CN107119031A - A kind of new thermophilic serobila DNA enzymatics of quaternary G tetra- - Google Patents
A kind of new thermophilic serobila DNA enzymatics of quaternary G tetra- Download PDFInfo
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- CN107119031A CN107119031A CN201710491309.9A CN201710491309A CN107119031A CN 107119031 A CN107119031 A CN 107119031A CN 201710491309 A CN201710491309 A CN 201710491309A CN 107119031 A CN107119031 A CN 107119031A
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- dna
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- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 26
- 238000006911 enzymatic reaction Methods 0.000 title abstract description 10
- 108020004414 DNA Proteins 0.000 claims abstract description 27
- 230000003197 catalytic effect Effects 0.000 claims abstract description 19
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims abstract description 13
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 13
- 230000000694 effects Effects 0.000 claims abstract description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 10
- 239000007853 buffer solution Substances 0.000 claims abstract description 7
- 229930024421 Adenine Natural products 0.000 claims abstract description 6
- 229960000643 adenine Drugs 0.000 claims abstract description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims abstract description 5
- 238000000137 annealing Methods 0.000 claims abstract description 4
- 230000008859 change Effects 0.000 claims abstract description 4
- 230000002708 enhancing effect Effects 0.000 claims abstract 2
- 239000002585 base Substances 0.000 claims 4
- 102000003992 Peroxidases Human genes 0.000 claims 1
- 239000003513 alkali Substances 0.000 claims 1
- 238000001816 cooling Methods 0.000 claims 1
- 108040007629 peroxidase activity proteins Proteins 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 description 9
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 6
- 229940025294 hemin Drugs 0.000 description 6
- 239000000243 solution Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- -1 ammonium ions Chemical class 0.000 description 1
- 229910052681 coesite Inorganic materials 0.000 description 1
- 229910052906 cristobalite Inorganic materials 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229910052682 stishovite Inorganic materials 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 229910052905 tridymite Inorganic materials 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
Abstract
There is the present invention relates to a kind of adenine ortho position enhancing catalytic activity and at high temperature the new thermophilic serobila DNA enzymatics of quaternary G tetra- of high catalytic activity.First in two terminal modified adenines of the DNA sequence dna rich in G bases, in the buffer solution of the ammonium ion containing 150mM, 95 DEG C of annealing 5min form the stranded structures of quaternary G tetra-.Then the structure and ferroheme are mixed, forms DNA enzymatic.By the temperature (25 95 DEG C) of programme-control reaction system, its catalytic activity is monitored, it was demonstrated that the catalytic performance of the enzyme at high temperature：10 hours at 75 DEG C, more than 50% activity can be kept, the enzyme shows the activity close to room temperature after 18 hours；5 minutes at 95 DEG C, activity does not change, and more than 50% activity can be kept after 1.5 hours, the enzyme shows the activity close to room temperature after 4 hours.The serobila DNA enzymatics of quaternary G tetra- that this is thermophilic are prepared simply, and cheap, heat endurance is good, are easy to storage, with certain application prospect.
Description
First, technical field
The present invention strengthens catalytic activity and new thermophilic with high catalytic activity at high temperature for a kind of adenine ortho position
Serobila DNA enzymatics of quaternary G tetra- and preparation method thereof.
2nd, background technology
Because protein is temperature sensitive, stability is poor, and protease is above restricted in application.Searching, discovery can be
Have the enzyme of high catalytic capability, high stability significant under the conditions of extreme environment such as high temperature.At present, it is most of artificial
The nano-particle with catalytic activity of synthesis, such as Au-SiO2Deng having shown stability more more preferable than protease.But this kind of material
Expect that building-up process is complicated, pattern requirement is strict, easily aggregation.In recent years, the DNA analogue enztmes with catalytic activity are closed extensively
Note.It can be formed after the serobilas of intramolecular G tetra- by the DNA sequence dna rich in G bases, simply be generated with reference to ferroheme (hemin), but
Catalytic activity is relatively low.By adding exogenous accelerator or can effectively being carried in groups such as the end modified cytimidines of DNA sequence dna
Its high catalytic activity.However, the serobila heat endurances of intramolecular G tetra- are poor, the DNA enzymatic of formation can not play it at high temperature and be catalyzed work(
Energy.The serobilas of quaternary G tetra- formed by four chains have good heat endurance, but due to the sedimentation of G planes, it is suppressed that
Hemin and the serobilas of quaternary G tetra- combination, cause its catalytic activity low.Thus, the catalytic action of DNA analogue enztmes at high temperature is fresh
Have been reported that.
The present invention first generates the chains of quaternary G tetra- using ammonium ion in the end modified adenine of the DNA sequence dna rich in G bases
Body structure, the serobila DNA enzymatics of quaternary G tetra- are combined to form with hemin.Adenine on ortho position can significantly improve the catalysis of DNA enzymatic
Activity, while remaining the heat endurance of the serobilas of quaternary G tetra-.This DNA enzymatic synthetic method is simple, catalytic activity is high, thermally-stabilised
Property it is good, be easy to preserve, had great application prospect in commercial Application.
3rd, the content of the invention
Present disclosure is：In two terminal modified adenines of the DNA sequence dna rich in G bases, in the ammonium ion containing 150mM
By 95 DEG C of stranded structures of annealing 5min formation quaternarys G tetra- in buffer solution, then the structure and ferroheme are mixed, formed thermophilic
The serobila DNA enzymatics of quaternary G tetra-.
The present invention is achieved through the following technical solutions：
(1) as shown in figure 1, in the terminal modified A bases of DNA sequence dna two rich in 4,5,6 or 7 G, by it in ammonium containing 150mM
Annealed 5 minutes for 95 DEG C in the buffer solution of ion, be cooled to room temperature, form the stranded structures of quaternary G tetra-.
(2) serobilas of quaternary G tetra- and hemin of formation are mixed, incubated 1 hour at room temperature, form the thermophilic chains of quaternary G tetra-
Body DNA enzymatic (Fig. 2).
(3) by the temperature (25-95 DEG C) of program calling and controlling reaction solution, H is catalyzed using DNA enzymatic2O2- ABTS (2 '-hydrazine-
Double -3- ethyl benzo thiazole phenanthrolines -6- sulfonic acid) reaction, monitor the catalytic activity under its different temperatures with AAS.
Compared with prior art, the invention has the characteristics that：
The present invention is by adenine modification in the end of rich G base sequences, and the serobila combination hemin of quaternary G tetra- of formation are built
Adenine on the thermophilic serobila DNA enzymatics of quaternary G tetra-, ortho position can significantly improve the catalytic activity of DNA enzymatic, while remaining four again
The heat endurance of first stranded structures of G tetra-.Relative to existing DNA enzymatic, have the characteristics that：
(1) DNA enzymatic has the advantages that building-up process is simple, cheap, heat endurance is good, is easy to preserve.
(2) DNA enzymatic has high catalytic activity at a wide range of temperatures, especially at a temperature of higher than 75 DEG C.
10 hours at (3) 75 DEG C, more than 50% activity can be kept, the enzyme shows the work close to room temperature after 18 hours
Property；5 minutes at 95 DEG C, activity does not change, and more than 50% activity can be kept after 1.5 hours, the enzyme, which is shown, after 4 hours connects
The activity of nearly room temperature.
4th, illustrate
Fig. 1 build the signal of the stranded structures of quaternary G tetra- with the rich G base sequences (exemplified by 5 G) of end modified adenine
Figure
The formation for the serobila DNA enzymatics of quaternary G tetra- that Fig. 2 are thermophilic and catalytic reaction schematic diagram
5th, embodiment
Embodiment 1：With reference to accompanying drawing 1, illustrate the formation of the stranded structures of quaternary G tetra-
The DNA sequence dna rich in G bases of two terminal modified adenines, in the Tris-HCl buffer solutions containing 150mM ammonium ions
In 95 DEG C of (0.01M buffer solutions, pH 7.0) annealing, room temperature is cooled to after 5 minutes, the stranded structures of quaternary G tetra- are formed.
Embodiment 2：With reference to accompanying drawing 2, illustrate the formation of the thermophilic serobila DNA enzymatics of quaternary G tetra- and its be catalyzed H at high temperature2O2-
ABTS reaction.
(1) preparation of enzyme solutions：The stranded structures of quaternary G tetra- and hemin of formation are blended in X- containing 0.05%Triton
In 100 and 1%DMSO 0.01M Tris-HCl buffer solutions (pH 7.0), ultimate density is respectively 0.86 μM and 1.72 μM, room
Temperature is incubated 1 hour.
(2) temperature of control enzyme solutions adds 1.28mMH between 25-95 DEG C2O2Reacted 5 minutes with 1.28mMABTS,
Its enzymatic activity, the resistance to elevated temperatures of studying enzyme are monitored with ultraviolet-uisible spectrophotometer.
Claims (6)
1. a kind of adenine ortho position enhancing catalytic activity and the new thermophilic serobilas of quaternary G tetra- at high temperature with high catalytic activity
DNA enzymatic.It is characterized in that the enzyme be using two terminal modified adenine bases the DNA sequence dna rich in G bases ammonium containing 150mM from
95 DEG C of annealing 5min in the buffer solution of son, the stranded structures of quaternary G tetra- formed after cooling are combined and formed with ferroheme.
2. DNA enzymatic according to claim 1, it is characterised in that the DNA sequence dna for forming enzyme contains 4,5,6 or 7 G alkali
Base, two terminal modified adenine bases.
3. DNA enzymatic according to claim 1, it is characterised in that the DNA sequence dna is annealed 5 minutes at 95 DEG C and is cooled to room
Temperature, forms the stranded structures of quaternary G tetra-.
4. DNA enzymatic according to claim 1, it is characterised in that it is formed by the stranded structure combination ferrohemes of quaternary G tetra-.
5. DNA enzymatic according to claim 1, it is characterised in that it still has peroxidase at a temperature of up to 95 DEG C
Characteristic, H can be catalyzed2O2- ABTS reactions produce color change.
6. DNA enzymatic according to claim 1,10 hours at 75 DEG C, more than 50% activity can be kept, should after 18 hours
Enzyme shows the activity close to room temperature；5 minutes at 95 DEG C, activity does not change, and more than 50% work can be kept after 1.5 hours
Property, the enzyme shows the activity close to room temperature after 4 hours.These features make it have certain application prospect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710491309.9A CN107119031B (en) | 2017-06-21 | 2017-06-21 | Novel thermophilic quaternary G quadruplex DNA enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710491309.9A CN107119031B (en) | 2017-06-21 | 2017-06-21 | Novel thermophilic quaternary G quadruplex DNA enzyme |
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| CN107119031A true CN107119031A (en) | 2017-09-01 |
| CN107119031B CN107119031B (en) | 2022-12-27 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111705044A (en) * | 2020-05-23 | 2020-09-25 | 南京大学 | Construction of novel controllable high-activity G quadruplex DNA enzyme |
| CN114703192A (en) * | 2022-05-18 | 2022-07-05 | 中南林业科技大学 | Application of adjacent base pair enhanced deoxyribozyme and biosensing and biosensor |
Citations (5)
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|---|---|---|---|---|
| CN104630363A (en) * | 2015-02-05 | 2015-05-20 | 山东大学 | Method for detecting activity of uracil-DNA glycosylase (UDG) based on fluorescence amplification strategy of label-free non-enzyme DNA machines |
| CN104774857A (en) * | 2015-04-21 | 2015-07-15 | 中国科学院上海高等研究院 | DNA (deoxyribonucleic acid) three-dimensional nano-structure artificial enzyme precursor, and preparation and application thereof |
| US20150197804A1 (en) * | 2013-12-30 | 2015-07-16 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Lt | Compositions, kits, uses and methods for amplified detection of an analyte |
| WO2016168656A1 (en) * | 2015-04-15 | 2016-10-20 | Ji Hoon Lee | Aptasensor and method of detecting target material |
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| US20150197804A1 (en) * | 2013-12-30 | 2015-07-16 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Lt | Compositions, kits, uses and methods for amplified detection of an analyte |
| CN104630363A (en) * | 2015-02-05 | 2015-05-20 | 山东大学 | Method for detecting activity of uracil-DNA glycosylase (UDG) based on fluorescence amplification strategy of label-free non-enzyme DNA machines |
| WO2016168656A1 (en) * | 2015-04-15 | 2016-10-20 | Ji Hoon Lee | Aptasensor and method of detecting target material |
| CN104774857A (en) * | 2015-04-21 | 2015-07-15 | 中国科学院上海高等研究院 | DNA (deoxyribonucleic acid) three-dimensional nano-structure artificial enzyme precursor, and preparation and application thereof |
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| TIANJUN CHANG等: "Activity Enhancement of G-Quadruplex/Hemin DNAzyme by Flanking d(CCC)", 《CHEM. EUR. J.》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111705044A (en) * | 2020-05-23 | 2020-09-25 | 南京大学 | Construction of novel controllable high-activity G quadruplex DNA enzyme |
| CN111705044B (en) * | 2020-05-23 | 2022-12-27 | 南京大学 | Construction of novel controllable high-activity G quadruplex DNA enzyme |
| CN114703192A (en) * | 2022-05-18 | 2022-07-05 | 中南林业科技大学 | Application of adjacent base pair enhanced deoxyribozyme and biosensing and biosensor |
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| CN107119031B (en) | 2022-12-27 |
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