A multi -price adaptation DNA nano ladder and its preparation methods and applications

A DNA molecule and aptamer technology, applied in the field of biomedicine, can solve problems such as multidrug resistance and drug resistance, and achieve the effects of strong biocompatibility, high affinity and selectivity, and easy functional modification.

Active Publication Date: 2022-06-07
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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  • A multi -price adaptation DNA nano ladder and its preparation methods and applications
  • A multi -price adaptation DNA nano ladder and its preparation methods and applications
  • A multi -price adaptation DNA nano ladder and its preparation methods and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Preparation method of rolling circle amplification (RCA) product

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

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

[0055]

[0056]

Embodiment 2

[0057] Example 2: Preparation method of DNA nano-ladder

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

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

[0060] The self-assembled products were characterized by transmission electron microscopy, such as image 3 As shown in (a), it can be seen that the s...

Embodiment 3

[0067] Example 3: Drug Loading Experiment

[0068] The concentration of immobilized Dox was 200nM, and the concentration of DNA nanoladder was 1nM, 0.4nM, 0.2nM, 0.1nM, 0.04nM, 0.02nM, 0.01nM, 0.005nM, 0.0025nM, respectively, mixed well, centrifuged, and incubated at 30°C for 30min.

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

[0070] Preparation method of DNA nanoladder-Dox complex: 200nM antitumor drug adriamycin hydrochloride (Dox) was mixed with 1nM, 0.4nM, 0.2nM, 0.1nM, 0.04nM, 0.02nM, 0.01nM, 0.005nM, 0.0025nM, respectively. DNA nanoladders were 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 binding to form a DNA nano-ladder-Dox complex, and ...

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Abstract

The invention provides a multivalent aptamer DNA nano-ladder and its preparation method and application. The present invention uses natural DNA molecules as raw materials, and on the basis of DNA origami theory, utilizes the special properties of rolling circle amplification products to construct a DNA nano-ladder, and introduces a large number of aptamers to construct a ladder with simple design and strong drug-loading capacity. , a drug delivery system with target specificity and biocompatibility, 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 multivalent aptamer DNA nano-ladder and a preparation method and application thereof. Background technique [0002] The incidence and mortality of cancer in my country are increasing year by year, and it has become one of the four major chronic diseases in my country. At present, chemotherapy drugs are widely used in cancer treatment, but chemotherapy drugs can induce cytotoxicity in both tumor cells and healthy cells; and due to the phenomenon of multidrug resistance, the special "protein pump" existing in some tumor cells will induce chemotherapy drugs. For example, doxorubicin hydrochloride (Dox) is pumped out of the cell and becomes resistant to the drug. Therefore, the development of a targeted drug carrier with a simple design that can specifically recognize tumor cells is a challenge in the field of biomedical technology. [0003] DNA nanostructures have become a new...

Claims

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Application Information

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Patent Type & Authority Patents(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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