Method for simulation of DNA nano origami structure as drug carrier by DAPI embedding and release

A kind of origami structure and nanotechnology, which is applied in the direction of pharmaceutical formula, medical preparations of non-active ingredients, fluorescence/phosphorescence, etc., can solve the problems of expensive reagent ordering, complicated experimental design and operation, etc.

Active Publication Date: 2015-10-28
智玺那诺(上海)生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In this process, the experimental design and operation are complicated, and the ordering of reagents is expensive

Method used

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  • Method for simulation of DNA nano origami structure as drug carrier by DAPI embedding and release

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] 16 12 μL of circular DNA (2 μM, sequence: 5'TATGCCCAGCCCTGTAAGATGAAGATAGCGCACAATGGTCGGATTCTCAACTCGTATTCTCAACTCGTATTCTCAACTCGTCTCTGCCCTGACTTC3'), dNTPs (4 μL, 10 mM phi2, RCA buffer (4 μL, 10 mM phi2) were added to μL primer DNA (1 μM, sequence: 5'CCAGCCTAAGAGTTGAGCA3') successively. polymerase 4μL, 10 U / μL), the final volume of the mixed solution was 40μL. Then amplified in a 30°C water bath for 30 min. 40 μL of the obtained product was subjected to 10% PAGE electrophoresis, and then recovered by slicing gel to remove small fragments of DNA, redundant circular DNA and primer DNA. Take 3 μL of the recovered product, add 12 μL of milliQ water, and add stapled strands 1-3 (100 μM, the sequence is as follows:

[0016] 5'-CAGCCCTGTAAGATGAAGATAGCGTCTATGCC-3'

[0017] 5'-CCCTGACTCACAATGGTCGGATTCCGTCTCTG-3'

[0018] 5’-TCTCAACTTCAACTCGTATTCTCAACTCGTAT-3’) 1 μL each, 2 μL TAE buffer (10×, 125 mM Mg 2+ ), the total volume is 20 μL, the solution is mixed well, placed in a hig...

Embodiment 2

[0020] 16 12 μL of circular DNA (2 μM, sequence: 5'TATGCCCAGCCCTGTAAGATGAAGATAGCGCACAATGGTCGGATTCTCAACTCGTATTCTCAACTCGTATTCTCAACTCGTCTCTGCCCTGACTTC3'), dNTPs (4 μL, 10 mM phi2, RCA buffer (4 μL, 10 mM phi2) were added to μL primer DNA (1 μM, sequence: 5'CCAGCCTAAGAGTTGAGCA3') successively. polymerase 4μL, 10 U / μL), the final volume of the mixed solution was 40μL. Then amplified in a 30°C water bath for 30 min. 40 μL of the obtained product was subjected to 10% PAGE electrophoresis, and then recovered by slicing gel to remove small fragments of DNA, redundant circular DNA and primer DNA. Take 3 μL of the recovered product, add 12 μL of milliQ water, and add stapled strands 1-3 (1 μM, the sequence is as follows:

[0021] 5'CAGCCCTGTAAGATGAAGATAGCGTCTATGCC3'

[0022] 5'CCCTGACTCACAATGGTCGGATTCCGTCTCTG3'

[0023] 5’TCTCAACTTCAACTCGTATTCTCAACTCGTAT3’, 1 μL each, 2 μL TAE buffer (10×, 125 mM Mg 2+ ), the total volume is 20 μL, the solution is mixed well, placed in a high temper...

Embodiment 3

[0025] 1612 μL of circular DNA (2 μM, sequence: 5'TATGCCCAGCCCTGTAAGATGAAGATAGCGCACAATGGTCGGATTCTCAACTCGTATTCTCAACTCGTATTCTCAACTCGTCTCTGCCCTGACTTC3'), dNTPs (4 μL, 10 mM phi2, RCA buffer (4 μL, 10 mM phi2) were added to μL primer DNA (1 μM, sequence: 5'CCAGCCTAAGAGTTGAGCA3') successively. polymerase 4μL, 10 U / μL), the final volume of the mixed solution was 40μL. Then amplified in a 30°C water bath for 30 min. 40 μL of the obtained product was subjected to 10% PAGE electrophoresis, and then recovered by slicing gel to remove small fragments of DNA, redundant circular DNA and primer DNA. Take 3 μL of the recovered product, add 12 μL of milliQ water, and add stapled strands 1-3 (1 μM, the sequence is as follows:

[0026] 5'CAGCCCTGTAAGATGAAGATAGCGTCTATGCC3'

[0027] 5'CCCTGACTCACAATGGTCGGATTCCGTCTCTG3'

[0028] 5’TCTCAACTTCAACTCGTATTCTCAACTCGTAT3’, 1 μL each, 2 μL TAE buffer (10×, 125 mM Mg 2+ ), the total volume is 20 μL, the solution is mixed well, placed in a high tempera...

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Abstract

The invention relates to a DNA nano-origami structure constructed by rolling circle amplification technology and DNA origami, at the same time utilizes fluorescent dye DAPI with DNA double-strand binding properties and cell membrane penetration properties to label and track the DNA nano-origami structure, thus realizing real-time monitoring and simulation of the drug loading and sustained release process of the DNA nano-origami structure as a drug carrier. And the method also can be applied as a new fluorescent quantitative analysis method to biomolecular detection, like tumor early detection, postoperative monitoring and evaluation, cell imaging and other fields.

Description

technical field [0001] The invention belongs to the field of DNA nanotechnology development, functionalization and application of nanomaterials, and relates to a DNA nano-origami structure constructed by DNA rolling circle amplification technology and DNA origami, and utilizes DNA double-strand binding characteristics and cell membrane penetration characteristics at the same time The fluorescent dye DAPI marks and tracks the DNA nano-origami structure, realizes real-time monitoring and simulates the drug-loading and sustained-release process of the DNA nano-origami composite structure as a drug carrier, and can also be used as a new fluorescent quantitative analysis method for biomolecular detection , such as early detection of tumors, postoperative monitoring and evaluation, and cell imaging. Background technique [0002] In recent years, malignant tumors have become one of the major diseases that seriously endanger human health and life worldwide. Survey data show that in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64A61K47/26
Inventor 何丹农颜娟胡冲娅金彩虹
Owner 智玺那诺(上海)生物科技有限责任公司
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