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An anticoagulant dna nanocomposite structure and its preparation method and application

A nanocomposite and nanostructure technology, applied in the field of anticoagulant DNA nanocomposite structure and its preparation, can solve the problem of not considering anticoagulation function and detoxification, inability to precisely control the spatial position, limited aptamer DNA load, etc. problem, to achieve the effect of good drug development potential, good biocompatibility, and controllable size

Active Publication Date: 2021-06-08
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there are also attempts by scholars to use gold nanorods to load nucleic acid aptamers or self-assemble micelles to form nanoscale anticoagulants, but they cannot precisely control the spatial positions of the two aptamers or only one aptamer is applicable. For assembly, the DNA load of aptamers is limited, and in vivo applications are potentially toxic. Some scholars extend several loop structures with nucleic acid aptamer sequences on the surface of DNA nanostructures to guide and control thrombin on the surface of nanostructures. Assembled, but did not consider its anticoagulant function and detoxification
Therefore, the preparation process of the above-mentioned prior art is complicated, the anticoagulant function is not significant, and there are many defects, which need to be further optimized and improved

Method used

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  • An anticoagulant dna nanocomposite structure and its preparation method and application
  • An anticoagulant dna nanocomposite structure and its preparation method and application
  • An anticoagulant dna nanocomposite structure and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Example 1 Preparation and purification of DNA origami nanostructures with capture sites

[0145] Firstly, the template strand, auxiliary folding sequence, and capture DNA strand were mixed at a ratio of 1:10:10, and a total of 18 capture strands hybridized with the nucleic acid aptamer TBA were designed (Fig. A total of 18 capture strands hybridized with the aptamer HD22 were designed (Fig. 1-A light dotted line), forming a total of 18 pairs of thrombin-binding sites (Fig. 1-B);

[0146] The captured DNA chain is exemplified as follows:

[0147] capture DNA strand I

[0148] SEQ ID NO.15:

[0149] AAAAAAAAAAAAAAA GCCAGCTGCCTGCAGGTCGACTCTGCAAGGCG.

[0150] SEQ ID NO.16:

[0151] AAAAAAAAAAAAAAA ATTAAGTTCGCATCGTAACCGTGCGAGTAACA.

[0152] SEQ ID NO.17:

[0153] AAAAAAAAAAAAAAAACCCGTCGTCATATGTACCCCGGTAAAGGCTA.

[0154] SEQ ID NO.18:

[0155] AAAAAAAAAAAAAAATCAGGTCACTTTTTGCGGGAGAAGCAGAATTAG.

[0156] SEQ ID NO.19:

[0157] AAAAAAAAAAAAAAACAAAATTAAAGTACGGTGTCTGGAAGA...

Embodiment 2

[0187] Example 2 Preparation and purification of DNA nanoarrays for assembling nucleic acid aptamers

[0188] The DNA origami structure designed and prepared according to Example 1 was mixed with the nucleic acid aptamer (site: nucleic acid aptamer TBA: nucleic acid aptamer HD22=1:5:5), at 1×TAE / Mg 2+ Annealing is carried out under the condition of buffer solution (pH8.0); annealing conditions: from 42°C to 25°C, every 5°C is a gradient, and the residence time of each gradient is 5min; six cycles are carried out; after annealing is completed, the nucleic acid loaded The DNA origami structure sample of the aptamer was added to the precipitation buffer (containing 15% PEG8000 (w / v), 5mM Tris, 1mM EDTA, and 505mM NaCl) at a volume ratio of 1:1. Centrifuge at 16000g for 25min to obtain the DNA nano anticoagulant.

Embodiment 3

[0189] Example 3 Capturing of thrombin by DNA nanoarrays loaded with nucleic acid aptamers

[0190] The DNA nanostructure loaded with nucleic acid aptamer constructed according to Example 2 was mixed with thrombin, nucleic acid aptamer TBA:nucleic acid aptamer HD22:thrombin=1:1:5, placed at room temperature for 30min; using atomic force Microscopic evaluation of the DNA structure loaded with nucleic acid aptamers and the ability to capture thrombin, the results are as follows figure 2 with image 3 shown;

[0191] Depend on figure 2 with image 3 It can be seen that after hybridization of the two aptamers, the size and height of the DNA nanostructure did not change significantly, and it was still a rectangular sheet of 90 × 60nm. After co-incubation with thrombin, the height of one side of the rectangular sheet increased significantly. That is, thrombin is captured by the structure with the aptamer, and the capture position coincides with the position of the pre-designed a...

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Abstract

The invention provides an anticoagulant DNA nanocomposite structure and its preparation method and application. The DNA nanocomposite structure includes a DNA nanostructure and an antithrombin nucleic acid aptamer. The DNA nanostructure consists of a DNA template chain , assisted folding DNA chain and capture DNA chain assembly, the antithrombin nucleic acid aptamer binds to the surface of the DNA nanostructure through the capture DNA strand; the present invention first self-assembles to obtain the DNA nanostructure, and through site design The short DNA strands of two anti-thrombin aptamers are assembled on the surface of the rectangular DNA structure, and the relative positions of the aptamers are precisely controlled to work synergistically. The assembly and preparation methods are optimized, and finally a DNA nanocomposite structure is obtained, which can effectively inhibit thrombin activity, and can use short DNA strands complementary to nucleic acid aptamers to rapidly detoxify them, which has broad application prospects and market value.

Description

technical field [0001] The invention relates to the technical field of nanometer medicines, in particular to an anticoagulant DNA nanocomposite structure and its preparation method and application. Background technique [0002] Thrombin performs the function of coagulation by recognizing various supramolecular substrates, and is a key enzyme in the coagulation system of organisms. Thrombin can rapidly and efficiently induce thrombus formation without the participation of many other coagulation factors. Thrombin can recognize soluble fibrinogen and convert it into insoluble fibrin, and can also induce platelet activation to form a platelet thrombus under the action of the fibrin gel network. When thrombin functions, it needs to recognize a specific substrate. There are two specific recognition sites on its surface, which are fibrinogen-recognition exosite and heparin-binding exosites. [0003] Antithrombin nucleic acid aptamer is a kind of nucleic acid sequence, and its sec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10A61K31/711A61P7/02
CPCA61K31/711A61P7/02C12N15/115C12N2310/16
Inventor 丁宝全赵帅蒋乔刘少利
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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