A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.
A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer.
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps.
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.
Cancer is still one of the leading causes of mortality worldwide1. Despite the significant improvement in cancer treatment in recent decades, anticancer drug development is still a highly debated topic. This is because chemotherapy, as the mainstay of cancer treatment, is accompanied by serious side effects that limit patient compliance with the treatment. Moreover, chemotherapy-induced cancer resistance to treatment has restricted its application as the sole choice of medical intervention. The application of monoclonal antibodies (mAbs) introduced an enhanced response to cancer treatments2. The rationale of using mAbs was to improve the efficacy of chemotherapeutics and minimize their adverse reactions. However, the administration of mAbs also became a challenge. This was not only because of the mAb-induced immunological reactions but also due to the animal-dependent and expensive production costs and difficult storage conditions3. Introduction of aptamers in the 1990s4 raised new hopes in cancer treatment, as the application of aptamers could address the challenges associated with mAbs.
Aptamers are short nucleic acid sequences that are specifically produced for a certain target. Systematic evolution of ligands by exponential enrichment (SELEX) is a common method in aptamer production. In SELEX, the protein of interest is incubated with a library of random nucleotide sequences, and through a series of iterative cycles, the aptamer specific for that protein is purified. Aptamers have similar target selectivity and specificity to mAbs, and therefore drug development in this field shows promising future applications. Aptamers specific for cancer biomarkers could be applied as single drugs and cancer diagnostic tools5,6,7. Due to their nano-sized structure, these aptamers could also act as drug carriers to deliver cytotoxic agents specifically to the tumor8. This would increase the efficacy of targeted drug delivery and decrease chemotherapy-associated, off-target adverse reactions. Moreover, these nanomedicines have a high tissue penetration, which makes them a desirable candidate for deep-tumor drug delivery and treatment. Aptamers can also be designed to target the transporters expressed on the blood-brain barrier (BBB) to improve drug delivery to brain tumors9. A good example of such an aptamer are bifunctional aptamers, targeting the transferrin receptor (TfR)10 to enhance drug delivery across the BBB, and delivering a cytotoxic drug payload to tumor cells11.
Despite all the advantages of aptamers, drug development in this field has not yet yielded a marketed, successful anticancer drug. One reason for this could be the lack of standard and reproducible methods that could be followed globally by researchers in the field. In this paper, a step-by-step protocol of an aptamer binding to a native protein expressed on the cell surface is demonstrated. This protocol is a prerequisite step in the preclinical assessment of anticancer aptamers. The assay is performed to show the selectivity and specificity of the purified aptamer collected from SELEX or a published aptamer sequence for confirmation of selectivity and specificity. This flow cytometric-based assay is a rapid, reliable, sensitive assay that accurately shows the selectivity and specificity of the aptamer, where the aptamer is being tested against proteins on the cell surface12,13,14. This method is demonstrated using the binding of an aptamer specific for EpCAM shown in this paper15. EpCAM, as a transmembrane glycoprotein, plays roles in tumor cell signaling, progression, migration, and metastasis16,17. To show the selectivity and specificity of this aptamer, EpCAM-positive and -negative cancer cells were used. The previously developed EpCAM specific aptamer, TEPP (5′-GC GCG GTAC CGC GC TA ACG GA GGTTGCG TCC GT-3′), and a negative control aptamer, TENN (5′-GC GCG TGCA CGC GC TA ACG GA TTCCTTT TCC GT-3), were used as EpCAM-binding and -non-binding aptamers, respectively10. The 3' end of both TEPP and TENN were labeled with a TYE665 fluorophore.
TEPP is a bifunctional aptamer that targets EpCAM from one end and TfR on the other. This has made TEPP a suitable candidate for drug delivery to EpCAM+ brain tumors. Using its TfR-specific end, TEPP traverses the blood-brain barrier, and using the EpCAM-specific end, finds the tumor and delivers its cargo (e.g., cytotoxic drugs) to the tumor. TENN has a similar length and 2D structure as TEPP, but it does not have affinity for the EpCAM or TfR, and hence is a suitable negative control aptamer. Using TEPP and TENN, testing the binding of an aptamer to the target protein using flow cytometry is shown in this paper. This protocol applies to the development of cell-specific aptamers. It is also applicable to further complementary and confirmation analyses of the aptamer sequences available in the literature. The protocol can also be used by those new to the aptamer field who are looking at using a previously published aptamer for their research and development (R&D) purposes. In this paper, two aptamer sequences available in the literature are studied.
NOTE: Prior to starting the experiment, wear personal protective equipment, including a lab coat, gloves, and goggles. See the Table of Materials for details about materials, reagents, equipment, and software used in this protocol.
1. Buffers required for the assay
Ingredients | Volume required | ||
Item | Concentration | ||
SELEX buffer | MgCl2 | 5 mM | 50 µL per sample + 10% pipetting error |
Blocking Buffer | MgCl2 | 5 mM | 500 µL per cell line |
BSA a | 1 mg/mL | ||
tRNA b | 0.1 mg/mL | ||
FBS c | 10% (v/v) | ||
Wash Buffer | MgCl2 | 5 mM | 1 mL for the first wash + 100 µL per test sample + 10% pipetting error |
Binding Buffer | MgCl2 | 5 mM | 50 µL per sample + 10% pipetting error |
BSA | 2 mg/mL | ||
tRNA | 0.2 mg/mL | ||
FBS | 20% (v/v) |
Table 1: Buffers required for the binding assay. aBovine Serum Albumin, bTransfer Ribonucleic Acid, cFetal Bovine Serum.
2. Preparation of aptamers
NOTE: The aptamers used in the assay are tagged with a fluorescence reporter molecule, and therefore care should be taken to protect them from light.
Figure 1: A diagram showing the steps in the preparation of aptamers. 1Stock 1 is stored at -20 °C for long-term preservation. 2Working concentrations are prepared in SELEX buffer and are not stored. Please click here to view a larger version of this figure.
3. Maintenance of cancer cells
NOTE: Prior to commencement of the study, make sure that the cells are at their early passage numbers, show their typical morphological features, and are mycoplasma free. To test the selectivity and specificity of the aptamer, cell lines that are high, moderate, and low/negative expressors of the protein of interest are ideally required.
4. Binding assay
NOTE: Figure 2 summarizes the steps required in the binding assay in adherent cells.
Figure 2: A diagram depicting the steps in performing an aptamer-protein-binding assay. Abbreviations: SELEX = Systematic Evolution of Ligands by EXponential Enrichment; BB = Blocking Buffer; WB = Wash Buffer; BiB = Binding Buffer. Please click here to view a larger version of this figure.
Figure 3: A diagram showing the different types of cells and aptamers required to perform the aptamer binding assay. Abbreviation: EpCAM = epithelial cellular adhesion molecule. This figure was created using Biorender.com. Please click here to view a larger version of this figure.
5. Flow cytometry and data analysis
NOTE: Before turning on the flow cytometer, make sure that there are no "bubbles" in the membrane filter units for the shut-down solution, cleaning solution, and sheath fluid (0.9% NaCl). "Bleed out" bubbles if there are bubbles in the capsules. Make sure that the waste container is empty, and containers of sheath fluid, water, and 1% bleach in ultrapure water are full.
An important aspect of new drug discovery and development is assuring the selectivity and specificity of the drug candidate. This means that the drug candidate should be able to discriminate between different cells and only affect the cell population of interest (selectivity). Selectivity is studied using cell lines that differ in terms of expression of the protein of interest. In this study, MDA-MB-231 and HEK 293T cell lines were chosen as EpCAM-positive and -negative cells. Specificity is another determinant that shows that the protein of interest only responds to a single drug candidate. Here, by using an EpCAM non-binding aptamer, TENN, it was shown that only TEPP attached to EpCAM. In EpCAM-positive cells (MDA-MB-231), overlaying the histograms representing cells treated with TEPP and TENN shows that the TEPP-treated cells are shifted to the right compared to the TENN-treated cells. This shows the binding of the aptamer, TEPP, to the protein of interest, EpCAM (Figure 4D). In negative control HEK 293T cells, overlaying histograms of TEPP and TENN does not reflect any shift (Figure 4E). This means that in EpCAM-expressing cells, TEPP as the EpCAM aptamer attached to its receptor, and furthermore, no binding was observed in EpCAM-negative cells. These results confirm the selectivity and specificity of the developed aptamer.
Figure 4: Gating and histograms showing the binding of the cells to the aptamer. (A) Selecting the population of cells, (B) the single cells, and (C) the histogram of the cells attached to the aptamer (200 nM). The binding of EpCAM aptamer (TEPP) versus a non-EpCAM-binding aptamer (TENN) in (D) EpCAM+ MDA-MB-231 cells and (E) EpCAM– HEK 293T cells is compared. Abbreviations: EpCAM = epithelial cellular adhesion molecule; FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; FSC-H = forward scatter-peak height; APC = allophycocyanin. Please click here to view a larger version of this figure.
The key challenge with developing new aptamers is the lack of standard guidelines that applies to different steps of this process. McKeague et al. have recently demonstrated some of the associated challenges, which lead to unclear presentations of data in publications and failure to replicate the research. They proposed fundamental guidelines necessary for consideration in characterizing aptamers19. An aptamer binding assay is a critical step in screening and/or characterizing aptamers23, which is widely used by researchers in the field. Since no single guideline exists to display the step-by-step protocol, a flow cytometry method, which is commonly used to study the aptamer-protein binding, is demonstrated in the accompanying video protocol.
There are several methods that measure the interaction of the aptamer and its target. Flow cytometry is one of these methods. Other examples include fluorescence polarization, surface plasmon resonance, capillary electrophoresis, and isothermal titration calorimetry24. Choosing the correct method depends on the application of the aptamer. However, it is important to know that each method has its limitations, and that the application of several assays is more beneficial for the characterization of small molecule aptamers24. The method described here has several advantages. It is one of the most reliable, precise, and accurate methods, and is also rapid and cost-effective. The flow cytometric analysis of aptamer binding can be applied in various steps of aptamer development, including candidate screening, truncation and optimization, characterization, and validation.
With fluorescence labeling, there are choices to either label or use the intrinsic fluorescence of the target or label the aptamer24. In the authors' experience, using labeled aptamers is reliable and easy to set up. Aptamers can be labeled with a fluorophore at any end (3' or 5')25; however, the end with less guanine is more favorable, as guanine can quench the fluorescence and its detection26. A disadvantage of this method is that the aptamer should be tagged to a fluorophore. Furthermore, although this method reflects the binding of the aptamer to the specific protein, it does not display the location of the interaction site. Hence, further studies, such as fluorescence microscopy, might be required to confirm the aptamer-protein interaction location25.
There are some critical notes to be considered while performing this assay. It is important to consider that each aptamer has specific requirements for handling and folding. This includes the choice of reconstitution and dilution buffers and the folding conditions. Reconstitution of lyophilized aptamers occurs in purified sterile pyrogen- and RNase-free ultrapure water. This is to minimize the concentration of ions around the nucleotides. Ion concentration highly affects the formation of 2D structures of aptamers and their affinity and stability. Hence, for further reconstitution of the aptamer stock, care should be taken in the correct preparation of other buffers and metal cationic solutions, such as MgCl225,27. The optimum concentration of metal cationic solution shields the negative charge of the aptamer yet, does not inhibit the interaction of the aptamer with its target.
Furthermore, because the aptamer has a potential for a non-specific, off-target binding, the application of blocking buffer plays a critical role in the binding assay. In addition to the negatively charged BSA, which is commonly used for antibodies, salmon sperm DNA or tRNA is also required here. This mixture blocks the positively charged proteins and nucleic acid binding sites. This blocking stage is specifically important for the selectivity and specificity of aptamers towards cancer cells, due to their negative charge compared to neutral and positively charged normal cells28. Furthermore, the 3D folding of aptamers is dependent on other factors such as temperature. The importance of conditions affecting the 3D formation of the aptamer, including the choice of tube and the duration of PCR phases, has been reviewed25. Moreover, the incubation time of the aptamer with the target should be optimized for every specific aptamer. The incubation temperature is also highly important. Maintenance of a temperature of 4 °C is especially critical for cell surface proteins that can easily be internalized, such as EpCAM25.
The other important factor in this assay is the application of proper controls. Cancer cells that either express or do not express the target protein should be used to evaluate the selectivity of the aptamer. In each experiment, in addition to the aptamer of interest, a negative control aptamer (a random sequence or a scrambled sequence) is required to show the specificity of the aptamer. Ideally, this control should have a similar length of nucleotides and undergo similar folding as the aptamer. In this experiment, a negative control (TENN), an aptamer with a similar structure to TEPP shown to have a low binding affinity with EpCAM, was used10.
The protocol presented here is a qualitative assay and can be further used for the quantitative assessment of binding affinity and the determination of the dissociation constant (Kd)29,30,31. However, it is important to show the reproducibility of the results using technical and biological replicates. To achieve this, it is highly important to consider major determinants to properly perform this assay. This could include, but is not limited to, using a low passage number of mycoplasma-free cells that are properly grown in their optimal conditions, using a constant number of cells to be exposed with the aptamer in each replicate, the application of proper temperature and folding conditions, maintaining similar experimental conditions for both the aptamer and the control, maintaining minimal exposure of the aptamers to environmental light, using optimized aptamer-cell exposure time and temperature, maintaining a temperature of 4 °C for the cells, which includes precooling the centrifuge, the 96-well plate, and the flow cytometry tubes, and accurately preparing the buffers, with an ionic concentration as close to the claimed concentrations as possible.
The authors have nothing to disclose.
The authors acknowledge the Institute for Mental and Physical Health and Clinical Translation (IMPACT) SEED funding, the "Alfred Deakin Postdoctoral Research Fellowship" program at Deakin University, and the "Australian Government Research Training Program Scholarship".
1.5 mL microcentrifuge tubes with attached lid | Sigma-Aldrich | T6649 | |
15 mL CellStar blue screw cap, conical bottom tube | Greiner Bio One | 188271 | |
5 mL serological pipettes | Greiner Bio One | 606180 | |
BD FACSCanto II Flow Becton Dickinson Cytometer | Becton Dickinson | N/A | |
BD FACSDiva V9.0 | BD Biosciences | N/A | |
Bovine Serum Albumin (BSA), Lyophilized powder | Sigma-AldrichTM | A7906-50G | |
Bright-line Hemocytometer | Sigma-Aldrich | Z359629 | |
Dulbecco’s Modified Eagle Medium (DMEM) High Glucose Media Powder | Life Technologies | 12100046 | |
Dulbecco’s Phosphate- Buffered Saline (DPBS) | Life Technologies | 21300025 | |
FlowJo, LLC 10.8.1 | BD Biosciences | N/A | |
Foetal Bovine Serum (FBS) | Bovogen | SFBS-F | |
HEK293T | American Type Culture Collection | ACS-4500 | |
Heracell 150i CO2 Incubator | Thermo Fisher Scientific | N/A | |
Heraeus Megafuge 16R Centrifuge | Thermo Fisher Scientific | N/A | |
Magnesium Chloride (MgCl2) | Sigma-Aldrich | M8266 | |
MDA-MB-231 | American Type Culture Collection | CRM-HTB-26 | |
Microplate, PS, 96 well, F-bottom (Chimney well), Black | Greiner Bio One | 655076 | |
MiniAmp Thermal Cycler | Thermo Fisher Scientific | A37834 | |
Phosphate-Buffered Saline (PBS) tablets | Life Technologies | 18912014 | |
Pyrogen- and RNase-free ultrapure water | Milli-Q | ||
T75 Cell Culture flask | Cellstar | 658170 | |
TENN | Integrated DNA Technologies | N/A | 5′-GC GCG TGCA CGC GC TA ACG GA TTCCTTT TCC GT-3 |
TEPP | Integrated DNA Technologies | N/A | 5′-GC GCG GTAC CGC GC TA ACG GA GGTTGCG TCC GT-3′ |
Transfer RNA (tRNA) | Sigma-Aldrich | R8508-5X1ML | |
Trypan Blue Solution | Life Technologies | 15250061 | |
Trypsin-EDTA | Gibco | 15400054 |