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Nano di-cyclic aptamer probe and application thereof

An aptamer and cyclic technology, applied in the field of nano-bicyclic aptamer probes, to achieve the effects of inhibiting activity, improving specificity, and improving specificity

Active Publication Date: 2017-03-22
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the presence of a large number of nucleases in the environment, the linear aptamer will be degraded

Method used

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  • Nano di-cyclic aptamer probe and application thereof
  • Nano di-cyclic aptamer probe and application thereof
  • Nano di-cyclic aptamer probe and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Design of linear aptamers

[0054] Using the tumor biomarker protein PTK7 highly expressed on the cell membrane surface of the target blood cancer cell RccF-CEM as a template, based on Cell-SELEX technology, design a linear ligand Ap that can bind to PTK7 protein with high specificity;

[0055] According to the principle of complementary base pairing, design the template T-Ap that can make the linear Ap loop closed, the linear C1 that can be complementary paired with the closed Ap partial sequence to form a small circle with a double ring structure, and the template T-C1 required for C1 loop closure, wherein , the partial sequences of the 3' and 5' ports of T-Ap and Ap are complementary, the partial sequences of the 3' and 5' ports of C1 and T-C1 are complementary, and the partial sequences of C1 and part of Ap are complementary , and, Ap contains two groups of Biotin and FAM.

[0056] The primer sequences used are as follows:

[0057] Among them, the linea...

Embodiment 2

[0065] Example 2: Transforming a linear aptamer into an aptamer probe with a functional region and a double loop region

[0066] Ap diluted to 10 μM was subjected to a monocyclic annulation reaction. The reaction steps are: (1) Phosphorylation reaction was carried out by shaking reaction in a constant temperature metal bath at 37°C for 30 minutes; (2) Ligase ligation reaction was carried out by shaking reaction at 55°C for 2 hours; (3) Template removal; (4) Product purification; Finally, a single, closed circular ApR was obtained, and C1 was obtained by the same method, and C1 was smaller than ApR. Then, ApR and C1 were complementary hybridized to obtain an aptamer probe with a double-loop structure and a functional region.

[0067] Specifically include the following steps:

[0068] (1) Add linear aptamer Ap or linear oligonucleotide C1 (10nM) to the buffer system containing 1 / 3 volume of ATP and 1 / 2 volume of Polynucleotide Kinase (T4 PNK) enzyme, use dd h 2 Dilute O ten ...

Embodiment 3

[0072] Example 3: Denaturing gel electrophoresis verifies the successful construction of aptamer probes with functional regions and double loop regions

[0073] The first part is to use denaturing gel electrophoresis to verify the successful synthesis of the single-ring closed product. First, prepare a 10% denaturing bulk gel (Urea-PAGE), and combine the synthesized single-ring aptamer ApR and the double-ring structure construction. The inner circle C1, and the corresponding linear single-stranded DNA, were subjected to denaturing gel electrophoresis under the same conditions, and the single-circular aptamer was verified according to the conclusion that the swimming speed of circular DNA is slower than that of linear DNA Whether the build job for is successful. like figure 1 As shown, the band of circular DNA is significantly higher than that of linear DNA, indicating that the swimming speed is slower, which proves the successful construction of single circular DNA.

[0074]...

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Abstract

The invention discloses a nano di-cyclic aptamer probe and application thereof. The nano di-cyclic aptamer probe (G-dApR) is very stable and is not degraded by in-vivo DNA. Meanwhile, the biological activity of target cells can be effectively inhibited by combining specific recognition with a leukemia cell surface highly-expressive biomarker. The design of the probe is researched and developed based on two advanced technologies in the field of tumors, namely a ligand technology and a nanotechnology. Firstly, a conventional linear aptamer structure is transformed into a structure including a functional zone (specific) and a di-cyclic DNA zone (stable) by adopting a nucleic acid modification means, and the biological stability is greatly improved; secondly, the probe is finally established by combining a cyclic aptamer and a dendritic nano material PAMAM. The shortcoming that an aptamer is unstable is greatly overcome, an effective means is provided for leukemia detection, and high-sensitivity diagnosis and treatment integrated clinical application is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a nano-bicyclic aptamer probe and its application. Background technique [0002] Due to its high incidence, great harm, difficult to cure, disease symptoms seriously affect the quality of life, and high cost of treatment, tumors have become a large class of diseases that humans urgently need to overcome. According to the latest statistics released by the World Health Organization, there are currently about 14 million cancer patients in the world, about 7 million new cases each year, and about 5 million people die of cancer every year, that is to say, about every 6 seconds. 1 person died of cancer. At the same time, the 2015 Chinese cancer statistics released by the National Cancer Registry of China (NCCR) also show that in 2015, China is expected to have 4.292 million new cancer cases and 2.814 million cancer deaths. [0003] Among the many types of cancer, blood cancer, also called ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
CPCC12Q1/6811C12Q1/6816C12Q2525/307C12Q2525/205C12Q2563/107
Inventor 贾力董海燕张婷霞
Owner FUZHOU UNIV
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