A nucleic acid aptamer targeting TSHR protein, a nucleic acid aptamer derivative and its application

Abstract

The present invention provides a nucleic acid aptamer targeting TSHR protein, a nucleic acid derivative and its application, and belongs to the field of molecular biology technology. The nucleic acid aptamer targeting TSHR protein in the present invention comprises a nucleotide sequence as shown in SEQ ID NO.1, and one or more modifications including radioactive labeling, therapeutic drug connection, fluorescent labeling or biotin labeling are performed on both ends or one end of the nucleic acid aptamer targeting TSHR protein to obtain a nucleic acid aptamer targeting TSHR protein and its derivatives. The nucleic acid aptamer and its derivatives in the present invention can identify and target cells expressing thyrotropin receptors, have high affinity, strong stability, low cost, and have the advantages of in vitro and in vivo imaging labeling functions, providing new molecular tools for the diagnosis and treatment of thyroid diseases and thyroid-related eye diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a nucleic acid aptamer targeting TSHR protein, a nucleic acid aptamer derivative and applications thereof. Background Art

[0002] The thyrotropin receptor (TSH), acting as the receptor for thyroid-stimulating hormone (TSH), plays a central role in controlling thyroid cell metabolism. The activity of this receptor is mediated by G proteins, which activate adenylate cyclase. Graves' disease (GD) is a common systemic autoimmune disease. Since its first description in medical literature in the early 19th century, the syndrome we now call GD has puzzled the medical community by its curious connection between an enlarged and overactive thyroid gland and inflammation and swelling of the connective tissue around the eye. The relationship between the TSHR and GD was not recognized until Adams and Purves discovered a long-acting thyroid stimulant and demonstrated that it stimulated adenylate cyclase activity in the thyroid gland. These antibodies are now known as thyrotropin immunoglobulins (TSIs). Immune tolerance to the TSHR is lost through an as-yet-unidentified mechanism. This misinterpretation of the TSHR as "non-self" results in the production of TSIs that target the TSHR on thyroid epithelial cells. TSIs are directly involved in the pathogenesis of Graves' disease and hyperthyroidism, interacting with the TSHR and leading to uncontrolled receptor stimulation. The clinical hallmark of GD is abnormal growth and overactivity of the thyroid gland, resulting in pathologically high thyroid hormone levels. These levels enhance oxygen consumption and metabolism in target tissues. In addition to the effects on the thyroid gland, approximately 20% of patients with true GD experience ocular manifestations of the disease, termed thyroid-associated ophthalmopathy (TAO). TAO represents a process of connective tissue activation and remodeling that can lead to disfigurement and blindness. Within this process, TSI, which acts through the thyroid gland receptor (TSHR), which is locally expressed in perithyroidal tissue, is associated with inflammation and dilatation within the orbit. Functional TSHR has been detected in orbital fat, extraocular muscles, and orbital fibroblasts. Therefore, there is strong evidence supporting a role for TSHR and TSI in thyroid overactivity and orbital pathology in GD. Furthermore, defects in TSHR have been widely reported as a cause of thyroid tumors (papillary and follicular carcinomas). (Terry Smith (2017): TSHR as atherapeutic target in Graves'disease, Expert Opinion on Therapeutic Targets, DOI: 10.1080 / 14728222.2017.1288215)

[0003] Currently, there is an antibody targeting the thyrotropin receptor, 5C9 antibody K1–70, but it has the disadvantages of high production cost, long cycle, poor batch stability, immunogenicity, and high price; (TurcuAF, Kumar S, Neumann S, et al. A small molecule antagonist inhibits thyrotropin receptor antibody-induced orbital fibroblast functions involved in the pathogenesis of Graves ophthalmopathy. J Clin Endocrinol Metab. 2013; 98(5): 2153-2159. doi: 10.1210 / jc.2013-1149) small molecule compound NCGC00229600 (ANTAG2) / NCGC00242364 (ANTAG3) / SMAS37a / b, but it has the disadvantages of low affinity (micromolar), short half-life (<3h), and low bioavailability (50%). (Marcinkowski P, Hoyer I, Specker E, et al. A New Highly Thyrotropin Receptor-Selective Small-Molecule Antagonist with Potential for the Treatment of Graves' Orbitopathy. Thyroid. 2019; 29(1):111-123. doi:10.1089 / thy.2018.0349).

[0004] Aptamers have the advantages of small size, strong specificity, high stability, low immunogenicity, ease of modification, and construction for targeted delivery, showing potential as target recognition and protein labeling. Aptamers are single-stranded DNA or RNA molecules 20-100 nucleotides in length. They are derived through systematic evolutionary selection of ligands using a method called Selective Electron Extraction (SELEX) (SELEX) and can form three-dimensional structures that specifically bind to target molecules. The most notable features of aptamers are as follows: 1) First, aptamer-mediated molecular recognition is highly specific, allowing it to distinguish subtle molecular differences. This proof of concept was demonstrated by using aptamers to distinguish three different but closely related, morphologically similar acute myeloid leukemia (AML) cell lines. This ability to accurately distinguish molecular features helps elucidate the molecular basis of pathogenesis. 2) Cell-SELEX was developed to simulate the real environment of living cells, enabling the generation of aptamers for any cell of interest without relying on prior knowledge of its molecular characteristics. Therefore, Cell-SELEX can be used to discover previously unknown biomarkers or their yet-to-be-recognized features in pathogenesis. 3) Aptamers are easily modified, which can enhance their in vivo stability. They can also be coupled to other drugs, molecules, or nanoparticles. After binding to receptors on the cell membrane, they can mediate their own entry or that of coupled particles into cells, making them ideal targeted molecular tools for drug delivery. However, no thyrotropin receptor-specific aptamers have been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide a nucleic acid aptamer targeting TSHR protein, a nucleic acid aptamer derivative and an application thereof.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a nucleic acid aptamer YC2 targeting TSHR protein, characterized in that it comprises a nucleotide sequence as shown in SEQ ID No.1.

[0008] Preferably, the nucleic acid aptamer YC2 targeting TSHR protein is heated at 22-27°C and 0.5-2.0 mM Na + , 0.1-1.0mMMg 2+ Under certain conditions, it has a stem-loop structure, such as Figure 1 shown.

[0009] Preferably, bases are added, deleted or replaced on the basis of the nucleic acid aptamer YC2 to obtain a nucleic acid aptamer with the same function.

[0010] Preferably, the TSHR protein is of human origin.

[0011] The present invention also provides a nucleic acid aptamer YC2 derivative targeting TSHR protein.

[0012] Preferably, the nucleic acid aptamer derivative is modified by one or more modifications including radioactive labeling, therapeutic drug linkage, fluorescent labeling or biotin labeling at both ends or one end of the nucleic acid aptamer YC2 targeting the TSHR protein.

[0013] The present invention also provides the use of the nucleic acid aptamer YC2 targeting TSHR protein and the nucleic acid aptamer YC2 derivative targeting TSHR protein in any of the following aspects:

[0014] (1) Preparation of TSHR protein detection reagent;

[0015] (2) preparing TSHR targeting vector reagents;

[0016] (3) Differentiation study between relatively high-expressing TSHR cells and relatively low-expressing TSHR cells;

[0017] (4) In vivo labeling study of TSHR protein;

[0018] (5) Prepare targeted drug-loaded preparations for thyroid-related diseases.

[0019] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention provides a nucleic acid aptamer targeting TSHR protein, and by characterizing the properties of the nucleic acid aptamer targeting TSHR protein and identifying the target molecule, it is helpful to discover one of the markers of Graves' disease (GD) - TSHR protein. The prepared nucleic acid aptamer can specifically recognize TSHR protein and target cells expressing thyrotropin receptor, and has the advantages of high affinity, non-immunogenicity, sequence stability, easy storage, convenient labeling, and small molecular weight; different parts of the nucleic acid aptamer are easy to modify and replace. When the nucleic acid aptamer of the present invention is used for TSHR protein detection, the operation is simpler and faster, and the synthesis cost of the nucleic acid aptamer of the present invention is lower than that of antibody preparation, and the cycle is short, the reproducibility is good, and there is no cytotoxicity, which provides a new idea for the diagnosis and treatment of thyroid diseases and thyroid-related eye diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For aptamer YC2, at 22-27°C, 0.5-2.0 mM Na+, 0.1-1.0 mM Mg 2+ Structural formula under conditions;

[0021] Figure 2 Flow cytometry analysis of the binding of the obtained nucleic acid aptamer YC2 to TSHR-293T and MOCK (the horizontal axis is the fluorescence intensity, and the vertical axis is the number of cells);

[0022] Figure 3The target type of nucleic acid aptamer YC2 was preliminarily identified as membrane protein for flow cytometry detection;

[0023] Figure 4 Aptamer-pull down demonstrated that the protein that YC2 may interact with TSHR-293T cells is TSHR (the first column is the positive control group, the second column is the blank group, the third column is the lib group, and the fourth column is the YC2 group). DETAILED DESCRIPTION

[0024] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

[0025] Example 1 Construction of TSHR-293T and MOCK cells

[0026] In this example, human embryonic kidney cell line (293T) was purchased from Wuhan Punosai Life Science Technology Co., Ltd., China.

[0027] (1) Construction of TSHR-293T

[0028] The human TSHR overexpression plasmid was transfected into the above 293T cells using lentivirus to obtain TSHR-293T. The steps are as follows:

[0029] 1. Construction of lentiviral expression vector containing TSHR target gene and plasmid purification and extraction;

[0030] 2. Co-transfect the lentiviral expression vector and packaging system into virus packaging 293T cells;

[0031] 3. Cell drug resistance screening.

[0032] (2) Construction of MOCK cells

[0033] The empty plasmid was transfected into 293T cells using lentivirus to generate MOCK cells.

[0034] Example 2 Screening of the nucleic acid aptamer YC2 using cell-SELEX technology

[0035] (1) Design of DNA library and primers used:

[0036] Random single-stranded DNA library:

[0037] 5'-ACCGACCGTGCTGGACTCA(N)42ACTATGAGCGAGCCTGGCG-3' (N represents any of the four bases A, T, G, and C) (SEQ ID NO. 2), which is a custom sequence randomly synthesized by Shanghai Bioengineering, China.

[0038] Upstream primer: 5'-fluorescein isothiocyanate-ACCGACCGTGCTGGACTCA-3' (SEQ ID NO. 3)

[0039] Downstream primer: 5'-biotin-CGCCAGGCTCGCTCATAGT-3' (SEQ ID NO. 4)

[0040] (2) Screening process:

[0041] The present invention uses TSHR-293T cells that highly express TSHR as a positive screening target and MOCK cells that do not express TSHR as a counter-screening target.

[0042] 2.1 Positive screening:

[0043] a. Incubation: Dissolve the randomized DNA library in binding buffer, denature at 95°C for 5 minutes, and anneal on ice for 10 minutes. Then, incubate with pretreated TSHR-293T cells that have been cultured for at least 48 hours and have reached approximately 90% confluency at 4°C for 1 hour. The binding buffer contains 5 mM MgCl2, 4.5 mg / ml glucose, 0.1 mg / ml tRNA, and 1 mg / ml BSA in DPBS. The incubation solution has the same composition as the binding buffer.

[0044] b. Isolation: Remove the incubation supernatant and rinse the cells several times with a wash buffer containing 5 mM MgCl2 and 4.5 mg / ml glucose in DPBS. Scrape the washed cells with sterile water and place them in a centrifuge tube. Denature at 95°C for 10 minutes, anneal on ice for 10 minutes, and centrifuge at 5500 rpm for 3 minutes. Aspirate the supernatant to isolate the first-round screening nucleic acid library of TSHR-293T cells.

[0045] c. PCR amplification of the library: Using the library obtained in step b as a template, perform amplification using the primers in step 1. Amplification conditions are: 95°C for 30 seconds, 55.9°C for 30 seconds, 72°C for 30 seconds (8 cycles), and 72°C for 5 minutes. A preliminary amplification product is obtained, and then the amplified product is used as a template for 8-12 amplification cycles for large-scale amplification.

[0046] The PCR system is:

[0047]

[0048] d. Preparation of single-stranded DNA: Use streptavidin-modified agarose beads to separate the biotin-labeled antisense strand of the PCR amplification product from step c. Then, denature the double-stranded DNA with 0.2M NaOH and collect the positive-sense single-stranded DNA library labeled with fluorescein isothiocyanate by desalting. The specific steps are as follows:

[0049] Mix the streptavidin-coated agarose beads by vibrating gently. Add 50 μl of beads to each of three 1.5 ml EP tubes (50 μl per 1 ml of DNA). Add 1 ml of the PCR product from 1.3 to each of the EP tubes containing the beads. Place the EP tubes on a Ferris wheel and incubate at 4°C for 1 hour.

[0050] SSDNA extraction - denaturing column

[0051] Remove the EP tube after incubation, centrifuge it slightly to concentrate the bottom, and place it in an ice box for use.

[0052] Mix the DNA-bead mixture by pipetting, then add it to the denaturing column. Collect the filtrate in the same EP tube. Once collected, add the filtrate to the denaturing column and filter. Repeat this process 3-4 times. Finally, rinse the EP tube with 5ml of DPBS and pass the wash solution through the column to thoroughly clean the tube and minimize DNA loss. Then, add 10ml of DPBS to the column to rinse the beads.

[0053] After washing, clean the column cap with DPBS, and then use the washed column cap to block the outflow port at the bottom of the column. Add about 460 μl 0.2 mol / L NaOH to the denaturing column, cover the column cap, and denature for 3 minutes.

[0054] After denaturation, take out a new 1.5ml EP tube to collect the filtrate until no liquid drips naturally. If the collected filtrate is less than 500μl, add sterile water to make up to 500μl.

[0055] ssDNA desalting - desalting column

[0056] When the last drop of sterile water is added to the desalting column, add the collected 500μl ssDNA filtrate. When the filtrate reaches the upper membrane surface of the desalting column, it indicates that all 500μl filtrate has entered the desalting area. At this time, use a new 1.5ml EP tube to collect the filtrate and add 1ml of sterile water to the column. Collect the dripping liquid to obtain 1ml of desalted ssDNA product and place it in an ice box for concentration measurement.

[0057] 2.2 Counter-screening: Incubate the single-stranded DNA library obtained in step d with counter-screening cells MOCK cells at 4°C for 30 min-2 h. Collect the supernatant after incubation to exclude non-specifically bound nucleic acid molecules. Collect the supernatant and continue incubating with positive screening cells for the next screening step.

[0058] 2.3 Cycle of screening process: Repeat the screening processes of 2.1 and 2.2 until a nucleic acid aptamer library with strong binding to target cells TSHR-293T cells is screened.

[0059] 2.4 High-throughput sequencing: The nucleic acid library with the largest binding site in the last round of screening was subjected to high-throughput sequencing, and the binding ability of the obtained sequences to TSHR-293T cells was detected by flow cytometry to determine the nucleic acid aptamer.

[0060] The final sequence of the nucleic acid aptamer YC2 was determined as follows:

[0061] 5'-ACCGACCGTGCTGGACTCACCATCTCTCCGAGAGCGCGAACTATGAGCGAGCCTGGCG-3' (SEQ ID NO. 1).

[0062] (3) Flow cytometry analysis of the binding of the obtained nucleic acid aptamer YC2 to TSHR-293T and MOCK

[0063] First, TSHR-293T and MOCK were cultured for 48 hours to make the cell density reach 90%, and then the attached cells were digested from the culture dish with 0.2% EDTA. 250nM synthetic FAM-labeled YC2 (synthesized by Shanghai Shenggong Bioengineering Co., Ltd.) was prepared with 200μl binding buffer, denatured at 95℃ for 5 minutes, and renatured on ice for 10 minutes. Incubated with 300,000 TSHR-293T or MOCK cells at 4℃ for 45 minutes. Wash the incubated cells 2-3 times with washing buffer, and then resuspend the cells in 300μl washing buffer. Fluorescence detection was performed by flow cytometry. The model of the flow cytometer was BD C6 plus, and the initial random DNA library was used as a control. The results are as follows. Figure 2 As shown by Figure 2 It can be seen that aptamer YC2 enhances the fluorescence signal of positive cells, but does not significantly enhance the fluorescence signal of negative cells, indicating that the nucleic acid aptamer only binds to the target cell TSHR-293T but not to MOCK.

[0064] Example 3 Preliminary Verification of Target Type of Aptamer YC2 by Flow Cytometry

[0065] First, culture TSHR-293T cells for 48 hours to make the cell density reach 90%, and then use 0.2% EDTA, 0.25% trypsin and 100μg / ml proteinase K to digest the adherent cells from the culture dish. Use 200μl binding buffer to prepare 250nM of synthesized YC2 with FAM labeling at the 5' end, denature at 95℃ for 5 minutes, and renature on ice for 10 minutes. Incubate with 300,000 TSHR-293T cells treated with different digestion methods at 4℃ for 1 hour. Wash the incubated cells 2-3 times with washing buffer, and then resuspend the cells in 300μl washing buffer. Fluorescence detection was performed by flow cytometry, and the initial random library of DNA with the same concentration gradient was used as a control. After treatment with proteinase K and trypsin, the fluorescence intensity of YC2 binding to the target cells returned to the corresponding position of the library, indicating that YC2 bound to the protein on the surface of the TSHR-293T cell membrane. The results are shown in the figure. Figure 3 As shown. Figure 3 It can be seen that after the cells were treated with trypsin or proteinase K and then incubated with the aptamer, there was no obvious enhancement in the fluorescence signal, indicating that the target of the aptamer was damaged; this is because 0.2% EDTA, trypsin and proteinase K treatment of cells will damage the membrane proteins on the cells. Therefore, the YC2 target can be preliminarily identified as a membrane protein.

[0066] Example 4: Aptamer-pull down demonstrated that the protein that interacts between YC2 and TSHR-293T cells is TSHR.

[0067] (1) TSHR-293T cells were cultured in 30 large dishes with a diameter of 10 cm in DMEM + 10% FBS + 1% penicillin-streptomycin double antibody in a 37°C, 5% CO2 incubator.

[0068] (2) When the confluence of cells reached 80%, the cells were digested with 0.2% EDTA, and then membrane proteins were extracted using a membrane protein extraction kit (brand: Feijing, product number: PH0710). 100 μg of membrane protein was taken and blocked with binding buffer supplemented with 20% fetal bovine serum and 0.1 mg / ml salmon sperm single-stranded DNA at 4°C for 1 hour. The mixture was then transferred to streptomycin-coated agarose beads and incubated for 1 hour. The supernatant and bead precipitate were obtained by centrifugation (blank group). The supernatant was further incubated with Biotin-library (biotin-labeled library, synthesized by Shanghai Biotech), and then centrifuged to obtain supernatant and bead precipitate (lib group); the supernatant was further incubated with Biotin-YC2 (YC2 nucleic acid aptamer labeled with biotin at the 5' end, synthesized by Shanghai Biotech), and then centrifuged to obtain bead precipitate (YC2 group).

[0069] (3) The beads in the blank, lib, and YC2 groups were washed five times with washing buffer, which was a DPBS solution containing 5 mM MgCl2 and 4.5 mg / ml glucose. Then, 2X loading buffer was added and denatured at 70°C for 10 min, renatured on ice for 10 min, and centrifuged at 5000 rpm for 3 min. The supernatant protein sample was carefully obtained.

[0070] (4) Prepare 10% SDS-PAGE gel and load the samples in the following order: marker, membrane protein positive control, blank group, lib group, and YC2 group. Transfer the membrane to NC membrane, block with 5% skim milk for 1 h, and incubate with TSHR antibody (brand: abcam, product number: ab27974) at a concentration of 1:1000 at 4°C overnight.

[0071] (5) Wash the NC membrane with TBST buffer for 10 min, repeat three times, and incubate the NC membrane with goat anti-mouse IgG secondary antibody at a working concentration of 1:5000 for 1 h before Odyssey imaging.

[0072] The results are as follows Figure 4 As shown, in Figure 4 In the figure, the second and third columns lack bands, while the fourth column does. This successfully demonstrates that YC2 can pull down TSHR, while the control group Lib cannot. Therefore, it is confirmed that the protein that YC2 interacts with TSHR-293T cells is TSHR.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A nucleic acid aptamer YC2 targeting thyrotropin receptor protein, characterized in that: The nucleotide sequence is shown as SEQ ID No.

1.

2. The nucleic acid aptamer YC2 according to claim 1, characterized in that The nucleic acid aptamer targeting the thyrotropin receptor protein is heated at 22-27° C. and 0.5-2.0 mM Na + , 0.1-1.0 mM Mg 2+ Under certain conditions, it has a stem-loop structure.

3. The nucleic acid aptamer YC2 according to claim 1, characterized in that The thyrotropin receptor protein is of human origin.

4. The nucleic acid aptamer YC2 according to claim 1, characterized in that One or more modifications including radioactive labeling, therapeutic drug connection, fluorescent labeling or biotin labeling are performed on both ends or one end of the nucleic acid aptamer.

5. Use of the nucleic acid aptamer YC2 according to any one of claims 1 to 4 in any of the following aspects: (1) Application in the preparation of detection reagents for thyrotropin receptor protein; (2) Application in the preparation of thyrotropin receptor-targeted carrier reagents; (3) Application in the preparation of labeling reagents for thyrotropin receptor protein; (4) Application in the preparation of targeted drug-loaded preparations for thyroid diseases.

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