阅读说明：本技术 使用适配体的生物分子成像方法 (Biomolecule imaging method using aptamers ) 是由 李仲桓 金钟仁 林钟勋 李钟旭 金镇羽 于 2018-04-25 设计创作，主要内容包括：本发明涉及用于对肿瘤疾病区域成像的组合物,包括荧光标记或放射性同位素标记的ERBB2适配体,其中用放射性同位素或荧光染料标记的ERBB2适配体用于对肿瘤区域进行体内成像。(The present invention relates to a composition for imaging a tumor disease region comprising a fluorescently labeled or radioisotope labeled ERBB2 aptamer, wherein ERBB2 aptamer labeled with a radioisotope or fluorescent dye is used for in vivo imaging of a tumor region.)

1. A composition for imaging a tumor disease region comprising a HER 2-specific ERBB2 aptamer comprising: a DNA sequence selected from the group consisting of SEQ ID NOs 1 to 35 of the sequence listing, wherein said aptamer is labeled with a radioisotope or a fluorescent dye.

2. The composition of claim 1, wherein the aptamer comprises a DNA sequence having the following SEQ ID NO: AP001-24 or SEQ ID NO: AP001-25:

AP001-24:5 '-A6G 66A GAG 666GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A-3', and

AP001-25:5 '-6 CC 6GG CA 6G 66 CGA 6GG AGG CC 666G a66ACA GCC CAG A-3', wherein 6 is NapdU [5- (N-naphthamide) -2 '-deoxyuridine ], a ═ 2' -deoxyadenosine, G ═ 2 '-deoxyguanosine, and C ═ 2' -deoxycytidine.

3. The composition of claim 1, wherein the aptamer is a chemically modified aptamer at least one of a 5 '-terminal position and a 3' -terminal position.

4. The composition of claim 3, wherein the modification is by cholesterol or PEG (polyethylene glycol).

5. The composition of claim 3, wherein the modification is by inverted deoxythymidine (idT), Locked Nucleic Acid (LNA), 2' -methoxynucleotide, 2' -aminonucleotide, or 2' F-nucleotide.

6. The composition of claim 1, wherein the radioisotope is selected from the group consisting of¹⁸F、³²P、¹²³I、⁸⁹Zr、⁶⁷Ga、²⁰¹Tl and¹¹¹In-111。

7. the composition of claim 6, wherein the radioisotope is¹⁸F。

8. The composition of claim 1, wherein the fluorescent dye is a cyanine fluorescent dye.

9. The composition of claim 8, wherein the fluorescent dye is Cy 5.

10. A method of providing cancer diagnostic information or cancer metastasis diagnostic information, comprising:

reacting a biological sample of a patient with a labeled aptamer according to any one of claims 1 to 9;

measuring the extent of binding of the aptamer in the biological sample of the patient; and

comparing the extent of binding of the aptamer in the biological sample of the patient to the extent of binding of the aptamer in its normal sample.

Technical Field

The present invention relates to a biomolecule imaging method using aptamers, and more particularly, to a method of obtaining images by using aptamers labeled with isotopes and binding them to a cell line expressing human epidermal growth factor receptor 2(HER 2).

Background

The source of the aptamer comes from latin, "aptus" and greek "meros. Aptamers are single-stranded nucleic acids having a DNA sequence consisting of about 20 to 90 bases. Aptamers with high specificity for and affinity for target molecules are typically screened by artificial evolution methods, such as in vitro SELEX (systematic evolution of ligands by exponential enrichment) as aptamer mining techniques. Thus, aptamers are considered to be very suitable agents for determining or discovering the extent of expression of a particular molecule targeted by the aptamer. Aptamers have advantages over antibodies in some respects, such as reduced production costs, ease of synthesis, low toxicity, no immune response, no generation of aptamers in animal systems other than antibodies, and the like. Aptamers are relatively newly developed reagents in the diagnostic field. A number of aptamers have been developed against a variety of targets, including thrombin, nucleolin, PSMA, TNC, and proteins of viral origin. In the therapeutic field, VEFG target aptamers were developed and approved by the FDA in 2004 as age-related macular degeneration therapeutics. Recently, such various types of aptamers are being developed in preclinical and clinical stages, and a large number of experiments related to diagnosis and treatment are being conducted.

HER2 is a well-known cancer gene in the art that is increased or overexpressed in about 15 to 30% of breast cancers. Furthermore, this is a factor associated with a high recurrence rate and poor prognosis of different cancers. HER2 activates two signaling systems, including the MAPK pathway that promotes cell proliferation and the PI3K-AKT pathway that increases cancer cell survival. Thus, the above factors are highly preferred targets for the treatment of cancer. In this regard, trastuzumab and pertuzumab for targeting HER2 currently exist as therapeutic monoclonal antibodies well known and available in the art and have been found to be effective in clinical applications. Heretofore, several DNA/RNA aptamers targeting HER2 were disclosed by the traditional SELEX method and cell-based SELEX. Furthermore, pharmaceutical examples exploiting the cancer suppressing properties of the HER2 aptamers have recently been reported.

Meanwhile, the molecular image may be a non-invasive method capable of visualizing biochemical events on the cellular molecular level of living cells or tissues or objects in real time without causing damage. Aptamers modified to magnetic nanomaterials or fluorescent materials may be provided as preferred substances for targeted fluorescence imaging or Magnetic Resonance Imaging (MRI). Several in vivo MRI studies have shown that cancer can be effectively targeted in mice with cancer. However, PET is clearly more advantageous diagnostically than anatomical techniques such as Computed Tomography (CT) and MRI, since metabolic changes occur before anatomical changes. In clinical applications, PET is widely used in basic research and preclinical fields. For example, PET can be used to verify or validate analysis of new radiation therapy, treatment efficacy of new therapeutic agents, and in vivo distribution of drugs. Advantages of PET may include probe depth from preclinical to clinical trials, excellent sensitivity, quantitative data, and convertibility (i.e., stage progression). That is, PET is a representative molecular imaging device that can detect biochemical changes in a target level of a living biomolecule and has high sensitivity, and thus is widely used for applications including basic science and preclinical fields. Targeting cancer using aptamers is one biomolecule imaging technique that has been proposed in recent years, for example, many researchers, including Hicke et al, have adopted aptamers in molecular imaging. They will already^99mTC bonded toTTA1, which is bound to tenascin C, an extracellular protein, by a covalent bond, and then the cancer is imaged in vivo using a gamma camera. Since then, PET imaging has also been performed by other groups of researchers.

However, the implementation of PET imaging using HER 2-specific ERBB2 aptamers has not been disclosed.

Disclosure of Invention

[ problem ] to provide a method for producing a semiconductor device

Aptamers are one type of nucleic acid and are materials with high specificity and affinity for target molecules. It is an object of the present invention to provide in vivo molecular imaging of aptamers labeled with radioisotopes or fluorescent dyes.

[ technical solution ] A

Fig. 1 is a schematic diagram showing the mechanism of a radioisotope or fluorescently labeled ERBB2 aptamer.

According to the present invention, HER2 aptamers labelled with a radioisotope or fluorescent dye are used for in vivo imaging.

[ PROBLEMS ] the present invention

In flow cytometry analysis, the ERBB2 aptamer bound hardly to MDA-MB231 cell line that did not express HER2, but had a high affinity for BT474 as a HER2 expressing cell line. Similarly, from images obtained by confocal microscopy, it was observed that aptamers bound to breast cancer cell lines expressing HER2, while showing only minimal binding to cells not expressing HER 2. Molecular images of positron emission tomography of mice transplanted with the BT474 cancer cell line in vivo have been demonstrated¹⁸The uptake of the F-labelled HER2 specific ERBB2 aptamer was significantly increased. ERBB2 aptamers may preferentially bind to HER 2-expressing breast cancer cell lines both in vivo and in vitro, because HER2 structures may be recognized on the cell surface.

Using radioactive isotopes (e.g.¹⁸F) Or a fluorescent dye-labeled ERBB2 aptamer can recognize the expression of HER2 in human breast cancer cells and achieve sufficient visualization. These results suggest the targeted therapeutic application of ERBB2 aptamers to HER2 positive breast cancer cells using such isotopes or fluorochromes labeledPotential methods of application for treating the same.

Drawings

Fig. 1 is a schematic diagram showing the mechanism of a radio-or fluorescently-labeled ERBB2 aptamer.

Fig. 2 shows the results of analysis of R- [ ERBB2 aptamer ] -X-hy (bp) -Cy5 using Typhoon FLA 70003% agarose gel, R- [ ERBB2 aptamer ] -X-hy (bp) -Cy5 being the product obtained by hybridization of R- [ ERBB2 aptamer ] -ODN-X (R ═ H, cholesterol or PEG, and X ═ H or idT) with ccodn-Cy 5.

FIG. 3 shows the results of identifying complementary base pairing between cholesterol- [ AP001-24] -ODN-idT or cholesterol- [ AP001-24] -ODN aptamer and fluorescently labeled cODN (cODN-Cy5) using 3% agarose gel at 50 ℃, 55 ℃ and 60 ℃ (black: aptamer, red: cODN-Cy 5).

FIG. 4 shows the results of identifying complementary base pairs between cholesterol- [ AP001-24] -ODN-idT, cholesterol- [ AP001-24] -ODN, PEGylated- [ AP001-25] -ODN-idT or PEGylated- [ AP001-25] -ODN aptamer and fluorescently labeled cODN (cODN-Cy5) using agarose gel heated to 95 ℃.

Figure 5 shows confocal image results of KPL4, N87 and SK-BR cell lines treated with R- [ ERBB2 aptamer ] -X-hy (bp) -Cy5, including confocal microscopy images of [ AP001-24] -hy (bp) -Cy5 and [ AP001-25] -hy (bp) -Cy5 aptamers in HER2 positive cell lines, in particular: (a) treating KPL4, HER2 positive breast cancer cell lines with Cy-labeled aptamers; (b) treatment with the same aptamer in the N87 cancer cell line; (c) the same aptamers were used for treatment in the SK-BR-3 cancer cell line (labeled DAPI: blue, Cy 5-aptamer: red).

FIG. 6 shows the results of FACS analysis of KPL4, N87 and SK-BR cell lines treated with R- [ ERBB2 aptamer ] -X-hy (bp) -Cy5, respectively.

In this regard, Table 3 shows the R- [ ERBB2 aptamers]-ODN-X (R ═ H, cholesterol or PEG, and X ═ H or idT) and crodn-L-F¹⁸(L ═ linker)) consisting of the R- [ ERBB2 aptamer]-X-hy(bp)-L-F¹⁸And (4) showing.

FIG. 7 shows [ AP001-24]]-hy(bp)-L-F¹⁸Results of microPET images of (a).

FIG. 8 shows [ AP001-24]]-idT-hy(bp)-L-F¹⁸Results of microPET images of (a).

FIG. 9 shows cholesterol- [ AP001-24]-hy(bp)-L-F¹⁸Results of microPET images of (a).

FIG. 10 shows cholesterol- [ AP001-24]-idT-hy(bp)-L-F¹⁸Results of microPET images of (a).

FIG. 11 shows PEGylated- [ AP001-24]-hy(bp)-L-F¹⁸Results of microPET images of (a).

FIG. 12 shows PEGylated- [ AP001-24]-idT-hy(bp)-L-F¹⁸Results of microPET images of (a).

FIG. 13 is the expression of [ AP001-24] in mice with KPL4 cancer]-hy(bp)-L-F¹⁸、[AP001-24]-idT-hy(bp)-L-F¹⁸Cholesterol- [ AP001-24]-hy(bp)-L-F¹⁸And cholesterol- [ AP001-24]-idT-hy(bp)-L-F¹⁸The comparison picture of (1).

Figure 14 shows the extent of HER2 expression in human breast cancer cell lines determined by western blot.

Figure 15 shows flow cytometric analysis of breast cancer cell lines using HER2 antibody and ERBB2 aptamer (AP001-25), wherein: (a) the viscosity table shows the fluorescence signals from the antibodies for BT474(HER2 positive cell line) and MDA-MB231(HER2 negative cell line) with ERBB2 aptamer (AP001-25) (red) or control DNA sequence (blue); (b) included are graphs for flow cytometric analysis of two cell lines using antibody, ERBB2 aptamer (AP001-25), and negative control.

Figure 16 shows confocal microscope images of the selected ERBB2 aptamer (AP001-25) in HER2 positive cell line, in particular: (a) treating BT474, HER2 positive breast cancer cell lines with FITC labeled aptamer; and (b) treated with the same ERBB2 aptamer (AP001-25) in the MDA-MB231 cancer cell line (marker DAPL: blue, FITC-ERBB2 aptamer: green).

FIG. 17 shows a representation of BT474 cancer in mice¹⁸F-labeled ERBB2 aptamer { [ AP001-25 { []-hy(bp)-L-F¹⁸In vivo PET image of (1) }, wherein Hy (bp) represents ODN/cODN as a hybridHybridization (base pairing).

FIG. 18 shows in mice with cancer¹⁸F-labeled ERBB2 aptamer { [ AP001-25 { []-hy(bp)-L-F¹⁸Results of in vivo distribution studies where data (% ID/g) are given as activity injections per gram of tissue in percent (error bars, SD (N-4)).

FIG. 19 shows representatives in mice with HER2 positive and negative cancers, respectively¹⁸F-labeled ERBB2 aptamer { [ AP001-25 { []-hy(bp)-L-F¹⁸In vivo PET image of, in particular: (a) HER2 overexpressed BT474 cancer (left axilla); (b) HER2 negative MDA-MB231 cancer (right axilla), wherein (a) shows more uptake than in (b); (c) use of¹⁸The F-labeled ERBB2 aptamer calculated the Counts Per Minute (CPM) and injected amount per gram (% ID/g) into the tumor tissue.

Figure 20 shows H & E and IHC staining of HER2 (original magnification 400-fold).

Detailed Description

The ERBB2 aptamer that specifically binds to the HER2 receptor associated with breast cancer used in the present invention has a DNA sequence of 5 '-TCAGCCGCCAGCCAGTTC- [ core sequence ] -GACCAGAGCACCACAGAG-3', wherein the number '6' in the core sequence or 'n' in the attached DNA sequence list represents naphthyl du (naptyldu).

[ Table 1]

6-NapdU [5- (N-naphthamide) - [0070 ═]2' -deoxyuridine](NapdU[5-(N-Naphthyl carboxyamide)-[0070]2’-deoxyuridine]). In the context of the present invention, radioactive isotopes ("radioisotopes") are used, for example¹⁸F、³²P、¹²³I、⁸⁹Zr、⁶⁷Ga、²⁰¹Tl and¹¹¹in-111, or fluorescent dyes such as cyanine fluorescent dyes like Cy3, Cy5, Cy7, etc. labeled HER2 aptamers are used for In vivo imaging. In embodiments of the invention, target specificity and potential clinical applications of in vivo molecular imaging have been evaluated using either radioisotope or fluorochrome labeled ERBB2 aptamers.

ERBB2 aptamer for human epidermal growth factor receptor 2(HER2)¹⁸F-fluoride isotope labeling. To confirm entry of the aptamer into HER2 expressing cancer cell lines, the aptamer was compared to control aptamers by flow cytometry and confocal microscopy. To pair¹⁸Positron emission tomography was performed on the F-labeled HER 2-specific ERBB2 aptamer, thereby obtaining biomolecular images over time of mice engrafted with BT474 or KPL4 cells.

[ preferred embodiment of the invention ]

Hereinafter, the present invention will be described in detail.

Cell culture

Human breast cancer cell lines expressing HER2, such as BT474, KPL4, N89 and SK-BR-3, were used for in vitro and in vivo experiments. In addition, human breast cancer cell line MDA-MB231 was used as a control group. All cell lines were purchased from ATCC and cultured and maintained in MEM medium containing 10% FBS.

Cell lysis, Western blotting

To extract intracellular proteins, cell lysates containing protease inhibitors were incubated on ice for 30 minutes. The resulting cell lysate was purified by centrifugation at 4 ℃ for 20 minutes. For protein quantification, cell lysates were quantified by the Bradford method, and then 30 μ g of protein extract was separated from each sample by electrophoresis using 10% SDS-PAGE. Then, the resultant was transferred onto a nitrocellulose membrane, and photosensitization was performed on an X-ray film with ECL using HER2 antibody and a control group, i.e., β -actin antibody, as probes.

ERBB2 aptamer synthesis

The DNA sequences of HER2- (+) targeting ERBB2 aptamers are shown in table 2 below.

[ Table 2]

The binding affinity (Kd) of the RBB2 aptamer, specifically AP001-24, to the target was 3.1nM and the binding affinity of AP001-25 to the target was 0.9 nM.

Here, 6 represents NapdU [5- (N-naphthamide) -2 '-deoxyuridine ], a ═ 2' -deoxyadenosine, G ═ 2 '-deoxyguanosine, and C ═ 2' -deoxycytidine, which are represented by the following formulae.

For aptamer hybridization, synthesis of ODN (5'-CAGCCACACCACCAG-3') comprising perfectly matched sequences, i.e., 3' to each of the ERBB2 aptamers { [ AP001-24] and [ AP001-25] }, was performed.

[ AP001-24] -ODN Synthesis

5'- [6CC 6GG CA 6G 66 CGA 6GG AGG CC 666G A66ACA GCC CAG A ] -CAG CCA CAC CAC CAG-3' { [ AP001-24] -ODN } was synthesized as follows.

Aptamer synthesis was performed by a phosphoramidite coupling reaction by solid phase synthesis method, and after synthesis, the product was synthesized at 70 ℃ under tert-butylamine: methanol: the reaction was carried out for 5 hours in a water (1:1:2v/v/v) solution to obtain an intact aptamer through cleavage and deprotection processes, which was then dried. The synthesized aptamers were separated by HPLC [ C18 column (Waters, Xbridge OST C1810X 50mm,260nm ], and then molecular weight measurements were performed by ESI MS mass spectrometry (Qtrap2000, ABI).

The 11 th aptamer (SEQ ID NO:11) in Table 1 corresponds to AP 001-24.

[ AP001-25] -ODN Synthesis:

5'- [ A6G 66A GAG 666GCC 6GA G6G CC6 CGC AAG GGC G6A ACA ] -CAG CCA CAC CAC CAG-3' { [ AP001-25] -ODN } was synthesized by the same synthetic procedure as described in the section above for the synthesis of { [ AP001-24] -ODN }.

The 12 th aptamer in Table 1 (SEQ ID NO:7) corresponds to AP 001-25.

In the same manner, each aptamer, i.e., CAG-3' { each aptamer in Table 1-ODN (SEQ ID NO: 1-35) }, was synthesized by the same procedure as described in the section for { [ AP001-24] -ODN } synthesis.

[ AP001-24] -ODN-idT Synthesis:

5'- [6CC 6GG CA 6G 66 CGA 6GG AGG CC 666G A66ACA GCC CAGA ] -CAG CCA CAC CAC CAG-idT-3' { [ AP001-24] -ODN-idT } was synthesized using idT (inverted dT) CPG (Glen,20-0302-10) by the same procedure as described in the section for { [ AP001-24] -ODN } synthesis.

[ AP001-25] -ODN-idT Synthesis:

5'- [ A6G 66A GAG 666GCC 6GA G6G CC6 CGC AAG GGC G6A ACAA ] -CAG CCA CAC CAC CAG-idT-3' { [ AP001-25] -ODN-idT } was synthesized using idT CPG (Glen,20-0302-10) by the same procedure as described in the section above for { [ AP001-24] -ODN } synthesis.

Cholesteryl- [ AP001-24] -ODN Synthesis:

5 '-cholesteryl- [6CC 6GG CA 6G 66 CGA 6GG AGG CC 666G A66ACA GCC CAG A ] -CAG CCA CAC CAC CAG-3' { cholesteryl- [ AP001-24] -ODN }, was synthesized using cholesterol-PA (Glen,10-1976-90) by the same procedure as described in the section above for the synthesis of { [ AP001-24] -ODN }.

Cholesteryl- [ AP001-25] -ODN Synthesis:

5 '-cholesteryl- [ A6G 66A GAG 666GCC 6GA G6G CC6 CGC AAGGGC G6A ACA A ] -CAG CCA CAC CAC CAG-3' { cholesteryl- [ AP001-25] -ODN }, was synthesized using cholesterol-PA (Glen,10-1976-90) by the same procedure as described in the section above for { [ AP001-24] -ODN } synthesis.

Cholesteryl- [ AP001-24] -ODN-idT Synthesis:

5 '-cholesteryl- [6CC 6GG CA 6G 66 CGA 6GG AGG CC 666G A66ACA GCC CAG A ] -CAG CCA CAC CAC CAG-idT-3' { cholesteryl- [ AP001-24] -ODN-idT }, was synthesized using idT CPG (Glen,20-0302-10) and cholesteryl-PA (Glen,10-1976-90) by the same procedure as described in the section above for { [ AP001-24] -ODN } synthesis.

Cholesteryl- [ AP001-25] -ODN-idT Synthesis:

synthesis of 5 '-cholesteryl [ A6G 66A GAG666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A ] -CAG CCA CAC CAC CAG-idT-3' { cholesteryl- [ AP001-25] -ODN-idT }, by the same procedure as described in the section above for { [ AP001-24] -ODN } Synthesis, using idT CPG (Glen,20-0302-10) and cholesterol-PA (Glen, 10-1976-90).

PEGylated- [ AP001-24] -ODN Synthesis:

5 '-PEGylated- [6CC 6GG CA 6G 66 CGA 6GG AGGCC 666G A66ACA GCC CAG A ] -CAG CCA CAC CAC CAG' { PEGylated- [ AP001-24] -ODN }, was synthesized using polyethylene glycol 2000 CED PA (Chemgenes, CLP-2119) by the same procedure as described in the section above for { [ AP001-24] -ODN } synthesis.

PEGylated- [ AP001-25] -ODN Synthesis:

5 '-PEGylated- [ A6G 66A GAG 666GCC 6GA G6GCC6 CGC AAG GGC G6A ACA A ] -CAG CCA CAC CAC CAG-3' { PEGylated- [ AP001-25] -ODN }, was synthesized using polyethylene glycol 2000 CED PA (ChemGenes, CLP-2119) by the same procedure as described in the section above for { [ AP001-24] -ODN } synthesis.

PEGylated- [ AP001-24] -ODN-idT Synthesis:

synthesis of 5 '-PEGylated- [6CC 6GG CA 6G 66 CGA 6GG AGG CC 666G A66ACA GCC CAG A ] -CAG CCA CAC CAC CAG-idT-3' { PEGylated- [ AP001-24] -ODN-idT } by the same procedure as described in the section above for { [ AP001-24] -ODN } Synthesis using idT CPG (Glen,20-0302-10) and polyethylene glycol 2000 CED PA (ChemGenes, CLP-2119).

PEGylated- [ AP001-25] -ODN-idT Synthesis:

synthesis of 5 '-PEGylated- [ A6G 66A GAG 666GCC 6GA G6G CC6 CGC AAG GGC G6 CC 396 6A ACA ] -CAG CCA CAC CAC CAG-3' { PEGylated- [ AP001-25] -ODN-idT }, by the same procedure as described in the section above for { [ AP001-24] -ODN } Synthesis, using idT CPG (Glen,20-0302-10) and polyethylene glycol 2000 CED PA (ChemGenes, CLP-2119).

cODN (complementary oligonucleotide) Synthesis of Cy5 conjugated cODN [ cODN-Cy5 ]:

the lower panels represent cODN-Cy5 and cODN-L-F¹⁸(L ═ linker) structure and synthesis thereof.

5 '-Cy 5- [ CTGGTGGTGTGGCTG ] -3' [ cODN-Cy5] was synthesized using Cy5-PA (Glen,10-5915-10) by the same procedure as described in the section above for the synthesis of { [ AP001-24] -ODN }.

Formation of cy 5-labeled ERBB2 aptamers

Table 3 shows the hybrid structure of R- [ ERBB2 aptamer ] -ODN-X (R ═ H, cholesterol, or PEG, and X ═ H or idT) and ctodn-Cy 5, which is represented by [ ERBB2 aptamer ] -X-hy (bp) -Cy 5.

[ Table 3]

A Cy 5-labeled ERBB2 aptamer, { R- [ ERBB2 aptamer ] -X-hy (bp) -Cy5}, was prepared in the following manner.

First, equimolar quantities of cODN-Cy5 and [ ERBB2 aptamer]ODN in annealing buffer (PBS). Here, MgCl is added₂Is controlled to a final concentration of 10 mM. The reaction product was left at 95 ℃ for 5 minutes and then slowly cooled at room temperature. cODN-Cy5 and [ ERBB2 aptamers]Hybridization efficiency of ODN was assessed by electrophoresis (Typhoon FLA 70003% agarose gel assay) and HPLC (XBridge OST analytical column (2.5 μm,4.6X 50mm, Waters,254nm,0.1 MTEAA/acetonitrile). FIG. 2 shows analysis of R- [ ERBB2 aptamer using Typhoon FLA 70003% agarose gel assay]Results of-X-hy (bp) -Cy5, R- [ ERBB2 aptamer]-X-hy (bp) -Cy5 is through [ ERBB2 aptamer]-ODN-X (R ═ H, cholesterol or PEG, and X ═ OH or idT) product hybridized to cod-Cy 5.

Complementary base pairing between a synthetic oligonucleotide labeled with a fluorescent dye, Cy5 (cODN-Cy5) and [ ERBB2 aptamer ] -ODN was evaluated. Cholesterol- [ AP001-24] -ODN-idT or cholesterol- [ AP001-24] -ODN and cODN-Cy5 were mixed at a ratio of 1: after mixing at ratio 1, the temperature was maintained and the ingredients were combined at 55, 60 and 65 ℃. To confirm binding, electrophoresis was performed in a 3% agarose gel, followed by fluorescence imaging of Cy5 by FLA 5000. Then, the whole aptamer was stained with EtBr and UV-imaged. The results are shown in fig. 3. To compare complementary base pairing between cholesteryl- [ AP001-24] -ODN-idT, cholesteryl- [ AP001-24] -ODN, PEGylated [ AP001-25] -ODN-idT, or PEGylated [ AP001-25] -ODN and cODN-Cy5, each of these aptamers was coupled with cODN-Cy5 at a ratio of 1:1 and heated at 95 c for 5 minutes to bond together, and then the bonding was evaluated in the same manner as described above. Complementary base pairing between cholesteryl- [ AP001-24] -ODN-idT, cholesteryl- [ AP001-24] -ODN, PEGylated- [ AP001-25] -ODN-idT, or PEGylated- [ AP001-25] -ODN and cODN-Cy5 was determined with or without heating at 95 ℃ and compared with each other. The comparison results are shown in fig. 4.

Form F¹⁸Radioisotope-labeled cODN (complementary oligonucleotide) [ cODN-L-F¹⁸]

Based on the method reported in the prior art¹⁸Synthesis of F-labeled cODN (see reference 24). Generation without addition of vector in the Synthesis apparatus (Tracerlab FXFN, GE Healthcare, Milwaukee, Wis., USA)¹⁸After F fluoride, it was reacted with methanesulfonate (at 100 ℃ for 10 min) and then purified by using HPLC¹⁸F-fluoro-PEG-azide(s) ((¹⁸F-FPA). 1M N, N-diisopropylethylamine in acetonitrile (10mL) and 100mM copper iodide in acetonitrile (20mL) were added to a 5 '-hexynyl complementary oligonucleotide (5' -hex-cODN; 200mg), to which was further added¹⁸F-FPA (750e1100MBq) was then subjected to click chemistry (20 min at 70 ℃). Synthesized¹⁸F-labeled cODN (cODN-L-F)¹⁸) Purification was performed using HPLC H (Xbridge OST C1810 x50mm, eluent 5:95 to 95:5 acetonitrile/0.1M TEAA at 20 min, flow rate 5mL/min, and UV (254 nm)).

Form F¹⁸Radioisotope-labeled ERBB2 aptamer { R- [ ERBB2 aptamer]-X-hy(bp)-L-F¹⁸]

Table 4 below shows the R- [ ERBB2 aptamers]-ODN-X (R ═ H, cholesterol or PEG, and X ═ OH or idT) and crodn-L-F¹⁸(L ═ linker) hybrid structures composed of [ ERBB2 aptamers]-X-hy(bp)-L-F¹⁸And (4) showing.

[ Table 4]

Preparation of F in the following manner¹⁸Radioisotope-labeled ERBB2 aptamer { R- [ ERBB2 aptamer]-X-hy(bp)-L-F¹⁸}。

First, equimolar cODN-L-F¹⁸And [ ERBB2 aptamer]ODN in annealing buffer (PBS). Here, MgCl is added₂Is controlled to a final concentration of 10 mM. The reaction product was left at 95 ℃ for 5 minutes and then slowly cooled at room temperature. cODN-L-F¹⁸And [ ERBB2 aptamer]The hybridization efficiency of ODN was evaluated using HPLC (Xbridge OST analytical column (2.5 μ M,4.6X 50mm, Waters,254nm,0.1M TEAA/acetonitrile). The products were pooled at a hybridization rate of 98% or more.

Confocal microscope

BT474, KPL4, N87, SK-BR-3, and MDA-MB231 cell lines were dispensed on cover slips and incubated overnight. When approximately 80% of the cell lines were grown, the grown cells were carefully washed and incubated by treatment with the fluorescently labeled ERBB2 aptamer { R- [ ERBB2 aptamer ] -hy (bp) -Cy5} at a concentration of 250 mM. After incubation, the product was washed carefully and then DAPI-loaded medium was loaded onto slides. Then, the fluorescence was observed by LSM700 confocal microscope. The microscope settings were as follows: FITC visualization with 488 laser; excitation and emission were observed using BP 490-555; 639 laser for texas red; and emission was observed using a LP640 filter.

Breast cancer cell lines that overexpress ERBB2, such as KPL4, N87, and SK-BR-3, were dispensed on coverslips and incubated overnight in the same manner as in previous experiments. When approximately 80% of the cell lines were grown, the grown cells were carefully washed and incubated by treatment with samples prepared using Cy5 fluorescently labeled ODN bound to ERBB2 aptamer using complementary base pairing. After incubation, the product was washed carefully and then DAPI-loaded medium was loaded onto slides. Then, fluorescence was observed by LSM700 confocal microscope.

The observation results are shown in fig. 5.

Flow cytometry

The specificity of the ERBB2 aptamer was verified by fluorescence activated cell isolation method using flow cytometry system (BD Biosciences). Appropriate numbers of BT474, KPL4, N87, SK-BR-3, or MDA-MB231 cancer cell lines were sub-cultured on Petri dishes to grow to approximately 80%. The grown cells were trypsinized and washed with PBS, and then fluorescently labeled ODN was bound to ERBB2 aptamer through complementary bases generated according to temperature. The cells are treated with the binding-completed sample. ERBB2 aptamer { R- [ ERBB2 aptamer ] -hy (bp) -Cy5} and a control group, namely an antibody containing 1% FES were treated at 4 ℃ for 30 minutes, respectively. The fully treated samples were washed and then bound ERBB2 aptamer was measured and analyzed by fluorescence activated cell isolation.

The results of the above measurements and analysis are shown in fig. 6.

In vivo experiments

The 17 β β -estradiol precipitate was subcutaneously implanted into the lateral cervical region of a four-week-old Balb/c nude mouse to release sufficient estrogen to potentially induce cancer. After several days, BT474 or KPL4 human breast cancer cell lines were implanted subcutaneously at 7x 10 per mouse⁶In individual cells. After 3 weeks of cancer development, the growth of the cancer was measured using calipers.

KPL4 cells as a human breast cancer cell line were subcutaneously implanted into 1x 10 cells per mouse in the right shoulder of Balb/C nude mice⁵In individual cells. Thereafter, the occurrence of cancer is induced.

F¹⁸PET imaging of radioisotope-labeled ERBB2 aptamers

F is to be¹⁸Radioisotope-labeled ERBB2 aptamer was injected into mice 60 minutes later, and passed through an Inveron MicroPET scanner (Siemens, Knoxv)ille, TN, USA) for 10 minutes to obtain a static image. For F¹⁸Radioisotope-labeled ERBB2 aptamer injection, mice anesthetized with 2% isoflurane, and 7.4MBq of F¹⁸The radioisotope-labeled ERBB2 aptamer was injected into the tail vein. The obtained list mode data were converted to sinograms and reconfigured by the 3D Ordered Subset Expectation Maximization (OSEM) algorithm and then evaluated using ASIpro (Concord Microsystems inc., Knoxville, TN).

F is to be¹⁸Radioisotope-labeled ERBB2 aptamer was intravenously injected into mice with tumors grown by injection of human breast tumor cells, followed by PET using siemens' invaon PET (Knoxville, TN). The injection volume was 13.7 ± 1.1MBq (370 ± 30uCi) and a dynamic PET study was performed for 30 minutes according to a ten 1 minute image and four 5 minute image protocol. These two static studies were performed 10, 60, 90 and 120 minutes after injection, respectively. Partial quantification of PET signals was performed by AMIDE software. The images were actually obtained on a pseudocolor scale proportional to the tissue concentration (% ID/g) of the positron-labeled probe. Red represents the highest concentration, while yellow, green and blue correspond to progressively lower concentrations.

The PET images are shown in fig. 7 to 13.

Results

Validation of HER2 expression and aptamer affinity for target tumor cells

Western blotting and flow cytometry were performed to investigate HER2 expression in the breast cancer cell line BT 474. Overexpression of BT474 and SKBR3 cell lines known to overexpress HER2 due to gene amplification was confirmed by western blot analysis. Furthermore, no signal was found to be detected in the corresponding portion of the negative control cell line MDA-MB231 (FIG. 14).

As shown in figure 15, flow cytometry was used to demonstrate that the HER2 antibody binds very specifically to HER2 positive BT474 cell line. In contrast to the antibody, it can be seen that the ERBB2 aptamer was very weak in the MDA-MB231 cell line, whereas it bound strongly to it in the BT474 cell line. Furthermore, no binding of aptamers to any cell line was seen in the random oligonucleotides. These results indicate that ERBB2 aptamer preferentially binds to HER2 positive cell line, and that this binding can be achieved by recognition of HER2 structure on the surface of the cell line. In the same manner, it was observed that KPL4, which is a breast cancer cell line, strongly bound to a fluorescently labeled aptamer of SK-BR-3 cell line.

Confocal microscopy analysis

Binding of ERBB2 aptamers to cells was further assessed by confocal microscopy (fig. 16). BT474 HER2 positive breast cancer cell line was treated with aptamers. Since the ERBB2 aptamer was fluorescently labeled, fluorescence was observed at the cell surface and the HER2 structure present at the cell surface was identified. The fluorescence exhibited by the aptamer was observed along the cell membrane, whereas the MDA-MB231 cell line as a negative control group did not exhibit any fluorescence signal and was therefore determined to not include HER 2. Thus, it was observed that ERBB2 aptamers could bind to HER2 positive breast cancer cell lines, but bind minimally to HER2 negative cells. After treating the breast cancer cell lines KPL4, N87 and SK-BR-3 with ERBB2 aptamers { [ AP001-24] and [ AP001-25] }, they formed complementary base pairing with the fluorescence-labeled ODN, and then fluorescence observation was performed by the same method as the above experiment using a confocal microscope. Both of the above types of ERBB2 aptamers were found to bind well to breast cancer cell lines, with [ AP001-24] showing fluorescence on the cell surface along the cell membrane, and [ AP001-25] showing fluorescence even inside the cell.

In vivo PET imaging, in vivo distribution, immunohistochemistry

In vivo biomolecular images of mice with BT474 or KPL4 cancer were given over time, according to animal mini-PET. Referring to fig. 17, a significant increase in the uptake of the 18F-labeled HER 2-specific ERBB2 aptamer was observed in tumor tissue present in the left axilla of mice. In the 120 min image, the ERBB2 aptamer clearly marked the cancer in the horizontal and coronal images. The apparent physiological uptake in the gut and bladder may reflect that these organs are the two major release pathways of radiology.

Injection of drugs¹⁸1 hour after the F-labeled ERBB2 aptamer, in mice with cancerThe in vivo distribution was verified. After sacrifice, radiation levels in individual tissues, including cancer, were measured by gamma counter and then expressed as% ID/g (fig. 18). The measurement results are also shown in table 5 below.

[ Table 5]

In cancer¹⁸The uptake of the F-labelled ERBB2 aptamer was 0.62 ± 0.04 per hour. The studies of in vivo distribution indicate that the kidney and the intestinal tract are¹⁸Two major release pathways for the F-labelled ERBB2 aptamer.

Figure 19 shows in mice with HER2 positive and negative cancer, respectively¹⁸Image of F-labeled ERBb2 aptamer. In contrast, BT474 cancers that overexpress HER2 have higher isotope uptake than HER2 negative MSA-MB231 cancers. For semi-quantification purposes, the total activity (nCi) in the VOI (voxel or volume of interest) is calculated. As a result of comparing T/M (tumor/muscle) uptake ratios between BT474 and MDA-MB231 cell lines, HER2 overexpressed in BT474 cancer showed higher T/M ratios and a comparative image (fig. 19). In immunohistochemistry, it was demonstrated that BT474 cancers resected from individual mouse groups showed high expression of HER2, whereas MDA-MB231 cells had lower HER2 expression (fig. 20). In contrast, BT474 cancer cells (upper panel) were observed to show higher HER2 staining in the cell membrane than the MDA-MB231 cell line (lower panel).

In accordance with the present invention, ERBB2 aptamers targeting HER2 have been successfully PET imaged in vivo. The present invention is the first case of HER2 target PET imaging using ERBB2 specific aptamers. In mice with BT474 cancer, PET images showed that ERBB2 aptamer might recognize HER2 in vivo and show the cancer relatively distinctive. Based on these results, the radiolabeled ERBB2 aptamer may be used for targeted therapy of HER2 positive breast cancer cell lines, or potentially for determining an appropriate therapeutic approach thereto.

As identified in the above embodiments, when the R- [ ERBB2 aptamer is prepared by]-ODN-X and cODN-L-F¹⁸Combined preparation of R- [ ERBB2 aptamer]-ODN-X/cODN-L-F¹⁸(in the above description by R- [ ERBB2 aptamers]-X-hy(bp)-L-F¹⁸Expressed), for example, aptamers chemically modified (i.e., protected) from R ═ H (no protecting group) and X ═ H (no protecting group) to R ═ cholesterol or PEG (polyethylene glycol) and X ═ idT (inverted deoxythymidine), LNA (locked nucleic acid), 2' -methoxynucleotide, 2' -aminonucleotide, 2' F-nucleotide, etc., for example, at the 5' end or 3' end position or both end positions, can ensure better images.

Since the increase in t can be improved due to the modification of the above-mentioned compounds_1/2(half-life) effect of blood clearance, i.e. increasing the half-life in blood in vivo, so the ERBB2 aptamer with radioisotope binding increasingly binds to tumors, thus increasing imaging efficiency [ with t when R ═ H and X ═ H_1/2If R and X are protected and modified, t is compared for 10 minutes_1/2It is increased to 1 hour to display a better image]。

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Sequence listing

<110> Neteorelbu Co

<120> PET imaging of HER2 expression Using radiolabeled aptamers

<130> 105903

<150> KR 2017/053456, KR 2018/

<151> 2017-04-26, 2018-04-

<160> 42

<170> KopatentIn 3.0

<210> 1

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-A01_ A05, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 1

angnnagagnnngccngagngccncgnaagggcgnaacaa 40

<210> 2

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-A02_ B05, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 2

nacngggcccgnnagccncnggcgcnccnncgcnngngcc 40

<210> 3

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-A03_ C05, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 3

nnancaacgcacngagggcgncagcnncnnnnnagg 36

<210> 4

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-A04_ D05, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 4

angnagagnnngccngagngccncgcaagggcgnaacag 39

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-A06_ E05, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 5

nccngncccggnnnacacaagnnaaggcagccgcnggana 40

<210> 6

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-B02_ F05, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 6

gncngaacaccgagannagcngaacgaacggnanggacgn 40

<210> 7

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-B03_ G05, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 7

nccnggcangnncganggaggccnnngannacagcccaga 40

<210> 8

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-B04_ H05, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 8

cgcgannagangaacgcacaanacccgnncngagnaaagn 40

<210> 9

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-B08_ A06, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 9

gncngaacaccgagannagccgaacgaacggnanggacgn 40

<210> 10

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-B09_ B06, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 10

gnnagacngaacgcacngagggccgcagccnancngaagg 40

<210> 11

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-B12_ C06, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 11

angnnagagnnngccngagngccncgcaagggcgnaacaa 40

<210> 12

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-C03_ E06, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 12

gncngagcancgcgnnnagccgaacgcncggngaggnagan 41

<210> 13

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-C05_ F06, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 13

ncanggcangnncganggaggccnnngannacagcccaga 40

<210> 14

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-C06_ G06, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 14

cnacacgaancaacnccccnccgcanacngaacancacaa 40

<210> 15

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-C08_ H06, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 15

nnagcaaaangccangngcgnccngncccggnnnacagc 39

<210> 16

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-C10_ A07, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 16

ngangnccccaacncagcngngaancnangcccccgccca 40

<210> 17

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-D01_ B07, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 17

cngagcggnnacnacaccaccgngagaccnnagnnacaaa 40

<210> 18

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-D02_ C07, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 18

annagangaaagcgcannccaacaacaganaancngaggg 40

<210> 19

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-D04_ E07, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 19

nnnggagngncnnacggnnggagnaancgaggangganga 40

<210> 20

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-D05_ F07, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 20

ccgnnaccnaccnccncgaccgngggngcccnnagnccca 40

<210> 21

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-D06_ G07, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 21

nccnggcangnncganggaggccnnngannacagccaga 39

<210> 22

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-D07_ H07, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 22

ccgnnaccnaccnccncgaccgngggngccnnnagnccca 40

<210> 23

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-D09_ A08, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 23

angnnagagnnngccngagngccncgcaagggcgnaacaa 40

<210> 24

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-D11_ B08, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 24

nccnggcangnncganggaggccnnngannacagcccagn 40

<210> 25

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-E04_ D08, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 25

annagangaaagcacannccaacaacaganaancngaggg 40

<210> 26

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-E11_ F08, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 26

angnnagagnnngccngagngcgncgcaagggcgnaacag 40

<210> 27

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-E12_ G08, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 27

ngagaagggcngngccnnacncaaaannngggancngaa 39

<210> 28

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-F05_ D09, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 28

nccnggnangnncganggaggccnnngannacagcccaga 40

<210> 29

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-F08_ E09, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 29

nagancncngannaggnagaacgcccnacncnaacggcag 40

<210> 30

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-F09_ F09, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 30

ngagaagggcngngccnnacncaaaannnggggancngaa 40

<210> 31

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-F11_ G09, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 31

ngagaagggcngngccnnacncaaaannnggggancngaa 40

<210> 32

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence was represented by clone No. 9-ER-N-G04_ B10, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 32

cgnccnnggngagnnngggncngagcaggagcacgngagn 40

<210> 33

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-H01_ E10, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 33

annagangaaagcacannccaacaacaganaancngaggg 40

<210> 34

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-H03_ G10, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 34

annagangaaagcacannccaacaacaganaancngaggg 40

<210> 35

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> the core sequence is represented by clone No. 9-ER-N-H09_ B11, wherein N is NapdU [5- (N-naphthamide) -2' -deoxyuridine ]

<400> 35

angnnagagncngccngagngccncgcaagggcgnaacag 40

17