CN115786351B

CN115786351B - A nucleic acid aptamer for detecting TSHR protein, derivatives and applications thereof

Info

Publication number: CN115786351B
Application number: CN202211273364.8A
Authority: CN
Inventors: 吴文灿; 吴恩德; 张嫣宸; 叶茂
Original assignee: Eye Hospital of Wenzhou Medical University
Current assignee: Eye Hospital of Wenzhou Medical University
Priority date: 2022-10-18
Filing date: 2022-10-18
Publication date: 2025-09-23
Anticipated expiration: 2042-10-18
Also published as: CN115786351A

Abstract

The present invention relates to the field of molecular biology, and more particularly to nucleic acid aptamers, derivatives, and applications thereof for detecting TSHR protein. The present invention provides a nucleic acid aptamer for detecting TSHR protein, the nucleotide sequence of which is shown in SEQ NO. 1. The present invention provides a DNA aptamer YC6 with high affinity for TSHR protein, which has been validated at the molecular, cellular, and tissue levels. The present invention discovers that the DNA aptamer YC6 has the ability to recognize and target cells expressing thyrotropin receptors, providing new insights into the diagnosis and treatment of thyroid diseases and thyroid-related eye diseases.

Description

Aptamer and derivative for detecting TSHR protein and application of aptamer and derivative

Technical Field

The invention relates to the technical field of molecular biology, in particular to a nucleic acid aptamer for detecting TSHR protein, a derivative and application thereof.

Background

Thyrotropin receptors act as receptors for Thyroid Stimulating Hormone (TSH), and play a central role in controlling thyroid cell metabolism. The activity of this receptor is mediated by the G protein to activate adenylate cyclase. Graves Disease (GD) is a common systemic autoimmune disease. Since the first description of the syndrome we now call GD in the medical literature early in the 19 th century, the odd link between augmentation and overactivity of the thyroid gland and inflammation and swelling of connective tissue around the eye has plagued the medical community. The relationship between TSHR and GD was not first recognized until Adams and Purves found a long-acting thyroid stimulating agent and demonstrated that it stimulated adenylate cyclase activity in the thyroid. These antibodies are now known as thyrotropin immunoglobulins (TSI). Wherein the immunological tolerance to TSHR is lost by a mechanism which has not yet been established. The result of this misinterpretation of TSHR as "non-self" is the production of thyrotropic immunoglobulins against thyrotropic receptors on thyroid epithelial cells.

Thyrotropin immunoglobulins are directly involved in the pathogenesis of Graves' disease and hyperthyroidism, and interaction with TSHR results in uncontrolled receptor stimulation. The clinical signs of GD are abnormal growth and overactivity of the thyroid, leading to high levels of pathogenic thyroid hormones. These enhance oxygen consumption and metabolism in the target tissue. In addition to the effect on the thyroid, about 20% of actual GD patients develop ocular manifestations of the disease, known as thyroid-related eye disease (TAO). TAO represents a connective tissue activation and remodeling process that can lead to destruction and blindness. In this process, TSI acts through TSHR expressed locally in the perithyroid tissue, and is associated with inflammation and dilation occurring in the orbit. Functional TSHR has been detected in orbital fat, extraocular muscle and orbital fibroblasts. Thus, there is strong evidence supporting the role of TSHR and TSI in the overactivity of the thyroid and orbital pathology of GD. In addition, it has been widely reported that the defect of TSHR is also a cause of thyroid tumors (papillary and follicular cancers) .(Terry Smith(2017)：TSHR as atherapeutic target in Graves'disease,Expert Opinion on Therapeutic Targets,DOI;10.1080/14728222.2017.1288215).

The existing thyrotropin receptor antibody 5C9 antibody K1-70 has the disadvantages of high production cost, long period, poor batch stability, immunogenicity and high price, namely ;(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 EndocrinolMetab.2013;98(5);2153-2159.doi;10.1210/jc.2013-1149) small molecular compound NCGC00229600 (ANTAG)/NCGC 00242364 (ANTAG 3)/SMAS 37a/b, but low affinity (micromolar), short half-life (3 h) and low bioavailability (50 percent) (Marcinkowski P,Hoyer I,Specker E,et al.A New Highly Thyrotropin Receptor-Selective Small-Molecule Antagonist with Potential forthe Treatment ofGraves'Orbitopathy.Thyroid.2019;29(1);111-123.doi;10.1089/thy.2018.0349).

The aptamer has the advantages of small volume, strong specificity, high stability, low immunogenicity, easy modification, targeted delivery construction and the like, and has the potential of being used as targeted recognition and labeling of TSHR proteins. The aptamer is single-stranded DNA or RNA with the length of 20-100 nucleotides, is obtained by systematic evolution screening of the ligand through an exponential enrichment (SELEX) method, and can form a three-dimensional structure to be specifically bound to a target molecule. The most remarkable characteristics of the aptamer are as follows:

1) First, aptamer-mediated molecular recognition has high specificity and can be used to distinguish subtle molecular differences. The proof of this concept is the use of aptamers to distinguish between three different but closely related and morphologically similar Acute Myeloid Leukemia (AML) cells. This ability to accurately distinguish molecular features helps elucidate the molecular basis of pathogenesis-related events.

2) Cell-SELEX was developed to be able to mimic living cells in a real environment, which is able to generate aptamers for any Cell of interest without relying on knowledge of its previous molecular characteristics. Thus, cell-SELEX can be used to discover previously unknown biomarkers, or features thereof that have not been recognized in pathogenesis;

3) The aptamer is easy to modify, can enhance the in vivo stability, can be coupled with other drugs, molecules or nano particles, can mediate itself or coupled particles to enter cells after being combined with receptors on cell membranes, and can be used as an ideal targeting molecule tool for drug delivery.

However, there is currently no report of thyrotropin receptor-specific nucleic acid aptamers.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and provides a aptamer for detecting TSHR protein, a derivative and application thereof, and the aptamer used in the method for constructing and identifying a target protein TSHR preparation and a targeting drug-carrying preparation is simple in preparation, low in cost, good in thyrotropin receptor protein selectivity, strong in binding force, high in detection sensitivity, simple in detection process operation and accurate in result.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a nucleic acid aptamer for detecting TSHR protein, and the nucleotide sequence of the nucleic acid aptamer is shown as SEQ NO 1.

The invention also provides a derivative of the aptamer for detecting the TSHR protein, wherein the derivative is obtained by carrying out radiolabel, therapeutic drug connection, fluorescent label or biotin label on two ends or one end of the aptamer sequence, so as to obtain the aptamer derivative with the same binding capacity to the TSHR as the aptamer.

The invention further provides a derivative of the aptamer for detecting the TSHR protein, wherein the derivative is obtained by deleting or adding one or more nucleotides to the aptamer sequence, so that the aptamer with the same function as the aptamer is obtained.

The invention further provides a nucleic acid aptamer for detecting the TSHR protein, a derivative of the nucleic acid aptamer for detecting the TSHR protein and application of the derivative in preparation of a TSHR protein detection reagent.

The invention further provides a nucleic acid aptamer for detecting the TSHR protein, a derivative of the nucleic acid aptamer for detecting the TSHR protein and application of the derivative in preparation of a TSHR targeting vector reagent.

The invention further provides the aptamer for detecting the TSHR protein, and the derivative of the aptamer for detecting the TSHR protein, and the application of the aptamer for detecting the TSHR protein in researching differentiation between relatively high-expression TSHR cells and relatively low-expression TSHR cells.

The invention further provides a nucleic acid aptamer for detecting the TSHR protein, and an application of the derivative of the nucleic acid aptamer for detecting the TSHR protein in researching the living body mark of the TSHR protein.

The invention further provides a nucleic acid aptamer for detecting the TSHR protein, and an application of the derivative of the nucleic acid aptamer for detecting the TSHR protein in construction of a thyroid disease targeted drug-carrying preparation.

Preferably, the thyroid disorder is one of thyroid nodule, hyperthyroidism, chronic lymphocytic thyroiditis, diffuse lesions of the thyroid, hypothyroidism and thyroid abnormality.

Compared with the prior art, the invention has the following beneficial effects:

1. The invention utilizes cell-SELEX screening technology, and uses slow virus to transfect TSHR plasmid into 293T cell (TSHR-293T) to construct model cell with high expression of TSHR protein, and uses slow virus to transfect empty plasmid into 293T cell (MOCK) as negative screen cell to eliminate DNA non-specifically combined with 293T cell, so as to ensure that the screened aptamer specifically recognizes TSHR protein. Screening with living cells as targets ensures that the aptamer obtained by screening recognizes the natural conformation of the target molecule.

2. The targeted TSHR aptamer has a unique stem-loop structure, has higher binding affinity and specificity through flow detection, can specifically identify TSHR-293T cells, and does not identify 293T cells. The aptamer obtained by screening can be further truncated and optimized, has small molecular weight, saves synthesis cost, improves affinity, is easy to modify and reform, has no cytotoxicity, strong binding specificity, no immunogenicity and high stability. The above advantages make the aptamer as a TSHR protein specific recognition molecular probe, and has important potential for diagnosing and targeted modulation of TSHR protein related diseases.

3. The invention provides a high affinity DNA aptamer YC6 that passes TSHR protein and is validated at the cellular level tissue level at the molecular level. The DNA aptamer YC6 is considered to have the effect of identifying and targeting cells expressing thyrotropin receptor, and provides a new idea for diagnosing and treating thyroid diseases and thyroid-related eye diseases.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the binding of aptamer YC6 to TSHR-293T and MOCK obtained by flow detection;

wherein the abscissa indicates fluorescence intensity and the ordinate indicates cell number.

FIG. 2 shows the fluorescence microscopy of the binding of aptamer YC6 to the orbital fat connective tissue of a thyroid-related eye patient;

TSHRab represents the TSHR antibody fluorescence signal, YC6-CY3 represents the CY3 fluorescein modified aptamer fluorescence signal, DAPI represents the nuclear fluorescence signal, and Merge represents the combined fluorescence signal of aptamer, antibody and cell binding.

FIG. 3 is a representation of the stability of agarose gel to aptamer YC 6.

FIG. 4 is a diagram of the stem-loop structure of the aptamer.

Detailed Description

The invention provides a nucleic acid aptamer for detecting TSHR protein, and a nucleotide sequence ACCGACCGTGCTGGACTCACTCGCAAGGGCACTTTTTTTAGGTCGACTATGAGCGAGCCTGGCG of the nucleic acid aptamer is shown as SEQ NO 1.

In the present invention, the aptamer YC6 for detecting TSHR protein has a unique stem-loop structure under the conditions of 25 ℃ and 1.0mM Na ⁺,0.5mM Mg²⁺, and the structural formula is shown in figure 4.

In the present invention, the thyroid disease is one of thyroid nodule, hyperthyroidism, chronic lymphocytic thyroiditis, diffuse lesions of the thyroid, hypothyroidism and thyroid abnormality.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Cell sources Primary cells used in the following examples were obtained by extraction from adipose connective tissue isolated from orbital decompression of thyroid-associated disease patients, and human embryonic kidney cell line (293T) was purchased from the Living technologies Co., gmbH, wuhanplague, china.

Example 1 the aptamer YC6 was screened by cell-SELEX technology and the binding of the resulting aptamer YC6 to the TSHR-293T and MOCK was detected in a flow assay.

(1) Design of the nucleic acid library and primers used:

Random single-stranded DNA library:

5'-ACCGACCGTGCTGGACTCA (N) 42ACTATGAGCGAGCCTGGCG-3' (N represents A, T, G, C four arbitrary bases, 42 represents 42N, i.e.42 random bases) (SEQNO. 2)

Upstream primer 5 '-fluorescein isothiocyanate-ACCGACCGTGCTGGACTCA-3' (SEQ NO. 3)

Downstream primer 5 '-Biotin-CGCCAGGCTCGCTCATAGT-3' (SEQ NO. 4)

(2) Screening:

The invention uses the TSHR-293T which expresses high TSHR as a positive screening target and uses MOCK cells which do not express TSHR as a negative screening target.

1. Positive screening:

a. incubation, namely dissolving the random DNA library by using a binding buffer solution, denaturing for 5min at 95 ℃, renaturating for 10min on ice, and then incubating for 1h with the pretreated cultured TSHR-293T with the cell fusion degree reaching about 90% at 4 ℃ for more than 48 h.

B. Separating, namely removing the supernatant after incubation, flushing the cells after incubation with a washing buffer solution for a plurality of times, then taking sterile water to scrape the washed cells and a centrifuge tube, denaturing at 95 ℃ for 10min, renaturating on ice for 10min, centrifuging at 5500rpm for 3min, and sucking the supernatant, namely separating to obtain the first round of screening nucleic acid library of the TSHR-293T cells.

And C, amplifying the library by PCR, wherein the library obtained in the step b is used as a template, and the primers are used as primers, and the amplification conditions are 95 ℃ for 30s, 55.9 ℃ for 30s, 72 ℃ for 30s, 8 cycles of amplification, 72 ℃ for 5min. Obtaining a preliminary amplification product, and then performing mass amplification by using the amplification product as a template to amplify a proper cycle number. DNA Single Strand preparation the antisense strand of the PCR amplified product in step c labeled with biotin was separated with streptavidin-modified agarose beads, then the DNA double strand was denatured with 0.2M NaOH, and a fluorescein isothiocyanate-labeled sense DNA single strand library was collected by desalting.

2. Reverse screening, namely incubating the DNA single-stranded library obtained in the step d with reverse screening cells MOCK cells, collecting supernatant after incubation to remove non-specifically bound nucleic acid molecules, and continuously incubating the collected supernatant with positive screening cells for the next screening step.

3. And (3) cycling the screening process, namely repeating the screening process of the step 1 and the step 2 until a nucleic acid aptamer library which is strong in binding with target cell TSHR-293T cells is screened. This process is repeated from several to tens of rounds.

4. High throughput sequencing the nucleic acid library with the highest binding to the last round of screening is subjected to high throughput sequencing, and the binding capacity of the sequence obtained by flow detection to the TSHR-293T cells is utilized to determine the nucleic acid aptamer.

First, TSHR-293T and MOCK were cultured for 48 hours to reach a cell density of 90%, and cells in an adherent state were digested from the dishes with 0.2% EDTA, respectively. Mu.l of binding buffer was used to prepare 250nM of synthesized FAM-labeled YC6, denatured at 95℃for 5min and renatured on ice for 10min, respectively. Incubate with 30 ten thousand TSHR-293T or MOCK cells for 45min at 4 ℃. The incubated cells were washed 2-3 times with wash buffer, and then resuspended in 300 μl wash buffer. Fluorescence detection was performed by flow cytometry, with the DNA initial random library as a control. The aptamer bound only to the target cell TSHR-293T and not to MOCK, and the results are shown in FIG. 1.

FIG. 1, abscissa FAM represents fluorescence signal, and ordinate is the number of cells. Aptamer-modified FAM fluorescence. The aptamer chains obtained by screening were incubated with positive and negative cells, respectively, and it can be seen from FIG. 1 that aptamer YC6 enhanced the fluorescence signal of positive cells, but not significantly enhanced for negative cells.

Example 2 fluorescence microscopy of aptamer YC6 binding to orbital fat connective tissue of thyroid-related eye patients

Paraffin sections are prepared by fixing, dehydrating, embedding and the like of orbital fat connective tissues of thyroid-related eye patients, after dewaxing, antigen retrieval and blocking and the like, the sections are incubated with TSHR antibody ((1:1000, abcam, abc974)) overnight, washed 3 times, incubated with secondary anti-goat anti-mouse second antibodyAF488 (1; 400, bios), prepared with 500 μl binding buffer into 250nM synthetic CY3 labeled YC6, denatured at 95 ℃ for 5min, renatured on ice for 10min, then incubated with sections for 45min at 4 ℃, washed 3 times, and sealed with DAPI-containing sealing liquid. Fluorescence signals at 340nm wavelength, 488nm wavelength, 570nm wavelength were detected by fluorescence microscopy, and the results are shown in FIG. 2.

FIG. 2 fluorescence microscopy detects binding of aptamer YC6 to orbital fat connective tissue of thyroid-related eye patients.

DAPI (DAPI-nuclear stain) reagent

TSHRab:goat-anti,mouse,TSHR,antibody

YC6-CY3, CY3 fluorescein modified YC6 aptamer

MERGE signal superposition

Example 3 characterization of stability of the aptamer YC6 by agarose gel

First, the synthesized aptamer YC6 was dissolved in DMEM complete medium containing 10% FBS at a final concentration of 3. Mu.M, and incubated at 37℃for 0h,1h,2h,4h,6h,12h,24h,48h,95℃for 5min, and then renatured on ice for 10min, and then stored at-80 ℃. The stability of the above samples was checked by 3% agarose gel electrophoresis. Compared with 0h, YC6 is incubated for 48h in the complete culture medium, and has no obvious degradation and stronger stability, and the result is shown in figure 3.

FIG. 3 is a graph showing stability of the aptamer YC6 to agarose gel.

The aptamer YC6 was dissolved in DMEM complete medium containing 10% FBS at a final concentration of 3. Mu.M, and incubated at 37℃for 0h,1h,2h,4h,6h,12h,24h,48h,95℃for 5min, and ice-renatured for 10min, and then stored at-80 ℃. The stability of the above samples was checked by 3% agarose gel electrophoresis. Compared with 0h, YC6 is incubated for 48h in a complete culture medium, and has no obvious degradation and high stability.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The aptamer for detecting thyrotropin receptor protein is characterized in that the nucleotide sequence of the aptamer is shown as SEQ ID NO. 1.

2. The aptamer of claim 1, wherein the aptamer sequence is radiolabeled, therapeutic drug linked, fluorescently labeled, or biotin labeled at both or one end of the sequence.

3. Use of the aptamer of claim 1 or 2 for the preparation of a detection reagent for thyrotropin receptor proteins.

4. Use of the aptamer of claim 1 or 2 for the preparation of a thyrotropin receptor targeting vector reagent.

5. Use of the aptamer of claim 1 or 2 for the preparation of a labelling reagent for thyrotropin receptor proteins.

6. Use of the aptamer of claim 1 or 2 for the preparation of a targeted drug-loaded formulation for thyroid diseases.

7. The use according to claim 6, wherein the thyroid disorder is one of thyroid nodules, hyperthyroidism, chronic lymphocytic thyroiditis, diffuse lesions of the thyroid, hypothyroidism and thyroid disorders.