CN112094846A - Modified base aptamer of specific targeting osteoarthritic synovial cell and application thereof - Google Patents

Abstract

The invention provides a modified base aptamer specifically targeting osteoarthritic synovial cells, and the aptamer has a nucleotide sequence shown in SEQ ID NO.1 or 2. The invention finally obtains the modified base aptamer of the specific targeted osteoarthritis synovial cells by positive screening of the modified aptamer library and the osteoarthritis synovial cells and negative back screening of various skeletal muscle source cells. The synovial cell aptamer sequence is screened from a modified aptamer library, the screening process is simple and convenient, the complicated steps of the traditional method are greatly reduced, the binding force with target cells is stronger due to the modified structure, the specificity of the synovial cell aptamer is greatly enhanced, the defect that the synovial cells from osteoarthritis diseases are bound with appropriate ligands in a targeted manner at present is overcome, and the specific treatment of synovitis can be directly carried out on different target effects such as drug delivery, interfering RNA (ribonucleic acid), miRNA (micro ribonucleic acid), exosome and the like through the modified group Amine-dU in the target aptamer.

Description

Modified base aptamer of specific targeting osteoarthritic synovial cell and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a modified base aptamer specifically targeting osteoarthritic synovial cells and application thereof.

Background

Osteoarthritis (OA) is a common type of arthritis in the elderly population, which can ultimately lead to the destruction and loss of joint function, severely affecting the quality of life of the elderly, and being a heavy burden on both the individual and society. Factors such as early occult nature of OA diseases and complex differential diagnosis are also reasons why the treatment time of these diseases is delayed and the prognosis is poor. The basic pathological features of OA are cartilage degeneration, subchondral bone hyperplasia, synovitis; in persistent chronic synovitis, synovial membrane hyperplasia is accompanied by bone invasion to destroy inflammation. The above-mentioned synovitis diseases, since there is no blood vessel on the surface of the articular cartilage, the consistent pathological change is the secretion of inflammatory cytokines by fibroblast-like synoviocytes (FLS) of the inner layer of the joint capsule, which normally can secrete lubricants to help joint movement, while FLS in an inflammatory state may aggravate chronic synovitis by increasing the production of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), interleukin 6(IL-6) and interleukin 1 (IL-1).

Currently, the most widely approved drugs for the treatment of RA are, for example: adalilimumab (Adalimumab), Infliximab (Infliximab), hormones and the like are also used for inhibiting cytokines TNF-a, IL-1 beta and IL-6 and treating OA, but obvious side effects such as allergy and monocyte reduction occur when the medicine is used systemically, and the short-term effects such as joint inflammation environment improvement, joint lubrication and the like can be achieved only by local injection of sodium hyaluronate. And the synovial inflammation is inhibited by injecting the medicament in the joint, and the synovial inflammation is not treated in a targeted way, so that the articular cartilage is seriously damaged. Overall, this does improve the inflammatory symptoms of some patients' joints by systemically suppressing immune responses or inflammatory cytokines, but does not completely address the issue that post-inflammatory activated FLS continue to secrete inflammatory cytokines, leading to OA disease progression. Therefore, the clinical treatment urgently needs a medicament which can be used for treating synovitis caused by OA and protecting the articular cartilage, is safe for a long time and can target the nuclear effector cell FLS in the pathogenesis of OA.

The aptamer technology is a nucleic acid technology appearing in recent years, has high specificity and high selectivity binding capacity with a target, and has no antigen-antibody property and no rejection reaction due to the fact that the aptamer belongs to DNA or RNA nucleic acid sequence fragments and is good in stability. Classical aptamer screening assays use the exponential enrichment of ligands by evolutionary evolution (SELEX), a screening and amplification technique first proposed by two groups of the U.S. in the early 90 s. It uses random oligonucleotide library with large capacity and combines with Polymerase Chain Reaction (PCR) in vitro amplification technology, and can obtain nucleic acid aptamer with high affinity and high specificity through several or tens of rounds of screening. The technology has no special requirement on the target substance, and various aptamers of the target substance, including metal ions, organic dyes, medicines, amino acids, nucleotides, polypeptides, cells and the like, are screened out at present. In addition, target proteins for aptamers are: enzymes, growth factors, antibodies, gene regulatory factors, cell adhesion factors, lectins, and the like. Because the aptamer has the characteristics of wide target molecule range, high affinity, strong specificity and the like, the screened aptamer can be specifically combined with target molecules playing an important role in the occurrence and development of diseases, and the function of the target molecules is blocked or closed, so that the aim of treating the diseases is fulfilled. Based on the above, the aptamer serving as a novel molecular probe has wide application prospects in aspects of drug screening, disease diagnosis, clinical treatment and the like, and has important significance in diagnosis and treatment of tumors. The Cell aptamer screening technology is a technology for enriching and screening aptamer libraries for target cells through multiple rounds, and in the Cell-SELEX technology (Cell-SELEX), living cells are used for selecting aptamers. In the field of tumor therapy, the Cell-SELEX method can select specific aptamers as cancer Cell targets, and aptamer-based probes have been developed for a variety of cancers, including cervical, ovarian, liver, prostate, breast, glioma, colorectal, and lung cancers. The Cell-SELEX target Cell screening technology mainly has the following advantages: the cell surface has a large number of molecules, which are likely to be targets, the native conformation of which is important for diagnostic and therapeutic applications; furthermore, since the whole cell is used for selection, no a priori knowledge of the biomarkers for the cell is required. An effector Cell of an inflammation mechanism in OA synovitis is FLS, Cell-SELEX is used for screening aptamers combined with FLS in a targeted mode, different medicines are carried for treating joint synovitis caused by different causes, and the method can be a key technology for solving diagnosis and treatment of the joint inflammation, shortening the course of disease and reducing side effects of treatment.

At present, in the research on the aspect of aptamer aiming at synovial cells, patent application No. 201510063418.1 entitled aptamer of specific targeting rheumatoid arthritis synovial cells and application thereof is based on a common aptamer library, adopts the traditional SELEX technology, and utilizes a human fibroblast synovial cell line MH7A positive sieve and a human hepatocyte line L-02 reverse sieve to screen out aptamer only suitable for the rheumatoid inflammation synovial cells. However, it was found in the patent that the aptamer selected based on it had no disclosure of dissociation constant, uncertain binding force, and was directed only to the aptamer of RA synovitis, so the aptamer directed to rheumatoid synovitis may not be suitable for OA synoviocytes. Meanwhile, the screening in the patent is based on the traditional common aptamer library, and the aptamers are not modified properly, so that the practical application of the aptamers to the diagnosis, treatment and the like of related diseases of synovial cells is limited. In addition, the aptamer is not freely loaded with technologies of detachable assembly of nano-sized cells, nucleic acids, proteins and the like, and the anti-screening cells only use hepatocytes, so that the specificity of the aptamer to distinguish fibroblast-derived lineage cells (such as chondrocytes, meniscus cells and tenocytes) cannot be proved.

Disclosure of Invention

The object of the present invention is to overcome the above-mentioned disadvantages of the prior art and to provide modified base aptamers. The invention screens two kinds of nucleic acid aptamer sequences aiming at synovial cells based on a modified aptamer library, and the aptamer can provide a reliable carrier for the subsequent use of the synovial cells for diagnosing or targeting treatment of joint inflammation.

The patent is based on a modified aptamer library, and a nucleic acid aptamer sequence aiming at synoviocytes of osteoarthritis is screened out, and the aptamer can provide a reliable carrier for the subsequent diagnosis or targeted treatment of joint inflammation by using the synoviocytes.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a modified base aptamer specifically targeting osteoarthritic synovial cells, which is characterized in that the aptamer has a nucleotide sequence shown in SEQ ID NO: 1 or 2.

The invention also provides a screening method of the modified base aptamer specifically targeting synovial cells, which is characterized by comprising the following steps of:

(1) preparing oligonucleotide library microspheres;

(2) negative screening: and (2) incubating the negative sieve cells with the oligonucleotide library microspheres obtained in the step (1), and then taking the non-adsorbed microspheres in the supernatant, wherein the non-adsorbed microspheres are the target aptamer microspheres of the invention.

(3) And (3) positive screening: combining the aptamer microspheres obtained in the step (2) with primary osteoarthritis synovial cells to obtain aptamer microspheres combined with the synovial cells;

(4) dissociation of aptamers from primary osteoarthritic synovial cells: and (3) adding an alkaline solution into the aptamer microsphere coupled with the target cell in the step (3), incubating, adding a buffer solution for neutralization and centrifuging, wherein the supernatant contains the oligonucleotide aptamer.

Preferably, the oligonucleotide library in the step (1) uses an X-Aptamer kit.

Preferably, the alkaline solution in step (4) is 1M NaOH, and the buffer solution is 2M Tris-Cl.

Preferably, the nucleotide sequence of the oligonucleotide aptamer in the step (4) is shown in SEQ ID NO: 1. 2, respectively.

The invention also provides an application demonstration of the modified base group Amine-dU in the specific targeting synovial membrane cell aptamer in the preparation of a medicine or a preparation for treating or diagnosing related diseases.

The invention has the beneficial effects that: the basic structure of the aptamer is completely different from that of the existing aptamer, the binding force of the aptamer and target cells is stronger, the blank that the target of synovial cells without osteoarthritis is bound with a suitable ligand at present is solved, and the aptamer can be directly labeled by a modifying group in the target aptamer, carried with nanoparticles, transmitted with drugs and the like. The screening process is simple and convenient in screening from the modified aptamer library, and the complicated steps of the traditional method are greatly reduced. And a plurality of synovial surrounding cells (chondrocytes, meniscal cells and tenocytes) are screened reversely, so that the specificity is greatly enhanced.

Drawings

Fig. 1 is a two-level schematic diagram (left) and a structural diagram (right) of a modified nucleotide molecule of two aptamers of the present invention, wherein in the diagram a, a base "T" in an original sequence is replaced by a modified base "X", "Y" or "W", and a replacement symbol "═ is shown; the base structure of X, Y, W in FIG. B.

FIG. 2 shows the binding of the aptamer library and 2 selected aptamers to synovial cells. From this figure, the SA1 and SA2 aptamers with high binding affinity to synoviocytes of osteoarthritis are obtained.

FIG. 3 is a specific analysis of two aptamers. By comparing the aptamer with human L02 hepatocyte and HK-2 primary, the aptamer is found to have strong specificity to osteoarthritis synovial cells and weak binding ability to other cells.

FIG. 4 shows the binding of two aptamers to human L02 hepatocytes, HK-2 kidney cells, and primary synoviocytes, respectively. It can be seen that the aptamer of interest has little binding to chondrocytes, while binding specifically to synoviocytes is good.

FIG. 5 is a graph showing the dissociation of a total of two aptamers from synoviocytes. By recording the binding of aptamers to synovial cells at different concentrations, according to the fitting equation: y ═ BmaxX/(Kd + X), and the dissociation constants Kd of the SA1-2 aptamers were calculated to be 35.86 ± 16.55 and 30.01 ± 8.85(nM), respectively. This figure shows that two aptamers bind well to synovial cells with dissociation constants in the nM range.

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.

Example 1 screening of osteoarthritic synovial cell aptamers

Step one, preparing an oligonucleotide library:

the oligonucleotide library is derived from the X-Aptamer kit (AM Biotechnologies, USA) comprising a library of microspheres specifically designed and produced by AM Biotech, USA, with a typical library volume of 109 microspheres, and is screened for high affinity modified aptamers by magnetic control of magnetic particles.

The X-Aptamer high-affinity modified Aptamer is an oligonucleotide containing modification, and can be combined with a specific cell, protein or small molecular target with high affinity and strong specificity. It is very different from traditional aptamer screening, and its library is designed by computer and contains a series of novel chemically modified nucleotides. These novel chemical modifications enhance the affinity of nucleotides to targets and the stability of aptamers and increase the efficiency of screening. These novel chemical modifications encompass phosphorodithioate backbone modifications, positively charged modifications, amino acid modified bases, and the like.

The specific operation is as follows:

two buffers a and B were prepared. Buffer A1 XPBS pH 7.4,1mM MgCl₂0.05% (V/V) Tween20,2mg/mL BSA. And (3) buffer solution B: 1 XPBS pH 7.4,1mM MgCl₂,0.05％(V/V)Tween20。

The dry microsphere bank was transferred to a 15mL centrifuge tube using a 1mL pipette and prepared buffer APBSTMB (1 XPBS pH 7.4,1mM MgCl, 1 XPBS)₂0.05% (V/V) Tween20,2mg/mL BSA), dry-loaded microsphere pools were completely wet-transferred to15mL centrifuge tubes (5 times 2mL each) and then vortexed on a vortex mixer.

Centrifugation was carried out at 3000CFR for 10min at room temperature in a floating bowl centrifuge (bucket bowl centrifuge).

The supernatant was carefully discarded using a 1mL pipette, and only about 100 μ L of supernatant remained in the tube (when the supernatant was discarded, it was ensured that the microspheres were all retained in the tube).

Add 3mL of buffer A to the previously prepared 15mL centrifuge tube and vortex on a vortex mixer.

The 15mL centrifuge tube prepared in the previous step is placed in a water bath kettle at 95 ℃ and kept stand for 5 min. Naturally cooling to room temperature (more than 30min is needed). (the purpose of this step is to allow the oligonucleotide to "anneal" to its lowest energy-consuming conformation, forming a stable tertiary structure). 7mL of buffer B was added. Then vortexed on a vortex mixer and centrifuged at 3000CFR for 10 min. The supernatant was carefully discarded using a 1mL pipette, and 100 μ L of the supernatant was retained in the tube. Fresh buffer B was added to a total volume of 1.8 mL. Finally, the microspheres containing the oligonucleotide library were transferred to 2mL tubes.

Step two, screening, comprising the following steps:

1. negative selection, the specific operation is as follows:

chondrocytes, meniscal cells, tenocytes were proliferated in T25 flasks to a density of about 80%. The cells were washed once with 2ml of buffer A and then 1ml of fresh buffer A was added to the cells. The step one prepared library was added to the cells and incubated with shaking for 1 hour, and then the supernatant was taken. These unbound beads were washed with buffer B and resuspended to about 1.5ml with selection buffer B, and the beads were retained for positive selection and screening.

2. Positive screening, the specific operation is as follows:

primary osteoarthritic synoviocytes were propagated in T25 flasks to a density of approximately 80%. The cells were washed once with 2ml of buffer A and then 1ml of fresh buffer A was added to the cells. Unbound beads from the negative screen were added to the cells and incubated for 1 hour with shaking. Unbound beads were removed by washing with buffer B, bound beads were recovered from the cells, scraped and centrifuged down. The particles contain beads that bind to the cells. The pellet was resuspended in 50. mu.L of buffer B.

3. Aptamer dissociation, specifically operating as follows:

to the above 50. mu.L of resuspension, 50. mu.L of 1M NaOH was added. Incubate at 65 ℃ for 30 min. 40 μ L of 2M Tris-Cl was added to neutralize the NaOH.

The cell material was pelleted and the supernatant containing the oligonucleotide aptamers that were screened against the target protein was transferred to a 1.5mL tube.

Example 2 sequencing and structural analysis of aptamers

PCR amplification

The supernatant was subjected to PCR amplification. A forward primer: 1 1.5mL centrifuge tube 5'-CAG GGG ACG CAC CAA GG-3', reverse primer 5'-ATC ACG CAG CAC GCG GGT CAT GG-3', stored at-20 ℃. The PCR reaction used 100. mu.L of 1 XPCR buffer (2.5mM MgCl2,0.2mM dNTP, 0.4. mu.M forward primer, 0.4. mu.M reverse primer and 1U Taq Polymerase) under the following amplification conditions: pre-denaturation at 94 deg.C for 1min, circulation at 94 deg.C for 30s, 50 deg.C for 30s and 72 deg.C for 1min, and final extension at 72 deg.C for 3 min. 20 cycles were set.

Second generation sequencing

The PCR product obtained above was sent to Biotechnology (Shanghai) Co., Ltd and subjected to second-generation sequencing analysis. After analysis, 2 aptamers with the highest sequencing frequency were synthesized against synoviocytes.

The second generation sequencing result is:

SA1:TTTTTAACACGACAACGCYGTGACXCXCGAACGYCATGCCGGTGGGCCCATG

SA2:TTTTTAAGCCCACXAWACYGTGCCCAXCACAGGGYGTWGCAGXCGYGCCATG

wherein X, Y and W are nucleotides modified by special modification groups in an X-aptamer library, the novel chemical modifications comprise phosphorodithioate framework modification, positive charge modification, amino acid modified base and the like, and the specific secondary structure of the aptamer is shown in figure 1.

Example 3 affinity verification of aptamers

Cell preparation: human originalPassage of the osteoarthritic synovial cells at 37 deg.C with 5% CO₂Culturing for 1-2 days in the medium to enable the medium to be in a logarithmic growth phase, covering the area of the bottom of a culture dish by cells by about 90-95%, ensuring the cells to have good activity, washing the cells by Phosphate Buffer solution (Phosphate Buffer Saline PBS) for three times, and adding 1mL of Buffer solution A to incubate for 10 minutes.

And (3) incubation process: selecting cells in a logarithmic growth phase, digesting by 0.25% pancreatin, taking a proper amount of cell suspension, washing for 2 times by combining with a buffer solution, respectively adding a fluorescence-labeled aptamer library with the concentration of 300nmol/L and the volume of 0.2mL and two aptamers (SA1 and SA2) sequenced in the embodiment 2, placing on ice, keeping out of the sun, incubating for 30 minutes on a shaking table, and washing the cells for 3 times by the buffer solution B after the incubation is finished;

and (3) loss analysis: and adding 0.2mL of buffer B, re-suspending the synovial cell aptamer complex, loading, and detecting the fluorescence value of each group of cells after the cells are combined with the aptamer by a flow cytometer. The results of Flowjo _ V10 software analysis flow cytometry analysis are shown in figure 2.

Example 4 specificity verification of aptamers (flow cytometry)

Cell preparation: the human hepatocyte line L02, the renal epithelial cell line HK-2, and the primary osteoarthritic synovial cells were passaged at 37 ℃ with 5% CO₂Culturing for 1-2 days in the medium to enable the medium to be in a logarithmic growth phase, covering the area of the bottom of a culture dish by cells by about 90-95%, ensuring the cells to have good activity, washing the cells by Phosphate Buffer solution (Phosphate Buffer Saline PBS) for three times, and adding 1mL of Buffer solution A to incubate for 10 minutes.

And (3) incubation process: respectively selecting the 4 cells in the logarithmic phase, digesting by 0.25% pancreatin, taking a proper amount of cell suspension, washing for 2 times by using a buffer solution A, adding fluorescent-labeled aptamers (SA1 and SA2) with the concentration of 300nmol/L and the volume of 0.2mL, placing on ice, keeping out of the sun, incubating on a shaking table for 30 minutes, and washing for 3 times by using a buffer solution B after the incubation is finished;

and (3) loss analysis: and adding 0.2mL of buffer B, re-suspending the synovial cell aptamer complex, loading, and detecting the fluorescence value of each group of cells after the cells are combined with the aptamer by a flow cytometer. The results of Flowjo _ V10 software analysis flow cytometry analysis are shown in figure 3.

Example 5 specificity verification of aptamers (immunofluorescence)

Respectively selecting human liver cell line L02, kidney epithelial cell line HK-2 and primary osteoarthritis synoviocytes in logarithmic growth phase, digesting with 0.25% pancreatin, uniformly spreading appropriate amount of cell suspension in a culture dish special for confocal microscope at 37 deg.C and 5% CO₂Culturing for 24 hours in a cell incubator, washing for 2 times, adding FAM fluorescence labeled aptamers (SA1 and SA2) with the concentration of 300nmol/L and the volume of 0.4mL, placing on ice, keeping out of the sun, incubating for 30 minutes on a shaking table, and after the incubation is finished, washing the cells for 3 times by using a buffer solution B; cell nuclei were stained with Hoechst solution at a concentration of 2. mu.g/mL for 5 minutes, and after the cells were washed again, 0.5mL of washing buffer was added, and imaging and photographing of the cells were performed under a confocal fluorescence microscope. The image results are shown in fig. 4.

Example 6 determination of dissociation constant KD values of aptamers

Cell preparation: the cell processing method was the same as in example 3.

And (3) incubation process: cell counting plate count 2X 10⁴Preparing synovial cells, FAM-labeled aptamers (SA1, SA2) and buffer A into solutions with concentration gradients of 0nmol/L, 10nmol/L, 20nmol/L, 50nmol/L, 60nmol/L, 80nmol/L and 100nmol/L and a volume of 200uL according to pre-calculated using amounts, placing the solutions on ice, keeping out of the light, incubating the solutions on a shaker for 30 minutes, removing unbound aptamer through a pipettor, and washing the cells for 3 times and 2 minutes each time with 0.5mL of precooled buffer B;

loss analysis and data processing: and adding 0.2mL of buffer B, re-suspending the synovial cell aptamer complex, loading, and detecting the fluorescence value of each group of cells after the cells are combined with the aptamer by a flow cytometer. Analysis was performed using sigmaplot14.0 software, according to the fitting formula: y ═ Bmax ×/(Kd + X), fitted curves and Kd values for aptamers were obtained. The curve of the fitting results is shown in fig. 5.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> grand era memorial Hospital of Zhongshan university

<120> modified base aptamer specifically targeting synoviocytes of osteoarthritis and application thereof

<130> 4.16

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 48

<212> DNA

<213> Synthesis

<400> 1

tttttaacac gacaacgcgt gacccgaacg catgccggtg ggcccatg 48

<210> 2

<211> 44

<212> DNA

<213> Synthesis

<400> 2

tttttaagcc cacaacgtgc ccacacaggg gtgcagcggc catg 44

Claims (6)

1. A modified base aptamer specifically targeting synoviocytes of osteoarthritis, said aptamer having the sequence as set forth in SEQ ID NO: 1 or 2.

2. A method of screening for a modified base aptamer specifically targeting synoviocytes of osteoarthritis according to claim 1, comprising the steps of:

(1) oligonucleotide library preparation: the oligonucleotide library forms a stable tertiary structure after annealing;

(2) negative screening: incubating negative sieve cells with the oligonucleotide library microspheres obtained in the step (1), and then taking unadsorbed microspheres in supernatant, wherein the unadsorbed microspheres are the target aptamer microspheres of the invention;

(3) and (3) positive screening: combining the aptamer microspheres obtained in the step (2) with primary osteoarthritis synovial cells to obtain aptamer microspheres combined with the synovial cells;

(4) dissociation of aptamers from primary osteoarthritic synovial cells: and (3) adding an alkaline solution into the aptamer microsphere coupled with the target cell in the step (3), incubating, adding a buffer solution for neutralization and centrifuging, wherein the supernatant contains the oligonucleotide aptamer.

3. The screening method according to claim 2, wherein the oligonucleotide library in the step (1) is obtained by using an X-Aptamer kit.

4. The screening method according to claim 2, wherein the alkaline solution in the step (4) is 1M NaOH and the buffer solution is 2M Tris-Cl.

5. The screening method according to claim 2, wherein the nucleotide sequence of the oligonucleotide adaptor in step (4) is as set forth in SEQ ID NO: 1 or 2.

6. Use of the modified base aptamer specifically targeting synovial cells as claimed in claim 1 in the preparation of a medicament or formulation for the treatment or diagnosis of osteoarthritic synovial cell related diseases.

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