Novel DNA aptamer and use thereof
阅读说明：本技术 新型dna适配体及其用途 (Novel DNA aptamer and use thereof ) 是由 金润姬 许钧 崔先一 金仁厚 于 2019-08-08 设计创作，主要内容包括：本公开涉及新型DNA适配体及其用途。具体地,本公开涉及使用Cell-SELEX从DNA文库中选择与癌细胞特异性结合的DNA适配体。本公开中经选择和优化以获得与癌细胞高结合亲和力的DNA适配体,由于其具有增强的对靶细胞和组织的靶向效率以及高血清稳定性,可有效用于癌症诊断。(The present disclosure relates to novel DNA aptamers and uses thereof. In particular, the present disclosure relates to the use of Cell-SELEX to select DNA aptamers that specifically bind to cancer cells from DNA libraries. The DNA aptamers selected and optimized in the present disclosure to obtain high binding affinity to cancer cells can be effectively used for cancer diagnosis due to their enhanced targeting efficiency to target cells and tissues and high serum stability.)
1. A DNA aptamer comprising a nucleotide sequence having at least 90% homology to the nucleotide sequence set forth in SEQ ID NO 6.
2. The DNA aptamer according to claim 1, comprising a nucleotide sequence shown as SEQ ID NO 6.
3. The DNA aptamer of claim 1, wherein the aptamer has been modified to be resistant to dnase.
4. The DNA aptamer of claim 3, wherein the modification is the replacement of the-OH group on the 2' carbon of the sugar group in one or more nucleotides with-Me (methyl), -OMe, -NH2-F (fluorine), -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -O-N-methylacetamido or-O-dimethylaminoyloxyethyl.
5. The DNA aptamer according to claim 3, wherein the modification is present in at least 10% of the nucleotides of SEQ ID NO 6.
6. The DNA aptamer according to claim 3, wherein the DNA aptamer has a nucleotide sequence as shown in SEQ ID NO 8, 12 or 14.
7. The DNA aptamer according to claim 1, which consists of a nucleotide sequence having at least 90% homology with the nucleotide sequence shown as SEQ ID NO. 4.
8. The DNA aptamer according to claim 1, consisting of the nucleotide sequence shown in SEQ ID NO. 4.
9. A composition for targeting a cancer tissue, the composition comprising the DNA aptamer according to any one of claims 1 to 8.
10. A composition for diagnosing cancer, comprising the DNA aptamer according to any one of claims 1 to 8.
11. A composition for treating cancer, comprising the DNA aptamer according to any one of claims 1 to 8.
12. The composition of claim 11, wherein the cancer is pancreatic cancer, colon cancer, liver cancer, lung cancer, brain tumor, oral cancer, ovarian cancer, or breast cancer.
13. The composition of claim 11, further comprising an anti-cancer agent coupled to the DNA aptamer.
14. The composition of claim 13, wherein the anticancer agent is one or more selected from the group consisting of MMAE (monomethylauristatin E ), MMAF (monomethylauristatin F, monomethylauristatin F), calicheamicin (calicheamicin), maytansine (mertansine), raloxib (ravtansine), tesiline (tesiline), doxorubicin (doxorubicin), cisplatin (cissplatin), SN-38, duocarmycin (duocarmycin), and pyrrolobenzodiazepine (pyrrobenozodiazepine).
15. The composition of claim 11, wherein the DNA aptamer is coupled to polyethylene glycol (PEG) or a derivative thereof, Diacylglycerol (DAG) or a derivative thereof, a dendrimer, an antibody, or a choline phosphate-containing polymer.
Technical Field
The present disclosure relates to novel DNA aptamers (dnaaptamers). In particular, the present disclosure relates to the use of Cell-SELEX to select DNA aptamers that specifically bind to cancer cells from a cancer DNA library. The present disclosure also relates to compositions comprising novel DNA aptamers that target cancerous tissues, diagnose cancer, or treat cancer. The present disclosure is based on studies conducted on original research projects of National Cancer Center (NCC) and a part of TIPS Program for Startup from the ministry of small and medium-sized enterprises and the division of the initiative enterprise.
[ item number: NCC-1210080, title: recognition of pancreatic cancer-specific metastasis factors Using Cell-SELEX ]
[ item number: NCC-1410270, title: development of novel anticancer drugs by establishing aptamer-antibody conjugation platform
[ item number: s2562351, title: development of therapeutic drugs for pancreatic cancer Using aptamer-antibody-drug conjugates
Background
An aptamer refers to a single-stranded DNA or RNA oligonucleotide having a unique three-dimensional structure and specifically binds to a target molecule in a manner similar to an antibody. In general, aptamers remain with high affinity at concentrations as low as nanomolar (nmol) to picomolar (pmol).
Aptamers are often compared to antibodies due to the target-specific binding properties of both. Compared to antibodies, aptamers are easy to produce by chemical synthesis without the use of biological processes of cells or animals, are relatively stable at high temperatures, and have advantages in terms of access to targets due to their small size. In addition, aptamers are superior to antibodies in their potential as therapeutic agents because they are easily modified during chemical synthesis and are not immunogenic and non-toxic. However, a disadvantage of aptamers is their short half-life, as they are susceptible to degradation by nucleases in vivo. This disadvantage can be overcome by various chemical modifications.
Cancer requires early detection and treatment. In particular, pancreatic cancer is one of the worst prognosis cancers with 1-year mortality being the highest of all cancers. The 2-year survival rate of pancreatic cancer is about 10%, and the 5-year survival rate is only 8% or less. Over the last two decades, the 5-year survival rate of almost all cancers has increased dramatically, but the results of pancreatic cancer are very poor, rising from 3% reported in 1997 to only about 8% in 2016.
In fact, only around 20% of pancreatic cancer patients are considered to be operable, even in this case, the survival rate after surgery may be as high as 90% or higher only when the tumor size is 1cm or less and there is no lymph node metastasis or distant metastasis. However, most patients have no surgery available at the time of diagnosis. When tumors are inoperable, patients often rely on chemotherapy or radiation therapy. However, since there is no clear standardized treatment currently, early detection of pancreatic cancer is very important to improve survival.
Pancreatic cancer has few early symptoms, and when patients become aware of the symptoms, in most cases, the cancer has progressed to an advanced stage. Therefore, early diagnosis of pancreatic cancer is very difficult. At present, few early detection markers suitable for pancreatic cancer are in practice.
Early stage pancreatic cancer is generally defined as a tumor that is less than 2cm in size and confined to the pancreas and has no invasion or lymph node metastasis. However, even if the size of pancreatic cancer is less than 2cm (threshold for early stage pancreatic cancer), metastasis is found in as many as 50% of such cases. Even in stage II (classified as early stage pancreatic cancer when staging pancreatic cancer according to tumor size, lymph node metastasis and distant metastasis), there is a common infiltration in the area connecting the superior mesenteric vein or portal vein, as well as early metastasis of small numbers of cells. In this case, the tumor is not resectable. In the case of surgery on a tumor in consideration of the fact that no distant metastasis is found in the scan and it is determined that the tumor is localized to the pancreas, it is not uncommon to find distant metastasis shortly after the surgery. In this case, the risk of recurrence is high even after surgery, and the median survival time is only 6 to 12 months because there is no anticancer agent having a significant therapeutic effect. In view of the above, there is a need to detect early stage pancreatic cancer that has not progressed to substantial distant metastasis or early metastasis of small numbers of cells, at least for early surgical resection (the only curative treatment for pancreatic cancer).
In developing probes targeting cell surface proteins, the extracellular domain of a cell surface protein can be isolated and purified as a recombinant protein and then used as an antigen to develop antibodies or select aptamers. The screening process for the selection of aptamers is commonly referred to as SELEX (systematic evolution of ligands by exponential enrichment) technology. Such techniques typically employ isolated recombinant proteins. However, during the isolation process, the three-dimensional structure of the cell surface protein may change, which if the three-dimensional structure of the protein is critical for binding to the target protein, may result in the selected aptamer actually failing to bind to the target protein on the cell surface.
Such limitations can be overcome by using the Cell-SELEX method, which involves specifically selecting aptamer Cell membranes using living cells, unlike the conventional SELEX technique.
In the present disclosure, a Cell-SELEX method is used to select DNA aptamers with high binding affinity for pancreatic cancer on pancreatic cancer cells, and then the selected aptamers are further studied to improve targeting efficiency of cells and tissues and serum stability. It has been found that the selected aptamers can specifically bind to pancreatic cancer as well as various cancers of colon, liver, lung, brain, oral, ovarian and breast.
[ Prior art documents ]
(patent document 1) KR 10-1458947
(patent document 2) KR 10-1250557
Brief description of the invention
It is an object of the present disclosure to provide novel DNA aptamers. In particular, the novel DNA aptamers are DNA aptamers that exhibit cancer-specific binding.
It is another object of the present disclosure to provide a method for diagnosing or treating cancer using a DNA aptamer exhibiting cancer-specific binding.
It is an object of the present disclosure to develop aptamers that are capable of detecting cancer cells. Aptamers that specifically bind to pancreatic cancer Cell membranes were selected using the Cell-SELEX technique. Specifically, in the present disclosure, an aptamer that specifically binds to pancreatic cancer cells was selected using CMLu-1 cells isolated from metastatic pancreatic cancer tissue as target cells and HPNE, a normal pancreatic tissue cell, as a control.
In one aspect, the present disclosure provides a DNA aptamer comprising a nucleotide sequence as set forth in SEQ ID NO 6. In another aspect, the present disclosure provides a DNA aptamer comprising a nucleotide sequence having at least 90% or 95% homology to the nucleotide sequence set forth as SEQ ID NO. 6. In one aspect of the disclosure, the DNA aptamer may exhibit specific binding for cancer. In one aspect of the disclosure, the DNA aptamer may consist of the nucleotide sequence shown as SEQ ID NO 6.
In another aspect, the present disclosure provides a DNA aptamer consisting of a nucleotide sequence having at least 90% or 95% homology to the nucleotide sequence set forth as SEQ ID No. 4. In one aspect of the disclosure, the DNA aptamer may consist of the nucleotide sequence shown as SEQ ID NO. 4.
As used herein, "a nucleotide sequence having at least 90% homology" refers to a nucleotide sequence comprising one or more additions, deletions, or substitutions of nucleotides to have greater than or equal to 90% but less than 100% nucleotide sequence identity to a reference sequence, and exhibits similar cancer-specific binding.
In one aspect of the present disclosure, having at least 90% homology to the nucleotide sequence set forth as SEQ ID NO. 4 may refer to a nucleotide sequence that differs from SEQ ID NO. 4 in a region of SEQ ID NO. 4, but not in a region corresponding to the nucleotide sequence of SEQ ID NO. 6.
The term "DNA aptamer" as used herein refers to a short single-stranded oligonucleotide that binds a corresponding target with high affinity and specificity and has a unique three-dimensional structure. Through an iterative in vitro selection and enrichment process, DNA molecules that specifically bind to a specific target, i.e., DNA aptamers, can be selected from a DNA aptamer library.
In an experimental example of the present disclosure, aptamers of one DNA pool enriched by the Cell-SELEX process were clustered into 11 aptamer families based on their sequence similarity (SQ1 to SQ 11). Among these families, SQ7 has been identified as having high binding affinity to metastatic cancer cell CMLu-1 of pancreatic cancer origin, and in addition the aptamers in SQ7 family have high sequence homology to each other, differing by only a few nucleotides (SQ 7a and SQ7b aptamers in table 3).
In addition, a truncated aptamer (32-mer) was prepared to increase synthesis yield and reduce synthesis cost using SQ7(SEQ ID NO:4) aptamer (80-mer) that specifically binds to pancreatic cancer tissue-derived cells as a template. Specifically, a SQ7-1 aptamer (SEQ ID NO:6) was prepared that reduced the aptamer size by more than half while maintaining binding affinity to CMLu-1 target cells. The inventors of the present disclosure concluded that the SQ7-1 aptamer is a region of the SQ7 aptamer sequence that is critical for its ability to target cancer cells and cancer tissues, and based on this further experiments were performed.
In one aspect of the present disclosure, there is provided a modified DNA aptamer, wherein the modification has been introduced into the DNA aptamer of the present disclosure such that it is dnase resistant and is present in at least 10% of the nucleotides of SEQ ID No. 6. In addition, the modified DNA aptamer has a nucleotide sequence shown as SEQ ID NO 8, 12 or 14.
In one aspect of the disclosure, the modification introduced to confer DNase resistance may be with-Me (methyl), -OMe, -NH2-F (fluoro), -O-2-methoxyethyl-O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -O-N-methylacetamido or-O-dimethylaminoyloxyethyl substituting the-OH group on the 2' carbon of the sugar group in one or more nucleotides.
In one working example of the present disclosure, internal 2' -O-methyl modified aptamers were prepared by modifying different regions in the secondary structure of SQ7-1 aptamer based on SQ7-1 aptamer as a template, and the serum half-life of the modified aptamers was determined to investigate their serum stability. The results indicate that the serum half-life of certain modified aptamers is increased by 90-fold or greater compared to the SQ7-1 aptamer.
The target cells for Cell-SELEX in the working examples of the present disclosure were CMLu-1 cells obtained from metastatic pancreatic cancer tissue by in situ transplantation experiments. Thus, the present disclosure may be particularly useful in the diagnosis of pancreatic cancer.
The present disclosure provides a composition for targeting cancer tissue, comprising the above DNA aptamer. In one aspect of the present disclosure, the composition comprising a DNA aptamer may comprise, in addition to the above-mentioned ingredients, an active ingredient having the same or similar function, or an ingredient that stabilizes the formulation of the composition, or enhances the stability of the aptamer. In one aspect of the disclosure, the composition may be a pharmaceutical composition.
In addition, the present disclosure provides a composition for diagnosing cancer, which comprises the aptamer according to one aspect of the present disclosure.
In one aspect of the disclosure, the DNA aptamer may be used in combination with an effector moiety.
In one aspect of the disclosure, the effector moiety may be a cytotoxic agent, an immunosuppressive agent, an imaging agent (e.g., a fluorescent moiety or a chelator), a nanomaterial, or a toxin polypeptide. The cytotoxic agent may be a chemotherapeutic agent.
In one aspect, the present disclosure relates to a composition for treating cancer, comprising a novel DNA aptamer according to one aspect of the present disclosure and an anticancer agent coupled to the DNA aptamer.
In one aspect of the present disclosure, the cancer may be, but is not limited to, pancreatic cancer, colon cancer, liver cancer, lung cancer, brain tumor, oral cancer, ovarian cancer, or breast cancer.
In one aspect of the present disclosure, the anticancer agent may be selected from one or more of, but not limited to, MMAE (monomethylauristatin E, monomethylauristatin F), MMAF (monomethylauristatin F ), calicheamicin (calicheamicin), maytansine (DM 1), raloxib (ravtanine, DM4), tesiline (SCX), doxorubicin (doxorubicin), cisplatin (cispin), SN-38, duocarmycin (duocarmycin), and Pyrrolobenzodiazepine (PBD).
In one aspect of the disclosure, the DNA aptamer may be coupled to polyethylene glycol (PEG) or a derivative thereof, Diacylglycerol (DAG) or a derivative thereof, an antibody, a dendrimer, or a zwitterion-containing biocompatible polymer (e.g., a choline phosphate-containing polymer).
In one aspect of the present disclosure, the composition may further contain a physiologically acceptable excipient, carrier or additive, which may include, but is not limited to, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, dibasic calcium phosphate, lactose, mannitol, raffinose (taffy), gum arabic, pregelatinized starch, corn starch, cellulose powder, hydroxypropyl cellulose, Opadry (Opadry), sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, glucose, sorbitol, and talc.
In one aspect of the disclosure, the composition can be administered to a subject in various forms according to a selected route of administration as understood by one of ordinary skill in the relevant art. For example, the compositions of the present disclosure may be administered by topical, enteral, or parenteral application. Topical applications include, but are not limited to, epidermal applications, inhalation, enema, eye drops, ear drops, and mucosal applications. Enteral applications include oral administration, rectal administration, vaginal administration, and gastric feeding tube administration. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intramedullary, epidural, intrasternal, intraperitoneal, subcutaneous, intramuscular, transepithelial, intranasal, intrapulmonary, intrathecal, rectal and topical administration.
Furthermore, in one aspect of the present disclosure, the composition may be formulated in any form suitable for the chosen route of administration. For formulation purposes, the compositions may be prepared using diluents or excipients including, but not limited to, fillers, binders, wetting agents, disintegrants, surfactants, and the like.
In one aspect of the present disclosure, the composition may comprise the DNA aptamer of one aspect of the present disclosure, the effective amount of which is determined by one of ordinary skill in the art in consideration of the administration route and the body weight, age, sex, health condition, diet, intake time, excretion rate, etc. of a subject in need thereof.
The DNA aptamers selected and optimized in the present disclosure to obtain high binding affinity to cancer cells can be effectively used for diagnosis and treatment of cancer due to their enhanced targeting efficiency to target cells and tissues and high serum stability.
Drawings
FIG. 1 shows the design of nucleotide sequences included in a DNA library of the present disclosure, as well as the design of forward and reverse primers that can be used to amplify or identify the nucleotide sequences described above. Specifically, the nucleotide sequences in the DNA library comprise 20 fixed nucleotides at the 5 'end, 40 random nucleotides in the middle, and another 20 fixed nucleotides at the 3' end. The forward primer was labeled with Cy5 at its 5 'end (5' -Cy 5-sequence-3 '), and the reverse primer was labeled with biotin at its 5' end (5 '-biotin-sequence-3').
FIG. 2 shows the results of enriching a ssDNA pool with binding affinity for pancreatic cancer cells from a DNA library of the present disclosure, and determining whether the cell binding affinity of the pool increases with the number of rounds using flow cytometry (fluorescence activated cell sorter; FACS).
Fig. 3 is a schematic diagram of a process of screening for aptamers using metastatic pancreatic cancer cells by the Cell-SELEX technique in the present disclosure.
Fig. 4 shows the results of measurement of Cell binding affinity of each Cy 5-labeled candidate aptamer obtained by screening aptamers using metastatic pancreatic cancer cells by the Cell-SELEX technique in the present disclosure.
Fig. 5 depicts the secondary structure of SQ7 aptamers obtained in the present disclosure by screening for aptamers using metastatic pancreatic cancer cells by the Cell-SELEX technique.
Figure 6a shows the results of determining target cell binding affinity of SQ7 aptamers of the present disclosure using flow cytometry (FACS). Specifically, the binding of SQ7 aptamer to target cells was determined and no treatment control (NT), DNA pool library and SQ8-Comp aptamer served as controls.
Figure 6b shows the results of determining the target cell binding affinity of SQ7-1 aptamers of the present disclosure using flow cytometry (FACS). Specifically, the binding of SQ7 aptamer and SQ7-1 aptamer to target cells was determined, and no treatment control (NT), DNA pool library and SQ8-Comp aptamer were used as controls.
FIG. 7 depicts the secondary structure of SQ7-1 aptamers prepared based on the SQ7 aptamers of the present disclosure.
Figure 8 shows the targeting distribution of SQ7 aptamers of the disclosure against target cells as determined by confocal microscopy. The gray ovals represent the nuclei and the brightest white areas represent the aptamers that bind to the cell surface or internalize into the cell.
Figure 9 shows the targeting profile of SQ7-1 aptamers of the disclosure against target cells as determined by confocal microscopy. The gray ovals represent the nuclei and the brightest white areas represent the aptamers that bind to the cell surface or internalize into the cell.
Figure 10 shows the targeting distribution of SQ7 aptamers of the disclosure against pancreatic cancer tissue as determined by bioluminescence imaging in a xenograft mouse model of human pancreatic cancer cell lines.
Figure 11 shows the targeting distribution of SQ7-1 aptamers of the disclosure against pancreatic cancer tissue as determined by bioluminescence imaging in a xenograft mouse model of human pancreatic cancer cell lines.
FIGS. 12a to 12d depict the secondary structures and modification positions of SQ7-1(1), SQ7-1(2), SQ7-1(3), SQ7-1(4), SQ7-1(5), SQ7-1(6), and SQ7-1(1,5) aptamers, which are internally 2' -O-methyl modified aptamers prepared based on the SQ7-1 aptamers of the present disclosure. Fig. 12a depicts the region of the internal 2 '-O-methyl modification with a rectangle, and fig. 12b to 12d specifically disclose the nucleotide sequence of each region of the internal 2' -O-methyl modification.
Figures 13 a-13 d show the measured SQ7-1, SQ7-1(1), SQ7-1(5) and SQ7-1(1,5) aptamer serum half-lives of the present disclosure.
FIG. 14 shows target cell binding affinities of the SQ7-1, SQ7-1(1), SQ7-1(5), and SQ7-1(1,5) aptamers of the present disclosure as determined by flow cytometry.
Figure 15 shows the targeting distribution of SQ7-1 aptamers of the disclosure to pancreatic cancer tissue as determined by bioluminescence imaging in xenograft mouse models of various pancreatic cancer cell lines of human pancreatic cancer patients.
FIGS. 16a and 16b show the binding affinities of SQ7-1 aptamers of the present disclosure to various pancreatic cancer cell lines as determined by flow cytometry (FACS).
FIG. 17 shows the geometric mean (in multiples) of the relative fluorescence intensity of the SQ7-1 aptamer versus the SQ7-1-Rev aptamer, based on the results obtained in FIG. 16.
FIG. 18 shows the geometric mean (in multiples) of the relative fluorescence intensity of SQ7-1 aptamer versus SQ7-1-Rev aptamer, based on the binding affinity of the SQ7-1 aptamer of the present disclosure to various cancer cell lines as determined by flow cytometry (FACS).
FIG. 19 shows the geometric mean (in multiples) of the relative fluorescence intensity of the SQ7-1 aptamer versus the SQ7-1-Rev aptamer, based on the binding affinity of the SQ7-1 aptamer of the present disclosure to various pancreatic cancer PDOX-derived cell lines as determined by flow cytometry (FACS).
Detailed Description
The present disclosure may be clearly understood from the above aspects and one or more experimental examples described below. Hereinafter, the present disclosure will be explained in detail so that those of ordinary skill in the art can easily understand and reproduce the present disclosure by referring to working examples described in the attached tables. However, the experimental examples or examples given below are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to such experimental examples or examples.
[ Experimental example 1]Screening of aptamers Using Cell-SELEX
The experimental procedure for screening for aptamers that specifically bind pancreatic cancer cells using the Cell-SELEX technique is shown in FIG. 3.
Specifically, in order to obtain a cell line expressing a cell membrane protein (characteristic of pancreatic cancer), pancreatic cancer cells were transplanted into an animal model, and a pancreatic cancer cell line CMLu-1 was isolated from a tissue in which tumors metastasized (fig. 3A).
ssDNA libraries were prepared and then screened using the pancreatic cancer cell line CMLu-1 cells as target cells (positive cells) and hTERT/HPNE cells (human pancreatic nestin expressing cells) as control cells (negative cells). Multiple rounds of repeated selection of ssDNA molecules that bind only to pancreatic cancer cells and not to control cells, and the resulting enriched ssDNA pools were cloned and sequenced, and then clustered.
(1) Construction of metastatic pancreatic cancer cell lines from xenografted mouse models of human pancreatic cancer cell lines
The method for obtaining the metastatic pancreatic cancer cell line CMLu-1 is as follows. NOD/SCID mice were used to create an orthotopic mouse model. First, in order to construct an animal model capable of simulating pancreatic cancer metastasis, a pancreatic cancer cell line stably expressing firefly luciferase (CFPAC-1-Luci) was established and used for noninvasive monitoring of tumorigenesis over time. CFPAC-1-Luci pancreatic cancer cells were transplanted in situ into NOD/SCID mice. After 43 days, tumor tissues were taken from lung tissues of mice with metastatic pancreatic cancer, and the isolated tumor tissues were genotyped, indicating that the genetic attributes of metastatic tumor cells are identical to those of pancreatic cancer cells. Then, single cells were prepared from the tumor tissue and cultured. CMLu-1 cells isolated from metastatic tumor tissue were cultured and maintained in RPMI-1640 medium (Hyclone, Logan, UT, USA) plus 10% FBS (Thermo Fisher Scientific, USA) and 100IU/mL of double antibody (antigen-antigen; Gibco).
The CMLu-1 cells obtained were used as positively selected cells in Cell-SELEX, and human pancreatic duct normal epithelial cells (HPNE) purchased from ATCC Inc. were used as negatively selected control cells.
(2) Preparation of ssDNA library and primers for Cell-SELEX
The DNA library for pancreatic cancer-specific aptamers in Cell-SELEX is a pool of DNA sequences consisting of fixed and specific combinations of nucleotides. The DNA sequence includes 20 fixed nucleotides at the 5 'end, 40 random nucleotides in the middle, and 20 fixed nucleotides at the 3' end. The 5 'end of the DNA sequence was labeled with Cy5 in order to monitor the selected enrichment using a fluorescence activated cell sorter ("FACS", i.e., "flow cytometer") and the 3' end was labeled with biotin (biotin) to purify the ssDNA molecules (fig. 1). Further, the forward primer was labeled with Cy5 at its 5 '-end (5' -Cy 5-sequence-3 '), and the reverse primer was labeled with biotin at its 5' -end (5 '-biotin-sequence-3'). The DNA composition of the DNA library and the forward and reverse primers are shown in Table 1.
[ Table 1]
PCR was used to amplify the eluted DNA pool. ssDNA was isolated by capturing the biotinylated complementary strand using a streptavidin-biotin bond and denaturing the double stranded DNA with NaOH. A PCR mixture was prepared and PCR was performed according to the supplier's instructions.
(3) Screening of libraries by Cell-SELEX
The ssDNA library prepared as described above was screened using CMLu-1 cells as target cells (positive cells) and hTERT/HPNE cells as control cells (negative cells). 10nmol of the DNA library was dissolved in 1000. mu.L of binding buffer (Dulbecco's PBS (Hyclone, USA) containing 5mM MgCl20.1mg/mL tRNA and 1mg/mL BSA). The DNA library or enriched pool was denatured at 95 ℃ for 10min, and cooled on ice for 10min, then incubated with CMLu-1 cells on an orbital shaker at 4 ℃ for 1 hour.
The CMLu-1 cells were then washed 3 times to remove unbound DNA sequences and the bound DNA molecules were eluted by a centrifuge for 15min at 95 ℃ using 1000 μ L binding buffer. For counter selection, the aptamer pool was incubated with hTERT/HPNE cells for 1 hour, and then the supernatant was collected for negative selection. The enriched pool was monitored using FACS and cloned into e.coli (Escherichia coli) using the quinagen clone sequencing kit (quinagen, Germany) to determine candidate aptamers.
(4) Cloning and sequencing of enriched ssDNA pools and multiple sequence alignment
To select candidate sequences, 5 rounds of enriched ssDNA libraries were cloned and sequenced. ssDNA pools were amplified by PCR using unmodified primers, ligated to pGEM-T easy vector (Promega, USA), and cloned into HITTMDH 5. alpha. competent cells (Promega, USA). Thereafter, 200 clones were analyzed for sequence using Cosmogenetech Inc (Seoul, Korea) and aligned using ClustalX 1.83.
The degree of enrichment according to the number of selection rounds is shown in FIG. 2.
The processes described in (1) to (4) above are shown in FIG. 3. Aptamer clustering by sequencing through the above process was classified into aptamers having similar sequences. Finally, 11 Cy 5-labeled candidate aptamer families were identified (SQ1 to SQ 11). The content of individual aptamer families throughout the pool is shown in table 2 below.
[ Table 2]
[ Experimental example 2]Determination of the specificity of binding of an enriched family of candidate aptamers to target cells
The enriched candidate aptamer family obtained in (4) of experimental example 1 was tested for specificity of binding to CMLu-1 target cells using flow cytometry (FACS).
Each Cy 5-labeled candidate aptamer family was pooled with 3105CMLu-1 cells and hTERT/HPNE cells were incubated together in Cell-SELEX binding buffer at 4 ℃ for 1 hour. With a solution containing 0.1% NaN3The cells were washed 3 times with binding buffer and the sequence-bound cells were resuspended in binding buffer. Using BD FacscaliburTMAnd FACSVerseseTM(BD Biosciences, USA) 10000 cells were subjected to fluorescence detection and the data were analyzed using the software FlowJo v10.0.7.
Figure 4 shows the results of determining the target cell binding affinity of a single Cy 5-labeled family of candidate aptamers. The aptamer with the highest binding specificity for metastatic pancreatic cancer cells (CMLu-1), the target cell, was identified as SQ7, the sequence of which is shown below.
SQ7 aptamer sequence
5’-AGCAGCACAGAGGTCAGATGATGTTGGTATATACTTCTTTAGCTTGGAACCAACTCTTGCCCTATGCGTGCTACCGTGAA-3’(SEQ ID NO:4)
The table below lists the aptamer sequences contained in the SQ7 family.
[ Table 3] sequences of SQ7 and SQ7 subtypes
[ Experimental example 3]Analysis of SQ7 aptamers and functional identification of aptamer fragments
The secondary structure of SQ7 aptamer selected in experimental example 2 was determined as shown in figure 5. Furthermore, in order to confirm cell binding affinity, SQ7 aptamer, as well as untreated control (NT), DNA pool library and SQ8-Comp aptamer (aptamer having nucleotide sequence partially complementary to SQ8 aptamer; SEQ ID NO:5) as controls, and target cell binding thereof was determined using FACS in the same manner as in Experimental example 2. As shown in fig. 6, the results indicate that, although the control group (i.e., NT, DNA pool library, and SQ8-Comp aptamer) all exhibited similarly low levels of binding affinity, SQ7 aptamer exhibited significantly superior target cell binding affinity.
To determine whether certain portions of the SQ7 aptamer were critical for pancreatic cancer specific binding, various aptamers comprising the portion SQ7 aptamer were prepared and their binding affinity to target cells was studied. If the length of the aptamer can be shortened while maintaining its cell-binding affinity, it is likely that the production cost of the aptamer will be reduced while enhancing cell permeation. The results show that the SQ7-1 aptamer, having the sequence shown in Table 4 below, has an advantage in endocytosis while almost completely retaining binding affinity for the target cells. In view of the above results, target cell binding affinities of the SQ7 aptamer, the SQ7-1 aptamer, NT, DNA pool library and the SQ8-Comp aptamer were determined using FACS in the same manner as in Experimental example 2. As shown in fig. 6b, the results indicate that the control group (i.e., NT, DNA library, and SQ8-Comp aptamer) had a similar level of low binding affinity, while the SQ7-1 aptamer exhibited significantly superior target cell binding affinity as did the SQ7 aptamer.
The nucleotide sequence of the SQ7-1 aptamer is shown in the following table (SEQ ID NO:6), and the corresponding secondary structure is shown in FIG. 7. In addition, an SQ7-1-Rev aptamer (aptamer having a sequence in the reverse orientation of SQ 7-1; SEQ ID NO:7) was prepared so as to be used as a control for the SQ7-1 aptamer in subsequent experiments.
[ Table 4]
[ Experimental example 4]Determination of the efficiency of targeting selected aptamers to cells and tissues
(1) Endocytosis efficiency of selected aptamers was determined by confocal microscopy imaging.
1X 10 hours before the experiment4Individual cell/well control cells (HPNE) and target cells (CMLu-1) were plated on 8-well chamber slides (Thermo scientific, USA) coated with poly-L-lysine (Sigma, USA). After washing with washing buffer, Cy 5-labeled aptamer (250nM) or DNA pool library was added and incubated at 4 ℃ in 200. mu.L of binding buffer. After two washes, 4% paraformaldehyde was usedThe cells were aldehyde fixed and then stained for nuclei with Hoechst 33342. Thereafter, the cells were imaged by confocal microscopy (LSM780, Carl Zeiss, Germany) and the images obtained were analyzed with the software Zen blue edition.
Confocal microscopy results for SQ7 and SQ7-1 aptamers are shown in fig. 8 and 9, where the brightest white areas represent areas of aptamer density. As shown in figures 8 and 9, SQ7 and SQ7-1 aptamers target pancreatic cancer cells but not control cells and show good levels of endocytosis (internalization).
(2) Aptamer-targeted pancreatic cancer determination by in vivo and in vitro fluorescence imaging
6 week old female Hsd Athymic nude-Foxn1 nude mice were purchased from Harlan Laboratories, Inc, France. Mice were housed under controlled light and humidity conditions in a Specific Pathogen Free (SPF) environment, with food and water provided by the NCC animal facility. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) (NCC-16-247) of the national cancer center institute (NCCRI). NCCRI is an agency approved by the International Association for assessment and acceptance of laboratory animals (AAALAC International).
By using CFPAC-1 cells (1X 10) purchased from ATCC6Individual cells) were injected into the tail of mouse pancreas to construct an orthotopic xenograft mouse model of pancreatic cancer. 3 weeks after inoculation, mice were divided into two groups according to the treatment protocol (Cy5.5-SQ8-Comp aptamer vs Cy5.5-SQ7 aptamer or Cy5.5-SQ7-1-Rev aptamer vs Cy5.5-SQ7-1 aptamer), and then the above Cy5.5-labeled aptamer (300 pmol/50. mu.L PBS) was administered intravenously.
For in vitro experiments, mice were sacrificed 15 minutes or 3 hours after administration and then dissected. Tumor tissues were isolated and bioluminescent imaging was performed using IVIS luminea (Caliper Life Science, Hopkinton, MA, USA). All Image data were analyzed using the software Living Image Acquisition and Analysis.
The grey scale plots in FIGS. 10 and 11 show the bioluminescent imaging results for the SQ7 and SQ7-1 aptamers. The total flux obtained from the imaging results is plotted in fig. 10 and 11.
In the imaging results shown at the top of the above figures, the brighter (whiter) the area, the greater the number of aptamers that bind to the cells in that area. It can be seen that the imaging results for SQ7 and SQ7-1 aptamers are whiter than those for the other aptamers.
Thus, it can be shown that the aptamer of the present disclosure migrates to and specifically binds to pancreatic cancer tissue and has significantly superior targeting efficiency to pancreatic cancer tissue, as compared to other control aptamers (SQ1 aptamer (SEQ ID NO:17), SQ8-Comp aptamer, and SQ7-1-Rev aptamer).
[ Experimental example 5]Preparation and characterization of aptamer modifications with enhanced serum stability
Based on the SQ7-1 aptamer prepared in the above experimental example, an internal 2 '-O-methyl (2' -O-methyl) modified aptamer was prepared to enhance the serum stability of the aptamer. Specifically, SQ7-1(1), SQ7-1(2), SQ7-1(3), SQ7-1(4), SQ7-1(5), SQ7-1(6) and SQ7-1(1,5) aptamers were prepared by modifying different regions in the secondary structure of the SQ7-1 aptamer (fig. 12a to 12 d).
The nucleotide sequences of the internal 2' -O-methyl modified aptamers produced are summarized in Table 5 below.
[ Table 5]
Mu.g of each of the internal 2' -O-methyl modified aptamers prepared above was incubated in mouse serum at 37 ℃ for 0, 0.1, 0.5, 2, 6 and 24 hours. At each time point described above, 0.5M EDTA was added to the sample to inhibit DNase activity, followed by addition of EtOH-NaOAc to effect precipitation. DNA aptamer pellet samples were analyzed by HPLC.
Chromatography was performed with a Waters e2695 HPLC system (MA, USA) equipped with a Variable Wavelength Detector (VWD) quatpupp. Chromatographic data was processed using a personal computer equipped with an Empower3 personal single system software for LC. Analytes were separated using a Venusil, XBP C18 column (250mm x 4.6mm, 5 μm) from Agela Technologies Inc. The mobile phase was a methanol-water (55:45, vol.) mixture with a flow rate of 0.5 mL/min. The column temperature was 30 ℃ and the measurement wavelength was 260 nm. For injection, 15 μ L LC microsyringes purchased from Shanghai GaoGe Industrial and tracking co., Ltd. (Shanghai, China).
The results show that the SQ7-1(1), SQ7-1(5) and SQ7-1(1,5) aptamers have significantly increased half-lives. Specifically, as shown in FIG. 13, the half-lives of the SQ7-1(1), SQ7-1(5), and SQ7-1(1,5) aptamers were increased 91-fold, 145-fold, and 760-fold, respectively, relative to the SQ7-1 aptamer. The binding affinity of the aptamers to the target cells was slightly lower than that of the SQ7-1 aptamers, but still significantly higher than those in the DNA pool library (thin left-most solid line in the figure) (FIG. 14).
In summary, as described above, the DNA aptamers of the present disclosure specifically bind pancreatic cancer with high binding affinity. It has been shown that the targeting efficiency of target cells or tissues can be increased by decreasing the size of the selected aptamer and that serum stability can be increased by modifications that enhance resistance to dnase.
[ Experimental example 6]Human pancreatic cancer tissue (Patient-treated) in mouse orthotopic xenograft model Orthotopic A Xenograft Model; PDOX model) targeting assay
To determine whether the aptamers of the present disclosure also exhibit excellent targeting efficiency in PDOX models that can replicate the complexity and heterogeneity of patient tumor tissues, in vivo validation experiments were performed using fluorescence imaging.
Subject-derived xenograft in situ (PDOX) models were established by collecting samples from patients who submitted informed consent, approved by the NCC ethical Review Board ("IRB"), and transplanting the samples directly into nude mouse pancreas (purchased from Harlan Laboratories, Inc. For a primary tumor sample (hereinafter referred to as "HPT") of a pancreatic cancer patient, direct surgical resection; for the advanced stage of inoperablePancreatic cancer patients, after collecting a biopsy sample of liver metastasis tissue (hereinafter referred to as "GUN"), were transferred to test tubes containing a culture medium and transplanted into female Hsd: Athymic nude-Foxn1 nude mice (obtained from the same source as experimental example 4) as soon as possible by cutting at the tail of pancreas and closing the incision after transplantation (PDOX 1 generation, F1). Thereafter, the tumor size was measured periodically by abdominal palpation and MRI when the tumor volume reached 3000mm3At that time, mice were sacrificed to obtain tumor tissue. Tumor tissue fragments of a certain size (3mm by 3mm) were then re-implanted in situ into a plurality of nude mouse subjects to generate offspring (F2, F3, F4 …) and to increase the number of subjects.
At passage 4 (F4), the established PDOX mouse model was divided into two groups of three subjects per group, i.v. injected with SQ7-1 aptamer or SQ7-1-Rev aptamer in the form of Cy5.5-labeled aptamer (300 pmol/50. mu.L PBS), respectively. After 15 minutes of administration, mice were sacrificed and dissected, tumor tissue was removed, and then bioluminescence imaging was performed using IVIS luminea (Caliper Life Science, Hopkinton, MA, USA). All Image data were analyzed using the software Living Image Acquisition and Analysis. The grey scale plot in fig. 15 shows the results of bioluminescent imaging. The total flux obtained from the imaging results is shown in fig. 15.
As with fig. 10 and 11, in the imaging results shown at the top of fig. 15, the brighter (whiter) the area, the greater the number of aptamers bound to the cells in that area. It can be seen that the SQ7-1 aptamer is whiter than the SQ7-1-Rev aptamer.
Thus, it was shown that the aptamers of the present disclosure have significantly superior targeting efficiency even in PDOX models that preserve the complexity and heterogeneity of patient tumor tissue.
[ Experimental example 7]Determination of binding affinities of different pancreatic cancer cell lines
Additional flow cytometry (FACS) analysis was performed to determine the binding affinity of the aptamers of the disclosure to various pancreatic cancer cell lines.
[ preparation of antibody binding to appropriate ligand ]
The 250nM digoxin (digoxigenin) -labeled SQ7-1 aptamer and 125nM anti-digoxin antibody (Abcam; Cat. No. ab420, USA) were mixed for 30 minutes at room temperature to prepare antibody binding partners.
Flow cytometry analysis was performed in the same manner as in experimental example 2, except that the antibody prepared as described above was used as a suitable ligand, and Alexa 488-labeled anti-mouse IgG (Invitrogen, USA) was used as a second antibody at a concentration of 4 μ g/mL. Cells from the CFPAC-1 cell line, the SNU-213 cell line, the SNU-410 cell line, the Capan-2 cell line, the HPAF-II cell line, the AsPC-1 cell line, the Capan-1 cell line, the MIA PaCa cell line, the BxPC-3 cell line and the PANC-1 cell line were used. All of the above cell lines were purchased from ATCC.
The results of determining the binding affinity of the aptamers to various pancreatic cancer cell lines are shown in fig. 16 and 17. FIG. 17 shows the geometric mean (expressed in multiples) of the relative fluorescence intensity of the SQ7-1 aptamer versus the SQ7-1-Rev aptamer.
According to the results shown in fig. 16 and 17, the SQ7-1 aptamer of the present disclosure binds all pancreatic cancer cell lines more efficiently and specifically than the SQ7-1-Rev aptamer. In combination with the results of experimental examples 4 and 6 discussed above, it can be seen that the aptamers of the present disclosure can specifically bind to various types of pancreatic cancer cells.
Furthermore, the SQ7 aptamer was able to similarly specifically bind to various types of pancreatic cancer cells, as it contains the SQ7-1 aptamer sequence.
[ Experimental example 8]Determination of binding affinities of various tumor cell lines
Additional flow cytometry (FACS) analysis was performed to determine the binding affinity of the aptamers of the disclosure to various tumor cell lines.
Flow cytometry analysis was performed in the same manner as in experimental example 2, but using the same antibody binding suitable ligand and secondary antibody as in experimental example 7. Cells from the U87 cell line, U251 cell line, CAL27 cell line, HEP3B cell line, A549 cell line, HCT116 cell line, SK-OV3 cell line, ES-2 cell line, MCF7 cell line, SK-BR3 cell line, NCI-N87 cell line, KPL4 cell line, BT-474 cell line, MDA-MB231 cell line and HCC1938 cell line were used. All of the above cell lines were purchased from ATCC.
The results of the measurement of binding affinity of the aptamers to various cancer cell lines are shown in FIG. 18. FIG. 18 shows the geometric mean (in multiples) of the relative fluorescence intensity of the SQ7-1 aptamer and the SQ7-1-Rev aptamer.
According to the results shown in fig. 18, the SQ7-1 aptamer of the present disclosure binds more efficiently and specifically to various cancer cell lines such as colon cancer, liver cancer, lung cancer, brain tumor, oral cancer, ovarian cancer and breast cancer cell lines, as compared to the SQ7-1-Rev aptamer. In combination with the results of experimental examples 4 and 6 discussed above, it can be seen that the aptamers of the present disclosure can specifically bind to various types of cancer cells.
In addition, the SQ7 aptamer will also specifically bind to various types of cancer cells as it contains the SQ7-1 aptamer sequence.
[ Experimental example 9]Determination of binding affinity of PDOX-derived cell line of pancreatic ductal adenocarcinoma
Additional flow cytometry (FACS) analysis was performed to determine whether the aptamers of the present disclosure are capable of specifically binding to pancreatic cancer cells of clinical patients.
Unlike normal cells, which have only a limited number of cell divisions before death, cancer cells are characterized by unlimited division. Therefore, cancer cells isolated from tumor tissue are thought to be able to form cell lines that can proliferate indefinitely even without transfection, and reproduce clinical and molecular biological characteristics of patients. In this experiment, tumor tissue taken from a pancreatic cancer PDOX mouse was divided into 3mm × 4mm fragments, mixed with a collagenase-containing human cell dissociation kit (Miltenyi Biotech Inc.) and placed in a tissue dissociator (Gentle Macs, Miltenyi Biotech Inc) for 1 hour to separate cells from connective tissue. After completion of the reaction, RPMI medium containing Fetal Bovine Serum (FBS) was added to inhibit the enzyme activity, followed by centrifugation to obtain cell pellets dissociated from the tissue. The pellet was suspended in RPMI medium containing FBS, and then the cells were spread evenly on 10cm dishes to 2X 106Level of individual cells. The culture medium was changed every other day while removing normal fibroblasts and dead cells, thereby establishing a PDOX-derived cancer cell line for each pancreatic cancer patient. Established cell lines are named in the same way as the PDOX from which they were derived.
Flow cytometry analysis was performed in the same manner as in experimental example 2 for cancer cell lines isolated from tumor tissues of PDOX model using liver metastasis patient biopsy tissue (GUN #13, GUN #16, GUN #20, GUN #34, GUN #38, GUN #41, and GUN #46), tumor cells isolated from tumor tissues of PDOX model using endoscopic ultrasound guided fine needle biopsy samples (EUS #16), and tumor cells isolated from tumor tissues of PDOX model using surgical resection samples (HPT #19, HPT #22, HPT #43, HPT #45, and HPT #48), except that the same antibody binding partner as in experimental example 7 was used.
The results of determining the binding affinity of the aptamers to various PDOX-derived cell lines are shown in figure 19. FIG. 19 shows the geometric mean (in multiples) of the relative fluorescence intensity of the SQ7-1 aptamer and the SQ7-1-Rev aptamer.
According to the results shown in figure 19, the SQ7-1 aptamers of the present disclosure bind more efficiently and specifically to various PDOX-derived cell lines derived from pancreatic cancer tissue collected from different patients, as compared to the SQ7-1-Rev aptamers. In combination with the results of experimental examples 4 and 6 discussed above, it can be seen that the aptamers of the present disclosure are capable of specifically binding to pancreatic cancer tissue of clinical patients.
In addition, the SQ7 aptamer will also specifically bind to pancreatic cancer tissue in patients because it contains the SQ7-1 aptamer sequence.
Although the technical idea of the present disclosure has been described above by referring to the embodiments provided in the working examples and illustrated in the drawings, it should be noted that various substitutions, modifications and changes may be made without departing from the technical idea and scope of the present disclosure as can be understood by those of ordinary skill in the art to which the present disclosure pertains. Further, it is noted that such alternatives, modifications, and variations are encompassed within the scope of the appended claims.
SEQUENCE LISTING
<110> national center for cancer
Peng organism A, Kao corporation
<120> novel DNA aptamer and use thereof
<130> P21116432WP
<150> KR10-2018-0167948
<151> 2018-12-21
<160> 17
<170> PatentIn version 3.5
<210> 1
<211> 76
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide design of DNA library
<220>
<221> Unsure
<222> (19)..(58)
<223> random sequence of DNA library N19-N58
<220>
<221> misc_feature
<222> (19)..(58)
<223> n is a, c, g, or t
<400> 1
ataccagctt attcaattnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnag 60
atagtaagtg caatct 76
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA library Forward primer
<220>
<221> 5'clip
<222> (1)..(1)
<223> 5' end with Cy5
<400> 2
ataccagctt attcaatt 18
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA library reverse primer
<220>
<221> 5'clip
<222> (1)..(1)
<223> 5' terminal addition of biotin
<400> 3
agattgcact tactatct 18
<210> 4
<211> 80
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA aptamer SQ7
<400> 4
agcagcacag aggtcagatg atgttggtat atacttcttt agcttggaac caactcttgc 60
cctatgcgtg ctaccgtgaa 80
<210> 5
<211> 80
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA aptamer SQ8-Comp
<400> 5
agcagcacag aggtcagatg gaacccgata aagaataagt acgacaaggt ggcgagagcc 60
cctatgcgtg ctaccgtgaa 80
<210> 6
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA aptamer SQ7-1
<400> 6
gttggtatat acttctttag cttggaacca ac 32
<210> 7
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA aptamer SQ7-1-Rev
<400> 7
caaccatata tgaagaaatc gaaccttggt tg 32
<210> 8
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<212> DNA
<213> Artificial Sequence
<220>
<223> Internally 2' -O-methyl-modified aptamer SQ7-1(1)
<220>
<221> modified_base
<222> (1)..(6)
<223> 2' -O-methyl modified base (1-6)
<400> 8
guugguatat acttctttag cttggaacca ac 32
<210> 9
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<212> DNA
<213> Artificial Sequence
<220>
<223> Internally 2' -O-methyl-modified aptamer SQ7-1(2)
<220>
<221> modified_base
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<400> 9
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<210> 10
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<212> DNA
<213> Artificial Sequence
<220>
<223> Internally 2' -O-methyl-modified aptamer SQ7-1(3)
<220>
<221> modified_base
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<400> 10
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<212> DNA
<213> Artificial Sequence
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<223> Internally 2' -O-methyl-modified aptamer SQ7-1(4)
<220>
<221> modified_base
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<400> 11
gttggtatat acttctttag cuuggaacca ac 32
<210> 12
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Internally 2' -O-methyl-modified aptamer SQ7-1(5)
<220>
<221> modified_base
<222> (27)..(32)
<223> 2' -O-methyl modified base (27-32)
<400> 12
gttggtatat acttctttag cttggaacca ac 32
<210> 13
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Internally 2' -O-methyl-modified aptamer SQ7-1(6)
<220>
<221> modified_base
<222> (7)..(26)
<223> 2' -O-methyl modified base (7-26)
<400> 13
gttggtauau acuucuuuag cuuggaacca ac 32
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<212> DNA
<213> Artificial Sequence
<220>
<223> Internally 2' -O-methyl-modified aptamer SQ7-1(1,5)
<220>
<221> modified_base
<222> (1)..(6)
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<220>
<221> modified_base
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<400> 14
guugguatat acttctttag cttggaacca ac 32
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<212> DNA
<213> Artificial Sequence
<220>
<223> DNA aptamer SQ7a
<400> 15
agcagcacag aggtcagatg atgttggtat atacttcttt agcttggaac caactcttct 60
cctatgcgtg ctaccgtga 79
<210> 16
<211> 80
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA aptamer SQ7b
<400> 16
agcaacacag aggtcagatg atgttggtat atacttcttt agcttggaac ccactcttgt 60
cctatgcgtg ctaccgtgaa 80
<210> 17
<211> 80
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA aptamer SQ1
<400> 17
agcagcacag aggtcagatg cttgggctat ctcttattca tgctgttcca ccgctctcgg 60
cctatgcgtg ctaccgtgaa 80