DNA nano flower-shaped composite structure as well as preparation method and application thereof

A composite structure, flower-shaped technology, which is applied in the directions of drug combinations, pharmaceutical formulations, and non-active ingredients medical preparations, etc., can solve the problems of complicated preparation process, low compatibility and safety, and difficult modification of materials.

Active Publication Date: 2019-11-29
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the preparation process of the above-mentioned prior art is cumbersome, the obtained materials are not easy to modify, and the compatibility and safety are not high, so further optimization is needed

Method used

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  • DNA nano flower-shaped composite structure as well as preparation method and application thereof
  • DNA nano flower-shaped composite structure as well as preparation method and application thereof
  • DNA nano flower-shaped composite structure as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0070] Example 1 Preparation of DNA nano flower-shaped composite structure

[0071] The DNA sequence used in this embodiment is as follows:

[0072]

[0073] 1. Hybridization of template and primer, circularization of template

[0074] Phosphorylated DNA template strand 1 and primer strand (each 10 μM, 2.8 μL), 10×T4 Buffer (4 μL), and deionized water 29.4 μL were mixed in equal proportions to form a 40 μL solution system, and placed in a programmed cooling device for denaturation (heat at 95°C for 5min), slowly anneal and cool down from 95°C to 4°C, every 1°C is a gradient, and the residence time of each gradient is 1min;

[0075] After the annealing process in the above steps, add T4 DNA ligase (400,000 units / ml, 1 μL) to the 40 μL solution system to circularize the template strand. The conditions are 24 ° C for 12 h, and then 65 ° C for 10 min to inactivate the enzyme, and then slowly anneal and cool down to 4°C; every 2°C is a gradient, and the residence time of each ...

Embodiment 2

[0078] Example 2 Preparation of DNA nano flower-shaped composite structure

[0079] The DNA sequence used in this embodiment is as follows:

[0080]

[0081] 1. Hybridization of template and primer, circularization of template

[0082] Phosphorylated DNA template strand 1 and primer strand (10 μM, 2.8 μL), 10×T4 Buffer (4 μL), and deionized water 29.4 μL were mixed in equal proportions to form a 40 μL solution system, and placed in a programmed cooling device for denaturation ( After heating at 95°C for 5min), slowly anneal and cool down from 95°C to 4°C, every 1°C is a gradient, and the residence time of each gradient is 1min; after the annealing process in the above steps, add 1μL (20000units 50 μL) of T4 DNA ligase to circularize the template strand, the condition is 24°C for 12 hours, then 65°C for 10 minutes to inactivate the enzyme, and then slowly anneal and cool down to 4°C; every 2°C is a gradient, and the residence time of each gradient is 2 minutes.

[0083] 2....

Embodiment 3

[0085] Example 3 Preparation of DNA nano flower-shaped composite structure

[0086] The DNA sequence used in this embodiment is as follows:

[0087]

[0088] 1. Hybridization of template and primer, circularization of template

[0089] Phosphorylated DNA template strand 2 and primer strand (10 μM, 2.8 μL), 10×T4 Buffer (4 μL), and deionized water 29.4 μL in equal proportions were mixed to form a 40 μL solution system, which was denatured in a programmed cooling device ( After heating at 95°C for 5min), slowly anneal and cool down from 95°C to 4°C, every 1°C is a gradient, and the residence time of each gradient is 1min;

[0090] After the annealing process in the above steps is over, add 1 μL (20000 units 50 μL) of T4 DNA ligase to circularize the template strand in 40 μL of the solution system. The conditions are 24 ° C for 12 hours, and then 65 ° C for 10 minutes to inactivate the enzyme, and then slowly anneal and cool down to 4 ° C ; Every 2°C is a gradient, and the r...

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Abstract

The invention provides a DNA nano flower-shaped composite structure as well as a preparation method and an application thereof. The DNA nano flower-shaped composite structure comprises a DNA nano flower-shaped structure formed by amplification of a single-stranded DNA template and an immunostimulatory nucleic acid fragment hybridized with the DNA nano flower-shaped structure, according to the invention, primers and template sequences are ingeniously designed, a rolling circle replication method is used for amplifying the template DNA to form a nano flower shape, creatively fuses a pH-responsive i-motif into a hybridization site to realize hybridization and acid response release of an immunostimulatory nucleic acid sequence, optimizes the reaction conditions, and finally successfully prepares the composite structure. The composite structure provided by the present invention can be used as a carrier for loading immune stimulating nucleic acid fragments to be efficiently transported inside immune cells, by responsively releasing the immune stimulating nucleic acid fragments, the immune cells can be activated, which provides a powerful novel adjuvant material for tumor immunotherapy, and has broad application prospects and market value.

Description

technical field [0001] The invention relates to the technical field of nano-medicines, in particular to a DNA nano-flower composite structure and a preparation method and application thereof. Background technique [0002] For the occurrence and metastasis of malignant tumors, it is now generally believed that it is related to the immune tolerance mechanism of the body. Although there are multiple immune surveillance mechanisms in the body, malignant cells may occur and continue to develop into tumors due to their ability to escape immune surveillance. Tumor immunotherapy is a therapy that stimulates the patient's immune system to attack its own tumor cells. Compared with traditional high-injury surgical treatment and radiotherapy and chemotherapy, tumor immunotherapy has more obvious effects and less side effects. It has been used as an adjuvant treatment for malignant tumors in clinical practice. [0003] Among the drugs used in many immunotherapies, a class of nucleic ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61K47/69A61K47/54A61P35/00A61P37/04
CPCA61K39/39A61K47/549A61K47/6901A61P35/00A61P37/04A61K2039/55561
Inventor 丁宝全孙伟蒋乔韦雨
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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