CN103910893A - Polypeptide-DNA hydrogel and preparation method - Google Patents
Polypeptide-DNA hydrogel and preparation method Download PDFInfo
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- CN103910893A CN103910893A CN201410101230.7A CN201410101230A CN103910893A CN 103910893 A CN103910893 A CN 103910893A CN 201410101230 A CN201410101230 A CN 201410101230A CN 103910893 A CN103910893 A CN 103910893A
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- dna molecule
- pidolidone
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- 238000002360 preparation method Methods 0.000 title abstract description 16
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- 238000004132 cross linking Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 45
- 229940005605 valeric acid Drugs 0.000 claims description 34
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- 238000006243 chemical reaction Methods 0.000 claims description 23
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- 150000002170 ethers Chemical class 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides a polypeptide-DNA hydrogel and a preparation method, wherein, the polypeptide-DNA hydrogel comprises the following components: polypeptide, wherein a single chain DNA molecule enables covalent binding on the polypeptide; a double chain DNA molecule, wherein the double chain DNA molecule has two cohesive ends; wherein the cohesive ends and the single chain DNA molecule are complementary, and polypeptide forms a crosslinking structure through the complementation of the cohesive ends of the double chain DNA molecule and the single chain DNA molecule. The mechanical strength of the polypeptide-DNA hydrogel can be adjusted, the biocompatibility is good, and the polypeptide-DNA hydrogel has reversible thermal responsiveness and specific enzyme responsiveness.
Description
Technical field
The invention belongs to biomedical sector, relate to particularly poly-polypeptide-DNA hydrogel and preparation method thereof.
Background technology
Hydrogel is that a class contains hydrophilic radical, can be by water-soluble swollen and keep the polymkeric substance of its complete three-dimensional net structure.It is significantly swelling that it can absorb a large amount of moisture in water, and after significantly swelling, can continue to keep its original structure and not dissolved, it can perception external stimulus subtle change, as temperature, pH value, ionic strength, electric field, magnetic field etc., and can stimulate to external world and produce response.Due to the structure of hydrogel and for cell provides bioenvironmental extracellular matrix quite similar, thereby hydrogel has wide practical use at biomedical sector.But preparation has the hydrogel of good biocompatibility and suitable physical strength, be still a major challenge of field of tissue engineering technology.
Thereby, still need deeply about the research of hydrogel at present.
Summary of the invention
The present invention is intended to solve to a certain extent one of technical problem in correlation technique.For this reason, it is good that one object of the present invention is to propose a kind of biocompatibility, adjustable mechanical intensity, and there is poly-polypeptide-DNA hydrogel of multiple response.
In one aspect of the invention, the invention provides a kind of poly-polypeptide-DNA hydrogel.According to embodiments of the invention, this poly-polypeptide-DNA hydrogel comprises: polypeptide, and on described polypeptide, covalent attachment has single strand dna; Double chain DNA molecule, wherein, described double chain DNA molecule has two sticky ends, wherein, described sticky end and described single strand dna complementation, described polypeptide forms crosslinking structure by the sticky end of described double chain DNA molecule and the complementation of described single strand dna.Contriver's discovery, poly-polypeptide-DNA hydrogel biocompatibility of the present invention is good, and physical strength is adjustable, and has multiple response, can be effective to the biomedical sector such as cell cultures, drug release.
According to embodiments of the invention, above-mentioned poly-polypeptide-DNA hydrogel also has following additional technical feature:
According to embodiments of the invention, two sticky ends of described double chain DNA molecule have identical nucleotide sequence.
According to embodiments of the invention, the length of two sticky ends of described double chain DNA molecule is identical with the length of described single strand dna.
According to embodiments of the invention, the length of two sticky ends of described double chain DNA molecule is respectively 8～12nt independently.Thus, be conducive to the formation of poly-polypeptide-DNA hydrogel, and the physical strength of the poly-polypeptide-DNA hydrogel forming is good, in addition, by regulating the length of two sticky ends of double chain DNA molecule, can form poly-polypeptide-DNA hydrogel with different physical strengths.
According to embodiments of the invention, described polypeptide has the aminoacid sequence as shown in SEQ ID NO:12.
According to embodiments of the invention, described single strand dna has the nucleotide sequence as shown in SEQ ID NO:1.
According to embodiments of the invention, described double chain DNA molecule has the first chain and the second chain, wherein, described the first chain and described the second chain meet one of following condition: described the first chain has the sequence shown in SEQ ID NO:2, and described the second chain has the sequence shown in SEQ ID NO:3; Described the first chain has the sequence shown in SEQ ID NO:4, and described the second chain has the sequence shown in SEQ ID NO:5; Described the first chain has the sequence shown in SEQ ID NO:6, and described the second chain has the sequence shown in SEQ ID NO:7; Described the first chain has the sequence shown in SEQ ID NO:8, and described the second chain has the sequence shown in SEQ ID NO:9; Described the first chain has the sequence shown in SEQ ID NO:10, and described the second chain has the sequence shown in SEQ ID NO:11.
According to embodiments of the invention, on each described polypeptide, be combined with multiple single strand dnas.Thus, be conducive to improve stability and the physical strength of poly-polypeptide-DNA hydrogel.
According to embodiments of the invention, on each described polypeptide, be combined with at least 4 described single strand dnas.Thus, stability and the physical strength of poly-polypeptide-DNA hydrogel are better.
According to embodiments of the invention, the mol ratio of described double chain DNA molecule and described single strand dna is 0.6～1.2:1.Thus, the physical strength of poly-polypeptide-DNA hydrogel is better.
According to embodiments of the invention, described polypeptide and described single strand dna form has the structure shown in following chemical formula:
Wherein, ssDNA represents described single strand dna, and * represents natural chiral amino acid.
In another aspect of this invention, the invention provides a kind of method of preparing poly-polypeptide-DNA hydrogel noted earlier.According to embodiments of the invention, the method comprises: polypeptide and double chain DNA molecule are provided, and wherein, on described polypeptide, covalent attachment has at least one single strand dna, described double chain DNA molecule has two sticky ends, and described sticky end and described single strand dna complementation; And described polypeptide is mixed with described double chain DNA molecule.Contriver finds, utilizes the method for the present invention, can fast and effeciently prepare foregoing poly-polypeptide-DNA hydrogel, and method is simple, processing ease.
According to embodiments of the invention, described in provide polypeptide further to comprise: the bromo-valeric acid of 5-is contacted, to obtain 5-nitrine-valeric acid with sodiumazide; Described 5-nitrine-valeric acid is contacted, to obtain 5-nitrine-valeric acid maloyl imines ester with maloyl imines; Described 5-nitrine-valeric acid maloyl imines ester is contacted, to obtain the single strand dna that contains azido group with amido modified single strand dna; γ-propargyl-Pidolidone is contacted, to obtain γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides with two (trichloromethyl) carbonic ethers; Described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides is contacted, to obtain γ-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer with γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides; Described-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer is contacted, to obtain Pidolidone γ-propargyl-Pidolidone ester copolymer with HBr with trifluoroacetic acid; The single strand dna that contains azido group described in making contacts with described Pidolidone γ-propargyl-Pidolidone ester copolymer, to obtain described polypeptide.
It should be noted that, the term " contact " that used in this article should be interpreted broadly, and it can be any mode that can make at least two kinds of reactant generation chemical reactions, for example, can be that two kinds of reactants are mixed under suitable condition.As required, can be under agitation, the reactant that needs are contacted mixes.
According to embodiments of the invention, further comprise: bromo-described 5-valeric acid is dissolved in to dimethyl sulfoxide (DMSO), be heated to after 40 degrees Celsius, add described sodiumazide, in 85 degrees Celsius, stir lower reaction and spend the night, obtained reaction solution is cooled to 40 degrees Celsius, add concentrated hydrochloric acid, stirring is spent the night, and through extracted with diethyl ether twice, obtains described 5-nitrine-valeric acid; Described 5-nitrine-valeric acid and described maloyl imines are dissolved in to methylene dichloride, under stirring, add 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), stirred overnight at room temperature, obtains described 5-nitrine-valeric acid maloyl imines ester; Described 5-nitrine-valeric acid maloyl imines ester is dissolved in to DMF, obtained solution is mixed with the aqueous solution and the phosphate buffer soln of described amido modified single strand dna, to obtain the single strand dna that contains azido group; γ-propargyl-Pidolidone is contacted, to obtain γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides with two (trichloromethyl) carbonic ethers; Make described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides and described γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides be dissolved in N, dinethylformamide, after adding title complex containing Ni as catalyzer, in 20 degrees Celsius of lower stirring reactions, obtain described γ-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer, wherein, the mol ratio of described γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides, described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides and described Ni is 10:3:0.05; Make described-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer be dissolved in trifluoroacetic acid, add after the acetum of HBr, stirring reaction 2 hours under room temperature, obtains described Pidolidone γ-propargyl-Pidolidone ester copolymer; And make described Pidolidone γ-propargyl-Pidolidone ester copolymer aqueous solution and CuSO
4solution, THTA(organic ligand), described in contain azido group single strand dna solution and sodium ascorbate solution mix, obtained mixing solutions is reacted and is spent the night under 50 degrees Celsius, oscillating condition, obtain described polypeptide.
According to embodiments of the invention, described single strand dna is prepared by solid phase phosphoramidite DNA synthetic method.
According to embodiments of the invention, described double chain DNA molecule obtains by following steps: by according to the described single strand dna of stoichiometric and TBE(Tris-boric acid-EDTA) damping fluid mixes, be heated to 95 degrees Celsius and be incubated after 3 minutes, in 2 hours, be cooled to room temperature, to obtain described double chain DNA molecule.
According to embodiments of the invention, described described polypeptide is mixed further and comprised with described double chain DNA molecule: the ratio that is 0.6-1.2:1 according to described double chain DNA molecule with the mol ratio of described single strand dna with described double chain DNA molecule by described polypeptide is mixed, to obtain the mixture of polypeptide and double chain DNA molecule; The ratio that is 0.3%-5% according to massfraction by the mixture of described polypeptide and double chain DNA molecule is dissolved in TBE(Tris-boric acid-EDTA) in damping fluid, to obtain poly-polypeptide-DNA hydrogel.
Accompanying drawing explanation
Fig. 1 has shown according to one embodiment of present invention, the polyacrylamide gel electrophoresis detection figure of ssDNA molecule and dsDNA molecule;
Fig. 2 has shown according to one embodiment of present invention, the frequency sweeping rheology result figure of Gel1-4;
Fig. 3 has shown according to one embodiment of present invention, the rheological property detected result figure of Gel4-11;
Fig. 4 has shown according to one embodiment of present invention, the photo of Gel2, Gel12 and Gel13;
Fig. 5 has shown according to one embodiment of present invention, the shear rheology experimental result picture of Gel2, Gel12 and Gel13;
Fig. 6 has shown according to one embodiment of present invention, the circular dichroism spectrum detection figure of Gel10 and Gel16;
Fig. 7 has shown according to one embodiment of present invention, the time scan rheology result figure of Gel10 and Gel16;
Fig. 8 has shown according to one embodiment of present invention, the rheological property detection figure of Gel2 between 25 degrees Celsius and 60 degrees Celsius;
Fig. 9 has shown according to one embodiment of present invention, the photo of poly-polypeptide-DNA hydrogel;
Figure 10 shown according to one embodiment of present invention, by after the polypeptide of different concns and double chain DNA molecule and NIH3T3 co-culture of cells, and the survival rate of NIH3T3 cell, wherein,
Figure 10 (a) is by after the polypeptide of different concns and NIH3T3 co-culture of cells, the survival rate of NIH3T3 cell,
Figure 10 (b) is by after the double chain DNA molecule of different concns and NIH3T3 co-culture of cells, the survival rate of NIH3T3 cell;
Figure 11 has shown according to one embodiment of present invention, utilizes poly-polypeptide-DNA hydrogel 3D to cultivate after 24h, the confocal fluorescent microphotograph of NIH3T3 cell, wherein,
The said three-dimensional body phase material that Figure 11 (a) forms for the attached NIH3T3 of poly-polypeptide-DNA hydrogel bag,
Figure 11 (b) is the confocal fluorescent microphotograph of the poly-attached NIH3T3 cell of polypeptide-DNA hydrogel bag; And
Figure 12 has shown according to one embodiment of present invention, the synthetic route chart of polypeptide.
Embodiment
Describe embodiments of the invention below in detail, it should be noted that embodiment described below is exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.In addition, if do not clearly not stated, all reagent of adopting are in the following embodiments can be buied on market, or can according to herein or known method synthetic, for the reaction conditions of not listing, be also that those skilled in the art easily obtain.
In one aspect of the invention, the invention provides a kind of poly-polypeptide-DNA hydrogel.According to embodiments of the invention, this poly-polypeptide-DNA hydrogel comprises: polypeptide, and on described polypeptide, covalent attachment has single strand dna; Double chain DNA molecule, wherein, described double chain DNA molecule has two sticky ends, wherein, described sticky end and described single strand dna complementation, described polypeptide forms crosslinking structure by the sticky end of described double chain DNA molecule and the complementation of described single strand dna.Contriver's discovery, poly-polypeptide-DNA hydrogel biocompatibility of the present invention is good, and physical strength is adjustable, and has multiple response, can be effective to the biomedical sector such as cell cultures, drug release.
According to embodiments of the invention, two sticky ends of described double chain DNA molecule have identical nucleotide sequence.
According to embodiments of the invention, the length of two sticky ends of described double chain DNA molecule is identical with the length of described single strand dna.
According to embodiments of the invention, the length of two sticky ends of described double chain DNA molecule is respectively 8～12nt independently.Thus, be conducive to the formation of poly-polypeptide-DNA hydrogel, and the physical strength of the poly-polypeptide-DNA hydrogel forming is good, in addition, by regulating the length of two sticky ends of double chain DNA molecule, can form poly-polypeptide-DNA hydrogel with different physical strengths.
According to embodiments of the invention, the kind of described polypeptide is not particularly limited.According to a concrete example of the present invention, described polypeptide is polyglutamic acid, has the aminoacid sequence as shown in SEQ ID NO:12: (L-Glu)
m(L-Glu-g-DNA)
n(SEQ ID NO:12), wherein, m=240, n=20.
According to embodiments of the invention, described single strand dna has the nucleotide sequence as shown in SEQ ID NO:2.
According to embodiments of the invention, described double chain DNA molecule has the first chain and the second chain, wherein, described the first chain and described the second chain meet one of following condition: described the first chain has the sequence shown in SEQ ID NO:2, and described the second chain has the sequence shown in SEQ ID NO:3; Described the first chain has the sequence shown in SEQ ID NO:4, and described the second chain has the sequence shown in SEQ ID NO:5; Described the first chain has the sequence shown in SEQ ID NO:6, and described the second chain has the sequence shown in SEQ ID NO:7; Described the first chain has the sequence shown in SEQ ID NO:8, and described the second chain has the sequence shown in SEQ ID NO:9; Or described the first chain has the sequence shown in SEQ ID NO:10, described the second chain has the sequence shown in SEQ ID NO:11.
According to embodiments of the invention, on each described polypeptide, be combined with multiple single strand dnas.Thus, be conducive to improve stability and the physical strength of poly-polypeptide-DNA hydrogel.In a concrete example of the present invention, on each described polypeptide, be combined with at least 4 described single strand dnas.Thus, stability and the physical strength of poly-polypeptide-DNA hydrogel are better.
According to embodiments of the invention, the mol ratio of described double chain DNA molecule and described single strand dna is 0.6～1.2:1.Thus, the physical strength of poly-polypeptide-DNA hydrogel is better.
According to embodiments of the invention, described polypeptide and described single strand dna form has the structure shown in following chemical formula:
Wherein, ssDNA represents described single strand dna, and * represents natural chiral amino acid.
In another aspect of this invention, the invention provides a kind of method of preparing poly-polypeptide-DNA hydrogel noted earlier.According to embodiments of the invention, the method comprises the following steps:
Polypeptide and double chain DNA molecule are provided
Polypeptide and double chain DNA molecule are provided, and wherein, on described polypeptide, covalent attachment has at least one single strand dna, and described double chain DNA molecule has two sticky ends, and described sticky end and described single strand dna complementation.
According to embodiments of the invention, with reference to Figure 12, described in provide polypeptide further to comprise: the bromo-valeric acid of 5-is contacted with sodiumazide, so as to obtain 5-nitrine-valeric acid (compound 1); Described 5-nitrine-valeric acid is contacted, to obtain 5-nitrine-valeric acid maloyl imines ester (compound 2) with maloyl imines; Described 5-nitrine-valeric acid maloyl imines ester is contacted, to obtain the single strand dna (compound 3) that contains azido group with amido modified single strand dna; γ-propargyl-Pidolidone is contacted, to obtain γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides (compound 4) with two (trichloromethyl) carbonic ethers; Described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides is contacted, to obtain γ-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer (compound 5) with γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides; Described-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer is contacted, to obtain Pidolidone γ-propargyl-Pidolidone ester copolymer (compound 6) with HBr with trifluoroacetic acid; The single strand dna that contains azido group described in making contacts with described Pidolidone γ-propargyl-Pidolidone ester copolymer, to obtain described polypeptide (compound 7).
According to embodiments of the invention, the described polypeptide that provides further comprises: bromo-described 5-valeric acid is dissolved in to dimethyl sulfoxide (DMSO), be heated to after 40 degrees Celsius, add described sodiumazide, in 85 degrees Celsius, stir lower reaction and spend the night, obtained reaction solution is cooled to 40 degrees Celsius, add concentrated hydrochloric acid, stirring is spent the night, and through extracted with diethyl ether twice, obtains described 5-nitrine-valeric acid; Described 5-nitrine-valeric acid and described maloyl imines are dissolved in to methylene dichloride, under stirring, add 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), stirred overnight at room temperature, obtains described 5-nitrine-valeric acid maloyl imines ester; Described 5-nitrine-valeric acid maloyl imines ester is dissolved in to DMF, obtained solution is mixed with the aqueous solution and the phosphate buffer soln of described amido modified single strand dna, to obtain the single strand dna that contains azido group; γ-propargyl-Pidolidone is contacted, to obtain γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides with two (trichloromethyl) carbonic ethers; Make described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides and described γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides be dissolved in N, dinethylformamide, after adding title complex containing Ni as catalyzer, in 20 degrees Celsius of lower stirring reactions, obtain described γ-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer, wherein, the mol ratio of described γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides, described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides and described Ni is 10:3:0.05; Make described-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer be dissolved in trifluoroacetic acid, add after the acetum of HBr, stirring reaction 2 hours under room temperature, obtains described Pidolidone γ-propargyl-Pidolidone ester copolymer; And make described Pidolidone γ-propargyl-Pidolidone ester copolymer aqueous solution and CuSO
4solution, THTA(organic ligand), described in contain azido group single strand dna solution and sodium ascorbate solution mix, by obtained mixing solutions reaction overnight under 50 degrees Celsius, oscillating condition, obtain described polypeptide.Thus, can fast and effeciently prepare polypeptide.
According to embodiments of the invention, the method for preparing described single strand dna is not particularly limited, and those skilled in the art can adopt any known means.According to a concrete example of the present invention, described single strand dna is prepared by solid phase phosphoramidite DNA synthetic method.
According to embodiments of the invention, described double chain DNA molecule obtains by following steps: will mix with tbe buffer liquid according to the described single strand dna of stoichiometric, be heated to 95 degrees Celsius and be incubated after 3 minutes, in 2 hours, be cooled to room temperature, to obtain described double chain DNA molecule.
Described polypeptide is mixed with described double chain DNA molecule
According to embodiments of the invention, described described polypeptide is mixed further and comprised with described double chain DNA molecule: the ratio that is 0.6-1.2:1 according to described double chain DNA molecule with the mol ratio of described single strand dna with described double chain DNA molecule by described polypeptide is mixed, to obtain the mixture of polypeptide and double chain DNA molecule; The ratio that is 0.3%-5% according to massfraction by the mixture of described polypeptide and double chain DNA molecule is dissolved in tbe buffer liquid, to obtain poly-polypeptide-DNA hydrogel.Wherein, the mol ratio of described double chain DNA molecule and described single strand dna is too high or too low, the physical strength of poly-polypeptide-DNA hydrogel is all undesirable, if and the massfraction of the mixture of polypeptide and double chain DNA molecule is too low, cannot form poly-polypeptide-DNA hydrogel, if the massfraction of the mixture of polypeptide and double chain DNA molecule is too high, gel-strength significantly increases.
Embodiment 1: single strand dna (ssDNA) is synthetic
Adopt solid phase phosphoramidite DNA synthetic method, utilize the single strand dna shown in ABI394DNA synthesizer synthetic table 1, specific as follows:
The solid phase carrier that contains particular bases is fixed on to DNA synthesizer, synthetic according to the sequence shown in table 1, then in 60 degrees Celsius with concentrated ammonia solution ammonia solution 3h, remove ammonia by vacuum concentration instrument, the thick product obtaining is passed through to high performance liquid chromatography (elutriant: acetic acid triethylamine buffer solution, 100mM, pH7.0) carry out after purification process, utilize 3% trifluoroacetic acid manually to remove and contain DMT(dimethoxytrityl) blocking group, then by filter deionized water wash, remove DMT, obtain target product, the structure of target product is through MALDI-TOF-MS(ionization time of flight mass spectrometry) checking is correctly.
Table 1
| Single strand dna | Base sequence |
| L1 | 5’-AAGTGTCCAGTT-3’（SEQ?ID?NO:1） |
| 12a | 5’-ACTATGCGTGAATCCGTTAGAACTGGACACTT-3’（SEQ?ID?NO:2） |
| 12b | 5’-CTAACGGATTCACGCATAGTAACTGGACACTT-3’（SEQ?ID?NO:3） |
| 8a | 5’-ACTATGCGTGAATCCGTTAGAACTGGAC-3’（SEQ?ID?NO:4） |
| 8b | 5’-CTAACGGATTCACGCATAGTAACTGGAC-3’（SEQ?ID?NO:5） |
| 8Ma | 5’-ACTATGCGTGAATCCGTTAGAACAGGAC-3’（SEQ?ID?NO:6） |
| 8Mb | 5’-CTAACGGATTCACGCATAGTAACAGGAC-3’（SEQ?ID?NO:7） |
| Ra | 5’-ACTATGCGTAGAATTCAATCCGTAAGAACTGGACACTT-3’（SEQ?ID?NO:8） |
| Rb | 5’-CTTACGGATTGAATTCTACGCATAGTAACTGGACACTT-3’（SEQ?ID?NO:9） |
| Ha | 5’-ACTATGCGTAGGATCCAATCCGTAAGAACTGGACACTT-3’（SEQ?ID?NO:10） |
| Hb | 5’-CTTACGGATTGGATCCTACGCATAGTAACTGGACACTT-3’（SEQ?ID?NO:11） |
Embodiment 2: double chain DNA molecule (dsDNA) is synthetic
According to the following step, take in embodiment 1 obtain ssDNA as raw material, the double chain DNA molecule shown in synthetic table 2, specific as follows:
By mixing with the 1 × tbe buffer liquid that contains 100mM NaCl according to the ssDNA of stoichiometric, then the solution obtaining is heated to 95 degrees Celsius, insulation 3min was then cooled to room temperature in 2 hours, obtained target double chain DNA molecule.Wherein, 12a12b, RaRb and HaHb have the sticky end that length is 12 bases, 8a8b has the sticky end that length is 8 bases, 8Ma8Mb has the sticky end that length is 8 bases, but in its sticky end, contain a mispairing site, RaRb and HaHb have restriction endonuclease sites independently of one another, are respectively Ecor I site (5 '-GAATTC-3 ') and BamH I site (5 '-GGATCC-3 ').
Table 2
| dsDNA | Raw material (ssDNA) |
| 12a12b | 12a，12b |
| 8a8b | 8a，8b |
| 8Ma8Mb | 8Ma，8Mb |
| RaRb | Ra，Rb |
| RaL1 | Ra，L1 |
| RbL1 | Rb，L1 |
| RaRbL1 | Ra，Rb，L1 |
| HaHb | Ha，Hb |
| HaHbL1 | Ha，Hb，L1 |
| HaL1 | Ha，L1 |
| HbL1 | Hb，L1 |
| 12aL1 | 12a，L1 |
| 12bL1 | 12b，L1 |
| 12a12bL1 | 12a，12b，L1 |
| 8a8bL1 | 8a，8b，L1 |
The polyacrylamide gel electrophoresis detection figure of ssDNA and dsDNA molecule is shown in Fig. 1, wherein, and in Fig. 1 (a), swimming lane 1-13 is followed successively by: HaHbL1, HaHb, HaL1, HbL1, Hb, Ha, the L1(that contains azido group is that the 5 ' end of L1 is connected with azido group, and preparation method is shown in embodiment 3), Ra, Rb, RaL1, RbL1, RaRb, RaRbL1; In Fig. 1 (b), swimming lane 1-7 is followed successively by: the L1 that contains azido group, 12a, 12aL1,12b, 12bL1,12a12b, 12a12bL1; In Fig. 1 (c), swimming lane 1-5 is followed successively by: the L1 that contains azido group, 8a, 8b, 8a8b, 8a8bL1.
Embodiment 3: the preparation of polypeptide
The preparation of 5-nitrine-valeric acid
By bromo-5-valeric acid (2.715g, 15mmol) be dissolved in 30ml dimethyl sulfoxide (DMSO) (DMSO), and be heated to 40 degrees Celsius, then add gradually sodiumazide (3.25g, 50mmol), then in 85 degrees Celsius, under agitation condition, reaction is spent the night, the temperature of obtained mixture is cooled to 40 degrees Celsius, add gradually concentrated hydrochloric acid (7mL), then stir and spend the night, by obtained extracted with diethyl ether for reaction product (5 × 80ml), collect ether phase, use successively 10% sodium hydrogen carbonate solution (2 × 100ml) and water (6 × 100ml) washing, then through anhydrous sodium sulfate drying, filter, evaporate to dryness, obtain yellow oil product, it is 5-nitrine-valeric acid.
The analytical results of yellow oil product is as follows:
1H-NMR(CDCl
3,400MHz):δ1.63-1.76(m,4H),2.40(t,J=7.4Hz,2H),3.30(t,J=6.9Hz,2H)；
13C?NMR(CDCl
3,400MHz):21.9,28.0,33.5,51.1,179.6；
ESI-MS?m/z?found:142.34[M]
-。
The preparation of 5-nitrine-valeric acid maloyl imines ester (5-azidopentanoic acid N-hydroxysuccinimide ester)
By 5-nitrine-valeric acid (885mg, 5.64mmol) with maloyl imines (780mg, 6.77mmol) be dissolved in 40ml methylene dichloride, under agitation condition, add 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (2.161g, 11.27mmol), stirred overnight at room temperature, the mixture obtaining is used successively to the hydrochloric acid soln of 1mol/L, water (4 × 100ml), salt solution washs, the organic layer obtaining is through anhydrous sodium sulfate drying, filter, concentrating under reduced pressure, obtain flaxen solution, the flaxen solution obtaining is concentrated by silica gel chromatographic column (elutriant: petrol ether/ethyl acetate=2:1) purifying final vacuum, obtain oily solution, it is 5-nitrine valeric acid maloyl imines ester.
The analytical results of oily solution is as follows:
1H-NMR(CDCl
3,400MHz):δ3.31(t,2H,J=6.8Hz),2.80(br?s,4H),2.63(t,2H,J=7.4Hz),1.77-1.83(m,2H),1.66-1.72(m,2H)；
13C-NMR(CDCl
3,400MHz)δ169.7(2C),168.7,51.4,31.0,28.4,26.1(2C),22.4；
ESI-MS?m/z?found:263.16[M+Na]
+。
The preparation of the single strand dna that contains azido group
Under room temperature, by the aqueous solution (4mM of amido modified L1 synthetic DNA synthesizer, 250 μ L), 5-nitrine-valeric acid maloyl imines ester (100mM), be dissolved in DMF (500 μ L) and phosphate buffer soln (pH8.0,100mM, 750 μ L) mix, by the mixing solutions shaken overnight obtaining, the product obtaining is through HPLC(elutriant: acetic acid triethylamine/acetonitrile=6:4) dense dry after purification process, must contain the L1 sequence of azido group.
The analytical results of the product obtaining is as follows:
Molecular weight: 3965;
Flight time: 3969.
The preparation of γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides (PLG-NCA)
γ-propargyl-Pidolidone (PLG) monomer and two (trichloromethyl) carbonic ethers are in tetrahydrofuran (THF), reaction under 50 degrees Celsius, obtain corresponding a-amino acid N-carbonyl acid anhydrides (NCA), the NCA obtaining is at room temperature viscous liquid, be dissolved in ethyl acetate, the NaHCO that is then 0.5% with the massfraction of frozen water and ice successively
3solution washing, institute's organic phase that obtains is through anhydrous Na
2sO
4dry, evaporation, obtain PLG-NCA(viscous liquid), yield is 20%.
The analytical results of the PLG-NCA obtaining is as follows:
1H-NMR(400MHz,CDCl
3):δ=2.17-2.29(m,2H),2.51(t,1H),2.60-2.71(m,2H),4.41(t,1H),4.72(d,2H),6.27(s,br,1H)。
The preparation of γ-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer (p (BLG-co-PLG))
Be the title complex that nickel, COD and depe form with Ni (COD) depe(, wherein, part COD represents 1,5-hexamethylene two octanes, part depe represents 1,2-bis--(diethyl phosphino-ethane)) be catalyzer, γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides (BLG-NCA) and PLG-NCA prepare p (BLG-co-PLG) by ring-opening polymerization.Specific as follows:
By γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides (BLG-NCA, 0.623g, 2.367mmol) and PLG-NCA(0.15g, 0.71mmol) be dissolved in N, in dinethylformamide (15.5mL), then in obtained solution, add Ni (COD) depe catalyzer (the 162.8 μ L that now join, 20mg/mL), wherein, BLG-NCA:PLG-NCA: catalyzer=10:3:0.05(mol ratio), by mixture obtained above in 20 degrees Celsius, under agitation condition, react, obtain reaction product, the sample that takes a morsel carries out GPC/LLS detection, the number-average molecular weight that obtains product is 52600Da, dispersion coefficient is 1.40, in reaction product obtained above, add ether to carry out recrystallization, then through centrifugal, room temperature vacuum-drying 24h, obtain p (BLG-co-PLG).
The preparation of Pidolidone γ-propargyl-Pidolidone ester copolymer (p (LGA-co-PLG))
P (BLG-co-PLG) is dissolved in trifluoracetic acid, then adding excessive concentration is the acetum of the HBr of 33wt%, by the mixing solutions obtaining stirring reaction 2 hours under room temperature, add ether to carry out recrystallization, by obtained solid through dialysis, freeze-drying, obtain yellow product, i.e. p (LGA-co-PLG), yield: 85%.The quantity warp of repeating unit (LGA and PLG)
1h-NMR(400MHz, D
2o) checking.
The preparation of THTA
Under agitation condition, bromo-3-propyl alcohol (1.75mL, 20.0mmol) is dissolved in 60mL water, and adds wherein NaN
3(2.60g, 40.0mmol), then, under 80 degrees Celsius, stirring reaction 24 hours, is cooled to after room temperature, and by extracted with diethyl ether twice, after salt water washing, organic phase is through anhydrous Na
2sO
4dry, evaporation, obtain 3-alkynyl-1-propyl alcohol crude product, it is dissolved in 40mL acetonitrile immediately, then add tripropyl-2-alkenyl amine (496 μ L, 3.5mmol), 2,6-lutidine (408 μ L, 3.5mmol) and CuBr(45mg, 0.316mmol), the mixture obtaining stirring reaction 64 hours under room temperature, nitrogen atmosphere, the product obtaining is through column chromatography (CH
2cl
2: CH
3oH, 10:1 → CH
2cl
2: CH
3oH, 3:1) carry out purifying, obtain THTA(tri--(3-hydroxypropyl triazole methyl) amine).
The analytical results of the THTA obtaining is as follows:
1H-NMR(400MHz,CD
3OD):δ=7.91(s,3H),4.48(t,6H),3.80(s,6H),3.55(t,6H),2.10(qn,6H)；
13C-NMR(400MHz,CD
3OD):δ=33.0,46.6,47.1,57.5,124.0,143.4；
ESI-MS?m/z?found:435.25[M+H]
+。
The preparation of polypeptide
By p (LGA-co-PLG), (22.1mg) in water-soluble (100 μ L), wiring solution-forming, by above-mentioned solution (25 μ L) and CuSO
4solution (25 μ L, 1M), THAT(150 μ L, L1 solution (the 1000 μ L that 200mM), contain azido group, 2.5mM) and sodium ascorbate solution (100 μ L, 2.5mM) mix, obtained mixing solutions is reacted and spent the night under 50 degrees Celsius, oscillating condition, the solution obtaining filters through filter (Millipore ' s Microcon YM-30), remove unreacted DNA and other micromolecular compounds, the product obtaining is reduced pressure dense dry, obtain p (LGA-co-PLG)-g-DNA, i.e. polypeptide.
Embodiment 4: the preparation of poly-polypeptide-DNA hydrogel
According to shown in table 3, the polypeptide that the dsDNA that embodiment 2 is prepared and embodiment 3 prepare mixes with the predetermined molar ratio mol ratio of single strand dna (double chain DNA molecule with), then with contain 200mM NaCl, pH be 8.0 or pH be 5.0 tbe buffer liquid is mixed with mixing solutions, the mixing solutions obtaining stirs the poly-polypeptide-DNA hydrogel that becomes transparent thickness through rifle head, to obtain final product.
Table 3
| Hydrogel | dsDNA | Mol ratio | Total mass mark | pH |
| Gel1 | 12a12b | 0.6：1 | 5% | 8.0 |
| Gel2 | 12a12b | 0.8：1 | 5% | 8.0 |
| Gel3 | 12a12b | 1.0：1 | 5% | 8.0 |
| Gel4 | 12a12b | 1.2：1 | 5% | 8.0 |
| Gel5 | 12a12b | 0.8：1 | 0.1% | 8.0 |
| Gel6 | 12a12b | 0.8：1 | 0.3% | 8.0 |
| Gel7 | 12a12b | 0.8：1 | 0.5% | 8.0 |
| Gel8 | 12a12b | 0.8：1 | 1% | 8.0 |
| Gel9 | 12a12b | 0.8：1 | 2% | 8.0 |
| Gel10 | 12a12b | 0.8：1 | 3% | 8.0 |
| Gel11 | 12a12b | 0.8：1 | 4% | 8.0 |
| Gel12 | 8a8b | 0.8：1 | 5% | 8.0 |
| Gel13 | 8Ma8Mb | 0.8：1 | 5% | 8.0 |
| Gel14 | RaRb | 0.8：1 | 5% | 8.0 |
| Gel15 | HaHb | 0.8：1 | 5% | 8.0 |
| Gel16 | 12a12b | 0.8：1 | 3% | 5.0 |
Note: total mass mark refers in the mixing solutions of dsDNA, polypeptide and tbe buffer liquid, the total mass mark of dsDNA and polypeptide; PH refers to tbe buffer liquid pH value.
The impact on poly-polypeptide-DNA hydrogel of embodiment 5:dsDNA and ssDNA proportioning
The Gel1-4 that embodiment 4 is prepared, at 25 degrees Celsius, carries out frequency sweeping rheology under 1% strained condition, frequency sweeping scope is 0.05Hz-100Hz, and test-results as shown in Figure 2.Wherein, 0.6/1 corresponding Gel1,0.8/1 corresponding Gel2,1.0/1 corresponding Gel3,1.2/1 corresponding Gel4, G ' represents to shear storage modulus, G ' ' represents to shear out-of-phase modulus, and in whole sweep limits, G ' shows to have formed poly-polypeptide-DNA hydrogel apparently higher than G ' '.
From the result of Fig. 2, when the mol ratio of double chain DNA molecule and single strand dna (being the sticky end of polypeptide) is 0.8:1 and 1.0:1, the physical strength of poly-polypeptide-DNA hydrogel is best, this is because can be substantially completely crosslinked between double chain DNA molecule and polypeptide, and when other ratios, the physical strength of poly-polypeptide-DNA hydrogel is poor, be because or double chain DNA molecule excessive, or polypeptide is excessive, between the two can not be completely crosslinked.Thus, show by regulating the mol ratio of double chain DNA molecule and single strand dna (being the sticky end of polypeptide), to regulate the physical strength of poly-polypeptide-DNA hydrogel.
Embodiment 6: the impact of total mass mark on poly-polypeptide-DNA hydrogel
The Gel4-11 that embodiment 4 is prepared carries out rheological property detection, and detected result as shown in Figure 3.
From the result of Fig. 3, total mass mark can remarkably influenced the physical strength of poly-polypeptide-DNA hydrogel, as can be seen from Figure 3, along with the rising of total mass mark, the physical strength of poly-polypeptide-DNA hydrogel strengthens gradually, this explanation adjusting total mass mark can effectively regulate the physical strength of poly-polypeptide-DNA hydrogel, can be according to different applied environment, prepare poly-polypeptide-DNA hydrogel with different physical strengths.
The impact of the length of embodiment 7:dsDNA sticky end on poly-polypeptide-DNA hydrogel
Gel2, Gel12 and the Gel13(photo implementing to prepare in 4 are shown in to Fig. 4) under 25 degrees Celsius, the condition of 1% strain, 1Hz, carry out shear rheology experiment, the results are shown in Figure 5.
As seen from Figure 4, Gel13(bp8M) be still flowing fluid, form poly-polypeptide-DNA hydrogel, and Gel2(bp12) and Gel12(bp8) all form transparent poly-polypeptide-DNA hydrogel.Contrast Gel12 and Gel13 are known, and the length of dsDNA sticky end is identical, if there is mispairing site in sticky end, are unfavorable for the formation of poly-polypeptide-DNA hydrogel.From the result of Fig. 5, G '/G ' ' value of Gel2, Gel12 and Gel13 is respectively 15.9,5.4 and 0.3, and G '/G ' ' value is higher, the physical strength of poly-polypeptide-DNA hydrogel is stronger, thus, show that the length of dsDNA sticky end is longer, the physical strength of poly-polypeptide-DNA hydrogel is better, thereby by adjusting the length of double chain DNA molecule sticky end, can obtain poly-polypeptide-DNA hydrogel with different physical strengths.
Embodiment 8: the impact of the secondary structure of polypeptide backbone on poly-polypeptide-DNA hydrogel
The Gel10 preparing in embodiment 4 and Gel16 are carried out respectively to circular dichroism spectrum detection and time scan rheology, and result is shown in respectively Fig. 6 and Fig. 7, wherein, and the corresponding Gel10 of pH8.0, the corresponding Gel16 of pH5.0.
From the result of Fig. 6, under the condition that is 5.0 at the pH of tbe buffer liquid, due to the protonation of carboxyl, polypeptide backbone is alpha helical conformation, and under the condition that is 8.0 at the pH of tbe buffer liquid, due to the deprotonation of carboxyl, polypeptide backbone is random-coil conformation.As seen from Figure 7, under two kinds of pH conditions, the G ' of poly-polypeptide-DNA hydrogel keeps identical substantially, but the G ' ' of Gel16 is apparently higher than the G ' ' of Gel10, show that poly-polypeptide-DNA hydrogel has good physical strength at sour environment, and then the secondary structure of explanation polypeptide backbone can affect the physical strength of poly-polypeptide-DNA hydrogel.
Embodiment 9: the thermo-responsive of poly-polypeptide-DNA hydrogel
The Gel2 preparing in embodiment 4, between 1% strain, 1Hz, 25 degrees Celsius-60 degrees Celsius, is carried out to rheology testing, the results are shown in Figure 8.
As seen from Figure 8, in the time of 25 degrees Celsius, G ' is apparently higher than G ' ', this is the characteristic feature that forms hydrogel, and in the time that temperature is increased to 60 degrees Celsius by 25 degrees Celsius, G ' is significantly lower than G ' ', this explanation hydrogel has become solution state, and be again down to 25 degrees Celsius and while keeping 15 minutes in temperature, solution can form again hydrogel again.And the conversion between this hydrogel and solution state can circulate repeatedly.Thus, show that poly-polypeptide-DNA hydrogel of the present invention has good, reversible thermo-responsive.
Embodiment 10: the enzyme responsiveness of poly-polypeptide-DNA hydrogel
According to the Gel2, the Gel14 that prepare in following steps detection embodiment 4 and the degradation property of Gel15, specific as follows:
Get 8 EP pipes, numbering a-h group, wherein organizes the Gel2 that a and the every pipe of group b add phosphate buffer soln (10 μ L, pH7.8) and 15 μ L, and the every pipe of group c-e adds and contains Tris-HCl(50mM, pH7.5), NaCl(100mM), MgCl
2(10mM), the solution of dithiothreitol (DTT) (1mM) and the Gel14 of 15 μ L, the every pipe of group f-h adds and contains Tris-HCl (20mM, pH8.5), KCl(100mM), MgCl
2(10mM), the solution of dithiothreitol (DTT) (1mM) and the Gel15 of 15 μ L, then according to shown in table 4, add respectively proteolytic enzyme Glu-C, EcoR I or BamH I then react 8 groups of EP pipes under room temperature condition, observe poly-polypeptide-DNA hydrogel in reaction process.Fig. 9 is shown in by the photo of the poly-polypeptide-DNA hydrogel of each group.
Table 4
As seen from Figure 9, after reaction for some time, add proteolytic enzyme Glu-C, poly-polypeptide-DNA hydrogel of EcoR I or BamH I is converted into solution morphology, and do not add poly-polypeptide-DNA hydrogel of enzyme still to keep the form of hydrogel, this shows, contain suitable restriction endonuclease sites by design double chain DNA molecule, poly-polypeptide-DNA hydrogel of the present invention can have special enzyme responsiveness, be that poly-polypeptide-DNA hydrogel of the present invention can be degraded under the effect of specific enzymes, this explanation poly-polypeptide-DNA hydrogel of the present invention can be used as pharmaceutical carrier.
Embodiment 11: the biocompatibility of poly-polypeptide-DNA hydrogel
The polypeptide that is 0.1-10mg/ml by concentration and concentration are that the double chain DNA molecule of 0.01-2.0mM adds respectively in the DMEM cell culture medium that contains NIH3T3 cell, then being placed in 37 degrees Celsius, the incubator of 5% carbonic acid gas cultivates 24 hours, through cultured cells after FDA/PI dyeing, calculate cell survival rate, the results are shown in Figure 10.
As seen from Figure 10, after cultivating in the substratum of the polypeptide that contains different concns and double chain DNA molecule, cell survival rate is all in 99 ± 0.5% left and right, show that polypeptide of the present invention and double chain DNA molecule do not have toxicity, there is good biocompatibility, and then poly-polypeptide-DNA hydrogel of the present invention prepared therefrom can be used as cell culture vector.
Embodiment 12:3D cell cultures
It is 5 × 10 that NIH3T3 cell is made into cell density with DMEM substratum
6the cell suspension of individual cell/ml, by the above-mentioned cell suspension of 4 μ L and the polypeptide solution (15wt% that contains 100mM sodium-chlor, 8 μ L) mix, then under slow agitation condition, add double chain DNA molecule solution (5mM, 8 μ L), make it to form the celliferous poly-polypeptide-DNA hydrogel of bag, above the celliferous poly-polypeptide-DNA hydrogel of bag, add 200 μ L cell culture mediums again, be placed in afterwards 37 degrees Celsius, in the incubator of 5% carbonic acid gas, cultivate 24 hours, then after FDA/PI dyeing, by the quantity of confocal fluorescent microscopic examination dead cell and viable cell, confocal fluorescent microphotograph is shown in Figure 11.
Can be seen by Figure 11, utilize poly-polypeptide-DNA hydrogel 3D to cultivate 24 hours, cell growth state is good, almost there is no dead cell, thus, shows that poly-polypeptide-DNA hydrogel biocompatibility of the present invention is good.
In the description of this specification sheets, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or example in conjunction with specific features, structure, material or the feature of this embodiment or example description.In this manual, to the schematic statement of above-mentioned term not must for be identical embodiment or example.And, specific features, structure, material or the feature of description can one or more embodiment in office or example in suitable mode combination.In addition,, not conflicting in the situation that, those skilled in the art can carry out combination and combination by the feature of the different embodiment that describe in this specification sheets or example and different embodiment or example.
Although illustrated and described embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, and those of ordinary skill in the art can change above-described embodiment within the scope of the invention, modification, replacement and modification.
Claims (10)
1. poly-polypeptide-DNA hydrogel, is characterized in that, comprising:
Polypeptide, on described polypeptide, covalent attachment has single strand dna;
Double chain DNA molecule, wherein, described double chain DNA molecule has two sticky ends;
Wherein, described sticky end and described single strand dna complementation;
Described polypeptide forms crosslinking structure by the sticky end of described double chain DNA molecule and the complementation of described single strand dna.
2. poly-polypeptide-DNA hydrogel according to claim 1, is characterized in that, two sticky ends of described double chain DNA molecule have identical nucleotide sequence,
Optionally, the length of two sticky ends of described double chain DNA molecule is identical with the length of described single strand dna,
Optionally, the length of two of described double chain DNA molecule sticky ends is respectively 8～12nt independently.
3. poly-polypeptide-DNA hydrogel according to claim 1, is characterized in that, described polypeptide has the aminoacid sequence as shown in SEQ ID NO:12,
Optionally, described single strand dna has the nucleotide sequence as shown in SEQ ID NO:1,
Optionally, described double chain DNA molecule has the first chain and the second chain, and wherein, described the first chain and described the second chain meet one of following condition:
Described the first chain has the sequence shown in SEQ ID NO:2, and described the second chain has the sequence shown in SEQ ID NO:3;
Described the first chain has the sequence shown in SEQ ID NO:4, and described the second chain has the sequence shown in SEQ ID NO:5;
Described the first chain has the sequence shown in SEQ ID NO:6, and described the second chain has the sequence shown in SEQ ID NO:7;
Described the first chain has the sequence shown in SEQ ID NO:8, and described the second chain has the sequence shown in SEQ ID NO:9;
Or described the first chain has the sequence shown in SEQ ID NO:10, described the second chain has the sequence shown in SEQ ID NO:11.
4. poly-polypeptide-DNA hydrogel according to claim 1, is characterized in that, is combined with multiple described single strand dnas on each described polypeptide,
Preferably, on each described polypeptide, be combined with at least 4 described single strand dnas.
5. poly-polypeptide-DNA hydrogel according to claim 1, is characterized in that, the mol ratio of described double chain DNA molecule and described single strand dna is 0.6～1.2:1.
6. poly-polypeptide-DNA hydrogel according to claim 1, is characterized in that, described polypeptide has the structure shown in following chemical formula:
Wherein, ssDNA represents described single strand dna, and * represents natural chiral amino acid.
7. a method of preparing poly-polypeptide-DNA hydrogel described in claim 1～6 any one, is characterized in that, comprising:
Polypeptide and double chain DNA molecule are provided, and wherein, on described polypeptide, covalent attachment has at least one single strand dna, and described double chain DNA molecule has two sticky ends, and described sticky end and described single strand dna complementation; And
Described polypeptide is mixed with described double chain DNA molecule.
8. method according to claim 7, is characterized in that, described in provide polypeptide further to comprise:
The bromo-valeric acid of 5-is contacted, to obtain 5-nitrine-valeric acid with sodiumazide;
Described 5-nitrine-valeric acid is contacted, to obtain 5-nitrine valeric acid maloyl imines ester with maloyl imines;
Described 5-nitrine valeric acid maloyl imines ester is contacted, to obtain the single strand dna that contains azido group with amido modified single strand dna;
γ-propargyl-Pidolidone and two-(trichloromethyl) carbonic ether is contacted, to obtain γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides;
Described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides is contacted, to obtain γ-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer with γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides;
Described-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer is contacted, to obtain Pidolidone γ-propargyl-Pidolidone ester copolymer with HBr with trifluoroacetic acid;
The single strand dna that contains azido group described in making contacts with described Pidolidone γ-propargyl-Pidolidone ester copolymer, to obtain described polypeptide,
Preferably, further comprise:
Bromo-described 5-valeric acid is dissolved in to dimethyl sulfoxide (DMSO), is heated to, after 40 degrees Celsius, add described sodiumazide, in 85 degrees Celsius, stir lower reaction and spend the night, obtained reaction solution is cooled to 40 degrees Celsius, add concentrated hydrochloric acid, stirring is spent the night, and through extracted with diethyl ether, obtains described 5-nitrine-valeric acid;
Described 5-nitrine valeric acid and described maloyl imines are dissolved in to methylene dichloride, under stirring, add 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide, stirred overnight at room temperature, obtains described 5-nitrine valeric acid maloyl imines ester;
Described 5-nitrine valeric acid maloyl imines ester is dissolved in to DMF, obtained solution is mixed with the aqueous solution and the phosphate buffer soln of described amido modified single strand dna, to obtain the single strand dna that contains azido group;
γ-propargyl-Pidolidone is contacted, to obtain γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides with two (trichloromethyl) carbonic ethers;
Make described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides and described γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides be dissolved in N, dinethylformamide, after adding title complex containing Ni as catalyzer, in 20 degrees Celsius of lower stirring reactions, obtain described γ-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer, wherein, the mol ratio of described γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides, described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides and described Ni is 10:3:0.05;
Make described-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer be dissolved in trifluoroacetic acid, add after the acetum of HBr, stirring reaction 2 hours under room temperature, obtains described Pidolidone γ-propargyl-Pidolidone ester copolymer;
Make described Pidolidone γ-propargyl-Pidolidone ester copolymer aqueous solution and CuSO
4solution, THTA, described in contain azido group single strand dna solution and sodium ascorbate solution mix, obtained mixing solutions is reacted and is spent the night under 50 degrees Celsius, oscillating condition, obtain described polypeptide.
9. method according to claim 7, is characterized in that, described single strand dna is synthetic by solid phase phosphoramidite DNA synthetic method,
Optionally, described double chain DNA molecule obtains by following steps:
To mix with tbe buffer liquid according to the described single strand dna of stoichiometric, be heated to 95 degrees Celsius and be incubated after 3 minutes, in 2 hours, be cooled to room temperature, to obtain described double chain DNA molecule.
10. method according to claim 7, is characterized in that, described described polypeptide is mixed further and comprised with described double chain DNA molecule:
The ratio that is 0.6-1.2:1 according to described double chain DNA molecule with the mol ratio of described single strand dna with described double chain DNA molecule by described polypeptide is mixed, to obtain the mixture of polypeptide and double chain DNA molecule;
The ratio that is 0.3%-5% according to massfraction by the mixture of described polypeptide and double chain DNA molecule is dissolved in tbe buffer liquid, to obtain poly-polypeptide-DNA hydrogel.
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