A method for embedding and releasing DNA nano-origami structures as drug carriers using DAPI embedding and releasing

An origami structure and nanotechnology, which can be used in pharmaceutical formulations, medical preparations with inactive ingredients, fluorescence/phosphorescence, etc., and can solve the problems of expensive reagent ordering, complicated experimental design and operation, etc.

Active Publication Date: 2018-01-16
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

AI Technical Summary

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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  • A method for embedding and releasing DNA nano-origami structures as drug carriers using DAPI embedding and releasing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Add 12 μL of circular DNA (2 μM, sequence: 5'TATGCCCAGCCCTGTAAGATGAAGATAGCGCACAATGGTCGGATTCTCAACTCGTATTCTCAACTCGTATTCTCAACTCGTCCTGCCCTμL) to 16 μL of primer DNA (1 μM, sequence: 5'CCAGCCTAAGAGTTGAGCA3'), dNTPs (4 μL, 10 mM), RCA buffer 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 high temperature of 95 °C, and then gradually cooled...

Embodiment 2

[0020] Add 12 μL of circular DNA (2 μM, sequence: 5'TATGCCCAGCCCTGTAAGATGAAGATAGCGCACAATGGTCGGATTCTCAACTCGTATTCTCAACTCGTATTCTCAACTCGTCCTGCCCTμL) to 16 μL of primer DNA (1 μM, sequence: 5'CCAGCCTAAGAGTTGAGCA3'), dNTPs (4 μL, 10 mM), RCA buffer 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×, 125mMMg 2+ ), the total volume is 20 μL, the solution is mixed well, placed in a high temperature of 95 °C, and then gradually cooled to 25 °C....

Embodiment 3

[0025]Add 12 μL of circular DNA (2 μM, sequence: 5'TATGCCCAGCCCTGTAAGATGAAGATAGCGCACAATGGTCGGATTCTCAACTCGTATTCTCAACTCGTATTCTCAACTCGTCCTGCCCTμL) to 16 μL of primer DNA (1 μM, sequence: 5'CCAGCCTAAGAGTTGAGCA3'), dNTPs (4 μL, 10 mM), RCA buffer 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×, 125mMMg 2+ ), the total volume is 20 μL, the solution is mixed well, placed in a high temperature of 95 °C, and then gradually cooled to 25 °C. ...

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Abstract

The invention relates to a DNA nano-origami structure constructed by DNA rolling circle amplification technology and DNA origami. At the same time, the fluorescent dye DAPI with DNA double-strand binding characteristics and cell membrane penetration characteristics is used to mark and track the DNA nano-origami structure. Real-time monitoring and simulating the drug loading and sustained release process of DNA nano-origami structure as a drug carrier can also be used as a new fluorescence quantitative analysis method for biomolecular detection, such as early detection of tumors, postoperative monitoring and evaluation, and cell imaging, etc. field.

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 Patents(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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