DNA (deoxyribonucleic acid) three-dimensional nano-structure artificial enzyme precursor, and preparation and application thereof

An artificial enzyme precursor and nanostructure technology, applied in the biological field, can solve the problems of weak catalytic ability, low catalytic activity, reduced stability and catalytic ability, and achieve the effect of stable structure and high sensitivity

Active Publication Date: 2015-07-15
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although ribozymes do not have the same highly diverse functional groups as proteins and have weak catalytic ability, because nucleic acids are more stable than proteins, simple in structure, easy to store and easy to prepare, people have been working on researching and improving these nucleases , it is expected to improve its substrate specificity and catalytic activity, so that it can be applied in industrial production
According to investigations, it is speculated that the catalytic activity of these nucleases is low because there is no delicate biomolecular skeleton to provide them with a sufficient chemical environment, thereby reducing their stability and catalytic ability

Method used

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  • DNA (deoxyribonucleic acid) three-dimensional nano-structure artificial enzyme precursor, and preparation and application thereof
  • DNA (deoxyribonucleic acid) three-dimensional nano-structure artificial enzyme precursor, and preparation and application thereof
  • DNA (deoxyribonucleic acid) three-dimensional nano-structure artificial enzyme precursor, and preparation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0068] 1. Materials and Reagents

[0069] The DNA used in the examples of the present invention was synthesized by Shanghai Sangong Company. hemin and TCEP were purchased from Sigma, and all other reagents were purchased from Sinopharm. The buffer solution used in the present invention comprises: TMK buffer solution (20mM tris buffer, 50mM MgCl 2 and 50mMKCl, pH 8.0); PBS buffer (10mM Na 2 HPO 4 , 2mM KH 2 PO 4 , 37mM NaCl and 2.7mM KCl, pH7.4); hemin was dissolved in DMSO and stored in a -20°C refrigerator for later use; 30mM TECP aqueous solution. All solutions were made up with Milli-Q water (18.2 MΩcm resistance).

[0070] 2. Design ideas

[0071] In order to improve the catalytic activity of G4-DNAzyme, the present invention uses DNA tetrahedron as the skeleton of G4-DNAzyme to provide a stable chemical environment for G4-DNAzyme, thereby constructing a novel artificial enzyme (Tetrazyme). The present invention utilizes the controllability of the DNA tetrahedron n...

Embodiment 2

[0108] The Tetrazyme precursors of various configurations prepared in the examples were immobilized on the working electrode, incubated with the coenzyme hemin to form a stable artificial enzyme Tetrazyme, and its activity was verified by electrochemical detection methods. Among them, Hemin and G4-DNAzyme not connected to the DNA tetrahedron backbone were used as controls. The G4-DNAzyme that is not connected to the DNA tetrahedron skeleton is a free G-quadruplex (Free G-quadruplex) that has been incubated with Hemin and modified sulfhydryl groups at the 3' end, containing 17 bases, and the nucleotide sequence is: GGGTAGGGCGGGTTGGG (SEQ ID NO. 1)

[0109] The overall structure of Free G-quadruplex is: GGGTAGGGCGGGTTGGG-C 6 -SH; SH means mercapto, C6 means 6 CH 2 .

[0110] 1. Clean the working electrode and fix different configurations of Tetrazyme precursors and Free G-quadruplex:

[0111] Take a gold electrode with a diameter of 2mm, first polish it with 0.3um and 0.5um ...

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Abstract

The invention relates to the technical field of biology, particularly a DNA (deoxyribonucleic acid) three-dimensional nano-structure artificial enzyme precursor, and preparation and application thereof. The invention provides a stable chemical environment by using a DNA tetrahedron as a nuclease framework; and by utilizing the controllability of the DNA tetrahedron nano structure, nucleases are respectively connected to different positions of the DNA tetrahedron framework, thereby forming the DNA tetrahedron nano-structure artificial enzyme precursor with different structures. Compared with the traditional nucleases, the enzyme formed by the DNA tetrahedron nano-structure artificial enzyme precursor with different structures has more stable structure and higher sensitivity, and the catalytic activity is 6-14 times of the traditional nuclease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA three-dimensional nanostructure artificial enzyme precursor and its preparation and application. Background technique [0002] Artificial enzymes have great application potential in the fields of biomedicine and chemical industry, and it is also an important tool for scientists to study the principles of protein catalysis. Natural enzymes are the result of long-term biological evolution, while artificial enzymes are constructed by scientists to achieve the purpose of transforming proteins by simulating the process of biological evolution and following the rules of protein formation. The construction of artificial enzymes not only enables us to make full use of the genes and proteins that exist in nature, but also to design and transform proteins at the molecular level, completing the process that can only be completed by evolution in nature for millions of years in a short peri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/11C12N9/22C12N15/10G01N27/327
Inventor 宓现强散丽黎曾冬冬张欢
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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