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Novel annular ternary aptamer and synthetic method and application thereof

A synthetic method and aptamer technology, applied in the field of molecular biology, can solve problems such as poor stability and low recognition efficiency, and achieve high stability, enhanced sensitivity, and improved stability

Active Publication Date: 2019-08-30
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a novel cyclic ternary aptamer with high stability and high specific recognition and its synthesis method and application, to solve the problem of single aptamer used in the diagnosis of tumor cells in the prior art. There are technical problems such as poor stability and low recognition efficiency, which can be used in the diagnosis and treatment of tumors to provide more efficient and lower cost diagnosis and treatment technology

Method used

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  • Novel annular ternary aptamer and synthetic method and application thereof
  • Novel annular ternary aptamer and synthetic method and application thereof
  • Novel annular ternary aptamer and synthetic method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The circular ternary aptamer in this example is a circular molecule formed by connecting three single-stranded aptamers through complementary base pairing at the ends. The specific synthesis method includes the following steps:

[0044] S1. Through literature search, select the aptamer single-chain SE15-8 that specifically recognizes the highly expressed antigen on the surface of tumor cells, wherein the nucleotide sequence of SE15-8 is SEQ No.1: AGC CGC GAG GGG AGG GAT AGG GTA GGGCGC GGCT-AGTCAAGA 1 . In this example, the aptamer single-chain SE15-8 can specifically recognize a transmembrane receptor-like protein with a relative molecular mass of 185,000 on tumor cells, which is produced by the proto-oncogene human epidermal growth factor receptor 2 (human epidermal growth factor receptor -2, HER2) gene, namely c-erbB-2 gene encoding.

[0045] S2. First, modify the sequence of SE15-8 so that it has a partial paired sequence, and obtain three nucleotide sequences of S...

Embodiment 2

[0056] The specific synthesis method of the cyclic ternary aptamer in this example includes the following steps:

[0057] S1. Through literature search, select the aptamer single-chain EPCAM-Apt that specifically recognizes the highly expressed antigen EPCAM on the surface of tumor cells, wherein the nucleotide sequence of EPCAM-Apt is SEQ No.5: ACGUAUCCCUUUUCGCGU 2 .

[0058] S2. First, the EPCAM-Apt sequence is modified to have a partial paired sequence, and three nucleotide sequences of SEQ No.6, SEQ No.7 and SEQ No.8 are respectively obtained (see the sequence listing for the specific base sequence), and then modified Add carboxyl fluorescent group 6-FAM and amino group, and phosphorylate the phosphate group at the 5' end to obtain three single-chain aptamers T1', T2' and T3' after transformation; sponsored by Sangon Bioengineering (Shanghai) Co., Ltd. Co., Ltd. synthesized three sequences of T1', T2' and T3';

[0059] T1':TTCGGAGTCAACTAT / iNH2C6dT / TATCAACTGAAGCCGCGAGGGGA...

experiment example 1

[0067] Experimental example 1 Enzymolysis stability test of cyclic ternary aptamer

[0068] Since the stability of the aptamer is mainly affected by the exonuclease in serum and blood after entering the organism, an experiment was designed to investigate the exonuclease (EXOⅠ ) stability under conditions. Configure the reaction solutions of the control group and the experimental group according to the following raw materials and proportioning.

[0069] Control group: purified water: 76 μl, T1: 9 μl (1 μM), EXOⅠ: 5 μl (5Μ), EXOⅠbμffer: 10 μl;

[0070] Experimental group: purified water: 55 μl, CTA: 30 μl (1 μM), EXOⅠ: 5 μl (5M), EXOⅠb μffer: 10 μl.

[0071] After the above experimental group and control group were reacted at 37°C for 0h, 1h, 3h, 5h, 8h, 12h, and 24h, 10 μl samples were taken respectively and placed in centrifuge tubes, and the enzyme inactivation treatment was carried out at 65°C for 10 minutes, and then Add DNA loading buffer, electrophoresis in 10% PAGE ge...

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Abstract

The invention provides a novel annular ternary aptamer. Three aptamer single chains carry base sequences being in mutual complementation, are connected to form a ring after being paired mutually through respectively-carried part complementary bases, and are the same or more aptamer single chains. The invention further discloses a synthetic method of the novel annular ternary aptamer. The syntheticmethod comprises the following steps of S1, according to antigen needing to be labeled and recognized, selecting three aptamer single chains matched with the antigen; S2, introducing a specific basesequence into the target aptamer single chains, so that the first aptamer single chain and the second aptamer single chain respectively carry the base sequence being complementary with the third aptamer; and S3, adding the three aptamer single chains being the same in quantity to a DNA ligase buffering solution, performing annealing, after lowering the temperature, adding DNA ligase, and performing a reaction for 1h or above, so as to obtain the novel annular ternary aptamer. The invention provides the novel aptamer being high in stability and high in specific recognition, and the aptamer canbe used for diagnosis and treatment of tumors.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a novel cyclic ternary aptamer and its synthesis method and application. Background technique [0002] Because it is often difficult to track and remove a small amount of tumor cells, it is difficult to detect early cancers, and by the time the diagnosis is made in the middle and late stages, the best time for treatment has been missed. Although surgery can remove 99% of malignant tumors, there are still a small number of tumor cells infiltrated in the tissue that are difficult to remove. A small number of remaining tumor cells will eventually enter the blood and lymphoid tissue through the primary tumor site to form circulating tumor cells (CTCs), and the existence of CTCs is the root cause of cancer metastasis and recurrence. Regardless of whether it is the tumor cells produced in the early stage or the tumor cells remaining after the operation, if timely monitoring...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/574A61K45/00A61K47/26A61K47/54A61P35/00
CPCA61K45/00A61K47/26A61K47/549A61P35/00C12N15/115C12N2310/16G01N33/574
Inventor 贾力王杰李书慧闵香
Owner FUZHOU UNIV