CN103411889B - A kind of method detecting endonuclease DNase I activity based on golden tetrahedron chirality nanostructured - Google Patents

A kind of method detecting endonuclease DNase I activity based on golden tetrahedron chirality nanostructured Download PDF

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CN103411889B
CN103411889B CN201310366140.6A CN201310366140A CN103411889B CN 103411889 B CN103411889 B CN 103411889B CN 201310366140 A CN201310366140 A CN 201310366140A CN 103411889 B CN103411889 B CN 103411889B
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dna1
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CN103411889A (en
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胥传来
郝昌龙
徐丽广
马伟
匡华
刘丽强
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Jiangnan University
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Abstract

A kind of based on golden tetrahedron chirality nanostructured detection endonuclease DNase? the method of I activity, belongs to the technical field of enzymology in molecular biology.Present invention process step is: the preparation of (one) detector probe: comprising: (1) 15nm particle diameter golden nanometer particle AuNP 1synthesis, (2) 25nm particle diameter golden nanometer particle AuNP 2synthesis, (3) AuNP 1with DNA1 coupling, (4) AuNP 2respectively with DNA2, DNA3, DNA4 coupling, (5) AuNP 1with AuNP 2assembling forms the golden tetrahedral structure with chirality; (2) justify two spectrum CD to detect, set up CD signal intensity and DNase? typical curve between I activity.The invention provides one and detect DNase based on circle two spectrum? the method of I activity, compared with traditional detection means, have highly sensitive, detectability is low, advantage easily and efficiently, has extraordinary actual application prospect.

Description

A kind of method detecting endonuclease DNase I activity based on golden tetrahedron chirality nanostructured
Technical field
The present invention relates to a kind of method detecting endonuclease DNaseI activity based on golden tetrahedron chirality nanostructured, belong to the technical field of enzymology in molecular biology.
Background technology
Endonuclease is the special enzyme of a class, and it can be hydrolyzed the phosphodiester bond destroyed between nucleic acid (comprising DNA and RNA) skeleton.This class of enzymes plays vital effect in biology field, comprises that DNA repairs, copies, Genotyping and molecular cloning, and it is widely used in genetic engineering field to prepare recombinant DNA molecules simultaneously.Along with the development of nanometer science and technology, endonuclease starts to be applied to assembling to obtain some novel nanometer package assemblies based on DNA, also the de-assembly to DNA Nanoscale assemblies is used to, and then some dynamic optics, electricity and magnetic properties of research nano material.DNaseI is a kind of endonuclease, and it plays an important role in many biochemical processes, and such as, in cellular programming death process, DNA molecular can be hydrolyzed to the basic composition unit of nucleosome by DNaseI.In addition, large quantifier elimination shows, when DNaseI activity reduces, can induce humans and animals generation systems lupus erythematosus, also likely can induce and produce cancer of the stomach or colon cancer; And when DNaseI hyperactivity, the generation of breast cancer likely can be caused.Therefore, be necessary very much to develop a kind of easy, sensitive method for detecting DNaseI activity.
The method of traditional detection DNaseI activity comprises high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA) etc.But it is not high that these methods have sensitivity, and detection time is long, need the shortcomings such as expensive instrument and equipment.In order to address these problems, novel detection means comprises fluorescent test, and electrochemical process and colourimetry are developed.In these methods, although colourimetry is simple, cost is low, its detectability is lower; Although fluorescence detection is convenient and swift, need the DNA probe that expensive fluorescein is modified; Although electrochemical method detectability is very low, need to modify electrode.So the present invention proposes a kind of novel, easy, sensitive detection method, its self-assembling technique based on nano material constructs the golden tetrahedron nanostructured with rival's property, and in this, as detection substrate.
The chiral gold tetrahedron formed by DNA hybridization, the content of its CD signal intensity and chirality assembly is closely related, by additional DNaseI, has been hydrolyzed the DNA molecular of connection function, destroys the tetrahedral steric configuration of chiral gold, will inevitably cause the reduction of CD signal.DNaseI activity is higher, and it is more that golden tetrahedron is dissociated, and it is more severe that CD signal reduces.By setting up the typical curve between CD signal intensity and DNaseI activity, the activity of DNaseI in solution can be detected.
Summary of the invention
The object of this invention is to provide a kind of method detecting DNaseI activity based on golden tetrahedron chirality nanostructured.
Technical scheme of the present invention: a kind of method detecting endonuclease DNaseI activity based on golden tetrahedron chirality nanostructured, processing step is:
(1) preparation of detector probe
(1) 15nm particle diameter golden nanometer particle (AuNP 1) synthesis
Adopt conventional method trisodium citrate reduction gold chloride synthesis 15nm particle diameter golden nanometer particle AuNP 1: the triangular flask chloroazotic acid of reaction is soaked 12h, after then using ultrapure water 3 times, dries, for subsequent use.Add the aqueous solution of chloraurate of 100mL0.25mM wherein, be heated with stirring to boiling, adding the freshly prepared massfraction of 2.5mL after 5min is the trisodium citrate aqueous solution of 1%, continue agitating heating, after 30min, terminate reaction, remove thermal source, be chilled to after room temperature, by AuNP until solution 1solution puts into 4 DEG C of refrigerators.
(2) 25nm particle diameter golden nanometer particle (AuNP 2) synthesis
Adopt conventional method trisodium citrate reduction gold chloride synthesis 25nm particle diameter golden nanometer particle AuNP 2: method and preparation AuNP 1identical, just the consumption of trisodium citrate is become 1.5mL, all the other conditions are all constant, can prepare AuNP 2solution.
(3) AuNP 1with DNA1 coupling
The AuNP that step (1) synthesizes 1carry out coupling with the DNA1 of sulfydryl modification and form AuNP 1-DNA1 complex: AuNP prepared by the step (1) of getting 50mL2nM 1add two (p-semi-annular jade pendant acyl phenyl) the Phenylphosphine di-potassium of 20mg bis-hydration, concussion mixing, hatch centrifugal segregation supernatant after 12h, will precipitate with 10mL0.5 × TBE resuspended; Add DNA1 wherein, control DNA1 and AuNP 1mol ratio be after 5:1,2h, add 2M sodium chloride and make sodium chloride final concentration be 50mM.After 12h, centrifugal, remove free DNA1 molecule, obtain AuNP 1-DNA1 complex.
DNA1：HS-5’-TTTGCCTGGAGATACATGCACATTACGGCTTTCCCTATTAGAAGGTCTCAGGTGCGCGTTTCGGTAAGTAGACGGGACCAGTTCGCC-3’。
(4) AuNP 2respectively with DNA2, DNA3, DNA4 coupling
The AuNP that step (2) synthesizes 2carry out coupling respectively with DNA2, DNA3, DNA4 of sulfydryl modification and form AuNP 2-DNA2, AuNP 2-DNA3, AuNP 2-DNA4 complex, coupling step is identical with step (3).
DNA2：HS-5’-TTTCGCGCACCTGAGACCTTCTAATAGGGTTTGCGACAGTCGTTCAACTAGAATGCCCTTTGGGCTGTTCCGGGTGTGGCTCGTCGG-3’；
DNA3：HS-5’-TTTGGCCGAGGACTCCTGCTCCGCTGCGGTTTGGCGAACTGGTCCCGTCTACTTACCGTTTCCGACGAGCCACACCCGGAACAGCCC-3’；
DNA4：HS-5’-TTTGCCGTAATGTGCATGTATCTCCAGGCTTTCCGCAGCGGAGCAGGAGTCCTCGGCCTTTGGGCATTCTAGTTGAACGACTGTCGC-3’。
(5) AuNP 1with AuNP 2assembling forms the golden tetrahedral structure with chirality
Get the AuNP of the 1mL2nM that step (4) is prepared respectively 2-DNA2, AuNP 2-DNA3, AuNP 2the AuNP of the 1mL2nM that-DNA4 complex and step (3) are prepared 1the mixing of-DNA1 complex is carried out hybridizing (hybridization buffer: 1 × TBE, hybridization time 24h), forms the golden tetrahedral structure with chirality; Carry out transmission electron microscope to this golden tetrahedral structure to characterize and circle two spectral characterizations;
(2) justify two spectrum CD to detect, set up the typical curve between CD signal intensity and DNaseI activity
The DNaseI of a series of different activities concentration is added: be respectively 0U/mL, 0.005U/mL, 0.01U/mL, 0.05U/mL, 0.1U/mL, 0.2U/mL, 0.5U/mL, 1U/mL, 10U/mL in chiral gold tetrahedron solution (1mL, 2nM) prepared by step (5); After reacting 10min at 37 DEG C, measure CD signal, active in horizontal ordinate with DNaseI, CD signal intensity is ordinate, Criterion curve.
Note: DNA used in the present invention all purchased from Chinese Shanghai Sheng Gong bioengineering company limited, and carries out purifying by polyacrylamide gel electrophoresis.
Beneficial effect of the present invention: the invention provides and a kind ofly detect the method for DNaseI activity based on circle two spectrum, compared with traditional detection means, have highly sensitive, detectability is low, advantage easily and efficiently, has extraordinary actual application prospect.
Accompanying drawing explanation
The transmission electron microscope photo of the golden tetrahedron chirality assembly that Fig. 1 the present invention is formed by nucleic acid hybridization.
In golden tetrahedron chirality assembly, the DNaseI of different activities is added, the variation diagram of the CD spectrum caused in Fig. 2 the present invention.
The canonical plotting of the circle two spectral detection DNaseI activity in Fig. 3 the present invention.
Embodiment
Embodiment 1
(1) preparation of detector probe
(1) 15nm particle diameter golden nanometer particle (AuNP 1) synthesis
Adopt trisodium citrate reduction gold chloride synthesis 15nm particle diameter golden nanometer particle: the triangular flask chloroazotic acid of reaction is soaked 12h, after then using ultrapure water 3 times, dry, for subsequent use.Add the aqueous solution of chloraurate of 100mL0.25mM wherein, be heated with stirring to boiling, adding the freshly prepared massfraction of 2.5mL after 5min is the trisodium citrate aqueous solution of 1%, continue agitating heating, after 30min, terminate reaction, remove thermal source, be chilled to after room temperature, by AuNP until solution 1solution puts into 4 DEG C of refrigerators.
(2) 25nm particle diameter golden nanometer particle (AuNP 2) synthesis
Adopt trisodium citrate reduction gold chloride synthesis 25nm particle diameter golden nanometer particle: method and preparation AuNP 1identical, just the consumption of trisodium citrate is become 1.5mL, all the other conditions are all constant, can prepare AuNP 2solution.
(3) AuNP 1with DNA1 coupling
The AuNP that step (1) synthesizes 1carry out coupling with the DNA1 of sulfydryl modification and form AuNP 1-DNA1 complex: AuNP prepared by the step (1) of getting 50mL2nM 1add two (p-semi-annular jade pendant acyl phenyl) the Phenylphosphine di-potassium of 20mg bis-hydration, concussion mixing, hatch centrifugal segregation supernatant after 12h, will precipitate with 10mL0.5 × TBE resuspended; Add DNA1 wherein, control DNA1 and AuNP 1mol ratio be after 5:1,2h, add 2M sodium chloride and make its final concentration be 50mM.After 12h, centrifugal, remove free DNA1 molecule, obtain AuNP 1-DNA1 complex.
DNA1：HS-5’-TTTGCCTGGAGATACATGCACATTACGGCTTTCCCTATTAGAAGGTCTCAGGTGCGCGTTTCGGTAAGTAGACGGGACCAGTTCGCC-3’。
(4) AuNP 2respectively with DNA2, DNA3, DNA4 coupling
The AuNP that step (2) synthesizes 2carry out coupling respectively with DNA2, DNA3, DNA4 of sulfydryl modification and form AuNP 2-DNA2, AuNP 2-DNA3, AuNP 2-DNA4 complex, coupling step is identical with step (3).
DNA2：HS-5’-TTTCGCGCACCTGAGACCTTCTAATAGGGTTTGCGACAGTCGTTCAACTAGAATGCCCTTTGGGCTGTTCCGGGTGTGGCTCGTCGG-3’；
DNA3：HS-5’-TTTGGCCGAGGACTCCTGCTCCGCTGCGGTTTGGCGAACTGGTCCCGTCTACTTACCGTTTCCGACGAGCCACACCCGGAACAGCCC-3’；
DNA4：HS-5’-TTTGCCGTAATGTGCATGTATCTCCAGGCTTTCCGCAGCGGAGCAGGAGTCCTCGGCCTTTGGGCATTCTAGTTGAACGACTGTCGC-3’。
(5) AuNP 1with AuNP 2assembling forms the golden tetrahedral structure with chirality
Get the AuNP of the 1mL2nM that step (4) is prepared respectively 2-DNA2, AuNP 2-DNA3, AuNP 2the AuNP of the 1mL2nM that-DNA4 complex and step (3) are prepared 1the mixing of-DNA1 complex is carried out hybridizing (hybridization buffer: 1 × TBE, hybridization time 24h), forms the golden tetrahedral structure with chirality.Carry out transmission electron microscope to this golden tetrahedral structure to characterize and circle two spectral characterizations.
(2) justify two spectrum CD to detect, set up the typical curve between CD signal intensity and DNaseI activity
The DNaseI of a series of different activities concentration is added: be respectively 0U/mL, 0.005U/mL, 0.01U/mL, 0.05U/mL, 0.1U/mL, 0.2U/mL, 0.5U/mL, 1U/mL, 10U/mL in chiral gold tetrahedron solution (1mL, 2nM) prepared by step (5); After reacting 10min at 37 DEG C, measure CD signal, active in horizontal ordinate with DNaseI, CD signal intensity is ordinate, Criterion curve.
DNA1：HS-5’-TTTGCCTGGAGATACATGCACATTACGGCTTTCCCTATTAGAAGGTCTCAGGTGCGCGTTTCGGTAAGTAGACGGGACCAGTTCGCC-3’；
DNA2：HS-5’-TTTCGCGCACCTGAGACCTTCTAATAGGGTTTGCGACAGTCGTTCAACTAGAATGCCCTTTGGGCTGTTCCGGGTGTGGCTCGTCGG-3’；
DNA3：HS-5’-TTTGGCCGAGGACTCCTGCTCCGCTGCGGTTTGGCGAACTGGTCCCGTCTACTTACCGTTTCCGACGAGCCACACCCGGAACAGCCC-3’；
DNA4：HS-5’-TTTGCCGTAATGTGCATGTATCTCCAGGCTTTCCGCAGCGGAGCAGGAGTCCTCGGCCTTTGGGCATTCTAGTTGAACGACTGTCGC-3’；

Claims (1)

1. detect a method for endonuclease DNaseI activity based on golden tetrahedron chirality nanostructured, it is characterized in that processing step is:
(1) preparation of detector probe
(1) 15nm particle diameter golden nanometer particle AuNP 1synthesis
Adopt trisodium citrate reduction gold chloride synthesis 15nm particle diameter golden nanometer particle AuNP 1;
(2) 25nm particle diameter golden nanometer particle AuNP 2synthesis
Adopt trisodium citrate reduction gold chloride synthesis 25nm particle diameter golden nanometer particle AuNP 2;
(3) AuNP 1with DNA1 coupling
The AuNP that step (1) synthesizes 1carry out coupling with the DNA1 of sulfydryl modification and form AuNP 1-DNA1 complex: AuNP prepared by the step (1) of getting 50mL2nM 1add two (p-semi-annular jade pendant acyl phenyl) the Phenylphosphine di-potassium of 20mg bis-hydration, concussion mixing, hatch centrifugal segregation supernatant after 12h, will precipitate with 10mL0.5 × TBE resuspended; Add DNA1 wherein, control DNA1 and AuNP 1mol ratio be after 5:1,2h, add 2M sodium chloride and make sodium chloride final concentration be after 50mM, 12h, centrifugal, remove free DNA1 molecule, obtain AuNP 1-DNA1 complex;
DNA1：HS-5’-TTTGCCTGGAGATACATGCACATTACGGCTTTCCCTATTAGAAGGTCTCAGGTGCGCGTTTCGGTAAGTAGACGGGACCAGTTCGCC-3’；
(4) AuNP 2respectively with DNA2, DNA3, DNA4 coupling
The AuNP that step (2) synthesizes 2carry out coupling respectively with DNA2, DNA3, DNA4 of sulfydryl modification and form AuNP 2-DNA2, AuNP 2-DNA3, AuNP 2-DNA4 complex, coupling step is identical with step (3);
DNA2：HS-5’-TTTCGCGCACCTGAGACCTTCTAATAGGGTTTGCGACAGTCGTTCAACTAGAATGCCCTTTGGGCTGTTCCGGGTGTGGCTCGTCGG-3’；
DNA3：HS-5’-TTTGGCCGAGGACTCCTGCTCCGCTGCGGTTTGGCGAACTGGTCCCGTCTACTTACCGTTTCCGACGAGCCACACCCGGAACAGCCC-3’；
DNA4：HS-5’-TTTGCCGTAATGTGCATGTATCTCCAGGCTTTCCGCAGCGGAGCAGGAGTCCTCGGCCTTTGGGCATTCTAGTTGAACGACTGTCGC-3’；
(5) AuNP 1with AuNP 2assembling forms the golden tetrahedral structure with chirality
Get the AuNP of the 1mL2nM that step (4) is prepared respectively 2-DNA2, AuNP 2-DNA3, AuNP 2the AuNP of the 1mL2nM that-DNA4 complex and step (3) are prepared 1the mixing of-DNA1 complex is hybridized, hybridization buffer: 1 × TBE, and hybridization time 24h forms the golden tetrahedral structure with chirality; Carry out transmission electron microscope to this golden tetrahedral structure to characterize and circle two spectral characterizations;
(2) justify two spectrum CD to detect, set up the typical curve between CD signal intensity and DNaseI activity
To chiral gold tetrahedron solution 1mL prepared by step (5), add the DNaseI of a series of different activities concentration in 2nM: be respectively 0U/mL, 0.005U/mL, 0.01U/mL, 0.05U/mL, 0.1U/mL, 0.2U/mL, 0.5U/mL, 1U/mL, 10U/mL; After reacting 10min at 37 DEG C, measure CD signal, active in horizontal ordinate with DNaseI, CD signal intensity is ordinate, Criterion curve.
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