KR101641920B1 - Nucleic acid aptamer specifically binding to EpCAM and their uses - Google Patents

Abstract

The present invention relates to a DNA aptamer exhibiting a high affinity specifically to EpCAM (epithelial cell adhesion molecule (EpCAM)) from a randomly synthesized ssDNA library using SELEX (Systematic Evolution of Ligands by Expectant Enrichment) The DNA aptamer that can specifically bind to EpCAM can be used to measure EpCAM more sensitively and accurately than antibody-based assays used in the past, and the DNA aptamer can be used as a marker of cancer stem cells or embryonic stem cells Stem cell or cancer research and diagnosis.

Description

[0001] Nucleic acid aptamers specifically binding to EpCAM and their uses [0002] Nucleic acid aptamers specifically bind to EpCAM and their uses [

The present invention relates to a nucleic acid aptamer which specifically binds to an epithelial cell adhesion molecule (EpCAM), and to a method for detecting EpCAM, stem cell or cancer stem cell using the same.

Aptamers are single-stranded nucleic acids (DNA, RNA, or modified nucleic acids) that can bind with high affinity and specificity to target molecules such as cells, proteins, small molecules, and chemicals, (Ellington and Szostak, 1990; Tuerk and Gold, 1990; Breaker, 2004). The aptamer is similar to the antibody in that it recognizes the target molecule (Hermann and Patel, 2000) that binds to the target molecule with a combination of structural suitability, electrostatic and van der Waals binding, and hydrogen bonding. However, aptamers have fewer molecular weight than antibodies and are capable of rapid and reproducible synthesis, are easily transformable, are stable over the long term, have low toxicity and low immunity, can penetrate rapidly into tissues, (Jayasena, 1999; Lee et al., 2006; Tan et al., 2011).

The epithelial cell adhesion molecule (EpCAM), a monocatalytic glycoprotein, is a carcinoma-associated antigen, which is expressed in several precursor cell populations and cancers (Dalerba et al., 2007; Dan et al. , 2006; 53 Maetzel et al., 2009; Stingl et al., 2005). EpCAM is a homologous, calcium-ion-independent cell adhesion molecule that is used as a target for treatment of human tumor immunotherapy. In addition, the maintenance of self-renewal of embryonic stem cells depends on the high level of expression of EpCAM and plays an important role in the proliferation and differentiation of human stem cells (Patriarca et al., 2012). Thus, EpCAM can be used as an important marker of tumor-initiated cells (cancer stem cells) or embryonic stem cells (Gonzalez et al., 2009; Lu et al., 2010; Ng et al., 2010; Sundberg et al., 2009 ).

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was first developed by the University of Colorado research team Larry Gold in 1990 as an aptamer digging technique. With SELEX, many aptamers that can bind to a variety of target molecules, such as metal ions, organic dyes and amino acids, antibiotics and peptides, as well as proteins of various sizes and functions, whole cell viruses and virus-infected cells, bacteria, Has come. In addition, the interaction between aptamer and various targets has been applied in various fields such as biotechnology, medicine, pharmacy, cell biology, microbiology and chemistry. For example, aptamers have been effectively applied as a means for treatment, detection and diagnosis of targets.

The Cell SELEX method used in this experiment is a cell-to-aptamer method, which allows direct binding to proteins present on the cell surface without purification of a high-purity protein, which is an advantageous method for locating target protein-specific aptamers. It is simple, fast, and reproducible by using intact cells to select effective aptamers that specifically bind to target cells. Using a protein purified as a target molecule is likely to obtain an aptamer for an accurate target molecule, but cell surface proteins often tend not to be readily purified. Also, although recombinant production and purification of the extracellular domain is possible, there is a concern that it will be able to maintain its shape in living cells, and aptamers selected from purified protein or protein domains do not recognize the type of protein present in living cells It also raises concerns about the failure. In order to reduce this risk, Cell-SELEX is a method of extracting an aptamer that can bind directly to a target protein having a structure in its original form in a cell. Thus, a recombinant protein produced and isolated from a conventional E. coli, It is possible to find an aptamer that is more likely to be used as an actual therapeutic agent.

Identification of selectively expressed cell surface markers is important in the purification and characterization of pluripotent stem cells, including induced pluripotent stem cells (iPSCs), which play an important role in stem cell research.

The present inventors have made efforts to find an aptamer specifically binding to EpCAM (EpCAM) expressed in most cancers. As a result, they have found DNA apps showing high affinity to EpCAM from chemically synthesized ssDNA library Tumor was selected using FACS cell-SELEX technology and the sequence and structure of the aptamer were established. The aptamer may be used as a stem cell marker and may be applied to stem cell and cancer cell research, cancer diagnosis / treatment.

It is an object of the present invention to provide a nucleic acid aptamer specifically binding to an epithelial cell adhesion molecule (EpCAM), and a method for detecting EpCAM, stem cell or cancer stem cell using the same.

In order to accomplish the above object, the present invention provides a nucleic acid aptamer which specifically binds to an epithelial cell adhesion molecule (EpCAM).

The present invention also provides an epithelial cell adhesion molecule (EpCAM) detection kit comprising the nucleic acid aptamer of the present invention.

In addition,

1) contacting the nucleic acid aptamer of the present invention with a biological sample; And

2) In step 1), a method for detecting a stem cell comprising the step of confirming whether a nucleic acid aptamer of the present invention binds to a biological sample is provided.

The present invention also provides a stem cell detection kit comprising the nucleic acid aptamer of the present invention.

In addition,

1) contacting the nucleic acid aptamer of the present invention with a biological sample; And

2) In step 1), a method for confirming expression of epithelial cell adhesion molecule (EpCAM) to provide information on cancer diagnosis, which comprises confirming whether or not the nucleic acid aptamer of the present invention binds to a biological sample.

In addition, the present invention provides a cancer diagnostic kit comprising the nucleic acid aptamer of the present invention.

The present invention relates to a DNA aptamer exhibiting a high affinity specifically to EpCAM (epithelial cell adhesion molecule (EpCAM)) from a randomly synthesized ssDNA library using SELEX (Systematic Evolution of Ligands by Expectant Enrichment) The DNA aptamer that can specifically bind to EpCAM can be used to measure EpCAM more sensitively and accurately than antibody-based assays used in the past, and the DNA aptamer can be used as a marker of cancer stem cells or embryonic stem cells Stem cell or cancer research and diagnosis.

FIG. 1A shows the expression levels of EpCAM in HEK 293, HepG2, Hela cells.
FIG. 1B shows the results of overexpression of EpCAM in HepG2 cells using Western blot and FACS (Fluorescence-Activated Cell Sorter) analysis.
FIG. 2A shows the degree of binding in HepG2 cells overexpressing EpCAM in SELEX 4, 6, 7, and 8 DNA pools.
FIG. 2B shows the degree of binding in HepG2 cells of SELEX 4, 6, 7 and 8 DNA pools.
FIG. 3 is a graph showing the binding affinity of 5 selected aptamers to HepG2 cells overexpressing EpCAM through FACS.
4A is a graph showing the binding affinity of EP166 aptamer in EpCAM-overexpressing HepG2 cells.
4B is a graph showing the binding affinity of EP166 aptamer in HepG2 cells.
4c is a view showing confocal microscopy of the binding specificity of EP166 in EpCAM overexpressed HepG2 cells.
Figure 4d compares the binding affinities of EP166 and SYL3C in EpCAM overexpressed HepG2 cells.
FIG. 5A is a diagram showing an expected secondary structure of EP166 obtained through the mfold program. FIG.
FIG. 5B is a graph showing the binding affinity of EP166 in EpCAM-overexpressing HepG2 cells using FACS according to the concentration of aptamer.
6A is a diagram showing the binding affinity of EP166 confirmed in J1ES cells.
FIG. 6B shows the binding affinity of EP166 identified in differentiated J1ES cells. FIG.
FIG. 7 is a diagram illustrating a FACS cell-SELEX process.

Hereinafter, the present invention will be described in detail.

The present invention provides a nucleic acid aptamer that specifically binds to an epithelial cell adhesion molecule (EpCAM) and comprises SEQ ID NO: 6.

The EpCAM is preferably expressed in cancer stem cells or embryonic stem cells.

The nucleic acid aptamer means a nucleic acid molecule capable of binding a specific molecule with high affinity and specificity, and the nucleic acid means DNA, RNA or a nucleic acid variant, and can be prepared by a method of Cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) But it is not limited thereto.

Preferably, the SELEX uses a single strand oligonucleotide pool or library composed of random sequences, and the oligonucleotide may be DNA or RNA or a DNA / RNA hybrid, which is not modified or modified, but is preferably DNA.

The oligonucleotide library may be a fully randomized or partially randomized oligonucleotide and preferably the oligonucleotide pool has at least one fixed sequence that can be composed of a sequence shared by all molecules of the oligonucleotide pool, RTI ID = 0.0 > and / or < / RTI > conserved sequences. The oligonucleotide pool preferably contains a random sequence region as well as a fixed sequence that is essential for effective replication. The oligonucleotide of the initial pool contains a fixed N-terminal and C-terminal sequence within which 35 to 50 random nucleotides are inserted. Random nucleotides may be produced by chemical synthesis and selection from randomly truncated intracellular nucleic acids, but are not limited thereto.

The fixed sequence of the oligonucleotide preferably comprises a primer hybridization site consisting of 19 nucleotides described in SEQ ID NOS: 1 and 2 at the N-terminus and C-terminus, respectively.

A more detailed SELEX method is preferably, but not limited to, performed in the following steps:

1) contacting the oligonucleotide library with the target under appropriate conditions;

2) separating an oligonucleotide specifically bound to a target molecule and an oligonucleotide not binding;

3) separating only the oligonucleotide from the oligonucleotide bound to the target molecule separated in the step 2);

4) amplifying the oligonucleotide separated in step 3); And

5) repeating steps 1) to 4) above to obtain oligonucleotides having high specificity and affinity for the target molecule.

In the step 5), it is preferably 5 to 10 repetitions, more preferably 7 repetitions, but is not limited thereto.

The nucleic acid aptamer of the present invention may comprise a chemically modified form. In general, wild-type RNA or DNA molecules that do not have chemical modifications are vulnerable to degradation by nucleic acid degrading enzymes. Thus, the nucleic acid aptamers of the present invention can introduce any form of chemical modification that confer various nuclease resistance known in the art. Preferably, the chemical modification includes 2'-aminopyrimidine, 2'-fluoropyrimidine, 2'-O-methyl ribose purine, and pyrimidine, and the phosphodiester linkage of the backbone is phosphorothioate bond . In the case of 2'-fluoro or 2'-O-methyl modification, mutant RNA polymerases capable of performing in vitro transcription using these modified nucleotide triphosphates are present, and therefore chemical transformations are introduced from the SELEX process . On the other hand, the 3'-end of the two nucleotides is 3'-3 'linked to increase the resistance to the nucleic acid degrading enzyme by capping the 3'-end. By attaching PEG (polyethyleneglycol) the renal filtration rate can be reduced.

The length of the nucleotide of the nucleic acid aptamer of the present invention is preferably 20 to 100, more preferably 40 to 80, and even more preferably 40 to 50 nucleotides.

The present invention also provides an EpCAM detection kit comprising the nucleic acid aptamer comprising SEQ ID NO: 6 and binding to an epithelial cell adhesion molecule (EpCAM).

The EpCAM is preferably expressed in cancer stem cells or embryonic stem cells.

The kit may additionally include essential elements necessary for detecting EpCAM. For example, a labeled secondary antibody, chromophores, an enzyme and its substrate or other material capable of binding to an antibody, and the like, capable of detecting an aptamer bound to the kit.

The kit preferably further comprises any one selected from the group consisting of a streptavidin-like phosphatease conjugate, a chemiluminescent substance, and a chemiluminescent substance, Not limited.

In a specific example of the present invention, in order to maximize the effect of EpCAM overexpression, the present inventors compared EpCAM expression levels in HEK 293, HepG2 and Hela cell lines in order to select a target cell line having low expression level of EpCAM, , And the expression level of EpCAM was lowest in HepG2 cells (see FIG. 1A).

In addition, a retroviral expression system (Retroviral expression system) was used to overexpress EpCAM in HepG2 cells in order to proceed with Cell-SELEX, a method of locating target protein-specific aptamers in a cell-to-aptamer manner. As a result, when EpCAM-HepG2 cells (EpCAM-HepG2) and HepG2 cells not overexpressed were over-expressed, EpCAM-HepG2 cells showed higher fluorescence by binding to anti-EpCAM antibody, Lt; / RTI > (FIG. 1B).

In addition, the present inventors used a single-stranded DNA (ssDNA) library labeled with fluorescein isothiocyanate (FITC) to construct an aptamer library by using Integrated DNA Technologies, Inc. (Coralville , synthesis was the library through the IA, USA) contains the primer hybridization site, consisting of 19 nucleotides at each side is described as a nucleotide (N ₄₀₎ and SEQ ID NO: 1 and 2, consisting of 40 randomly (see Table 1 ).

In order to select an aptamer specifically binding to EpCAM, the present inventors used ssDNA (ssDNA library) having 40 DNA sequences randomly selected as an initial pool as a cell SELEX method (FACE-SELEX) using flow cytometry, Lt; / RTI > As a result, it was confirmed that the proportion of aptamers specifically binding to EpCAM-HepG2 cells was increased by increasing the fluorescence intensity of EpCAM-HepG2 cells as the number of SELEXs progressed. And the binding efficiency between the cell and the ssDNA pool was decreased at the eighth time (see FIG. 2). In addition, about 200 aptamers obtained from the seventh aptamer pool were cloned and sequenced.

In addition, 200 aptamers subjected to the sequence analysis were predicted by using the mfold program (http://mfold.rit.albany.edu), and 33 aptamers having different structures were selected . Their binding affinity to EpCAM-HepG2 cells was confirmed by FACS and the dissociation constants (K _d ) were determined. As a result, EP166 (5'-AAC AGA GGG ACA AAC GGG GGA AGA TTT GAC GTC GAC GAC A-3 ') of SEQ ID NO: 6 among the above selected aptamers showed the highest binding affinity to EpCAM 3). EP166 bound in EpCAM-HepG2 cells but not in negative control HepG2 cells (see Figures 4a and 4b). Also, SYL3C, which is a known apcam-specific aptamer, and EP166 of the present invention were compared with each other. As a result, EpCAM-HepG2 cells showed higher affinity than SYL3C (Fig. The EP166 aptamer was found to have a dissociation constant of 5.48 + 0.84 mM (Fig. 5A). The EP166 aptamer had a dissociation constant of 5.48 + 0.84 mM (See FIG. 5B).

Accordingly, the present invention selected a DNA aptamer exhibiting high affinity to EpCAM from a chemically synthesized ssDNA library using FACS cell-SELEX technology and established the sequence and structure of the DNA aptamer, Or as a marker for embryonic stem cells, and can be applied to stem cell or cancer research and diagnosis.

In addition,

1) contacting the nucleic acid aptamer comprising SEQ ID NO: 6 and binding to an epithelial cell adhesion molecule (EpCAM) to a biological sample; And

2) confirming whether or not the nucleic acid aptamer binds to the biological sample in the step 1).

The present invention also provides a stem cell detection kit comprising the nucleic acid aptamer comprising SEQ ID NO: 6 and binding to an epithelial cell adhesion molecule (EpCAM).

The EpCAM is preferably expressed in cancer stem cells or embryonic stem cells.

The nucleotide length of the nucleic acid aptamer is preferably 20 to 100, more preferably 40 to 80, and even more preferably 40 to 50 nucleotides.

The stem cells are preferably cancer stem cells or embryonic stem cells.

The term " biological sample " in the present invention refers to all samples obtained from human or mammals such as tissues, cells, blood, serum, plasma, lymph, bone marrow, saliva, milk, urine, feces, , Spinal fluid, joint fluid, thymus fluid, ascites, amniotic fluid and cell tissue fluids.

The binding confirmation can be performed by any method known in the art as long as the binding of the aptamer to the biological sample can be confirmed. According to the embodiment of the present invention, a fluorescence-activated cell sorter (FACS) microscopic.

The stem cell detection is preferably performed by confirming the expression of epithelial cell adhesion molecule (EpCAM).

In a specific embodiment of the present invention, we used a mouse embryonic stem cell line, J1ES, to determine if the selected aptamer EP166 could be used as an embryonic stem cell marker and found that the aptamer EP166 of the invention specifically binds Respectively. As a result, it was confirmed that EP166 bound to undifferentiated J1ES cells but not to J1ES cells differentiated into embryoid bodies (see FIG. 6). As a result, it was confirmed that EP166, which is an aptamer for EpCMA, could be used as a specific marker for embryonic stem cells.

Accordingly, the present invention selected a DNA aptamer exhibiting high affinity to EpCAM from a chemically synthesized ssDNA library using FACS cell-SELEX technology and established the sequence and structure of the DNA aptamer, Or as a marker for embryonic stem cells, and can be applied to stem cell or cancer research and diagnosis.

In addition,

1) contacting the nucleic acid aptamer comprising SEQ ID NO: 6 and binding to an epithelial cell adhesion molecule (EpCAM) to a biological sample; And

2) a step of confirming whether or not the nucleic acid aptamer is bound to the biological sample in the step 1), and a method for confirming whether the epithelial cell adhesion molecule (EpCAM) is expressed to provide cancer diagnosis information.

In addition, the present invention provides a cancer diagnostic kit comprising the nucleic acid aptamer comprising SEQ ID NO: 6 and binding to an epithelial cell adhesion molecule (EpCAM).

The EpCAM is preferably expressed in cancer stem cells or embryonic stem cells.

The nucleotide length of the nucleic acid aptamer is preferably 20 to 100, more preferably 40 to 80, and even more preferably 40 to 50 nucleotides.

The cancer is preferably selected from the group consisting of liver cancer, prostate cancer, breast cancer, colon cancer, lung cancer, gallbladder cancer, pancreatic cancer and stomach cancer, but is not limited thereto.

Accordingly, the present invention selected a DNA aptamer exhibiting high affinity to EpCAM from a chemically synthesized ssDNA library using FACS cell-SELEX technology and established the sequence and structure of the DNA aptamer, Or as a marker for embryonic stem cells, and can be applied to stem cell or cancer research and diagnosis.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> EpCAM ( epithelial cell adhesion molecule ) Production of overexpressed cells

<1-1> Selection of target cells

In order to maximize the effect of EpCAM overexpression, a target cell line with low expression level of EpCAM was selected. In order to compare the expression level of EpCAM in HEK 293, HepG2 and Hela cell lines, anti-EpCAM antibody Were subjected to Western blotting experiments.

Specifically, the cell lines were purchased from American Type Cuticle Collection (ATCC). Proteins were extracted from each cell line and quantitated. The same amounts of proteins were separated by molecular weight using SDS-PAGE, and the amount of EpCAM expressed in each cell line was measured using Western blot using EpCAM antibody And finally confirmed.

As a result, it was confirmed that the expression amount of EpCAM was the lowest in HepG2 cells as shown in Fig. 1 (Fig. 1A). Thus, HepG2 cells were designated as target cells to overexpress EpCAM.

<1-2> Using Retroviral Expression System EpCAM Overexpression of

Since it is important that the target protein, EpCAM, is overexpressed on the surface of the cell in order to proceed with Cell-SELEX, which is a method of discovering a target protein-specific aptamer by a cell-to-aptamer method that binds to a protein existing on the cell surface, A retroviral expression system (Retroviral expression system) was used to overexpress EpCAM in HepG2 cells.

Specifically, the gene encoding EpCAM was inserted into the pRetroX-IRED-Ds red vector (Clontech) multicloning site, and the FLAG tag was inserted at the C-terminus of the EpCAM gene. For the preparation of EpCAM over-stabilized cell line, GP2-293 cells were dispensed in a 100 mM culture dish at a ratio of about 70%, and DMEM (Gibco) medium without antibiotics and containing 10% FBS was used for efficient gene transfer . (Gibco) at a ratio of 60 μl: 12 μg: 12 μg / ml in a volume of 12 μg / ml, followed by pipetting of the cells for 24 hours with Lipofectamine 2000 ^™ (Invitrogen), pIRES / DsRed / EpCAM vector and pVSV- After 5 hours, the culture medium was changed to DMEM containing 10% FBS. After 48 hours, the virus-containing viral soup was transferred to a 15 ml tube and centrifuged at 3,000 rpm for 15 minutes. The viral soup was separated from the supernatant by filtration through a 0.45 ㎛ filter and stored at -80 ° C Respectively. In order to infect cells with retrovirus, HepG2 cells were dispensed at a ratio of 50% in a 100 mm culture dish. After 24 hours, the viral soup and the culture solution were mixed at a ratio of 1: 1 and polybrene was added at a concentration of 8 ㎍ / ml . After 12 hours of infection, the cells were replaced with culture medium containing 10% FBS. After 48 hours, only infected cells were separated using FACSA (FACSAria cell sorter, BD Bioscience). Cells overexpressing EpCAM were analyzed by Western blot using anti-EpCAM antibody.

As a result, as shown in Fig. 1, when HepC2 cells (EpCAM-HepG2) overexpressing EpCAM were compared with HepG2 cells not overexpressed, EpCAM-HepG2 cells were more likely to bind to anti-EpCAM antibody and showed high fluorescence As a result, it was confirmed that EpCAM was over-expressed on the surface of cells (Fig. 1).

< Example  2> Aptamer ( aptamer Creating the Library

A single-stranded DNA (ssDNA) library labeled with fluorescein isothiocyanate (FITC) was prepared from Integrated DNA Technologies, Inc. (Coralville, IA, USA) . The library contains a nucleotide (N _40), and a primer hybridization site, consisting of 19 nucleotides which are respectively listed in SEQ ID NO: 1 and 2 to the N-terminal and C-terminal of the oligonucleotide consisting of 40 randomly. The 5'-FITC-labeled 5'-FITC primer (SEQ ID NO: 3) and the 3'-primer (SEQ ID NO: 5) were used for PCR and the other 5'-primer Were used to clone selected DNA pools (Table 1).

Design of oligonucleotides for the production of aptamer library 5'-FITC-CGCGGAAGCGTGCTGGGCC-N ₄₀ -CATAACCCAGAGGTCGAT-3 ' SEQ ID NO: 1 CGCGGAAGCGTGCTGGGCC (N-terminal primer hybridization site) SEQ ID NO: 2 CATAACCCAGAGGTCGAT (C-terminal primer hybridization site) SEQ ID NO: 3 FITC-labeled 5 'primer (27 mer) 5'-FITC-GGGGAATTCGCGGAAGCGTGCTGGGCC-3 SEQ ID NO: 4 5 'primer (27 mer) 5'-GGGGAATTCGCGGAAGCGTGCTGGGCC-3 SEQ ID NO: 5 3 'primer (28 mer) 5'-GGGGGGATCCATCGACCTCTGGGTTATG-3

< Example  3> FACS ( Fluorescence - Activated Cell Sorter ) - Cell  SELEX ( Systematic  Evolution of Ligands by EXponential enrichment ) EpCAM Specific to Of app tamer  Selection

Cell SELEX method (FACE-SELEX) using flow cytometry was applied to screen for aptamers specifically binding to EpCAM. SsDNA (ssDNA library) with 40 randomly selected DNA sequences was used as the initial pool.

Specifically, for the SELEX, EpCAM-overexpressed HepG2 cells (EpCAM-HepG2) prepared in the above Example <1-2> were cultured and washed once with PBS. To prevent cell membrane proteins from being damaged, 2% EDTA (ethylene diamine tetra After the cells were removed from the culture dish, 100 μl of the reaction solution (4.5 g / L glucose, 5 mM MgCl ₂ and 10% FBS in Dulbecco's PBS [Sigma]) was added to 1 × 10 ⁶ cells Released. FITC-labeled ssDNA library (10 nmol, initial DNA pool size 10 ¹⁶ ) was dissolved in 20 ml of distilled water, denatured at 95 ° C for 5 minutes, and then cooled on ice for 10 minutes to form secondary structure. During this process, salmon sperm DNA (0.1 mg / ml; Sigma, St. Louis, MO, USA) dissolved in the reaction solution was reacted with EpCAM-HepG2 cells (1 × 10 ⁶ ) to prevent nonspecific binding. The cells were then incubated with 10 nmol ssDNA library and bovine serum albumin (1 mg / ml) for 30 minutes at 37 ° C. After centrifugation, the supernatant was removed and the cells were washed 5 times with 300 μl of the reaction solution. Cells bound with FITC - labeled ssDNA were classified as FACSAria and heated at 95 ° C for 5 minutes to isolate ssDNA bound to the cells. The separated ssDNAs were purified by phenol-chloroform (Sigma) extraction, Sephadex G-25 column (Sigma) and ethanol precipitation method. The purified ssDNA was amplified by PCR using FITC-labeled primers (SEQ ID NO: 3 and SEQ ID NO: 5).

A total of 8 SELEXs were performed for screening the aptamer. To remove the aptamer that recognizes other proteins in HepG2 cells other than EpCAM, HepC2 cells that did not overexpress EpCAM were subjected to 4 negative, 4th, 6th, Screening. The 7th ssDNA pool was cloned into DH5α using TA cloning kit and sequenced (Enzynimics). A total of 200 aptamers were analyzed and 33 aptamers were selected and synthesized.

As a result, as shown in FIG. 2, it was confirmed that the proportion of aptamers specifically binding to EpCAM-HepG2 cells was increased by increasing the fluorescence intensity of EpCAM-HepG2 cells as the number of SELEXs increased 2a). However, since the intensity of fluorescence decreased from 7th, the binding efficiency between cell and ssDNA pool decreased in 8th. Whereas no signal was observed in the negative control group (Fig. 2B).

In addition, about 200 aptamers obtained from the seventh aptamer pool were cloned and sequenced. However, a sequence common to the 200 aptamers could not be obtained.

< Example  4> EpCAM Specific to Of app tamer  Combination analysis and Dissociation constant value ( K _d ) decision

After sequential analysis, 200 aptamers were predicted using a mfold program (http://mfold.rit.albany.edu) and 33 aptamers having different structures were selected. Their binding affinity to EpCAM-HepG2 cells was confirmed by FACS and the dissociation constants (K _d ) were determined.

Specifically, the most thermodynamically stable structures among the anticipated structures for the aptamer sequence were selected through the mfold program (The RNA Institute), and each aptamer was selected from EpCAM-overexpressed cells (EpCAM-HepG2) (HepG2), and the mean fluorescence intensity (MFI) of the cells bound to the aptamer was measured by FACS. The measured MFI values were used to calculate the equilibrium dissociation constant (K _d ) of the aptamer and intercellular interaction via the equation Y = BmaxX / (Kd + X) (Sigma Plot).

In addition, aptamer binding analysis was performed using FACS and a confocal microscope. To evaluate the enrichment of the aptamer pool during SELEX, 1 × 10 ⁶ of each of the 100 pmol ssDNA pool, EpCAM-HepG2 cells and HepG2 cells of each screening step was added to 300 μl of the <Example 3> At 37 &lt; [deg.] &Gt; C for 15 minutes. For EP166, 10 μM aptamer was reacted with 5 × 10 ⁵ cells in 200 μl of the reaction solution at 37 ° C for 15 minutes. After the reaction, the reaction solution was washed five times with 300 μl of the reaction solution. The cell pellet was resuspended in 20 μl of the reaction solution, and 10 μl of the cell pellet was dropped on a slide glass to be measured. Then, FITC fluorescence was measured on a LSM 510 META confocal microscope (CalZeiss). The remaining 10 [mu] l cells were resuspended in 500 [mu] l of the reaction solution and fluorescence was measured with a FACS calibur flow cytometer (BD Bioscience).

As a result, as shown in FIG. 3 and FIG. 4, EP166 (5'-AAC AGA GGG ACA AAC GGG GGA AGA TTT GAC GTC GAC GAC A-3 ') of SEQ ID NO: (Fig. 3). &Lt; tb &gt; &lt; TABLE &gt; EP166 bound in EpCAM-HepG2 cells but not in negative control HepG2 cells (Figures 4a and 4b).

Previously known EpCAM-specific aptamers, SYL3C, had a high affinity for the breast cancer cell line MDA-MB-231 and the gastrointestinal cell line KaToIII with nanomolar K _d values (Song et al., 2013). Thus, EP166 and SYL3C aptamer showed affinity for EpCAM-HepG2 cells, indicating that EP166 exhibited higher affinity than SYL3C (Fig. 4D).

Also, as shown in FIG. 5, analysis of the second-order structure prediction data of EP166 using the mfold program revealed that two hairpin structures were located at a very short distance (FIG. 5A). These two hairpin structures are expected to play an important role in binding EpCAM, but additional testing is required to demonstrate this. In addition, it was confirmed that EP166 aptamer had a dissociation constant value of 5.48 + 0.84 mM (Fig. 5B). EP166 aptamer has a high binding affinity in EpCAM-HepG2 cells, whereas the very weak binding to HepG2 cells is due to the low expression of EpCAM in normal HepG2 cells, and therefore EP166 aptamer specifically binds to EpCAM .

< Example  5> Mouse Embryonic Stem Cells J1ES For EP166 Of app tamer  Confirm binding

Various cell surface markers and gene molecule markers have been identified as markers of undifferentiated embryonic stem cells (Zhao et al., 2012). EpCAM is also used as a marker of human embryonic stem cells, mouse embryonic stem cells, embryonic carcinoma cells and hepatocellular carcinoma. In order to confirm whether the selected aptamer EP166 can be used as an embryonic stem cell marker, the following experiment was carried out using mouse embryonic stem cell line J1ES.

Specifically, J1ES (American Type Culture Collection; ATCC) derived from mouse embryonic stem cell line 129s4 / Jae was cultured in ESC medium [15% ES145 tested fetal bovine serum (FBS) (PAA, Colbe, Germany), 1% penicillin-streptomycin (Invitrogen), 2 mM of L-glutamine (Invitrogen), 1% non-essential amino acids (NEAA, Invitrogen), 0.1 mM of β-mercaptoethanol (Invitrogen), and 1 × 10 ³ U / mL of leukemia inhibitory factors (Dulbecco's modified Eagle's Medium (DMEM) (Invitrogen, Carlsbad, Calif.) Containing 5% CO ₂ at 37 ° C in a high-glucose Dulbecco's modified Eagle's medium (Millipore, Billerica, And maintained with fibroblasts (mouse embryonic fibroblasts; MEFs).

For cell differentiation, the cells were cultured in a medium containing no leukemia inhibitory factors (Milipore), and the embryoid bodies (EBs) were cultured in a LIF-free ES medium supplemented with 90 mM non-cell-culture- (SPL Lifesciences, Pocheon, Korea) with 3 × 10 ⁶ cells / cm 2 of ES cells. The medium was replaced every two days (2EB, 4EB, 6EB and 8EB) and trans-RA (10 mM; Sigma-Aldrich, St. Louis, Mo.) was added to the 4 day (4EB) ). On day 8, the cultures were treated with trypsin, and the cells were cultured in a tissue culture (poly-D-lysine-laminin (Sigma-Aldrich) coated at a concentration of 1.5 × 10 ⁵ cells / And the test was conducted for binding test in the same manner as in < Example 4 &gt;.

As a result, as shown in Fig. 6, it was confirmed that EP166 bound to undifferentiated J1ES cells but not to J1ES cells differentiated into embryoid bodies (Fig. 6). As a result, it was confirmed that EP166, which is an aptamer for EpCMA, could be used as a specific marker for embryonic stem cells.

<110> Korea Research Institute Bioscience and Biotechnology <120> Nucleic acid aptamers specifically bind to EpCAM and their uses <130> 2014P-08-004 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> N-hybrid_primer <400> 1 cgcggaagcg tgctgggcc 19 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> C-hybrid_primer <400> 2 cataacccag aggtcgat 18 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 5-FITC-primer <400> 3 ggggaattcg cggaagcgtg ctgggcc 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 5-primer <400> 4 ggggaattcg cggaagcgtg ctgggcc 27 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> 3-primer <400> 5 ggggggatcc atcgacctct gggttatg 28 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> EP166 <400> 6 aacagaggga caaacggggg aagatttgac gtcgacgaca 40

Claims (14)

A nucleic acid aptamer specifically binding to an epithelial cell adhesion molecule (EpCAM) and comprising SEQ ID NO: 6.
The nucleic acid aptamer according to claim 1, wherein the nucleic acid aptamer is selected by the method of Cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment).
An epithelial cell adhesion molecule (EpCAM) detection kit comprising the nucleic acid aptamer of claim 1.
The epitaxial cell adhesion molecule (EpCAM) detection kit according to claim 3, wherein the epithelial cell adhesion molecule (EpCAM) is expressed in embryonic stem cells.
The kit according to claim 3, wherein the kit further comprises any one selected from the group consisting of a streptavidin-like phosphatease conjugate, a chemiluminescent substance, and a chemiluminescent substance, (EpCAM) detection kit.
1) contacting the nucleic acid aptamer of claim 1 with a biological sample; And
2) In the step 1), it is confirmed whether the nucleic acid aptamer of claim 1 is bound to the biological sample.
[Claim 7] The method according to claim 6, wherein the stem cells are embryonic stem cells.
[Claim 7] The method according to claim 6, wherein the binding is confirmed by a fluorescence-activated cell sorter (FACS) or a confocal microscopic.
[Claim 7] The method according to claim 6, wherein the stem cell detection is by confirming the expression of epithelial cell adhesion molecule (EpCAM).
A stem cell detection kit comprising the nucleic acid aptamer of claim 1.
[Claim 11] The stem cell detection kit according to claim 10, wherein the stem cells are embryonic stem cells.
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