KR101759209B1 - Nucleic Acid Aptamer Capable of Specifically Binding to HER-2-Overexpressing Breast Cancer Cell or Tissue and Use Thereof - Google Patents

Abstract

The present invention relates to a nucleic acid plasmid that can be used for the diagnosis and treatment of breast cancer by specifically recognizing and binding to HER-2 overexpressing breast cancer cells or tissues and its use. The nucleic acid aptamer according to the present invention specifically recognizes and binds to HER-2 expressed on the cell surface, thereby binding HER-2 overexpressing breast cancer cells to in can be used as a target for vivo imaging or drug delivery, and can be useful as a composition for diagnosis and treatment of breast cancer, thereby contributing to an increase in the survival rate of breast cancer patients.

Description

[0001] Nucleic Acid Aptamer capable of specifically binding to HER-2 overexpressing breast cancer cells or tissues and its use [0002] Nucleic Acid Aptamer Capable of Specific Binding to HER-2 Overexpressing Breast Cancer Cell or Tissue and Use Thereof [

The present invention relates to a nucleic acid plasmid that can be used for the diagnosis and treatment of breast cancer by specifically recognizing and binding to HER-2 overexpressing breast cancer cells or tissues and its use.

Ligand materials capable of binding specifically to cancer cells with high affinity for cancer cell surface proteins are important for the development of cancer diagnosis and therapeutic agents. On the other hand, these ligand materials can also be used as cancer biomarkers. Understanding these biomarkers can further accelerate the development of new cancer therapies.

HER-2 / ErbB2 / Neu (HER-2) is a transmembrane receptor tyrosine kinase, one of the epithelial growth factor receptor family (Roskoski, R., Jr. Biochem . Biophys . Res . Commun. , 319 : 1, 2004 ). Thus, HER-2 is a receptor-like protein composed of an extracellular domain that binds to ligands and an intracellular domain that causes protein activation. Overexpression of HER-2 is observed in 20-30% of breast cancer cell lines. HER-2 ectodomain-directed monoclonal antibody (trastuzumab, or Herceptin) has been approved to treat breast cancer (Roskoski, R., Jr. Biochem Biophys Res Commun, 319:.... 1, 2004), HER-2 is also targeted drug delivery for cancer (Chiu, SJ et al, J. Control Release, 97:.. 357, 2004)... and attractive target for in vivo imaging (Tolmachev, V., Curr Pharm Des , 14 : 2999, 2008).

Aptamers are single-stranded DNA or RNA oligonucleotides that can be folded into specific three-dimensional structures to bind with high affinity and specificity to the target. Targets for platameras can be small molecules, peptides, proteins, or cells themselves (Dua, P. et al., Recent Pat . DNA Gene Seq . , 2: 172, 2008).

Such aptamers have advantages such as smaller size, better tissue permeability, ease of chemical manipulation, and no immune response compared to antibodies. Also, in vivo imaging applications, the aptamer has a faster response rate and a better signal-to-noise ratio than the antibody due to its small size. The aptamer can also be attached to magnetic nanoparticle surfaces and used in magnetic resonance imaging (MRI) to improve image specificity.

As cancer therapies, aptamers can be used directly as "escort aptamers" that inhibit the cancer growth signal pathway by blocking growth factors or growth factor receptors, or deliver therapeutic molecules such as cytotoxic agents, radionuclides, or siRNA to target cells . Therefore, the obtaining of a specific cancer cell type or a specific platemma targeting cancer cell surface proteins is helpful in the development of novel cancer diagnostic and therapeutic agents.

However, in many cancer cell types, specific cell surface biomarker proteins are not known and it has been difficult to isolate platamers targeting specific cancer cell types. In addition, even if the diarrheal cell surface biomarker protein is known, the membrane protein is typically difficult to express and separate, or the separated membrane protein will not have the original structure such as being present in the cell membrane. Therefore, the apta had a dimmer cell difficulties that can not recognize the same protein in the surface (Cerchia, L. et al, PLoS Biol, 3:.. e123, 2005).

Therefore, the present inventors have made extensive efforts to isolate aptamer that can be used for diagnosis and treatment of breast cancer by specifically binding to HER-2 overexpressing breast cancer cells or tissues. As a result, by EXponential enrichment), the selected aptamer is selectively bound only to the HER-2 overexpressing breast cancer cell line. Then, the selected aptamer is selected from the breast cancer cells overexpressing HER-2 or the cells overexpressing HER-2 Specific binding, thus completing the present invention.

It is an object of the present invention to provide a nucleic acid plasmid that specifically binds to HER-2 overexpressing breast cancer cells or tissues.

It is still another object of the present invention to provide a composition for diagnosing or treating breast cancer using the nucleic acid platemaker.

In order to achieve the above object, the present invention provides a nucleic acid sequence selected from the group consisting of the nucleic acid sequences represented by SEQ ID NOS: 9, 12, 13, and 17, and specifically comprising HER-2 overexpressing breast cancer cells or tissues Wherein U is T in the nucleic acid sequence, wherein the nucleic acid abstamer is DNA, wherein the nucleic acid abstamer is 15 to 60 nt.

The present invention also relates to a nucleic acid sequence which comprises any one of the nucleic acid sequences selected from the group consisting of the nucleic acid sequences represented by SEQ ID NOs: 9, 12, 13 and 17, and which can specifically bind to HER-2 (Where U is T in the nucleic acid sequence if the nucleic acid aptamer is DNA).

The present invention also provides a method for detecting breast cancer, which uses the nucleic acid abstamator.

The present invention also provides a composition for diagnosing or treating breast cancer containing the nucleic acid platemaker.

The present invention also provides a sensor for breast cancer diagnosis in which the nucleic acid aptamer is immobilized.

The present invention also provides a kit for the diagnosis of breast cancer containing the nucleic acid platemaker.

The present invention also provides a method for detecting breast cancer using the breast cancer diagnostic sensor or the breast cancer diagnostic kit.

The present invention also provides a breast cancer specific drug delivery composition containing the nucleic acid abortamer.

The present invention provides a nucleic acid plasmid capable of specifically binding to HER-2 overexpressing breast cancer cells or tissues, and a composition for diagnosing or treating breast cancer using the same.

The nucleic acid aptamer according to the present invention specifically recognizes and binds to HER-2 expressed on the cell surface, thereby binding HER-2 overexpressing breast cancer cells to in can be used as a target for vivo imaging or drug delivery, and can be useful as a composition for diagnosis and treatment of breast cancer, thereby contributing to an increase in the survival rate of breast cancer patients.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a screening procedure for an umbilical cord specifically binding to HER-2 overexpressing breast cancer cells or tissues according to the present invention.
FIG. 2 is a result of analyzing the sequence of the final compressor pool obtained by performing the Cell-SELEX process according to the schematic diagram of FIG.
3A is a graph showing the results of inhibition of HER-2 expression using final siRNAs (m15R) and HER-2 specific siRNAs obtained by negative selection using an initial RNA library pool and an MDA-MB-231 cell line 3B is a graph showing the binding affinity of breast cancer cell line of uterum pool (si15R) obtained through negative selection process using SK-BR-3 cell line by quantitative PCR. SK-BR-3 cell line and MDA- Western blot analysis of HER-2 protein in MB-231 cell line is shown.
FIG. 4 is a photograph showing the affinity of the initial library pool and selected platamer for the SK-BR-3 and MDA-MB-231 cell lines using fluorescence detection method.
FIG. 5A shows the secondary structure of the S6 erythromycin and FIG. 5B shows the binding affinity of the S6 erythromycin to the SK-BR-3 cell line by the equilibrium filtration method to obtain the HER-2-overexpressing breast cancer cell line SK-BR-3 ≪ / RTI >
FIG. 6 is a photograph showing the binding affinity of the S6 caldimmer to the NIH-3T6.7 cell line (NIH-3T3 / HER-2) and the NIH-3T3 cell line using fluorescence detection method.
7A is a graph showing the binding affinity of NIH-3T6.7 cell line (NIH-3T3 / HER-2) and NIH-3T3 cell line of S6 caldimater using quantitative PCR, and 7B is a graph showing the binding affinity of NIH- And the Western blot analysis of HER-2 protein in the NIH-3T3 cell line.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The definitions of the main terms used in the description of the present invention and the like are as follows.

As used herein, the term " nucleic acid plasmid "refers to a small single-stranded oligonucleotide capable of specifically recognizing a target substance with high affinity.

As used herein, the term "sample " refers to a composition to be assayed and presumed to contain a breast cancer marker, and may include breast tissue, breast cells, blood, serum, plasma, saliva, sputum and urine.

As used herein, the term "nucleic acid sequence having 90% or more and less than 100% homology" means a nucleic acid sequence having 90% or more and less than 100% Quot; means a nucleic acid sequence.

In one aspect, the present invention relates to a nucleic acid molecule comprising any one of the nucleic acid sequences selected from the group consisting of the nucleic acid sequences represented by SEQ ID NOs: 12, 13 and 17, and capable of specifically binding to HER-2 overexpressing breast cancer cells or tissues It is about plumbers.

In the present invention, the nucleic acid abdomen may include any one of nucleic acid sequences selected from the group consisting of the nucleic acid sequences shown in SEQ ID NOS: 1 to 12 below.

M11, S4: 5'-UCUAGCUCUCCUCUAGAGUGGGCCUCUAGAGUUUGACUG-3 '(SEQ ID NO: 1)

S16: 5'-UC-AGCUCUCUUCUAGAGUGGGCCUCUAGAGUUUGACUGG-3 '(SEQ ID NO: 2)

M9: 5'-AGAGAGGGAUUGGAUAGGCGUCU GCGUGUCUCUCUGCCA C - UACUAUGGGG -3 ' (SEQ ID NO: 3)

M2, M5, M29, S1: 5' - AGAGAGAGAUUGGAUAGGCGUCU GCGUGUCUCUCUGCCA C - UACUAUGGGG -3 ' (SEQ ID NO: 4)

M8: 5'-UCGAGUGGGCCUUGAGAUUGGAUAACUG GCGUGUCUCUCUGCCA C - UACUAUGGGG -3 ' (SEQ ID NO: 5)

S15, S17: 5'-UGGAUGGGGAGAUCCGUUGAGUAAGCGG GCGUGUCUCUCUGCC A C-- UACUAUGGGG -3 '( SEQ ID NO: 6)

S6: 5'-UGGAUGGGGAGAUCCGUUGAGUAAGCGG GCGUGUCUCUCUGCC G C CU UGCUAUGGGG-3 ' (SEQ ID NO: 7)

M1, S14:

   5'-GUAAGACGAGGUUCGCCGGCCUCGCGAUUGGAUUUGCUGGUCAGUCUGCUUACUAUGGGG-3 '(SEQ ID NO: 8)

M16, S5, S10, S27:

    5'-GUAAGACGAGGUUCGCCCGCCUCGCGAUUGGAUUUGCUGGUCAGUCUGCUUACUAUG-3 '(SEQ ID NO: 9)

S9: 5'-UUCGAAUGGUUCCUCGUCUUCCAAGAUCUUACUAUGGGG-3 '(SEQ ID NO: 10)

M13: 5'-CUAGUGGGCCUAGGAUUGUAGGCGAGUCUCCUUUCUUACUAUGGGG-3 '(SEQ ID NO: 11)

S13: 5'-AAGCAACUCCUGCCUUUCUAGCUGCAAAGUGGGCCUUUGU-3 '(SEQ ID NO: 12)

In the above, the nucleic acid sequences of SEQ ID NOS: 1 and 2 commonly have UCUAGAGUGGGCCUCUAGAGUUUGACUU (SEQ ID NO: 13), and the nucleic acid sequences of SEQ ID NOS: 3 to 7 are commonGCGUGUCUCUCUGCCACUACUAUGGGG (SEQ ID NO: 14) orGCGUGUCUCUCUGCCGCCUUGCUAUGGGG(SEQ ID NO: 15). At this time, the nucleic acid sequences of SEQ ID NOS: 3 to 7 vary in the 5'-side sequence, but the 3'-side half is conservedGCGUGUCUCUCUGCC(SEQ ID NO: 16) andURCUAUGGGG (R is A or G; SEQ ID NO: 17). Further, the nucleic acid sequences of SEQ ID NOS: 8, 10, and 11URCUAUGGGG (R is A or G; SEQ ID NO: 17). In addition, it was confirmed that the nucleic acid sequence of SEQ ID NO: 9 has the sequence of URCUAUG (R is A or G) in which the GGG was truncated at the 3 'end in the nucleic acid sequence of SEQ ID NO: 17. On the other hand, the nucleic acid sequence of SEQ ID NO: 12 has a nucleic acid sequence of AGUGGGCCU in common with the nucleic acid sequences of SEQ ID NOs: 1 and 2.

At this time, the total number of nucleotides constituting the nucleic acid strutrimer may be 15 to 200 nts, preferably 15 to 60 nts. When the number of total nucleotides is small, chemical synthesis and mass production are easier and the cost advantage is also great. In addition, the chemical modification is easy, the in-vivo stability is high, and the toxicity is also low. In addition, each nucleotide contained in the nucleic acid strutrimer may include one or more chemical modifications that are the same or different. For example, at the 2 'position of the ribose, the nucleotide in which the hydroxyl group is substituted with any atom or group have. Examples of such an arbitrary atom or group include a hydrogen atom, a fluorine atom or an -O-alkyl group such as -O-CH ₃ , an -O-acyl group such as -O-CHO, ₂ ). ≪ / RTI > In the present invention, when the nucleic acid is DNA, U in the nucleic acid sequence is represented by T, and such sequence is included in the scope of the present invention. And will be apparent to those skilled in the art.

The nucleic acid abdomen according to the present invention is URCUAUGGGG (R is A or G; SEQ ID NO: 17), nucleic acid sequences of SEQ ID NOS: 13 to 16, etc. The presence of such a commonly conserved region is indicated by SEQ ID NOS: 1 to 12 A nucleic acid sequence having 90% or more and less than 100% of homology with any one of the nucleic acid sequences selected from the nucleic acid sequences of the present invention is a nucleic acid amplicter capable of specifically binding to a breast cancer cell line. 1 to 12, one to several nucleotides are added to, deleted or substituted for any one selected from the nucleic acid sequences of SEQ ID NOS: 1 to 12, Or more and less than 100%, it will be obvious that the HER-2 overexpressing breast cancer cell line binding ability similar to the nucleic acid abortamer according to the present invention will be exhibited.

In the example of the present invention, as shown in FIG. 1, the plasmids of the nucleic acid sequences shown in SEQ ID NOS: 1 to 12 are selected through the Cell-SELEX process and then subjected to real-time PCR and fluorescence detection It was confirmed that the nucleic acid sequences shown in SEQ ID NOS: 1 to 12 were specifically bound to the HER-2 overexpressing breast cancer cell line. A specific binding to such a breast cancer cell line indicates that the nucleic acid aptamer according to the present invention can be usefully used for diagnosis of a substantial breast cancer.

Accordingly, the present invention relates to a composition for diagnosing breast cancer containing the nucleic acid abortamer from a different viewpoint.

In addition, the present invention relates to a method for detecting breast cancer using the nucleic acid platemater from a different viewpoint. The method may further comprise the step of contacting the nucleic acid strand with the sample selected from breast tissue, breast cells, blood, serum, plasma, saliva, sputum and urine. The sample is not limited to the above, provided that it is a sample separated from a mammal, preferably a human body, which may contain HER-2, which is a breast cancer marker such as a sample or secretory fluid capable of being obtained by minimal invasion, It is self-evident.

When the nucleic acid platemaker is brought into contact with a sample, specific binding between HER-2, which is a breast cancer marker existing in the sample, and the nucleic acid excretion occurs. Therefore, it is possible to detect breast cancer by identifying and binding a nucleic acid tympanic membrane with fluorescence or the like and then identifying the presence or absence of a signal.

On the other hand, the nucleic acid < RTI ID = 0.0 > abtamers specifically binding to breast cancer cells or tissues can be attached to a solid support such as beads, particles, dipsticks, fibers, filters, membranes and glass slides and silane or silicate supports The present invention relates to a sensor for diagnosing breast cancer in which nucleic acid aptamers specifically binding to the breast cancer cells or tissues are immobilized.

The solid support comprises at least one substantially rigid surface on which the nucleic acid platelets can be immobilized immovably. At this time, the nucleic acid aptamer can be immobilized by any conventional chemical coupling method. For example, by binding biotin to the end of the nucleic acid plamper to form a complex, immobilizing streptavidin on the surface of the chip or the like, and reacting the biotin with streptavidin immobilized on the surface of the substrate, An abutment can be immobilized on the substrate surface.

The present invention may also be provided in the form of a kit for the diagnosis of breast cancer, which comprises an extramammary that specifically binds to the breast cancer cell or tissue. This breast cancer diagnostic kit can include buffer solutions and containers for performing and analyzing the detection as needed, such as bottles, tubs, sachets, envelopes, tubes, ampoules, and the like. They may take the same form and may be formed partly or wholly of plastic, glass, paper, foil, wax, and the like. The container may be fitted with a cap which is initially part of the container or can be fully or partially detachable, which may be attached to the container by mechanical, adhesive, or other means. The container may also be equipped with a stopper, which is accessible to the contents by the injection needle. The kit may include an external package, and the external package may include instructions for use of the components.

It is well known in the art that the present invention can be used to treat cancer by inhibiting the mechanism of cancer by binding an umbilical cord specifically binding to a cancer cell line to a cancer cell line, It is apparent to those skilled in the art that a composition containing the aptamer according to the present invention can be provided as a composition for the treatment of breast cancer since the aptamer according to the present invention specifically binds to breast cancer cells or tissues and inhibits the breast cancer mechanism . In particular, HER-2 ectodomain-directed monoclonal antibody (trastuzumab, or Herceptin) has been approved for the treatment of breast cancer as a bar (Roskoski, R., Jr. Biochem Biophys Res Commun, 319:.... 1, 2004), the It can be seen that the nucleic acid abtamers according to the invention can be used for the treatment of breast cancer.

In addition, a known breast cancer-specific drug or a known apoptosis gene such as Herpes simplex virus-thymidine kinase (HSV-TK), cytosine deaminase (CD) And small interfering RNA (siRNA), which inhibits the expression of genes that play an important role in metastasis, can be transferred to breast cancer cell lines. HER-2 has also been used as a target for drug delivery to breast cancer (Chiu, SJ et al . , J. Control . Release , 97 : 357, 2004), it is apparent that the present invention can be provided in the form of a breast cancer specific drug delivery composition containing the nucleic acid platemer.

The pharmaceutical dosage forms of the compositions of the present invention may also be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

The pharmaceutical composition according to the present invention may be formulated according to a conventional method, for example, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, Can be used. Examples of carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. The compositions of the present invention may be administered to mammals, such as rats, mice, livestock, humans, and the like in a variety of routes, all manner of administration being contemplated, for example, rectal or intravenous, muscular, subcutaneous, Or by intracerebral injection.

Meanwhile, in one embodiment of the present invention, it was confirmed that aptamers according to the present invention can specifically bind HER-2 overexpressing breast cancer cell lines as well as HER-2 overexpressed cell lines by fluorescence detection and quantitative PCR , It can be used as a HER-2 targeting platemer.

Accordingly, in another aspect, the present invention also provides a nucleic acid sequence comprising any one of the nucleic acid sequences selected from the group consisting of the nucleic acid sequences represented by SEQ ID NOs: 12, 13, and 17, and specific for HER-2 overexpressing breast cancer cells or tissues (Where U is T in the nucleic acid sequence if the nucleic acid abstamer is DNA), which is capable of binding to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are for illustrating the present invention only and that the scope of the present invention is not construed as being limited by these embodiments.

Example  One: HER -2 overexpressing breast cancer cell line Abtamer's  detach

1-1: ssRNA  Library and PCR  Amplification and cDNA  For synthesis Primer  Ready

A random ssDNA library having the following sequence was chemically synthesized and separated into PAGE (Genotech Inc., Korea).

5'-ATA CCA GCT TAT TCA ATT NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NAG ATA GTA AGT GCA ATC T-3 '(SEQ ID NO: 18)

The initial pool contained 3 x 10 < ¹³ > molecules. The N40 upstream primer of SEQ ID NO: 19 and the N40 downstream primer of SEQ ID NO: 20 were used for PCR amplification and cDNA synthesis.

Upstream primer: 5'-GGTAATACGACTCACTATAGGGAGATACCAGCTTATTCAATT-3 '(SEQ ID NO: 19)

Downstream primer: 5'-AGATTGCACTTACTATCT-3 '(SEQ ID NO: 20)

The amplified library was transformed into RNA using Durascribe T7 RNA polymerase (Eqicentre). In this case, the U and C of RNA can be made to have 2'-F instead of 2'-OH by using 2'-F UTP and 2'-F CTP instead of UTP and CTP, respectively, So that the biomedical application can be performed.

1-2: HER -2 overexpressing breast cancer cell line Abtamer's  Selection

As shown in FIG. 1, positive selection was first performed on SK-BR-3 (American Tissue Culture Collection), a breast cancer cell line known to overexpress HER-2.

The SK-BR-3 cell line was cultured in McCoy's 5A medium (Welgene) supplemented with 10% fetal bovine serum at 37 ° C and antibiotics (100 U / mL penicillin and 100 μg / mL stepomycin; Gibco BRL) Respectively. The cells were then recovered by trypsinization and then 1x10 ⁶ cells were recovered in complete medium at 37 ° C.

Meanwhile, 160 pmole N40 RNA library of Example 1-1 was added to binding buffer (4.5 g / L glucose, 5 mM MgCl ₂ , 0.1 mg / ml yeast tRNA, 1 mg / ml BSA in Dulbecco's PBS ), And then quenched on ice. The recovered cell line was then washed twice with wash buffer (4.5 g / L glucose, 5 mM MgCl ₂ in Dulbecco's PBS) and the prepared RNA library on the binding buffer was incubated at 4 ° C for 45 minutes. Uncoupled sequences were fractionated through two successive washes with a wash buffer solution. Then, by heating at 95 ° C for 5 minutes, the bound sequences on the cell surface were eluted and separated by PCI extraction (phenol: chloroform: isoamyl alcohol etraction: Bioneer, Korea). The obtained RNA was reverse transcribed using the ImProm-II Reverse Transcription System (Promega, USA) and PCR amplified. The isolated PCR product was subjected to in vitro transcription using T7 polymerase (Ambion, USA).

After the positive selection process was performed 14 times, the concentration of the RNA library to the binding platamera reached a saturation state. As shown in FIG. 1, two speech selection processes are performed in parallel. At this time, the positive selection process was repeated for the SK-BR-3 cell line overexpressing HER-2 when the negative selection process was performed.

In one screening procedure, MDA-MB-231 (American Tissue Culture Collection), a cell line that hardly expresses HER-2, was used as a cell line for negative selection to remove the tympanic membrane binding to the cell line. MDA-MB-231 cell line was cultured in RPMI-1640 medium (Gibco BRL) supplemented with 10% fetal bovine serum at 37 ° C and antibiotics (100 U / mL penicillin and 100 μg / mL stepomycin; Gibco BRL) , Recovered, and treated in the same manner as above. The platamer bound to the cell line was removed, and the unbound platemer was collected.

The other screening procedure was a new negative screening strategy using an SK-BR-3 cell line that inhibited HER-2 expression using HER-2 specific siRNAs to remove the tympanum binding to it. Transfection of siRNA was performed as follows. First, the SK-BR-3 cell line was plated on a 60 mm plate with complete medium without antibiotics and then cultured for 24 hours until a 50% confluency was reached. Then, 10 nM siRNA (bioneer, Korea) was transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Twenty-four hours later, the transfected cells were counted, and 1 x 10 ⁶ cells were treated in the same manner as above, and the platamer bound to the cell line was removed and the unbonded platemer was collected.

The products of each screening were cloned using TA vector (RBC, Korea), and the clones were sequenced using multialign software (http://bioinfo.genotoul.fr/multalin/ multalin.html) , A common sequence on the clone was analyzed.

The sequences on the clones collected and collected from the platelet pool obtained through the obtained negative selection process are shown in FIG. 2, and the sequences of the RNA plasmids according to the present invention obtained therefrom are as follows.

M11, S4: 5'-UCUAGCUCUCCUCUAGAGUGGGCCUCUAGAGUUUGACUG-3 '(SEQ ID NO: 1)

S16: 5'-UC-AGCUCUCUUCUAGAGUGGGCCUCUAGAGUUUGACUGG-3 '(SEQ ID NO: 2)

M9: 5'-AGAGAGGGAUUGGAUAGGCGUCU GCGUGUCUCUCUGCCA C - UACUAUGGGG -3 ' (SEQ ID NO: 3)

M2, M5, M29, S1: 5' - AGAGAGAGAUUGGAUAGGCGUCU GCGUGUCUCUCUGCCA C - UACUAUGGGG -3 ' (SEQ ID NO: 4)

M8: 5'-UCGAGUGGGCCUUGAGAUUGGAUAACUG GCGUGUCUCUCUGCCA C - UACUAUGGGG -3 ' (SEQ ID NO: 5)

S15, S17: 5'-UGGAUGGGGAGAUCCGUUGAGUAAGCGG GCGUGUCUCUCUGCC A C-- UACUAUGGGG -3 '( SEQ ID NO: 6)

S6: 5'-UGGAUGGGGAGAUCCGUUGAGUAAGCGG GCGUGUCUCUCUGCC G C CU UGCUAUGGGG-3 ' (SEQ ID NO: 7)

M1, S14:

   5'-GUAAGACGAGGUUCGCCGGCCUCGCGAUUGGAUUUGCUGGUCAGUCUGCUUACUAUGGGG-3 '(SEQ ID NO: 8)

M16, S5, S10, S27:

    5'-GUAAGACGAGGUUCGCCCGCCUCGCGAUUGGAUUUGCUGGUCAGUCUGCUUACUAUG-3 '(SEQ ID NO: 9)

S9: 5'-UUCGAAUGGUUCCUCGUCUUCCAAGAUCUUACUAUGGGG-3 '(SEQ ID NO: 10)

M13: 5'-CUAGUGGGCCUAGGAUUGUAGGCGAGUCUCCUUUCUUACUAUGGGG-3 '(SEQ ID NO: 11)

S13: 5'-AAGCAACUCCUGCCUUUCUAGCUGCAAAGUGGGCCUUUGU-3 '(SEQ ID NO: 12)

In the above, the nucleic acid sequences of SEQ ID NOS: 1 and 2 were confirmed to have UCUAGAGUGGGCCUCUAGAGUUUGACUU (SEQ ID NO: 13) in common, and the nucleic acid sequences of SEQ ID NOS: 3-7 were commonly found in GCGUGUCUCUCUGCC A CUACUAUGGGG (SEQ ID NO: 14) or GCGUGUCUCUCUGCC G C CU UGCUAUGGGG (SEQ ID NO: 15). That is, the nucleic acid sequences of SEQ ID NOS: 3 to 7 differ in the 5'- side sequence, but the 3'- side half is conserved and GCGUGUCUCUCUGCC (SEQ ID NO: 16) and URCUAUGGGG (R is A or G; SEQ ID NO: 17). In addition, the nucleic acid sequences of SEQ ID NOS: 8, 10, and 11 are also URCUAUGGGG (R is A or G; SEQ ID NO: 17). In addition, it was confirmed that the nucleic acid sequence of SEQ ID NO: 9 has the sequence of URCUAUG (R is A or G) in which the GGG was truncated at the 3 'end in the nucleic acid sequence of SEQ ID NO: 17. On the other hand, the nucleic acid sequence of SEQ ID NO: 12 was confirmed to have the nucleic acid sequence of AGUGGGCCU in common with the nucleic acid sequences of SEQ ID NOs: 1 and 2. On the other hand, several sequences have been inserted during the SELEX cycle, indicating that some aptamers have a sequence of 40 mer or more.

Example  2: Cell - SELEX Done Abtamer  Grass HER -2 overexpression breast cancer cell line

Using the quantitative PCR method, the eluate obtained by the negative selection process using the initial ssRNA pool before the SELEX method and the MDA-MB-231 cell line and the HER-2 specific siRNA were used for the HER -2 expression in the SK-BR-3 cell line. The binding affinity of the eluate was measured.

That is, the RNA pool of the initial library and the final round was treated with the SK-BR-3 cell line, which is a HER-2 overexpressing breast cancer cell line. At this time, MDA-MB-231 cell line, which is a breast cancer cell line that hardly expresses HER-2, was used as a control group.

Both the in-put plum pool and the out-put plum pool were used as templates for cDNA synthesis. The cDNA synthesis was performed using the ImProm-II Reverse Transcription System (Promega) according to the manufacturer's protocol Respectively. An aliquot (1/250) of the cDNA reaction mixture was analyzed by quantitative PCR according to the manufacturer's protocol using a Step-One real-time PCR machine (Applied Biosystems) and the ratio of the output signal to the input was calculated. At this time, the primers used in the PCR are the same as the primers of SEQ ID NO: 19 and SEQ ID NO: 20.

As a result, as shown in FIG. 3A, in the case of the pool of the initial library, neither the SK-BR-3 nor the MDA-MB-231 cell line showed affinity, and the negative selection process using the MDA-MB- HER-2 expression was inhibited by using the ultimatum pool and HER-2 specific siRNA obtained from the SK-BR-3 cell line. Unlike the MDA-MB-231 cell line, which is not expressed, it has been confirmed that HER-2 overexpressing cell line SK-BR-3 cell line exhibits high binding affinity.

In other words, it was confirmed that HER-2 overexpressing breast cancer cell line was specifically enriched into a platelet aggregate that specifically recognized the cell-SELEX process.

FIG. 3B shows the expression of HER-2 protein by Western blotting analysis, showing that HER-2 is overexpressed in SK-BR-3 cell line but hardly expressed in MDA-MB-231 cell to be. Western blot analysis for confirmation of expression of HER-2 protein was performed as follows. Cells were washed twice with cold PBS (phosphate-buffedred saline) and then eluted with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS). Cells were eluted by vortexing at 4 ° C for 30 minutes, centrifuged at 12000 rpm for 10 minutes, and the supernatant was collected and quantified by Protein Assay Reagent (Pierce). 50 의 of the protein was separated by SDS-PAGE, and electrophoresed with PVDF membrane (Millipore). The blot was blocked with 5% skim milk on TBS for 1 hour at room temperature and then incubated for 16 hours at 4 ° C with a primary antibody diluted 1: 500 (mouse monoclonal anti-c-ErbB2 / c-Neu antibody, Calbiochem) Lt; / RTI > Blots were washed three times for 5 minutes with TBS-Tween 20 and then incubated with horse-radish peroxidase-labeled goat anti-mouse IgG (BIO-RAD) diluted 1: 2000 on TBS-Tween for 1 hour at room temperature . After 5 washes with TBS-Tween for 6 min, the proteins were visualized with ECL plus reagent (Amersham Bioscience).

Example  3: Separated angle Abtamer's HER -2 overexpressing breast cancer cell line

The binding affinity of MDA-MB-231, a breast cancer cell line that hardly expresses HER-2 overexpressing breast cancer cell lines SK-BR-3 and HER-2, isolated from Example 1 was measured by fluorescence detection For this, 5 'TAMRA (tetramethylrhodamine) labeled downstream primer was used. 5'-TAMRA-AGATTGCACTTACTATCT-3 '(SEQ ID NO: 21) was used as the TAMRA-labeled 3' primer.

First, TAMRA labeled primers on an annealing buffer (30 mM HEPES-KOH pH 7.4, 100 mM KCl, 2 mM MgCl2, 50 mM NH4Ac) were added to the initial RNA library (Pool) Followed by thermal denaturation at 90 ° C for 2 minutes, followed by gradual cooling, followed by incubation at 37 ° C for 60 minutes. Cells grown on a petri dish bottom were inoculated with 1 μM of the labeled RNA library or platelets in binding buffer (4.5 g / L glucose, 5 mM MgCl ₂ , 0.1 mg / ml yeast tRNA, 1 mg / ml BSA in Dulbecco's PBS) The cells were incubated at 4 ° C for 45 min and then rapidly washed twice with a washing buffer (4.5 g / L glucose, 5 mM MgCl ₂ in Dulbecco's PBS). Then, fluorescence was observed with a fluorescence microscope (Olympus) Respectively.

As a result, unlike the case of SK-BR-3 and MDA-MB-231 cell lines showing no strong affinity for the pool of the initial library, as shown in Fig. 4, -231 cell line, negative selection was carried out using SK-BR-3 cell line in which HER-2 expression was inhibited by using final tympanic pool and HER-2 specific siRNA obtained through negative selection process, Unlike the MDA-MB-231 cell line, which has almost no HER-2 expression in both the hamster and the hamster, the HER-2 over-expressing cell line SK-BR-3 cell line showed high binding affinity. That is, the aptamer according to the present invention specifically recognizes the HER-2 over-expressing cell line and thus can be used for diagnosis and treatment of breast cancer.

In contrast, among the tested platamers, S6 aptamer of SEQ ID NO: 7 was weakest against MDA-MB-231 cell line, while the binding force to SK-BR-3 cell line was the strongest. Experiments were performed.

Example  4: S6 Abtamer's  By equilibrium filtration HER -2 overexpression of breast cancer cell line Kd ) Measure

The secondary structure of the S6 extruder identified in Example 3 was predicted using the Mfold program, which is shown in Figure 5A.

On the other hand, nonlinear regression analysis was performed to measure dissociation constants of the S6 compressors. (1 μM concentration range from 10 nM) and RNA library pools were incubated for 45 min at 4 ° C in a 60 mm culture dish with 1 × 10 ⁵ HER-2-overexpressing SK-BR-3 cell line on the binding buffer Then, the cells were collected after two washing steps, and RNAs bound in the same manner as in the Cell-SELEX process of Example 1 were eluted. Thereafter, the amount of platemer bound to the cells was quantified using the quantitative PCR of Example 2 above.

To calculate the dissociation constants, the percentage of abomasum first put into the breast cancer cell line in the following equilibrium form was calculated using Sigmaplot 10.0 software and the dissociation constant was calculated by nonlinear regression analysis.

_{y = (B max · ssRNA)} / (K d + ssRNA)

Where y is the degree of saturation, B _max is the number of maximum coupling points, and K _d is the dissociation constant.

As a result, as shown in FIG. 5B, the dissociation constant of the S6 caldimater was measured to be 94.6 nM, indicating strong binding force to the HER-2-overexpressing breast cancer cell line SK-BR-3.

However, S6-aptamer does not target HER-2, but rather confirms that it targets any other receptor that is more expressed in SK-BR-3 than MDA-MB-231 cells that rarely express HER-2 The following experiment was further performed.

Example  4: S6 Abtamer's HER -2 for binding affinity measurement

4-1: Through fluorescence detection NIH -3 T3 / HER -2 ( NIH -3 T6 .7) for binding affinity measurement

In order to confirm whether S6 aptamer targets HER-2, NIH-3T6.7 cells (ATCC CCL-96), which is an NIH-3T3 cell line that overexpresses human HER-2 and NIH-3T3 The binding affinity for the cell line (American Tissue Culture Collection) was measured in the same manner as in Example 3 using fluorescence detection using a 5 'TAMRA-labeled 3' primer. The NIH-3T3 cell line was cultured in DMEM medium (Gibco BRL) supplemented with 10% fetal bovine serum at 37 ° C and antibiotics (100 U / mL penicillin and 100 μg / mL stepomycin; Gibco BRL) And NIH3T6.7 cells were maintained in DMEM medium.

As a result, as shown in Fig. 6, the S6 aptamer was found to express NIH-3T6.7 cells (NIH-3T3 cells overexpressing HER-2; NIH- 3T3 / HER2). ≪ / RTI >

4-2: Quantification PCR through NIH -3 T3 / HER -2 ( NIH -3 T6 .7) for binding affinity measurement

In addition, the binding affinity of NIH-3T3.7 cell line overexpressing human HER-2 to the NIH-3T3 cell line not expressing HER-2 was measured by the quantitative PCR method in the same manner as in Example 2 Respectively.

As a result, as shown in FIG. 7A, the S6 aptamer was also found to express NIH-3T6.7 cells (NIH-3T3 cells overexpressing HER-2; NIH-3T6 cells -3T3 / HER2). ≪ / RTI > This suggests that, with the results of Example 4-1, the target of the S6 uterus is HER-2, not another protein on the surface of the SK-BR-3 cell line.

FIG. 7B shows the result of Western blot analysis showing that the NIH-3T3 cell line does not express HER-2 but the NIH-3T6.7 cell line expresses HER-2, and this was performed in the same manner as in Example 2. FIG.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> Nucleic Acid Aptamer Capable of Specifically Binding to          HER-2-overexpressing Breast Cancer Cell or Tissue and Use Thereof <130> P09-B203 <160> 33 <170> Kopatentin 1.71 <210> 1 <211> 39 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer M11, S4 <400> 1 ucuagcucuc cucuagagug ggccucuaga guuugacug 39 <210> 2 <211> 39 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer S16 <400> 2 ucagcucucu ucuagagugg gccucuagag uuugacugg 39 <210> 3 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer M9 <400> 3 agagagggau uggauaggcg ucugcguguc ucucugccac uacuaugggg 50 <210> 4 <211> 50 <212> RNA <213> Artificial Sequence <220> RNA aptamers M2, M5, M29 and S1 <400> 4 agagagagau uggauaggcg ucugcguguc ucucugccac uacuaugggg 50 <210> 5 <211> 55 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer M8 <400> 5 ucgagugggc cuugagauug gauaacuggc gugucucu gccacuacua ugggg 55 <210> 6 <211> 55 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer S15 and S17 <400> 6 uggaugggga gauccguuga guaagcgggc gugucucu gccacuacua ugggg 55 <210> 7 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer S6 <400> 7 uggaugggga gauccguuga guaagcgggc gugucucu gccgccuugc uaugggg 57 <210> 8 <211> 60 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer M1 and S14 <400> 8 guaagacgag guucgccggc cucgcgauug gauuugcugg ucagucugcu uacuaugggg 60                                                                           60 <210> 9 <211> 57 <212> RNA <213> Artificial Sequence <220> RNA aptamers M16, S5, S10 and S27 <400> 9 guaagacgag guucgcccgc cucgcgauug gauuugcugg ucagucugcu uacuaug 57 <210> 10 <211> 39 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer S9 <400> 10 uucgaauggu uccucgucuu ccaagaucuu acuaugggg 39 <210> 11 <211> 46 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer M13 <400> 11 cuagugggcc uaggauugua ggcgagucuc cuuucuuacu augggg 46 <210> 12 <211> 40 <212> RNA <213> Artificial Sequence <220> <223> RNA aptamer S13 <400> 12 aagcaacucc ugccuuucua gcugcaaagu gggccuuugu 40 <210> 13 <211> 28 <212> RNA <213> Artificial Sequence <220> <223> Common region of aptamer <400> 13 ucuagagugg gccucuagag uuugacuu 28 <210> 14 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> Common region of aptamer <400> 14 gcgugucucu cugccacuac uaugggg 27 <210> 15 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> Common region of aptamer <400> 15 gcgugucucu cugccgccuu gcuaugggg 29 <210> 16 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> Common region of aptamer <400> 16 gcgugucucu cugcc 15 <210> 17 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> Common region of aptamer <220> <221> misc_feature <222> (2) <223> R means A or G. <400> 17 urcuaugggg 10 <210> 18 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> ssDNA library for Cell-SELEX <400> 18 ataccagctt attcaattnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnag 60 atagtaagtg caatct 76 <210> 19 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> N40 upstream primer <400> 19 ggtaatacga ctcactatag ggagatacca gcttattcaa tt 42 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> N40 downstream primer <400> 20 agattgcact tactatct 18 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TAMRA-labeled 3 'primer <400> 21 agattgcact tactatct 18 <210> 22 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> cDNA coding RNA aptamer M11, S4 <400> 22 tctagctctc ctctagagtg ggcctctaga gtttgactg 39 <210> 23 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> cDNA coding RNA aptamer S16 <400> 23 tcagctctct tctagagtgg gcctctagag tttgactgg 39 <210> 24 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> cDNA coding RNA aptamer M9 <400> 24 agagagggat tggataggcg tctgcgtgtc tctctgccac tactatgggg 50 <210> 25 <211> 50 <212> DNA <213> Artificial Sequence <220> CDNA coding RNA aptamers M2, M5, M29 and S1 <400> 25 agagagagat tggataggcg tctgcgtgtc tctctgccac tactatgggg 50 <210> 26 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> cDNA coding RNA aptamer M8 <400> 26 tcgagtgggc cttgagattg gataactggc gtgtctctct gccactacta tgggg 55 <210> 27 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> cDNA coding RNA aptamer S15 and S17 <400> 27 tggatgggga gatccgttga gtaagcgggc gtgtctctct gccactacta tgggg 55 <210> 28 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> cDNA coding RNA aptamer S6 <400> 28 tggatgggga gatccgttga gtaagcgggc gtgtctctct gccgccttgc tatgggg 57 <210> 29 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> cDNA coding RNA aptamer M1 and S14 <400> 29 gtaagacgag gttcgccggc ctcgcgattg gatttgctgg tcagtctgct tactatgggg 60                                                                           60 <210> 30 <211> 57 <212> DNA <213> Artificial Sequence <220> CDNA coding RNA aptamers M16, S5, S10 and S27 <400> 30 gtaagacgag gttcgcccgc ctcgcgattg gatttgctgg tcagtctgct tactatg 57 <210> 31 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> cDNA coding RNA aptamer S9 <400> 31 ttcgaatggt tcctcgtctt ccaagatctt actatgggg 39 <210> 32 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> cDNA coding RNA aptamer M13 <400> 32 ctagtgggcc taggattgta ggcgagtctc ctttcttact atgggg 46 <210> 33 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> cDNA coding RNA aptamer S13 <400> 33 aagcaactcc tgcctttcta gctgcaaagt gggcctttgt 40

Claims (19)

An RNA plasmid represented by any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOS: 3 to 8, 10 and 11 and capable of specifically binding to HER-2 overexpressing breast cancer cells or tissues.
delete delete delete delete The method according to claim 1, wherein the hydroxyl group at the 2'-position of the ribosome of at least one kind of nucleotide contained in the plummeter is any one of a hydrogen atom, a fluorine atom, -O-alkyl group, -O- Lt; RTI ID = 0.0 &gt; RNA &lt; / RTI &gt;
3 to 8, 10 and 11, and is capable of specifically binding to HER-2.
delete delete delete delete 8. The method of claim 7, wherein the hydroxyl group at the 2 'position of the ribosomes of at least one kind of nucleotide contained in the plummeter is any one of a hydrogen atom, a fluorine atom, an -O-alkyl group, Lt; RTI ID = 0.0 &gt; RNA &lt; / RTI &gt;
A method for detecting breast cancer cells or breast cancer tissue, comprising contacting the squamometer of claim 1 or 6 with a separate biological sample.
14. The detection method according to claim 13, wherein the sample is selected from breast tissue, breast cells, blood, serum, plasma, saliva, sputum and urine.
A composition for diagnosing breast cancer, comprising the composition of claim 1 or 6.
A sensor for diagnosing breast cancer, wherein the aptamer according to claim 1 or 6 is fixed.
A kit for diagnosing breast cancer, which comprises the squamometer of claim 1 or 6.
7. A composition for treating breast cancer, comprising the composition of claim 1 or 6.
7. A breast cancer-specific drug delivery composition containing the plaster of claim 1 or 6.

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