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Modified base aptamer for specifically targeting osteoarthritis synovial cells and application of modified base aptamer

A technology for osteoarthritis and synoviocytes, applied in the field of modified base aptamers, can solve the problems of uncertain binding force, inability to prove specificity, inapplicability to OA synoviocytes, etc., to reduce cumbersome steps, The screening process is simple and the effect of specificity enhancement

Active Publication Date: 2020-12-18
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is found in the patent that the dissociation constant of the aptamer screened out is not disclosed, the binding force is uncertain, and it is only for the aptamer of RA synovitis, so the aptamer for rheumatoid synovitis may be Not applicable to OA synoviocytes
At the same time, the screening in this patent is based on the traditional general aptamer library, and the aptamer has not been properly modified, which limits its practical application in the diagnosis and treatment of synoviocyte-related diseases
In addition, the aptamer is not loaded with detachable assembly nano, nucleic acid, protein and other technologies at will, and the anti-screening cells only use hepatocytes, which cannot be proved to distinguish fibroblast-derived lineage types of cells (such as chondrocytes, meniscus cells, tendon cells, etc.) specificity between cells)

Method used

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  • Modified base aptamer for specifically targeting osteoarthritis synovial cells and application of modified base aptamer
  • Modified base aptamer for specifically targeting osteoarthritis synovial cells and application of modified base aptamer
  • Modified base aptamer for specifically targeting osteoarthritis synovial cells and application of modified base aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Screening of Osteoarthritis Synoviocyte Aptamers

[0027] Step 1. Preparation of oligonucleotide library:

[0028] The oligonucleotide library is derived from the X-Aptamer kit (AM Biotechnologies, USA), which includes a microsphere library specially designed and produced by the American AM Biotech Company. The storage capacity is generally 109 microspheres. Magnetic control of particles to screen for high-affinity modified aptamers.

[0029]X-Aptamer high-affinity modified aptamers contain modified oligonucleotides, which can bind to specific cells, proteins or small molecule targets with high affinity and strong specificity. It is very different from traditional nucleic acid aptamer screening in that 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. Thes...

Embodiment 2

[0045] Example 2 Sequencing and structural analysis of aptamers

[0046] PCR amplification

[0047] PCR amplification was performed on the supernatant. Forward primer: one 1.5mL centrifuge tube: 5’-CAG GGG ACG CAC CAAGG-3’, reverse primer: 5’-ATC ACG CAG CAC GCG GGT CAT GG-3’, store at -20°C. The PCR reaction uses 100 μL of 1×PCR buffer (2.5mM MgCl2, 0.2mM dNTP, 0.4μM forward primer, 0.4μM reverse primer and 1U Taq Polymerase), the amplification conditions are: pre-denaturation 94°C, 1min, cycle conditions 94°C for 30s, 50°C for 30s and 72°C for 1min, and finally 72°C for 3min. Set for 20 loops.

[0048] next generation sequencing

[0049] Send the PCR products obtained above to Sangon Bioengineering (Shanghai) Co., Ltd. for next-generation sequencing analysis. After analysis, the 2 most frequently sequenced aptamers were synthesized against synoviocytes.

[0050] The next generation sequencing results are:

[0051] SA1:TTTTTAACACGACAACGCYGTGACXCXCGAACGYCATGCCGGTGGGCCCA...

Embodiment 3

[0054] Example 3 Affinity Verification of Aptamers

[0055] Cell preparation: Human primary osteoarthritic synoviocytes were passaged at 37°C, 5% CO 2 Culture in medium for 1 to 2 days, so that it is in the logarithmic growth phase, and let the cells cover about 90%-95% of the bottom area of ​​the culture dish, and at the same time ensure that the cells have good viability. ) was washed three times, and 1 mL of buffer A was added to incubate for 10 minutes.

[0056] Incubation process: select cells in the logarithmic growth phase, digest with 0.25% trypsin, take an appropriate amount of cell suspension, wash with binding buffer twice, add fluorescent markers with a concentration of 300nmol / L and a volume of 0.2mL The aptamer library and the two aptamers (SA1, SA2) sequenced in Example 2 were placed on ice, protected from light, and incubated on a shaker for 30 minutes. After the incubation, the cells were washed 3 times with buffer B;

[0057] Loss analysis: add 0.2mL buffer...

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Abstract

The invention provides a modified base aptamer for specifically targeting osteoarthritis synovial cells. The aptamer has a nucleotide sequence as shown in SEQ ID NO.1 or 2. Through modifying an aptamer library, performing positive selection on the osteoarthritis synovial cells and performing negative reverse screening on various kinds of skeletal muscle derived cells, the modified base aptamer forspecifically targeting the osteoarthritis synovial cells is finally obtained. The synovial cell aptamer sequence is screened from the modified aptamer library; the screening process is simple and convenient; complicated steps of a traditional method are greatly reduced; due to the modified structure, the binding force with target cells is higher; the specificity of the synovial cell aptamer is specifically and greatly enhanced; the aptamer targeted binding defect of existing osteoarthritis disease derived synovial cells is overcome; and in the subsequent process, modified groups AminedU in the target aptamer can directly carry nanometer particles to deliver drugs, interfering RNA, miRNA, exosomes and the like to act on different target points to perform specific treatment on synovitis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a modified base aptamer specifically targeting osteoarthritis synoviocytes and an application thereof. Background technique [0002] Osteoarthritis (OA) is a common type of arthritis in the elderly population, which may eventually lead to the destruction and loss of joint function, seriously affecting the quality of life of the elderly, and is a heavy burden on individuals and society. The occult nature of early OA diseases and the complexity of differential diagnosis are also the reasons for the delay in treatment and poor prognosis of these diseases. The basic pathological features of OA are cartilage degeneration, subchondral bone hyperplasia, and synovitis; while persistent chronic synovitis, synovial membrane hyperplasia accompanied by bone invasion and destruction inflammation. In the above-mentioned synovitis diseases, since there are no blood vessels on the surface of articu...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10A61K47/26A61P19/02A61P19/08
CPCC12N15/115C12N15/1048A61K47/26A61P19/02A61P19/08C12N2310/16C12N2320/13C12N2320/31C12N2310/313
Inventor 陈仲丁悦林创鑫周苗苗宋斌罗文强
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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