0.2 M). Then, the PCR products on beads were quantified by the aforementioned method with FAM-labeled reverse primer (FAM-R-Primer) to be 70 amol/ bead. Nearly 4 × 107 aptamer candidates displaying on each bead surface after 35 PCR cycles can guarantee sufficient affinity to form bead/target complexes by the multivalent effect. MSD-SELEX for Aptamer Selection Against EpCAM. To demonstrate the feasibility of MSD-SELEX for simple and efficient aptamer enrichment and screening, a cancer biomarker EpCAM protein was chosen as the target for aptamer selection.31,32 Due to the difference in capacity between the bead library (107−1010) and a typical initial random DNA library (1014−1016), five cycles of conventional SELEX with EpCAM-beads as targets were performed to enrich the DNA
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RESULTS AND DISCUSSION Working Principle of MSD-SELEX. The working principle of MSD-SELEX is schematically shown in Figure 1. A statistically diluted random ssDNA library at the single copy level is encapsulated into individual emulsion droplets containing a single bead covalently coupled with the forward primers for emulsion PCR to generate monoclonal beads each displaying millions of unique DNA sequences.28−30 The resulting monoclonal bead library is then incubated with targets for enrichment and identification. The interaction of DNA with targets is therefore converted to the interaction of beads and targets, which can be directly visualized as bead/target complexes under the microscope for large target particles such as cells or molecules functionalized on microbeads. Because nonbinding or weak binding beads can be easily removed through a washing process to enrich the library, only beads with high binding affinity sequences are retained and easily isolated to yield aptamer candidates. Due to the one-bead−one-sequence nature of the monoclonal beads, the binding ability of DNA sequences in each individual high affinity bead clone is immediately confirmed via flow cytometry without the need for DNA sequencing and chemical synthesis. The bead clones with low Kd values can be sequenced and synthesized for further application. The processes of amplifying individual sequences from the DNA library onto the bead surfaces, visualizing the binding events of beads with targets, and individually isolating high affinity monoclonal beads are the keys to the proposed method. Compared to conventional SELEX methods, the MSDSELEX approach avoids many rounds of selection, large-scale 5884
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Figure 3. Interaction of fifth library beads (A) and F-Primer beads (B) with target Kato III cells. Flow cytometry analysis results of the obtained clones from candidate beads (C) and negative beads (D) interacting with EpCAM-beads. sClone indicates the negative clones (beads) from the supernatant.
library for matching with the bead library. Using smaller microbeads, the capacity of the bead library can be significantly improved and the preselection cycle could be further reduced, possibly to a single round. The enriched fifth ssDNA library was statistically diluted at 0.3 template/droplet and isolated into individual emulsion droplets containing one DNA molecule for each F-Primer bead. Droplet emulsion PCR was then performed to amplify individual DNA sequences on beads to generate millions of monoclonal beads with unique DNA sequences. Since only one DNA template was present in each droplet, the PCR bias (unequal amplification among different sequences),33 which normally exists in bulk solution PCR, can be efficiently avoided.34 Every aptamer candidate has the same chance to be amplified in the distinct emulsion droplets. After bead recovery and removal of the antisense ssDNA, the resulting beads with sense ssDNA were used as the bead library to incubate with the targets. Since the target EpCAM is a membrane protein of cancer cells, EpCAM positive cells, human gastric cancer cells line Kato III, were chosen to incubate with the bead library. After incubation and several washing steps, the beads that did not bound to the adherent Kato III cells were efficiently removed, while the beads forming complex with Kato III cells were retained in the culture dish as highlighted by the rectangle in Figure 3A. Since millions of unique aptamer candidates are present on each bead, the bead can bind strongly with EpCAM protein and remain in the culture dish. In contrast, the beads with only F-Primers yielded no binding to the target cells as the negative control (Figure 3B). Moreover, the binding strength could be easily tuned by adjusting the washing strength to retain relatively higher or lower binding sequences. Seven positive clonal beads were extracted using a capillary tube. Each of these positive clones was amplified with FAM-labeled FPrimers (FAM-F-Primer) by conventional PCR to generate FAM-labeled ssDNA for further confirmation.
To evaluate the binding ability of the DNA sequence from each clonal bead, we first used flow cytometry to test the binding of these sequences with the EpCAM-beads, which were prepared by attaching His-tagged recombinant EpCAM proteins to Ni-beads. The right-shift of the signal intensity from that of the unselected library to the fifth library showed an obvious enrichment (Figure 3C). By setting the fluorescence intensity of the fifth library as the criterion for binding affinity evaluation, all seven positive clones exhibited fluorescence shifts significantly larger than the fifth library against EpCAM-beads (Figure 3C), and the negative clones in the supernatant (sClones) that did not bind to Kato III cells showed smaller shifts than the fifth library (Figure 3D). The results demonstrated that the MSDSELEX method can efficiently eliminate the low affinity clones and enrich the high affinity sequences, thus eliminating the need for a tedious screening process. The Kd values of all seven positive clones were determined to be in the nanomolar range (Table S3). Clone 5 with the smallest Kd value (30 ± 8 nM) was subjected to cloning and sequencing to identify sequence information of aptamer EpCAM-5 (see Table S1). Characterization of Selected Aptamer. To confirm that EpCAM-5 was indeed a good aptamer, FAM-labeled EpCAM-5 was chemically synthesized, and a series of experiments was performed to investigate its binding affinity and specificity toward EpCAM protein and EpCAM positive cell lines. The synthesized EpCAM-5 bound selectively to EpCAM-beads and showed no affinity toward the control Ni-beads (Figure S2). The Kd value of EpCAM-5 toward EpCAM protein was found to be 33 ± 3 nM (Figure S2), similar to that of clone 5. Using conventional SELEX process, the fifth library was further subjected to seven additional rounds of enrichment leading to the identification of four aptamer sequences.32 We further compared the sequences obtained by these two methods and found that EpCAM-5 is highly similar to the sequence of SYL4 5885
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Figure 4. Binding of FAM-labeled EpCAM-5 or random sequence (RS) against different cell lines including EpCAM-negative cell lines HEK-293T (A) and Ramos (B), and EpCAM-positive cell lines T47D (C) and Kato III (D). (E) Confocal images of cultured Ramos and Kato III cells stained with FAM-labeled EpCAM-5 and random sequence. The final DNA concentration was 200 nM.
(Kd = 86 ± 17 nM).32 Because the SYL4 family accounts for about 23% of the 12th library sequenced,32 finding similar sequences in the earlier fifth round is not surprising. This comparison reveals the advantage of the new method to identify aptamer sequences with few enriching cycles. To test whether the aptamer was able to bind to native EpCAM protein on the surface of human cancer cells, we studied the interaction of the aptamer with EpCAM-positive cell lines using flow cytometry. Besides Kato III, ductal breast epithelial tumor-derived cell line T47D was chosen as the positive cell line, with human embryonic kidney 293T cell line HEK-293T and human Burkitt’s lymphoma cell line Ramos as negative controls. As shown in Figure 4A−D, the EpCAM-5 was able to bind to the different types of human EpCAM-positive cancer cell lines, but they did not bind to normal cell line (HEK-
293T) or EpCAM-negative cancer cell line (Ramos), indicating that the selected aptamer can specifically recognize the recombinant EpCAM protein as well as the native EpCAM protein on live cell membranes. To further illustrate the potential of EpCAM-5 for clinical applications, fluorescence imaging of cancer cells was performed. As shown in Figure 4E, the EpCAM-positive Kato III cells displayed high fluorescence signals after incubating with FAM-labeled EpCAM-5, while the EpCAM-negative cell line Ramos displayed a negligible fluorescence signal. In contrast, none of these cell lines displayed any significant fluorescence after incubating with the random sequence. These results further confirmed that EpCAM-5 can selectively differentiate EpCAMpositive cell lines from the negative ones. Since EpCAM has been detected in most adenocarcinomas and has also been 5886
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Figure 5. (A) The structure of AFB1. (B) Observation of complex formed by AFB1-beads and library displaying beads. (C) Flow cytometry results of the obtained clones with AFB1-beads. (D) The Kd measurement of aptamer AFB1-1 against the target toxin.
AFG1 and AFM1, since they have a very similar structure. But it did not recognize other toxins with obvious structural difference, such as sterigmstocystin, T-2 toxin, patulin, citrinin, and zearelenone. The results clearly verified the generality of our MSD-SELEX method for selecting aptamers with excellent binding affinity and specificity. For large targets, such as cells, the visualization of library beads with targets and separation of unbound beads with bound beads can be directly realized. For small targets, such as small molecules and proteins, the targets can be immobilized on certain microbeads for easy visualization and isolation.
found in metastases, malignant effusions, and cancer stem cells,35,36 the selected EpCAM binding aptamer has great potential for use in targeted cancer therapy, cancer imaging, and circulating tumor cell enrichment. Previously, the conventional SELEX method had been applied to isolate aptamers against EpCAM, which required 12 cycles of selection and a tedious and expensive cloning− sequencing−synthesis−screening workflow.32 Our agarose droplet microfluidic approach avoids large-scale DNA sequencing and expensive and time-consuming DNA synthesis of large populations of aptamer candidates, but it still takes 10 cycles for enrichment and labor-intensive effort for candidate screening.26 In contrast, with MSD-SELEX, only five cycles of optional preselection were performed while there was no need for largescale DNA sequencing, time-consuming DNA synthesis, and labor-intensive screening of large populations of aptamer candidates, signifying the advantages of the new method. Generality Demonstration by MSD-SELEX against AFB1. To further demonstrate the generality of our MSDSELEX method, we applied the method for rapid identification of aptamers against small molecule toxin aflatoxin B1 (AFB1, Figure 5A).37,38 In the selection, AFB1 was immobilized on magnetic beads (AFB1-beads, Figure S3) and incubated with the bead library displaying the pre-enriched fifth library. The beads with aptamer candidates can form the complex with AFB1beads, as highlighted by the circle in Figure 5B. The clones obtained from displaying beads all showed better binding affinity against AFB1-beads than that of the fifth library (Figure 5C), which were identified as AFB1 aptamers. The Kd values of three clones were determined (Table S3) and clone 1 with the smallest Kd as 0.42 ± 0.15 μM was subjected to cloning and sequencing for identification of aptamer sequence AFB1-1. Then the Kd value of aptamer AFB1-1 toward AFB1 was confirmed to be 0.65 ± 0.11 μM by fluorescence anisotropy analysis (Figure 5D). As the specificity test showed in Figure S4, the aptamer AFB1-1 exhibited strong recognition to AFB1 analogues, such as
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CONCLUSIONS In conclusion, we have developed a novel MSD-SELEX method for efficient enrichment and identification of aptamers. Taking advantage of single-molecule droplet PCR to generate monoclonal beads each displaying millions of identical DNA sequences, the interaction of aptamer candidates on beads with targets can be visualized while unbound beads can be easily removed by washing. As a result, only the beads with high affinity sequences are identified, which can be further confirmed without the need of its sequence information because of the monoclonal nature of beads. The method has been successfully demonstrated to rapidly isolate aptamers against cancer biomarker protein EpCAM and small toxin molecule AFB1 with only a few cycles of preselection and without the need for extensive DNA sequencing, synthesis, and screening of a large population of aptamer candidates. The simplicity and high selection efficiency of MSD-SELEX are achieved by several key factors. First, the large number of sequences on the surface of each bead ensure strong formation of the bead/target complex by the multivalent effect. Second, the tunable washing strength allows controllable selection to isolate DNA clones against targets with desired binding affinity. Third, the interaction of DNA with targets is converted to the interaction of beads and targets, which can be directly visualized as bead/target 5887
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Analytical Chemistry
Article
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complexes under the microscope. More importantly, the onebead-one-sequence nature of the monoclonal beads allows rapid isolation and characterization of the binding affinity of individual sequences without sequence information. Compared with the conventional enrichment−cloning−sequencing−synthesis− screening work flow, the MSD-SELEX method based on highly parallel single-molecule emulsion PCR is simple, rapid, highly efficient, and cost-effective for aptamer enrichment and identification, which will greatly accelerate the aptamer discovery process to meet the growing demands for bioanalytical, biotechnological, and biomedical applications.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental details and additional characterization data. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: (+86) 592-218-7601. Fax: (+86) 592-218-9959. E-mail:
[email protected]. Author Contributions †
These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the National Basic Research Program of China (2010CB732402, 2013CB933703), the National Science Foundation of China (91313302, 21205100, 21275122, 21075104), National Instrumentation Program (2011YQ03012412), Natural Science Foundation of Fujian Province, China (2013J01061), the Fundamental Research Funds for the Central Universities (2012121025), and the National Science Foundation for Distinguished Young Scholars of China (21325522) for their financial support.
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dx.doi.org/10.1021/ac501423g | Anal. Chem. 2014, 86, 5881−5888
