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CN109536503B - A nucleic acid aptamer targeting and binding to CD133 protein and its screening method and application - Google Patents

A nucleic acid aptamer targeting and binding to CD133 protein and its screening method and application Download PDF

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CN109536503B
CN109536503B CN201811484443.7A CN201811484443A CN109536503B CN 109536503 B CN109536503 B CN 109536503B CN 201811484443 A CN201811484443 A CN 201811484443A CN 109536503 B CN109536503 B CN 109536503B
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朱栩杭
葛明华
那仁满都拉
黄萍
杨畅
李清林
兰霞斌
曹君
温庆良
黄煜庆
周菁楠
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Zhejiang Cancer Hospital
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Abstract

The invention provides an aptamer of targeted binding CD133 protein and a screening method and application thereof, wherein the sequence of the aptamer is shown as SEQ ID NO.1, the aptamer of targeted binding CD133 protein is screened from a ssDNA library by adopting a cell screening method, the screened ssDNA is detected by a high-throughput sequencing method, and finally, the aptamer AP-1-M is selected by analyzing the stability and the affinity of the obtained sequence by methods such as flow cytometry and the like. Then, an immunofluorescence method proves that the aptamer AP-1-M can be specifically combined with the CD133 protein in a targeted mode and is combined with ATC cells expressing the CD133 protein to achieve the effect of targeted delivery, and a new thought and method are provided for ATC targeted therapy.

Description

Aptamer combined with CD133 protein in targeted manner and screening method and application thereof
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to a nucleic acid aptamer capable of being combined with CD133 protein in a targeted manner.
Background
Thyroid cancer (thyroid carcinoma) is the most common thyroid malignancy, accounting for approximately 1% of all malignancies. They are classified into Differentiated Thyroid Carcinoma (DTC), Poorly Differentiated Thyroid Carcinoma (PDTC), and undifferentiated thyroid carcinoma (ATC) according to their malignancy. ATC, because of its rapid expansion to the neck, causes respiratory distress and esophageal obstruction, is highly susceptible to lymph nodes and distant metastases, with survival at this level being less than 6 months, and even only 10% within 1 year. However, the molecular mechanisms of ATC are not clear and the methods of diagnosis and treatment are limited, so that there is an urgent need for better methods of diagnosis and treatment.
The CD133 protein is a member of cell membrane protein superfamily, contains 865 amino acids, glycoprotein with the molecular weight of about 120kDa, and the molecular structure thereof comprises: an extracellular N-terminus, 2 extracellular Loop loops, a 5-transmembrane domain, a 59 amino acid cellular C-terminus and 8N-glycosylated termini. The predicted size of the CD133 protein is 97kDa, but the actual molecular weight of glycosylated CD133 is 120 kDa. In recent years, researchers have found that CD133 protein is present on the surface of a variety of stem cell-like tumor cells, including liver, colon, and ovarian cancers.
The aptamer, also called chemical antibody, has precise targeting property and is composed of single-chain ribonucleotide or deoxyribonucleotide. Although most of the aptamers have no treatment effect, the aptamers can be connected with toxic drugs to form a targeted aptamer conjugate, and the drugs can be accurately delivered to target cells, so that the aptamer conjugate drugs are endocytosed by the target cells, and the drugs are released inside the cells to induce apoptosis, thereby effectively improving the drug concentration of local tumors, greatly reducing the toxicity of other tissues and organs in vivo, and achieving the effect of real targeted synergism and attenuation.
The CD133 protein exists on the surfaces of various stem cell-like tumor cells, including liver cancer, colon cancer and ovarian cancer, and provides a better specific target for clinically screening and researching novel drugs, thereby providing a thought for developing safer and more effective molecular targeted drugs.
In addition, there are also studies reporting that CD133 protein is expressed in undifferentiated thyroid cancer (ATC), but there is currently a lack of suitable aptamers for treatment studies of undifferentiated thyroid cancer.
Disclosure of Invention
The membrane protein CD133 of the invention is an aptamer and can be combined in a targeted mode. Can be used for ATC targeted therapy and research.
The invention aims to provide a single-stranded DNA aptamer for targeted binding to CD133 protein.
According to one aspect of the invention, an aptamer targeted to bind to a CD133 protein is provided, and the aptamer targeted to bind to the CD133 protein is a single-stranded DNA aptamer.
According to one aspect of the invention, an aptamer which can be combined with CD133 protein in a targeted manner is provided, and the sequence of the aptamer which can be combined with CD133 protein in a targeted manner is shown as SEQ ID NO.1 in a sequence table
The sequence is as follows:
5’-TACCAGCCGTTTCCCCGGAGGGTCACCCCTGACGCATTCGGTTGAC-3’。
according to one aspect of the present invention, there is provided a method of screening for aptamers that target binding to a CD133 protein, the method comprising the steps of:
a. screening aptamers which are targeted to be combined with CD133 protein from the ssDNA library by adopting a cell screening method;
b. b, detecting the ssDNA screened in the step a by a high-throughput sequencing method, analyzing the stability and the affinity of the sequence, and screening a nucleic acid aptamer which is targeted and combined with the CD133 protein;
c. and (c) verifying that the aptamer which is screened out in the step (b) and is targeted to bind the CD133 protein can be specifically targeted to bind the CD133 protein through an immunofluorescence method, and the aptamer can be bound with ATC cells expressing the CD133 protein.
According to one aspect of the invention, a method for screening aptamers capable of targeted binding to CD133 protein is provided, wherein the sequence of one aptamer capable of targeted binding to CD133 protein screened by the screening method is shown as SEQ ID NO.1 in the sequence table, namely AP-1-M.
The 5' end of the AP-1-M is connected with 8 pairs of GC base sequences, and the AP-1-M can directly carry cytotoxic drugs (such as adriamycin) to form an AP-1-M-Dox drug conjugate and selectively kill ATC cells with CD133 positivity. Therefore, the invention provides the application of the AP-1-M-Dox in preparing a preparation for treating CD133 positive tumors (such as ATC).
According to one aspect of the invention, the application of the aptamer capable of targeting and binding to the CD133 protein is provided, and the application of the aptamer (AP-1-M) capable of targeting and binding to the CD133 protein and having a sequence shown as SEQ ID NO.1 in a sequence table in a treatment drug for undifferentiated thyroid cancer.
According to one aspect of the invention, the application of the aptamer targeted to bind to the CD133 protein is provided, and the aptamer (AP-1-M) coupled toxic drug targeted to bind to the CD133 protein forms a targeted therapeutic drug for undifferentiated thyroid cancer.
According to one aspect of the present invention, there is provided a use of an aptamer targeted to bind to a CD133 protein, the aptamer-conjugated toxic drug construct targeted to bind to the CD133 protein using a cytotoxic drug bound by attaching 8 pairs of GC base sequences to the 5' end of the above aptamer targeted to bind to the CD133 protein.
The aptamer AP-1-M is obtained by coupling classical chemotherapeutic drug adriamycin with aptamer AP-1-M to ensure that the aptamer AP-1-M has a good cytotoxicity effect, and the aptamer AP-1-M can be selectively bound with CD133 positive tumor cells and is weakly bound with CD133 negative cells, so that the invention also provides an application of the aptamer as a CD133 positive tumor chemotherapy targeted drug carrier, the chemotherapeutic drug is bound on the aptamer and can selectively kill the CD133 positive tumor cells without toxicity to normal cells when tumor radiotherapy and chemotherapy are carried out, and the chemotherapeutic drug can be selected from metabolic drugs such as methotrexate, fluorouracil, floxuridine, gemcitabine and raltitrexed, and anticancer drugs such as mitomycin C and bleomycin, Doxorubicin, epirubicin, pirarubicin, plant alkaloids such as vinblastine, paclitaxel, hydroxycamptothecin, antitumor hormones such as tamoxifen, letrozole, prednisone, heterosides such as cisplatin, carboplatin, mitoxantrone, antitumor small molecule targeted drugs such as gefitinib, imatinib or lapatinib, more preferably the above chemotherapeutic drugs are selected from fluorouracil, floxuridine, methotrexate, cisplatin, gemcitabine, raltitrexed, mitomycin, bleomycin, vinblastine, paclitaxel, hydroxycamptothecin, carboplatin, tamoxifen, letrozole, prednisone, gefitinib, imatinib or lapatinib.
According to one aspect of the present invention, there is provided the use of an aptamer that binds to a CD133 protein in a targeted manner, the cytotoxic agent being doxorubicin.
The overall idea of the aptamer combined with the CD133 protein in a targeted mode is as follows:
the aptamer which is targeted and combined with the CD133 protein is screened from the ssDNA library by adopting a cell screening method, the screened ssDNA is detected by a high-throughput sequencing method, and finally, the aptamer is selected by analyzing the stability and the affinity of the obtained sequence by methods such as flow cytometry and the like. Then, the aptamer is proved to be capable of specifically targeting and binding to the CD133 protein through an immunofluorescence method, and the aptamer is combined with ATC cells expressing the CD133 protein to play a role in targeted delivery. This provides new ideas and methods for the targeted therapy of ATC.
The aptamer targeted to bind to the CD133 protein has the following advantages: 1. the screening in vitro can be carried out quickly and artificially; 2. the molecular weight is not large, no immunoreaction occurs, the molecular weight is easy to be specifically combined with target molecules, the affinity is strong, and the dissociation constant can reach pmol to nmol; 3. good chemical stability, reversible denaturation and renaturation, easy room temperature transportation, long-term storage and the like.
Drawings
FIG. 1 is a graph showing the expression and localization of CD133 protein in different cell lines according to one embodiment of the present invention;
FIG. 2 is a specific detection map of aptamer AP-1-M according to one embodiment of the present invention;
FIG. 3 is a graph showing the detection of the binding of aptamer AP-1-M to FRO cells according to one embodiment of the present invention.
FIG. 4 is a schematic diagram of the coupling synthesis of aptamers AP-1-M and Dox according to one embodiment of the present invention;
FIG. 5 is a specific assay for the aptamer AP-1-M according to one embodiment of the present invention.
Detailed Description
The invention will be further described with reference to the accompanying drawings.
Example 1: screening for aptamers that target binding to CD133 protein
a. Screening aptamers which are targeted to be combined with CD133 protein from the ssDNA library by adopting a cell screening method;
b. b, detecting the ssDNA screened in the step a by a high-throughput sequencing method, analyzing the stability and the affinity of the sequence, and screening a nucleic acid aptamer which is targeted and combined with the CD133 protein;
c. and c, verifying that the aptamer which is screened out in the step b and is targeted to bind the CD133 protein can be specifically targeted to bind the CD133 protein through an immunofluorescence method, and the aptamer can be bound with ATC cells expressing the CD133 protein.
The sequence of one of the screened aptamer which is targeted to bind with the CD133 protein is shown as SEQ ID NO.1 in the sequence table, namely AP-1-M.
Example 2: expression and localization of CD133 protein in different cell lines
As shown in FIG. 1, Caco-2: colon adenocarcinoma cells; FRO: undifferentiated thyroid cancer cells; NTH: normal thyroid cells; 293T: human embryonic kidney cell line.
The expression of CD133 protein in different cell lines was observed by Confocal Microscopy (Confocal Microscopy) (FIG. 1), red fluorescence (i.e., bright spots in the four lower left micrographs in FIG. 1) represents the expression distribution of the membrane protein CD133 protein, and blue fluorescence (i.e., bright spots in the nuclei of each micrograph in FIG. 1) represents DAPI, indicating the cell nucleus. The results showed that CD133 protein was expressed on the cell membrane in FRO cells of undifferentiated thyroid cancer and Caco-2 cells of colon adenocarcinoma cells as a positive control group, while HEK-293T cells and normal thyroid cells Nthy-ori3-1(NTH) as a negative control group did not express CD133 protein.
Example 3: aptamer AP-1-M binds to CD133 positive and negative cells.
As shown in FIG. 2, AP-1-M aptamer was used to label AP-1-M aptamer by FITC, HEK-293T cells over-expressing CD133 protein were used as positive cells, wild-type HEK-293T cells were used as negative cells, AP-1-M aptamer, positive cells and negative cells were incubated at 4 ℃ and observed under confocal laser microscopy to find that AP-1-M and positive cells were combined and localized on the cell membrane (FIG. 2-A) and not combined with negative cells (FIG. 2-B), which also indicates that AP-1-M is targeted aptamer for CD133 protein.
The results showed that AP-1-M is an aptamer that specifically binds to CD133 protein, and that AP-1-M has a Kd value of 101.4nM as determined by flow cytometry for the affinity curve of the aptamer.
Example 4: binding of aptamer CD133 and undifferentiated thyroid cancer cell FRO.
As shown in FIG. 3, AP-1-M and FRO cells were incubated at 4 ℃ and 37 ℃ for 30 minutes, respectively, and it was found that AP-1-M and FRO cells were bound and localized on the cell membrane due to the inhibition of endocytosis of the cells at 4 ℃ (FIG. 3-A); whereas AP-1-M was engulfed intracellularly by FRO cells due to endocytosis of the cells at 37 deg.C (FIG. 3-B).
The result shows that the AP-1-M can be combined with the FRO cell, is positioned on the cell membrane and can be endocytosed into the cell so as to achieve the aim of targeted delivery.
Example 5: method for coupling aptamer AP-1-M with drug
As shown in FIG. 4, the DNA sequence rich in GC base pairs is preferably studied, and because the DNA sequence rich in GC base pairs can polymerize by itself to form a pocket structure, drugs containing loops, such as adriamycin, erythromycin and the like, are physically embedded, and the drug loading of the Aptamer is detected and calculated by utilizing the characteristic that the adriamycin has a fluorescence spectrum of itself and is quenched in the GC base pairs.
Firstly connecting 8 pairs of GC base sequences to the 5' end of AP-1-M, then incubating AP-1-M and Dox in different concentration ratios, after incubating for 2 hours, comparing the absorbance at 590nM by using a spectrophotometer, finally determining 1:10 as the optimal concentration ratio, and obtaining the aptamer drug conjugate-AP-1-M-Dox.
Based on this, the AP-1-M-Dox of the invention can be prepared into preparations for treating CD133 positive tumors (such as ATC).
Example 6: drug toxicity effect of AP-1-M-Dox on normal thyroid cells and undifferentiated thyroid cancer cells
FIG. 5 shows a specific assay diagram of aptamer AP-1-M (A: normal thyroid cell Nthy-ori 3-1; B: undifferentiated thyroid cancer cell: FRO)
Respectively taking a normal thyroid cell strain Nthy-ori3-1(NTH) and an undifferentiated thyroid (ATC) cell strain FRO to pave in a 96-well plate with 5000 per well (90 ul of RPMI-1640 culture solution containing 10% fetal bovine serum); NTH cell strain is control group A, FRO cell strain is experimental group B, and is divided into group A1 (AP-1-M-Dox treatment group) and group A2 (Dox treatment group), group B1 (AP-1-M-Dox treatment group) and group B2 (Dox treatment group) according to different cell treatment modes; then adding adriamycin (Dox) or AP-1-M-Dox (10 uL/well) with different concentrations into cells in different wells of each group, wherein 9 groups are provided, the first group of cells are not treated and are used as negative control, and the concentration of the rest 8 groups is sequentially increased by 2 times, the minimum concentration is about 0.016umol/mL, and the maximum concentration is 50 umol/mL; culturing in a saturated water vapor carbon dioxide incubator with 5% CO2 at 37 deg.C for 3 hr, and changing the culture solution of all the cells in the wells after 3 hr (90 ul of RPMI-1640 culture solution containing 10% fetal calf serum); and finally culturing in an incubator for 48 hours, adding 10 mu L of CCK into each hole, culturing in the incubator for 3 hours, and measuring the OD value of 450nm by using an enzyme-labeling instrument.
The result shows that AP-1-M-Dox has good cytotoxicity on the FRO of ATC cells, but the toxicity is slightly weaker than Dox, and the toxicity is obviously weaker than Dox on NTH of normal thyroid cells which do not express CD 133.
Sequence listing
<110> Zhejiang province tumor hospital
<120> aptamer combined with CD133 protein in targeting manner, and screening method and application thereof
<130> CN-CN-2018-0675-1
<141> 2018-12-06
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 46
<212> DNA
<213> Artificial Sequence
<400> 1
taccagccgt ttccccggag ggtcacccct gacgcattcg gttgac 46

Claims (5)

1.一种靶向结合CD133蛋白的核酸适配体，其特征在于，所述靶向结合CD133蛋白的核酸适配体为单链DNA核酸适配体，所述靶向结合CD133蛋白的核酸适配体序列如序列表中SEQ ID NO.1所示。1. A nucleic acid aptamer for targeting and binding to CD133 protein, wherein the nucleic acid aptamer for targeting and binding to CD133 protein is a single-stranded DNA nucleic acid aptamer, and the nucleic acid aptamer for targeting and binding to CD133 protein is a single-stranded DNA nucleic acid aptamer. The ligand sequence is shown in SEQ ID NO. 1 in the sequence listing. 2.根据权利要求1所述的一种靶向结合CD133蛋白的核酸适配体，其特征在于，所述靶向结合CD133蛋白的核酸适配体序列5’端还连接有8对GC碱基序列。2. a kind of nucleic acid aptamer targeting binding CD133 protein according to claim 1, is characterized in that, the 5' end of the nucleic acid aptamer sequence targeting binding CD133 protein is also connected with 8 pairs of GC bases sequence. 3.根据权利要求1所述的靶向结合CD133蛋白的核酸适配体和细胞毒性药物的偶联物在制备治疗未分化型甲状腺癌的药物中的应用。3 . The application of the conjugate of the nucleic acid aptamer that targets and binds to CD133 protein and a cytotoxic drug according to claim 1 in the preparation of a drug for the treatment of undifferentiated thyroid cancer. 4 . 4.根据权利要求3所述的应用，其特征在于，所述的靶向结合CD133蛋白的核酸适配体偶联毒性药物通过在5’端连接8对GC碱基序列从而结合细胞毒性药物。4. The application according to claim 3, wherein the nucleic acid aptamer-conjugated toxic drug targeting and binding to CD133 protein binds to the cytotoxic drug by connecting 8 pairs of GC base sequences at the 5' end. 5.根据权利要求4所述的靶向结合CD133蛋白的核酸适配体的应用，其特征在于，所述的细胞毒性药物为阿霉素。5 . The application of the nucleic acid aptamer targeting and binding to CD133 protein according to claim 4 , wherein the cytotoxic drug is doxorubicin. 6 .
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