Multivalent aptamer DNA nano ladder and preparation method and application thereof

A DNA molecule and aptamer technology, applied in the field of biomedicine, can solve problems such as multidrug resistance and drug resistance

Active Publication Date: 2020-11-17
CHINA UNIV OF PETROLEUM (EAST CHINA) +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, chemotherapeutic drugs are widely used in cancer treatment, but chemotherapeutic drugs can induce cytotoxicity in both tumor cells and healthy cells; and due to the phenomenon of multidrug resistance, special "protein pumps" in some tumor cells will induce chemotherapeutic drugs For example, doxorubicin hydrochloride (Dox) is pumped out of the cell and develops drug resistance

Method used

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  • Multivalent aptamer DNA nano ladder and preparation method and application thereof
  • Multivalent aptamer DNA nano ladder and preparation method and application thereof
  • Multivalent aptamer DNA nano ladder and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: the preparation method of rolling circle amplification (RCA) product

[0053] Carry out rolling circle amplification (RCA) reaction with circular DNA templates A and B respectively, in which 2 μL primers (10 μM), 2 μL circular DNA templates (10 μM), and 2 μL 10×Phi29 DNA polymerase buffer were sequentially added to each reaction system and 2 μL of dNTPs (7mM), supplemented with ddH 2 O to a total volume of 20 μL, add 0.8 μL Phi29 DNA polymerase (10U / μL) to initiate the reaction, mix well, centrifuge at a low speed, place in a constant temperature shaker at 30°C for 10min, and then keep the temperature at 65°C for 10min to inactivate Phi29 DNA Polymerase to produce chain A and chain B of rolling circle amplification (RCA) products. Using agarose gel electrophoresis to characterize the rolling circle amplification (RCA) product, the length is about 8000nt.

[0054] Table 1: Oligonucleotide sequences involved in Example 1

[0055]

[0056]

Embodiment 2

[0057] Embodiment 2: the preparation method of DNA nano-ladder

[0058] The rolling circle amplification (RCA) product prepared in Example 1 was 1 μL each for chain A and chain B, and 1 μL each for short chains 1-6 (concentration 100 μM), and added to 42 μL 2×TAE-Mg 2+ (24mM) centrifuge tube, add ddH 2 0 to 100 μL, mix well, centrifuge at low speed properly, heat-treat at 95°C for 5min, and anneal naturally to 25°C to prepare a DNA nano-ladder.

[0059]Transmission electron microscopy was tested at an accelerating voltage of 200KV. Sample preparation for transmission electron microscopy: 5 μL DNA nano-ladder samples were dropped onto copper grids, left to stand for 5 minutes, stained with 1% uranyl acetate for 1 minute, and tested after the samples were completely dry.

[0060] The self-assembled products were characterized by transmission electron microscopy, as image 3 As shown in (a), it can be seen that the shape of the product is linear, and the width is about 10 nm, ...

Embodiment 3

[0067] Embodiment 3: Drug loading experiment

[0068] The fixed Dox concentration was 200nM, and the DNA nano-ladder concentrations were 1nM, 0.4nM, 0.2nM, 0.1nM, 0.04nM, 0.02nM, 0.01nM, 0.005nM, 0.0025nM, mixed evenly, centrifuged, and incubated at 30°C for 30min.

[0069] The detection method of the microplate reader is fluorescence intensity detection, the detection type is endpoint scanning mode, the excitation wavelength is set to 485nm, the emission wavelength is set to 590nm, and the detection temperature is 30°C.

[0070] The preparation method of DNA nano-ladder-Dox complex: 200nM antitumor drug doxorubicin hydrochloride (Dox) was mixed with 1nM, 0.4nM, 0.2nM, 0.1nM, 0.04nM, 0.02nM, 0.01nM, 0.005nM, 0.0025nM respectively The DNA nanoladder was incubated at 30°C for 30min.

[0071] The antitumor drug doxorubicin hydrochloride (Dox) is inserted between the G-C bases of the DNA double-strand by physical combination to form a DNA nano-ladder-Dox complex, and the autofluo...

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Abstract

The invention provides a multivalent aptamer DNA nano ladder and a preparation method and application thereof. Natural DNA molecules are used as raw materials, on the basis of the DNA origami theory,the DNA nano ladder is constructed by utilizing the special property of a rolling circle amplification product, a large number of aptamers are introduced, a drug delivery system which is simple in design and high in drug loading capacity and has target specificity and biocompatibility is constructed, and through aptamer or drug replacement, the carrier can also be applied to various types of target cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a polyvalent aptamer DNA nano-ladder and its preparation method and application. Background technique [0002] The incidence and mortality of cancer in my country are on the rise year by year, and it has become one of the four major chronic diseases in my country. At present, chemotherapeutic drugs are widely used in cancer treatment, but chemotherapeutic drugs can induce cytotoxicity in both tumor cells and healthy cells; and due to the phenomenon of multidrug resistance, special "protein pumps" in some tumor cells will induce chemotherapeutic drugs For example, doxorubicin hydrochloride (Dox) is pumped out of the cell, resulting in drug resistance. Therefore, it is a challenge in the field of biomedical technology to develop a targeted drug carrier that is simple in design and can specifically recognize tumor cells. [0003] DNA nanostructures have become a new type of drug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/26A61K31/704A61P35/00C12Q1/6844
CPCA61K47/26A61K31/704A61P35/00C12Q1/6844C12Q2531/125
Inventor 张志庆张子辰杨春天王芳张国栋王秀凤周亭
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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