CN100406466C - Nucleic acid hydrolysis cutting agent based on cucurbituril - Google Patents
Nucleic acid hydrolysis cutting agent based on cucurbituril Download PDFInfo
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Abstract
The invention relates to pseudorotaxane molecular compound produced by cucurbit[6]uril and 1, 6-di-imidazole group di-acid salt whose chemical molecular formula is C36H36N24O12*C12H20N4*2X*nH2O whose X equals to Cl, H2PO4, HSO4, NO3, Br; n is not less than 1. The compound can be used in nucleic acid cutting whose condition is that the cutting reagent is dissolved in buffer system solution with given pH value, then mixed with cutting substrate; their final density is kept in given range; and their temperature is also kept in given range. The cutting reagent can be used to cut nucleic acid in short time at physiology condition, further study the application in gene therapy medicine preparation to develop into gene therapy medicine.
Description
Technical field
The present invention relates to a kind of nucleic acid cleavage agent, specifically belong to a kind of nucleic acid hydrolysis cutting agent based on the melon ring.
Background technology
As the carrier of genetic information and the basis of genetic expression, adjust in the vital movements such as growth at biology, growth, breeding and have a very important role.HGP in 2000 finishes, indicate that human medicine and pharmacology will enter gene molecule epoch, secret, inherited disease, molecular disease, cancer, acquired immune deficiency syndrome (AIDS) and the blood supply of the life in human several thousand of puzzlement, organ supply etc. all can be found the outlet of the annotation and the solution of science.And the core of everything be to the DNA nucleic acid molecule identification, cut out and be connected.Therefore, the research of artificial nucleic acid cutting reagent all is most active focus preface research topic in biological chemistry and the molecular biology all the time.The nucleic acid cutting fragments into than short fragment by the nucleic acid molecule of chemical reaction with long-chain exactly.The research nucleic acid cutting reagent has very important theory and using value.At first, the research to the cutting mechanism of artificial nucleic acid cutting reagent will go far towards our thorough to the nuclease catalytic mechanism.Secondly, cutting reagent has crucial application in the gene therapy of disease.And nucleic acid cutting reagent can be used as important biology tool, can be used for studying the probe of nucleic acid higher structure.
About the artificial nucleic acid cutting reagent,, mainly be divided into free radical cutting reagent, ester hydrolysis cutting reagent and cancellation type cutting reagent, following classification according to the difference of cutting mechanism:
1, the reagent of free radical mechanism cutting nucleic acid can both produce the sugar ring or the base of certain free radical attack nucleic acid, seizes hydrogen atom and causes its oxidation to destroy, and then nucleic acid chains is ruptured.For example, EDTA-Fe
2+/ H
2O
2System (Roki ta, S.E.Romero-Fredes, L Nucleic.Acids.Res.1992,20,3069), Cu
2+/ reductive agent system (Reed, C.J.Douglas, K.T.Biochem.Biphys.Res.Commun.1989,162,1111), photochemistry cutting system (Carter, P.J.Breiner, K.M.Thorp, H.H.Biochemistry.1998,37,13736), microbiotic (Burger, R.M.Chem.Rev, system such as 1998,98,1153).Such cutting reagent destroys some pentose ring and even the Nucleotide on the nucleic acid chains, and the result loses original message part.Therefore, such cutting reagent exists the fragment that cuts to be difficult to by poor, the many shortcomings such as toxic side effect is strong, system complexity of specificity of further utilizing, cutting.(Wanrong, Zhao Gang, Chen Jing, fiber crops are far away, Zhao Yufen, Science Bulletin, 2000,45,785).
2, eliminate the reagent of mechanism cutting nucleic acid: Lai Selai tripeptides KWK (Behmoaras, T.Toulme, J.J.Helene, C.Nature, 1981,292,858) and can cut nucleic acid by the mode of eliminating.Such cutting reagent is extremely rare, does not possess ubiquity.
3, with the reagent of ester hydrolysis mechanism cutting nucleic acid: the direct attack of cutting reagent provides the phosphorus atom of phosphodiester bond in certain nucleophilic group attack nucleic acid, makes nucleic acid hydrolysis thereby nucleophilic substitution reaction takes place.For example Cu (II)-L-Histidine title complex is by hydrolysis mechanism cutting nucleic acid (Ren, R.Yang, P.Zheng, W.J.Hua, Z.C.Inorg.Chem.2000,39,5454); The complex compound hydrolysed nucleic acid of double-core metal ion (soaring cloudy rosy clouds, Yang Pin, Science Bulletin, 2004,49,1471) seryl histidine dipeptide can be at the DNA of hydrolysis superhelix type under the neutrallty condition.(Li, X.H.Wan, R.Zhang, Q.et.al.Clncinnati Section American Chemical Society, Cininnati, Ohio, 1998,130) the hydrolysis cutting reagent has been got rid of defective among free radical cutting reagent many, and possesses ubiquity, can be described as the diced system of simulation natural acid enzyme of the best of artificial lytic enzyme.But up to now, effectively artificial enzyme's system of the hydrolysis of catalytic hydrolysis DNA is less relatively.And, the overwhelming majority is the complex compound of metal ion, these contain the Chemistry Nuclease of metal ion, majority is that requirement has the activity of the best pH of tart and these enzymes controlled by metal ion, and these have all hindered them in chelation buffer or the application when having metal chelator to occur.Therefore, need to seek dissimilar nucleases, this kind of enzyme has the pH an of the best near pH=7.0 or pH=7.0, and its performance activity does not need metal ion.Get the hydrolysis cutting reagent with respect to metal ion, the nucleic acid cutting reagent of metal ion does not have its even more important relatively theory significance and application potential (Zhong Rugang, Zhao Lijiao, Zhao Yufen, chemical journal, 2004,62,2444).Here, we have developed a kind of DNA artificial nuclease based on the melon ring of novel not metal ion.
Summary of the invention
The object of the present invention is to provide a kind of cutting agent of nucleic acid hydrolysis efficiently of not metal ion.
Nucleic acid hydrolysis cutting agent provided by the invention is pseudorotaxane molecular compound (pseudorotaxane), its chemical molecular formula: C
36H
36N
24O
12C
12H
20N
42XnH
2O, wherein X=Cl, H
2PO
4, HSO
4, NO
3, Br; N 〉=1; Chemical structural formula (I):
Nucleic acid cleavage agent of the present invention, the artificial nuclease that specifically can be used as molecular biology research is used for the nucleic acid cutting, also can further research and develop into gene therapy medicine in addition.
The preparation method of nucleic acid hydrolysis cutting agent of the present invention: comprise the steps:
(1) with glycoluril, formaldehyde mixes with mol ratio at 1: 2, is the sulfuric acid H of 9 mol in concentration
2SO
4Solution, 70-75 ℃ was reacted 24 hours, was warming up to 95-100 ℃ then, continued reaction 12 hours, be cooled to room temperature, reaction solution poured in the water of 10 times of reaction solution volumes, add the acetone that 6-10 doubly measures volume, separate out white solid, leave standstill, suction filtration is collected the gained solid.The gained solid dispersed adds isopyknic acetone in the water of certain volume, stirring is left standstill, suction filtration gained white solid is used small amount of acetone drip washing after with 60 ℃ water thorough washing, after draining, vacuum-drying gets white solid, is melon ring host molecule (cucurbit[6] uril).Reaction formula:
(2) imidazoles is added in the tetrahydrofuran (THF) dissolve, adding sodium hydride again mixes, imidazoles and sodium hydride mol ratio 1: 1.2-2, stirred 1-2 hour, and added 1 of 0.3-0.5 times of imidazoles molal quantity, the 6-dibromo-hexane, stirred 12-24 hour, removal of solvent under reduced pressure gets pasty solid, adds entry and makes the solid dissolving, this solution chloroform (CHCl
3) dividing three fully extractions, combined chloroform liquid is used anhydrous Na
2SO
4Drying is filtered, and chloroform is removed in decompression, gets the tawny oily liquids, and this liquid is 1,6-diimidazole base hexane; With 1, it is 1: 1 strong acid solution that 6-diimidazole base hexane is dissolved in volume ratio, backflow 1-3 hour, the decompression concentrated solution volume is to 1/3-1/5, add tetrahydrofuran (THF) to separating out white solid, filtration drying gets guest molecule 1,6-diimidazole base hexane diacid salt (representing with G).Reaction formula:
(3) with melon ring host molecule and 1,6-diimidazole base hexane diacid salt guest molecule G mixes according to mol ratio 1: 1-1.2, adds dissolved in distilled water, stirs 12-24 hour; Be evaporated to the 1/3-1/5 that liquor capacity is initial volume, add the tetrahydrofuran (THF) that 6-10 doubly measures volume, separate out white solid, suction filtration is collected this solid, uses small amount of acetone drip washing, and vacuum-drying gets pseudorotaxane molecular compound (representing with P).Reaction formula:
Pseudorotaxane molecular compound of the present invention is comprising melon ring (C
36H
36N
24O
12Cucurbit[6] uril) host molecule and guest molecule 1,6-diimidazole base hexane dihydrochloride (G1), 1,6-diimidazole base hexane diphosphate (G2), 1,6-diimidazole base hexane dithionate (G3), 1,6-diimidazole base hexane dinitrate (G4), 1,6-diimidazole base hexane two hydrobromates (G5) the pseudorotaxane molecular compound (representing with P1, P2, P2, P4, P5 respectively) that forms respectively.
The artificial nuclease that pseudorotaxane molecular compound of the present invention can be used as molecular biology research is used for the nucleic acid cutting, the condition of cutting is cutting reagent to be dissolved in the certain pH value buffer body be tied to form solution, mix with the cutting substrate then, and the final concentration of buffer reagent and substrate is kept within the specific limits, place under the certain temperature to be incubated, realize cutting substrate nucleic acid.Cutting reagent of the present invention is the not cutting agent of nucleic acid hydrolysis efficiently of metal ion of a class, under physiological condition, under lower concentration, can cut nucleic acid in the short time.Cutting reagent of the present invention also can be used in the preparation gene therapy medicament, is further used for the research and development of gene therapy medicament.
Nucleic acid hydrolysis cutting agent pseudorotaxane molecular compound of the present invention is to the cutting of DNA
The preparation of solution
(1) preparation of 0.5M EDTA (pH 8.0) solution
In 800ml distilled water, add 181.6g two water disodium ethylene diamine tetraacetate (EDTA-Na
22H
2O), vigorous stirring on magnetic stirring apparatus is regulated pH value to 8.0 with NaOH, be settled to the 1000ml. packing then after autoclaving standby.
(2) the Tris-acetate (preparation of 50 * TAE) electrophoretic buffers
242gTris (Tutofusin tris) alkali and 57.1ml third acetate are dissolved in the EDTA solution of 100ml 0.5M, add deionized water and transfer to 1000ml.
(3) preparation of reaction buffer
1.21gTris be dissolved in the 800ml deionized water.Add 0.363gNaCl, transfer pH 7.18 to be settled to 1000ml and to be mixed with 10mM Tris, 6.2mM NaCl solution for standby with concentrated hydrochloric acid.
(4) EB solution
Take by weighing EB (pyridine of bromination second) 0.1g in brown bottle, with 100ml deionized water dissolving and constant volume, it is standby to be stored in 4 ℃ of preservations of refrigerator.
(5) preparation of enzymolysis stop buffer
0.05% tetrabromophenol sulfonphthalein, 50% (v/v, volume ratio) aqueous glycerin solution, 0.1M EDTA
(6) preparation of cutting reagent
Cutting reagent solution with the buffer preparation 5mM that is prepared.
Cutting experiment
(1) hybrid reaction: get clean Eppendorf pipe, the buffered soln distilled water, cutting agent, the plasmid DNA (pBR 322DNA) that add predetermined amount respectively successively, with whizzer all compositions are got rid of to Eppendorf pipe bottom, make final concentration be respectively Tris-HCl:10mM; Cutting reagent: 150 μ M; PBR 322DNA:0.014mg/mL was placed in the water-bath of 37 ℃ of constant temperature the lucifuge reaction 4 hours.
(2) preparing gel: take by weighing 0.32gAgarose (agarose) and add 50 * TAE0.8ml, add bi-distilled water to 40ml, microwave oven heating 2min dissolves it fully, when treating that solution temperature is reduced to about 70 ℃, add EB solution 25 μ L, make the EB final concentration be about 0.5 μ g/ml, shake up.When temperature is further reduced to the 40-50 ℃ of left and right sides, pour into rapidly in the disk electrophoresis bed of putting comb hole and baffle plate well, after waiting to solidify, inject electrophoretic buffer, slowly take out comb and baffle plate.
(3) electrophoresis: reaction system to be measured is in 37 ℃ of constant temperature water bath grooves behind the constant temperature certain hour, to take out in its thermostatic bath, each adds 4 μ L tetrabromophenol sulfonphthalein indicator, point sample is aerial in the point sample of disk electrophoresis bed respectively with the solution in the Eppendorf pipe with microsyringe, run 2-3h under the voltage 50V condition, see that indicator has moved to the gel middle part, cuts off the electricity supply, take out gel, on SYNGENE GENE GENIUS BIO IMAGING SYSTEM gel imaging system instrument, make a video recording.
Description of drawings:
The electrophorogram of the cutting reagent P1 cutting pBR 322DNA of Fig. 1 different concns.
10mM Tris-HCl, 6.2mM NaCl, pH 7.18 is at 37 ℃ of constant temperature 4h; Swimming lane 1:DNA contrast; Swimming lane 2-5: difference 10 μ M, 25 μ M, 50 μ M, the cutting reagent of 150 μ M.Analytical results sees Table 1.
Fig. 2 concentration is that cutting reagent P1, P2, P3, P4, the P5 of 150 μ M cuts electrophorogram to pBR 322DNA.10mM Tri s-HCl, 6.2mM NaCl, pH 7.18 is at 37 ℃ of constant temperature 4h; Swimming lane 1:DNA contrast.
Embodiment:
Embodiment 1-5 is that cutting agent prepares embodiment; Embodiment 6-10 cutting agent cutting DNA embodiment.
Embodiment 1
Step 1. synthesizing of main body melon toroidal molecule:
11.4g glycoluril (80mmol) is in 100ml four-hole round-bottomed flask, adding 40ml concentration is the sulfuric acid H of 9M (mol)
2SO
4Solution, oil bath are warming up to 70 ℃, begin to drip 14ml, 37% formalin is noted during dropping maintaining the temperature between 70-75 ℃, dropwises 70-75 ℃ of reaction of back insulation 24 hours, be warming up to 95-100 ℃ then, after continuing reaction 12h, be cooled to room temperature, reaction solution is poured in the 400ml water, add acetone 2L, separate out white solid, leave standstill 20min after, suction filtration is collected the gained solid.Gained polity is scattered in 200 water, adds acetone 200ml, stirs 20min, leaves standstill, and suction filtration gained white solid is with the about 50ml acetone of usefulness drip washing after 60 ℃ the water 300ml washing three times, after draining, and the vacuum drying 6.5g of white solid, productive rate, 48%.
Step 2. guest molecule 1,6-diimidazole base hexane dihydrochloride (G1) synthetic:
Add 20ml exsiccant tetrahydrofuran (THF) (THF) liquid in the single necked round bottom flask of 50ml, add the 1.5g imidazoles, after the stirring and dissolving, slowly gradation adds 95% NaH, and behind the stirring at room 2h, reaction system is suspension liquid, add 2.44g 1 then, 6-dibromo-hexane, stirring at room 24h aftertreatment.Removal of solvent under reduced pressure gets pasty solid, adds 100ml water, and the solid dissolving is yellow solution, this solution 120ml chloroform (CHCl
3) dividing three extractions, combined chloroform liquid is used anhydrous Na
2SO
4Dry 4h filters, and the chloroformic solution decompression is removed, and gets the tawny oily liquids, and this liquid is 1,6-diimidazole base hexane.With 1,6-diimidazole base hexane is dissolved in the 10ml water, adds the 10ml concentrated hydrochloric acid, and backflow 2h, decompression concentrated solution volume are about 5ml, adds tetrahydrofuran (THF) 30ml, separates out white solid, gets guest molecule G1, and 1,6-diimidazole base hexane dihydrochloride.
1HNMR nucleus magnetic resonance (300MHz, 25 ℃, D
2O, TMSP): δ 8.71 (s, 2H), 7.50 (s, 1H), 7.46 (s, 1H), 4.23 (t, 4H, CH
2), 1.89 (m, 4H, CH
2), 1.33 (m, 4H, CH
2);
13C NMR (300MHz, D
2O): δ 134.13,121.56, and 119.48,49.05,28.94,24.75; Ultimate analysis (calcd.%) for C
12H
20N
4Cl
2: C, 49.48; N, 19.24; H, 6.87.Found:C, 49.86; N, 19.44; H, 6.53.
Step 3. cutting reagent super molecular compound pseudorotaxane molecule P1's is synthetic:
The single necked round bottom flask of 50ml, the melon ring of adding 121mg (0.1mmol), the guest molecule G1 of 29.2mg (0.1mmol) adds 20ml distilled water, 80 ℃ of insulated and stirred 24h.Cooling, the decompression concentrated solution volume is about 5ml, adds tetrahydrofuran (THF) 30ml, separates out white solid, and suction filtration is collected this solid, uses small amount of acetone drip washing, and vacuum-drying gets pseudorotaxane molecular compound P1, productive rate 86%.
1HNMR nucleus magnetic resonance (300MHz, 25 ℃, D
2O, TMSP): δ 9.16 (s, 2H), 7.92 (s, 2H), 7.37 (s, 2H), 5.78 (d, 12H), 5.58 (s, 12H), 4.32 (d, 12H), 3.99 (t, 4H), 1.18 (m, 4H), 0.57 (m, 4H);
13C NMR (300MHz, D
2O): δ 155.8,135.2, and 123.3,116.8,71.1,51.2,48.6,28.6,27.3; The ESI-MS electrospray ionization mass spectrum: 608.7 is the double charge positively charged ion, and 1215.4 is single electric charge positively charged ion, ultimate analysis (calcd.%) for C
36H
36N
24O
12C
12H
20N
42Cl8H
2O:C, 40.22; N, 27.37; H, 5.03.Found:C, 39.99; N, 27.12; H, 4.97. crystal are oblique system, P2
1/ c spacer, a=12.369 (4)
, b=20.115 (6)
, c=12.578 (4)
, β=113.664 °, V=2866.3 (15), Z=2, Dc=1.659gcm
-3, R
Int=0.0223, F (000)=1500, M=1432, deviation factors is R
1=0.0664, wR
2=0.1900
Embodiment 2
Step 1. repeat embodiment 1 step 1, get used main body melon ring.
Step 2. obtain guest molecule G2,1,6-diimidazole base hexane diphosphate with the used concentrated hydrochloric acid of strong phosphoric acid alternate embodiment 1 step 2.Ultimate analysis (calcd.%) for C
12H
24N
4P
2O
8: C, 34.78; N, 13.53; H, 5.80.Found:C, 34.91; N, 13.74; H, 5.73.
Step 3. substitute guest molecule G1 with guest molecule G2, repeat embodiment 1 step 3, get pseudorotaxane molecular compound P2, productive rate 86%.Ultimate analysis (calcd.%) for C
36H
36N
24O
12C
12H
20N
42H
2PO
412H
2O:C, 35.42; N, 24.11; H, 5.17.Found:C, 35.59; N, 24.32; H, 5.07.
Embodiment 3
Step 1. repeat embodiment 1 step 1, get used main body melon ring.
Step 2. obtain guest molecule G3,1,6-diimidazole base hexane dithionate with the used concentrated hydrochloric acid of vitriol oil alternate embodiment 1 step 2.Ultimate analysis (calcd.%) for C
12H
22N
4S
2O
8: C, 34.78; N, 13.53; H, 5.31.Found:C, 34.78; N, 13.64; H, 5.22.
Step 3. substitute guest molecule G1 with guest molecule G3, repeat embodiment 1 step 3, get pseudorotaxane molecular compound P2, productive rate 84%.Ultimate analysis (calcd.%) for C
36H
36N
24O
12C
12H
20N
42HSO
410H
2O:C, 36.23; N, 24.65; H, 4.91.Found:C, 36.39; N, 24.72; H, 4.88.
Embodiment 4
Step 1. repeat embodiment 1 step 1, get used main body melon ring.
Step 2. obtain guest molecule G4,1,6-diimidazole base hexane dinitrate with the used concentrated hydrochloric acid of concentrated nitric acid alternate embodiment 1 step 2.Ultimate analysis (calcd.%) for C
12H
20N
6O
6: C, 41.86; N, 24.41; H, 5.81.Found:C, 41.96; N, 24.44; H, 5.93.
Step 3. substitute guest molecule G1 with guest molecule G4, repeat embodiment 1 step 3, get pseudorotaxane molecular compound P4, productive rate 88%.Ultimate analysis (calcd.%) for C
36H
36N
24O
12C
12H
20N
42NO
39H
2O:C, 38.35; N, 27.96; H, 4.93.Found:C, 38.39; N, 28.02; H, 5.03.
Embodiment 5
Step 1. repeat embodiment 1 step 1, get used main body melon ring.
Step 2. obtain guest molecule G5,1,6-diimidazole base hexane two hydrobromates with the used concentrated hydrochloric acid of Hydrogen bromide alternate embodiment 1 step 2.Ultimate analysis (calcd.%) for C
12H
20N
4Br
2: C, 37.89; N, 14.74; H, 5.26.Found:C, 37.86; N, 14.69; H, 5.35.
Step 3. substitute guest molecule G1 with guest molecule G5, repeat embodiment 1 step 3, get pseudorotaxane molecular compound P5, productive rate 91%.Ultimate analysis (calcd.%) for C
36H
36N
24O
12C
12H
20N
42Br8H
2O:C, 37.89; N, 25.78; H, 4.73.Found:C, 37.48; N, 25.42; H, 4.61.
Embodiment 6:
Under physiological condition, as plasmid pBR 322DNA and cutting reagent P1,37 ℃ of constant temperature are after 4 hours, and super coiled DNA is degraded to three kinds of forms, become by superhelix supercoiled (S) and to incise nicked circular (C), degraded changes line style linear (L) into then.As shown in Figure 1: the initial concentration of DNA is 0.05mg/ml, and the concentration range of cutting reagent P1 is 0～150 μ M.Along with the increase of P1 concentration, can see that plasmid pBR 322DNA changes into gradually from superhelix S to incise C and line style L that final superhelix S almost all changes into and incises C and line style L.What change the results are shown in Table 1.The result is the optimum concn of cutting as can be seen when cutting agent P1 concentration is 150 μ M thus.
Embodiment 7:
On the basis of embodiment 1, choose best experiment condition, the concentration of cutting reagent is decided to be 150 μ M, remaining condition repeats condition among embodiment 1, with the P1 in the P2 alternate embodiment 1 cutting drawing see Fig. 2 swimming lane P2.
Embodiment 8:
On the basis of embodiment 1, choose best experiment condition, the concentration of cutting reagent is decided to be 150 μ M, remaining condition repeats condition among embodiment 1, with the P1 in the P3 alternate embodiment 1 cutting drawing see Fig. 2 swimming lane P3.
Embodiment 9:
On the basis of embodiment 1, choose best experiment condition, the concentration of cutting reagent is decided to be 150 μ M, remaining condition repeats condition among embodiment 1, with the P1 in the P4 alternate embodiment 1 cutting drawing see Fig. 2 swimming lane P4.
Embodiment 10:
On the basis of embodiment 1, choose best experiment condition, the concentration of cutting reagent is decided to be 150 μ M, remaining condition repeats condition among embodiment 1, with the P1 in the P5 alternate embodiment 1 cutting drawing see Fig. 2 swimming lane P5.
The cutting reagent P1 cutting pBR 322DNA of table 1, different concns
| Cutting reagent P1/ μ M | Superhelix S% | Incise C% | Line style L% |
| Conrol | 64 | 36 | 0 |
| 10 | 58 | 40 | 2 |
| 25 | 51 | 43 | 6 |
| 50 | 32 | 55 | 13 |
| 150 | 7 | 69 | 24 |
Based on above-mentioned experimental result, the artificial nuclease that the cutting reagent of indication of the present invention can be used as molecular biology research is used for the nucleic acid cutting, can further research and develop into gene therapy medicine.
Claims (4)
1. a nucleic acid hydrolysis cutting agent is characterized in that it is melon ring host molecule and 1, the pseudorotaxane molecular compound that 6-diimidazole base hexane diacid salt guest molecule constitutes, its chemical molecular formula: C
36H
36N
24O
12C
12H
20N
42XnH
2O, wherein X=Cl, H
2PO
4, HSO
4, NO
3, Br; N 〉=1; Its chemical structural formula (I):
2. according to the preparation method of the described a kind of nucleic acid hydrolysis cutting agent of claim 1, it is characterized in that comprising the steps:
(1) use glycoluril, formaldehyde prepares melon ring host molecule under the acid solution condition;
(2) imidazoles is added in the tetrahydrofuran (THF) dissolve, add sodium hydride again and mix imidazoles and sodium hydride mol ratio 1: 1.2-2, stirred 1-2 hour, and added 1 of 0.3-0.5 times of imidazoles molal quantity, the 6-dibromo-hexane, stirred 12-24 hour, remove desolvate pasty solid, be dissolved in water, fully extract with chloroform, drying is filtered, and removes chloroform, get 1,6-diimidazole base hexane; With 1,6-diimidazole base hexane is dissolved in strong acid solution, refluxes, and concentrates, and adds tetrahydrofuran (THF), separates out white solid, filters, and is dry 1,6-diimidazole base hexane diacid salt guest molecule;
(3) with melon ring host molecule and 1,6-diimidazole base hexane diacid salt guest molecule mixes according to mol ratio 1: 1-1.2, adds dissolved in distilled water, stirs 12-24 hour; Concentrate, add tetrahydrofuran (THF) and separate out white solid, suction filtration is collected this solid, and drying gets the pseudorotaxane molecular compound.
3. according to the purposes of the described a kind of nucleic acid hydrolysis cutting agent of claim 1, the artificial nuclease that is used as molecular biology research is used for the nucleic acid cutting.
4. according to the purposes of the described a kind of nucleic acid hydrolysis cutting agent of claim 1, the application in the preparation gene therapy medicament.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| Title |
|---|
| 新笼状化合物—五、七、八元瓜环. 姚晓青，申永强，陶，朱，祝黔江，薛赛凤，罗绪强.贵州大学学报(自然科学版)，第20卷第1期. 2003 |
| 新笼状化合物—五、七、八元瓜环. 姚晓青，申永强，陶，朱，祝黔江，薛赛凤，罗绪强.贵州大学学报(自然科学版)，第20卷第1期. 2003 * |
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