CN103910893B - Poly-polypeptide-DNA hydrogel and preparation method thereof - Google Patents
Poly-polypeptide-DNA hydrogel and preparation method thereof Download PDFInfo
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Abstract
The invention provides poly-polypeptide-DNA hydrogel and preparation method thereof, wherein, 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 the complementation of described single strand dna; Described polypeptide becomes crosslinking structure by the sticky end of described double chain DNA molecule with the complementary of described single strand dna.Poly-polypeptid DNA hydrogel physical strength of the present invention is adjustable, and biocompatibility is good, has reversible thermo-responsive and specific enzyme responsiveness.
Description
Technical field
The invention belongs to biomedical sector, relate to poly-polypeptide-DNA hydrogel and preparation method thereof particularly.
Background technology
Hydrogel is that a class contains hydrophilic radical, can be water-swellable and keep the polymkeric substance of its Complete three-dimensional network structure.It is significantly swelling that it can absorb a large amount of moisture in water, and can continue to keep its original structure after significantly swelling and do not dissolved, it can the subtle change of perception external stimulus, as temperature, pH value, ionic strength, electric field, magnetic field etc., and can stimulate to external world and produces response.Because the structure of hydrogel provides bioenvironmental extracellular matrix quite similar with for cell, thus, hydrogel has wide practical use at biomedical sector.But preparation has the hydrogel of good biocompatibility and suitable physical strength, is still a major challenge of field of tissue engineering technology.
Thus, still need deeply about the research of hydrogel at present.
Summary of the invention
The present invention is intended to solve one of technical problem in correlation technique to a certain extent.For this reason, it is good that one object of the present invention is to propose a kind of biocompatibility, adjustable mechanical intensity, and the poly-polypeptide-DNA hydrogel with 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 the complementation of described single strand dna, described polypeptide becomes crosslinking structure by the sticky end of described double chain DNA molecule with the complementary of described single strand dna.Contriver finds, 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 separately 8 ~ 12nt.Thus, be conducive to the formation of poly-polypeptide-DNA hydrogel, and the physical strength of the poly-polypeptide-DNA hydrogel formed is good, in addition, by regulating the length of two sticky ends of double chain DNA molecule, the poly-polypeptide-DNA hydrogel with different physical strength can be formed.
According to embodiments of the invention, described polypeptide has the aminoacid sequence as shown in SEQIDNO:12.
According to embodiments of the invention, described single strand dna has the nucleotide sequence as shown in SEQIDNO:1.
According to embodiments of the invention, described double chain DNA molecule has the first chain and the second chain, wherein, described first chain and described second chain meet one of following condition: described first chain has the sequence shown in SEQIDNO:2, and described second chain has the sequence shown in SEQIDNO:3; Described first chain has the sequence shown in SEQIDNO:4, and described second chain has the sequence shown in SEQIDNO:5; Described first chain has the sequence shown in SEQIDNO:6, and described second chain has the sequence shown in SEQIDNO:7; Described first chain has the sequence shown in SEQIDNO:8, and described second chain has the sequence shown in SEQIDNO:9; Described first chain has the sequence shown in SEQIDNO:10, and described second chain has the sequence shown in SEQIDNO:11.
According to embodiments of the invention, each described polypeptide is combined with multiple single strand dna.Thus, the stability and the physical strength that improve poly-polypeptide-DNA hydrogel is conducive to.
According to embodiments of the invention, each described polypeptide is combined with at least 4 described single strand dnas.Thus, stability and the physical strength of gathering 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 are formed 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 preparing poly-polypeptide-DNA hydrogel noted earlier.According to embodiments of the invention, the method comprises: provide polypeptide and double chain DNA molecule, 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 the complementation of described single strand dna; And described polypeptide is mixed with described double chain DNA molecule.Contriver finds, utilizes the method for the present invention, fast and effeciently can prepare foregoing poly-polypeptide-DNA hydrogel, and method is simple, processing ease.
According to embodiments of the invention, described in provide polypeptide to comprise further: 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 hydroxysuccinimide ester with hydroxysuccinimide; Described 5-nitrine-valeric acid hydroxysuccinimide ester is contacted, to obtain the single strand dna containing 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 ether; 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 with HBr with trifluoroacetic acid, to obtain Pidolidone γ-propargyl-Pidolidone ester copolymer; The described single strand dna containing azido group is contacted, to obtain described polypeptide with described Pidolidone γ-propargyl-Pidolidone ester copolymer.
It should be noted that, term " contact " 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, such as, can be mixed under suitable condition by two kinds of reactants.As required, can under agitation, mix needing the reactant carrying out contacting.
According to embodiments of the invention, comprise further: bromo-for described 5-valeric acid is dissolved in dimethyl sulfoxide (DMSO), after being heated to 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 hydroxysuccinimide are dissolved in methylene dichloride, under stirring, add 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), stirred overnight at room temperature, obtains described 5-nitrine-valeric acid hydroxysuccinimide ester; Described 5-nitrine-valeric acid hydroxysuccinimide ester is dissolved in DMF, the aqueous solution of obtained solution and described amido modified single strand dna and phosphate buffer soln are mixed, to obtain the single strand dna containing azido group; γ-propargyl-Pidolidone is contacted, to obtain γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides with two (trichloromethyl) carbonic ether; Described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides and described γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides is made to be dissolved in N, dinethylformamide, add title complex containing Ni as after 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, after adding the acetum of HBr, react 2 hours in stirred at ambient temperature, obtain 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 single strand dna solution containing azido group and sodium ascorbate solution mixing, by obtained mixing solutions in 50 degrees Celsius, react under oscillating condition and spend the night, 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 is obtained 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 after being incubated 3 minutes, room temperature is cooled to, to obtain described double chain DNA molecule in 2 hours.
According to embodiments of the invention, described mixing with described double chain DNA molecule by described polypeptide comprises further: mixed, to obtain the mixture of polypeptide and double chain DNA molecule according to the ratio that described double chain DNA molecule and the mol ratio of described single strand dna are 0.6-1.2:1 with described double chain DNA molecule by described polypeptide; The mixture of described polypeptide and double chain DNA molecule is dissolved in TBE(Tris-boric acid-EDTA according to the ratio that massfraction is 0.3%-5%) in damping fluid, to obtain poly-polypeptide-DNA hydrogel.
Accompanying drawing explanation
Fig. 1 shows according to one embodiment of present invention, and the polyacrylamide gel electrophoresis of ssDNA molecule and dsDNA molecule detects figure;
Fig. 2 shows according to one embodiment of present invention, the frequency sweeping rheology result figure of Gel1-4;
Fig. 3 shows according to one embodiment of present invention, the rheological property detected result figure of Gel4-11;
Fig. 4 shows according to one embodiment of present invention, the photo of Gel2, Gel12 and Gel13;
Fig. 5 shows according to one embodiment of present invention, the shear rheology experimental result picture of Gel2, Gel12 and Gel13;
Fig. 6 shows according to one embodiment of present invention, and the circular dichroism spectrum of Gel10 and Gel16 detects figure;
Fig. 7 shows according to one embodiment of present invention, the time scan rheology result figure of Gel10 and Gel16;
Fig. 8 shows according to one embodiment of present invention, and the rheological property of Gel2 between 25 degrees Celsius and 60 degrees Celsius detects figure;
Fig. 9 shows according to one embodiment of present invention, the photo of poly-polypeptide-DNA hydrogel;
Figure 10 shows according to one embodiment of present invention, 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 shows according to one embodiment of present invention, after utilizing poly-polypeptide-DNA hydrogel 3D to cultivate 24h, and the confocal fluorescent microphotograph of NIH3T3 cell, wherein,
The said three-dimensional body phase material that Figure 11 (a) is formed 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 shows according to one embodiment of present invention, the synthetic route chart of polypeptide.
Embodiment
Be described below in detail embodiments of the invention, it should be noted that embodiment described below is exemplary, only for explaining the present invention, and can not limitation of the present invention be interpreted as.In addition, if do not clearly not stated, adopted in the following embodiments all reagent are, and market can be buied, or can according to herein or known method synthesis, for the reaction conditions do not listed, 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 the complementation of described single strand dna, described polypeptide becomes crosslinking structure by the sticky end of described double chain DNA molecule with the complementary of described single strand dna.Contriver finds, 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 separately 8 ~ 12nt.Thus, be conducive to the formation of poly-polypeptide-DNA hydrogel, and the physical strength of the poly-polypeptide-DNA hydrogel formed is good, in addition, by regulating the length of two sticky ends of double chain DNA molecule, the poly-polypeptide-DNA hydrogel with different physical strength can be formed.
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 SEQIDNO:12: (L-Glu)
m(L-Glu-g-DNA)
n(SEQIDNO:12), wherein, m=240, n=20.
According to embodiments of the invention, described single strand dna has the nucleotide sequence as shown in SEQIDNO:2.
According to embodiments of the invention, described double chain DNA molecule has the first chain and the second chain, wherein, described first chain and described second chain meet one of following condition: described first chain has the sequence shown in SEQIDNO:2, and described second chain has the sequence shown in SEQIDNO:3; Described first chain has the sequence shown in SEQIDNO:4, and described second chain has the sequence shown in SEQIDNO:5; Described first chain has the sequence shown in SEQIDNO:6, and described second chain has the sequence shown in SEQIDNO:7; Described first chain has the sequence shown in SEQIDNO:8, and described second chain has the sequence shown in SEQIDNO:9; Or described first chain has the sequence shown in SEQIDNO:10, described second chain has the sequence shown in SEQIDNO:11.
According to embodiments of the invention, each described polypeptide is combined with multiple single strand dna.Thus, the stability and the physical strength that improve poly-polypeptide-DNA hydrogel is conducive to.In a concrete example of the present invention, each described polypeptide is combined with at least 4 described single strand dnas.Thus, stability and the physical strength of gathering 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 are formed 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 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
There is provided polypeptide and double chain DNA molecule, 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 the complementation of described single strand dna.
According to embodiments of the invention, with reference to Figure 12, described in provide polypeptide to comprise further: the bromo-valeric acid of 5-is contacted, to obtain 5-nitrine-valeric acid (compound 1) with sodiumazide; Described 5-nitrine-valeric acid is contacted, to obtain 5-nitrine-valeric acid hydroxysuccinimide ester (compound 2) with hydroxysuccinimide; Described 5-nitrine-valeric acid hydroxysuccinimide ester is contacted, to obtain the single strand dna (compound 3) containing 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 ether; 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 with HBr with trifluoroacetic acid, to obtain Pidolidone γ-propargyl-Pidolidone ester copolymer (compound 6); The described single strand dna containing azido group is contacted 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 comprises further: bromo-for described 5-valeric acid is dissolved in dimethyl sulfoxide (DMSO), after being heated to 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 hydroxysuccinimide are dissolved in methylene dichloride, under stirring, add 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), stirred overnight at room temperature, obtains described 5-nitrine-valeric acid hydroxysuccinimide ester; Described 5-nitrine-valeric acid hydroxysuccinimide ester is dissolved in DMF, the aqueous solution of obtained solution and described amido modified single strand dna and phosphate buffer soln are mixed, to obtain the single strand dna containing azido group; γ-propargyl-Pidolidone is contacted, to obtain γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides with two (trichloromethyl) carbonic ether; Described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides and described γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides is made to be dissolved in N, dinethylformamide, add title complex containing Ni as after 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, after adding the acetum of HBr, react 2 hours in stirred at ambient temperature, obtain 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 single strand dna solution containing azido group and sodium ascorbate solution mixing, by obtained mixing solutions in 50 degrees Celsius, reaction overnight under oscillating condition, obtain described polypeptide.Thereby, it is possible to fast and effeciently prepare polypeptide.
According to embodiments of the invention, the method 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 is obtained by following steps: mixed with tbe buffer liquid by the described single strand dna according to stoichiometric, be heated to 95 degrees Celsius and after being incubated 3 minutes, room temperature is cooled to, to obtain described double chain DNA molecule in 2 hours.
Described polypeptide is mixed with described double chain DNA molecule
According to embodiments of the invention, described mixing with described double chain DNA molecule by described polypeptide comprises further: mixed, to obtain the mixture of polypeptide and double chain DNA molecule according to the ratio that described double chain DNA molecule and the mol ratio of described single strand dna are 0.6-1.2:1 with described double chain DNA molecule by described polypeptide; The mixture of described polypeptide and double chain DNA molecule is dissolved in tbe buffer liquid according to the ratio that massfraction is 0.3%-5%, 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, poly-polypeptide-DNA hydrogel cannot be formed, 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) synthesizes
Adopt solid phase phosphoramidite DNA synthetic method, utilize the single strand dna shown in ABI394DNA synthesizer synthetic table 1, specific as follows:
Solid phase carrier containing particular bases is fixed on DNA synthesizer, according to the sequent synthesis shown in table 1, then in 60 degrees Celsius with concentrated ammonia solution ammonia solution 3h, ammonia is removed by vacuum concentration instrument, by the thick product that obtains by high performance liquid chromatography (elutriant: acetic acid triethylamine buffer solution, 100mM, pH7.0) after carrying out purification process, utilize the trifluoroacetic acid of 3% manually to remove containing DMT(dimethoxytrityl) blocking group, then by filter deionized water wash, removing 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) synthesizes
According to the following step, with the ssDNA obtained in embodiment 1 for raw material, the double chain DNA molecule shown in synthetic table 2, specific as follows:
Mixed with the 1 × tbe buffer liquid containing 100mMNaCl by ssDNA according to stoichiometric, then the solution obtained is heated to 95 degrees Celsius, insulation 3min, was then cooled to room temperature, obtains target double chain DNA molecule in 2 hours.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 containing a mismatch site in its sticky end, RaRb and HaHb has restriction endonuclease sites independently of one another, is respectively EcorI site (5 '-GAATTC-3 ') and BamHI 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, in Fig. 1 (a), swimming lane 1-13 is followed successively by: HaHbL1, HaHb, HaL1, HbL1, Hb, Ha, 5 ' the end of L1(and L1 containing azido group 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 containing azido group, 12a, 12aL1,12b, 12bL1,12a12b, 12a12bL1; In Fig. 1 (c), swimming lane 1-5 is followed successively by: the L1 containing azido group, 8a, 8b, 8a8b, 8a8bL1.
Embodiment 3: the preparation of polypeptide
The preparation of 5-nitrine-valeric acid
By bromo-for 5-valeric acid (2.715g, 15mmol) be dissolved in 30ml dimethyl sulfoxide (DMSO) (DMSO), and be heated to 40 degrees Celsius, then sodiumazide (3.25g is added gradually, 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 concentrated hydrochloric acid (7mL) gradually, then stir and spend the night, by obtained reaction product with extracted with diethyl ether (5 × 80ml), collect ether phase, use sodium hydrogen carbonate solution (2 × 100ml) and water (6 × 100ml) washing of 10% successively, then through anhydrous sodium sulfate drying, filter, evaporate to dryness, obtain yellow oil product, i.e. 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)；
13CNMR(CDCl
3,400MHz):21.9,28.0,33.5,51.1,179.6；
ESI-MSm/zfound:142.34[M]
-。
The preparation of 5-nitrine-valeric acid hydroxysuccinimide ester (5-azidopentanoicacidN-hydroxysuccinimideester)
By 5-nitrine-valeric acid (885mg, 5.64mmol) with hydroxysuccinimide (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 obtained is used successively the hydrochloric acid soln of 1mol/L, water (4 × 100ml), salt solution washs, the organic layer obtained is through anhydrous sodium sulfate drying, filter, concentrating under reduced pressure, obtain flaxen solution, the flaxen solution obtained is concentrated by silica gel chromatographic column (elutriant: petrol ether/ethyl acetate=2:1) purifying final vacuum, obtain oily solution, i.e. 5-nitrine valeric acid hydroxysuccinimide ester.
The analytical results of oily solution is as follows:
1H-NMR(CDCl
3,400MHz):δ3.31(t,2H,J=6.8Hz),2.80(brs,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-MSm/zfound:263.16[M+Na]
+。
The preparation of the single strand dna containing azido group
Under room temperature, by the aqueous solution (4mM of the amido modified L1 that DNA synthesizer synthesizes, 250 μ L), 5-nitrine-valeric acid hydroxysuccinimide ester (100mM), be dissolved in DMF (500 μ L) and phosphate buffer soln (pH8.0,100mM, 750 μ L) mixing, by the mixing solutions shaken overnight obtained, the product obtained is through HPLC(elutriant: triethylamine acetate/acetonitrile=6:4) dense dry after purification process, obtain the L1 sequence containing azido group.
The analytical results of the product obtained 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 ether are in tetrahydrofuran (THF), react under 50 degrees Celsius, obtain corresponding a-amino acid N-carbonyl acid anhydrides (NCA), the NCA obtained is at room temperature viscous liquid, being dissolved in ethyl acetate, is then the NaHCO of 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, obtains PLG-NCA(viscous liquid), yield is 20%.
The analytical results of the PLG-NCA obtained 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))
With the title complex that Ni (COD) depe(and nickel, COD and depe are formed, wherein, part COD represents 1,5-hexamethylene two octane, part depe represents 1,2-bis--(diethylphosphino ethane)) be catalyzer, γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides (BLG-NCA) and PLG-NCA prepares 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 now joined, 20mg/mL), wherein, BLG-NCA:PLG-NCA: catalyzer=10:3:0.05(mol ratio), by mixture obtained above in 20 degrees Celsius, react under agitation condition, obtain reaction product, the sample that takes a morsel carries out GPC/LLS detection, the number-average molecular weight obtaining product is 52600Da, dispersion coefficient is 1.40, in reaction product obtained above, add ether carry out recrystallization, then through centrifugal, the dry 24h of room temperature in vacuo, 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 the acetum that excessive concentration is the HBr of 33wt% is added, the mixing solutions obtained is reacted 2 hours in stirred at ambient temperature, add ether and 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) verify.
The preparation of THTA
Under agitation condition, bromo-for 3-propyl alcohol (1.75mL, 20.0mmol) is dissolved in 60mL water, and adds NaN wherein
3(2.60g, 40.0mmol), then under 80 degrees Celsius, stirring reaction 24 hours, after being cooled to room temperature, 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 immediately in 40mL acetonitrile, then tripropyl-2-alkenyl amine (496 μ L, 3.5mmol), 2,6-lutidine (408 μ L are added, 3.5mmol) and CuBr(45mg, 0.316mmol), the mixture obtained stirring reaction 64 hours under room temperature, nitrogen atmosphere, the product obtained 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 obtained 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-MSm/zfound:435.25[M+H]
+。
The preparation of polypeptide
By in water-soluble to p (LGA-co-PLG) (22.1mg) (100 μ L), wiring solution-forming, by above-mentioned solution (25 μ L) and CuSO
4solution (25 μ L, 1M), THAT(150 μ L, 200mM), L1 solution (the 1000 μ L containing azido group, 2.5mM) with sodium ascorbate solution (100 μ L, 2.5mM) mix, by obtained mixing solutions in 50 degrees Celsius, react under oscillating condition and spend the night, the solution obtained filters through filter (Millipore ' sMicroconYM-30), remove unreacted DNA and other micromolecular compounds, the product obtained is carried out decompression 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 prepare embodiment 2 and embodiment 3 prepare mixes with the predetermined molar ratio mol ratio of single strand dna (double chain DNA molecule with), then with containing 200mMNaCl, pH be 8.0 or pH be 5.0 tbe buffer liquid be mixed with mixing solutions, the mixing solutions obtained stirs the poly-polypeptide-DNA hydrogel becoming clear viscous 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.
Embodiment 5:dsDNA and ssDNA proportioning are on the impact of poly-polypeptide-DNA hydrogel
By the Gel1-4 that embodiment 4 prepares, at 25 degrees Celsius, carry out frequency sweeping rheology under 1% strained condition, frequency sweeping ranges 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 ' represent shearing storage modulus, G ' ' represents shearing out-of-phase modulus, and in whole sweep limit, G ' shows to define 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 (i.e. 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 during 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 (i.e. the sticky end of polypeptide), the physical strength of poly-polypeptide-DNA hydrogel to be regulated.
Embodiment 6: total mass mark is on the impact of poly-polypeptide-DNA hydrogel
Gel4-11 embodiment 4 prepared carries out rheological property detection, and detected result as shown in Figure 3.
From the result of Fig. 3, total mass mark remarkably influenced can gather the physical strength of 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 illustrates the physical strength regulating total mass mark effectively can regulate poly-polypeptide-DNA hydrogel, according to different applied environments, can prepare the poly-polypeptide-DNA hydrogel with different physical strength.
The length of embodiment 7:dsDNA sticky end is on the impact of poly-polypeptide-DNA hydrogel
Gel2, Gel12 and Gel13(photo prepared in enforcement 4 is shown in Fig. 4) in 25 degrees Celsius, 1% strain, 1Hz condition under 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.Gel12 and Gel13 is known in contrast, and the length of dsDNA sticky end is identical, if there is mismatch site in sticky end, is unfavorable for the formation of poly-polypeptide-DNA hydrogel.From the result of Fig. 5, G '/the 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, thus by adjusting the length of double chain DNA molecule sticky end, the poly-polypeptide-DNA hydrogel with different physical strength can be obtained.
Embodiment 8: the secondary structure of polypeptide backbone is on the impact of poly-polypeptide-DNA hydrogel
Gel10 and Gel16 prepared in embodiment 4 is carried out respectively circular dichroism spectrum to detect and time scan rheology, result is shown in Fig. 6 and Fig. 7 respectively, wherein, and the corresponding Gel16 of pH8.0 corresponding Gel10, pH5.0.
From the result of Fig. 6, under the pH of tbe buffer liquid is the condition of 5.0, due to the protonation of carboxyl, polypeptide backbone is alpha helical conformation, and under the pH of tbe buffer liquid is the condition of 8.0, 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 then apparently higher than the G ' ' of Gel10, show that poly-polypeptide-DNA hydrogel has good physical strength at sour environment, and then illustrate that the secondary structure of polypeptide backbone can affect the physical strength of poly-polypeptide-DNA hydrogel.
Embodiment 9: the thermo-responsive of poly-polypeptide-DNA hydrogel
By the Gel2 for preparing in embodiment 4 in 1% strain, 1Hz, between 25 degrees Celsius-60 degrees Celsius, carry out rheology testing, the results are shown in Figure 8.
As seen from Figure 8,25 degrees Celsius time, G ' is apparently higher than G ' ', this is the characteristic feature forming hydrogel, and when temperature is increased to 60 degrees Celsius by 25 degrees Celsius, G ' is significantly lower than G ' ', this illustrates that hydrogel has become solution state, and when temperature is again down to 25 degrees Celsius and is kept 15 minutes, solution can form hydrogel again again.Further, 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
The degradation property of Gel2, Gel14 and Gel15 of preparing in embodiment 4 is detected according to following steps, specific as follows:
Get 8 EP pipe, numbering a-h group, wherein organize a and group b often pipe add the Gel2 of phosphate buffer soln (10 μ L, pH7.8) and 15 μ L, organize c-e often pipe add containing Tris-HCl(50mM, pH7.5), NaCl(100mM), MgCl
2(10mM), the solution of dithiothreitol (DTT) (1mM) and the Gel14 of 15 μ L, group f-h often pipe adds containing 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 proteolytic enzyme Glu-C respectively, then 8 groups of EP pipes are reacted by EcoRI or BamHI under room temperature condition, observe poly-polypeptide-DNA hydrogel in reaction process.Fig. 9 is shown in by each group of photo gathering polypeptide-DNA hydrogel.
Table 4
As seen from Figure 9, after reaction for some time, add proteolytic enzyme Glu-C, poly-polypeptide-DNA the hydrogel of EcoRI or BamHI is converted into solution morphology, and the poly-polypeptide-DNA hydrogel not adding enzyme still keeps the form of hydrogel, this shows, suitable restriction endonuclease sites is contained by design double chain DNA molecule, poly-polypeptide-DNA hydrogel of the present invention can have special enzyme responsiveness, namely poly-polypeptide-DNA hydrogel of the present invention can be degraded under the effect of specific enzymes, and this illustrates that poly-polypeptide-DNA hydrogel of the present invention can be used as pharmaceutical carrier.
Embodiment 11: the biocompatibility of poly-polypeptide-DNA hydrogel
The double chain DNA molecule of to be the polypeptide of 0.1-10mg/ml and concentration by concentration be 0.01-2.0mM adds in the DMEM cell culture medium containing NIH3T3 cell respectively, then be 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 containing different concns and double chain DNA molecule, cell survival rate is all about 99 ± 0.5%, 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 DMEM substratum is made into cell density
6the cell suspension of individual cell/ml, by the above-mentioned cell suspension of 4 μ L and the polypeptide solution (15wt% containing 100mM sodium-chlor, 8 μ L) mixing, 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, 200 μ L cell culture mediums are added again above the celliferous poly-polypeptide-DNA hydrogel of bag, be placed on 37 degrees Celsius, cultivate 24 hours in the incubator of 5% carbonic acid gas, 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, does not almost have dead cell, thus, shows that poly-polypeptide-DNA hydrogel biocompatibility of the present invention is good.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not must for be identical embodiment or example.And the specific features of description, structure, material or feature can combine in one or more embodiment in office or example in an appropriate manner.In addition, when not conflicting, the feature of the different embodiment described in this specification sheets or example and different embodiment or example can carry out combining and combining by those skilled in the art.
Although illustrate and describe 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, revises, replace and modification.
Claims (11)
1. a 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 the complementation of described single strand dna;
Described polypeptide becomes crosslinking structure by the sticky end of described double chain DNA molecule with the complementary of described single strand dna,
Described polypeptide has the structure shown in following chemical formula:
Wherein, ssDNA represents described single strand dna, and * represents natural chiral amino acid.
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 sticky ends of described double chain DNA molecule is separately 8 ~ 12nt.
3. poly-polypeptide-DNA hydrogel according to claim 1, it is characterized in that, described polypeptide has the aminoacid sequence as shown in SEQIDNO:12,
Optionally, described single strand dna has the nucleotide sequence as shown in SEQIDNO:1,
Optionally, described double chain DNA molecule has the first chain and the second chain, and wherein, described first chain and described second chain meet one of following condition:
Described first chain has the sequence shown in SEQIDNO:2, and described second chain has the sequence shown in SEQIDNO:3;
Described first chain has the sequence shown in SEQIDNO:4, and described second chain has the sequence shown in SEQIDNO:5;
Described first chain has the sequence shown in SEQIDNO:6, and described second chain has the sequence shown in SEQIDNO:7;
Described first chain has the sequence shown in SEQIDNO:8, and described second chain has the sequence shown in SEQIDNO:9;
Or described first chain has the sequence shown in SEQIDNO:10, described second chain has the sequence shown in SEQIDNO:11.
4. poly-polypeptide-DNA hydrogel according to claim 1, is characterized in that, each described polypeptide is combined with multiple described single strand dna.
5. poly-polypeptide-DNA hydrogel according to claim 4, is characterized in that, each described polypeptide is combined with at least 4 described single strand dnas.
6. 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.
7. prepare a method for poly-polypeptide-DNA hydrogel described in any one of claim 1 ~ 6, it is characterized in that, comprising:
There is provided polypeptide and double chain DNA molecule, 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 the complementation of described single strand dna; 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 to comprise further:
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 hydroxysuccinimide ester with hydroxysuccinimide;
Described 5-nitrine valeric acid hydroxysuccinimide ester is contacted, to obtain the single strand dna containing 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 with HBr with trifluoroacetic acid, to obtain Pidolidone γ-propargyl-Pidolidone ester copolymer;
The described single strand dna containing azido group is contacted, to obtain described polypeptide with described Pidolidone γ-propargyl-Pidolidone ester copolymer.
9. method according to claim 8, is characterized in that, comprises further:
Bromo-for described 5-valeric acid is dissolved in dimethyl sulfoxide (DMSO), after being heated to 40 degrees Celsius, adds described sodiumazide, in 85 degrees Celsius, stir lower reaction and spend the night, obtained reaction solution is cooled to 40 degrees Celsius, adds 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 hydroxysuccinimide are dissolved in methylene dichloride, add 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide under stirring, stirred overnight at room temperature, obtain described 5-nitrine valeric acid hydroxysuccinimide ester;
Described 5-nitrine valeric acid hydroxysuccinimide ester is dissolved in DMF, the aqueous solution of obtained solution and described amido modified single strand dna and phosphate buffer soln are mixed, to obtain the single strand dna containing azido group;
γ-propargyl-Pidolidone is contacted, to obtain γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides with two (trichloromethyl) carbonic ether;
Described γ-propargyl-Pidolidone ester N-carbonyl acid anhydrides and described γ-benzyl-Pidolidone ester N-carbonyl acid anhydrides is made to be dissolved in N, dinethylformamide, add title complex containing Ni as after 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 Ni is 10:3:0.05;
Make described-benzyl-Pidolidone ester γ-propargyl-Pidolidone ester copolymer be dissolved in trifluoroacetic acid, after adding the acetum of HBr, react 2 hours in stirred at ambient temperature, obtain described Pidolidone γ-propargyl-Pidolidone ester copolymer;
Make described Pidolidone γ-propargyl-Pidolidone ester copolymer aqueous solution and CuSO
4solution, THTA, described single strand dna solution containing azido group and sodium ascorbate solution mixing, by obtained mixing solutions in 50 degrees Celsius, react under oscillating condition and spend the night, obtain described polypeptide.
10. method according to claim 7, is characterized in that, described single strand dna is synthesized by solid phase phosphoramidite DNA synthetic method,
Optionally, described double chain DNA molecule is obtained by following steps:
Described single strand dna according to stoichiometric is mixed with tbe buffer liquid, is heated to 95 degrees Celsius and after being incubated 3 minutes, in 2 hours, is cooled to room temperature, to obtain described double chain DNA molecule.
11. methods according to claim 7, is characterized in that, mixed by described polypeptide comprise further with described double chain DNA molecule:
Described polypeptide is mixed, to obtain the mixture of polypeptide and double chain DNA molecule according to the ratio that described double chain DNA molecule and the mol ratio of described single strand dna are 0.6-1.2:1 with described double chain DNA molecule;
The mixture of described polypeptide and double chain DNA molecule is dissolved in tbe buffer liquid according to the ratio that massfraction is 0.3%-5%, to obtain poly-polypeptide-DNA hydrogel.
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