CN1045448C - Using seryl histidine, phosphorylated serine, phosphorylated threonine as nucleic acid chipping agent - Google Patents
Using seryl histidine, phosphorylated serine, phosphorylated threonine as nucleic acid chipping agent Download PDFInfo
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- CN1045448C CN1045448C CN96114313A CN96114313A CN1045448C CN 1045448 C CN1045448 C CN 1045448C CN 96114313 A CN96114313 A CN 96114313A CN 96114313 A CN96114313 A CN 96114313A CN 1045448 C CN1045448 C CN 1045448C
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Abstract
The present invention relates to a cutting agent using seryl histidine, phosphorylation serine and phosphorylation threonine as DNA and RNA. The cutting agent has the following cutting conditions: the cutting agent is dissolved in sterile high purity water for being prepared into solutions; the solutions are mixed with cutting substrates; the pH value of the solutions and the cutting substrates is adjusted by buffer solutions; the final concentration of buffering agents and the substrates is kept within a certain range; the buffering agents and the substrates are placed at certain temperature, and temperature is kept; then, the cutting to substrates is realized. The cutting agent of the present invention can cutting ribonucleic acid at wide temperature and within a wide pH value range and can be used artificial tool enzymes and gene treating medicines studied by molecular biology after being coupled with nucleic acid specific sequence recognition systems.
Description
The present invention relates to the new purposes of seryl Histidine, phosphorylated Serine, phosphorylated threonine as nucleic acid cutting agent, belong to biological technical field.
DNA (thymus nucleic acid) and RNA (Yeast Nucleic Acid) are the genetic material of organism.To the research of its 26S Proteasome Structure and Function, be one of main contents of molecular biology research.Study nucleic acid construct and function relationship, just need carry out various operations nucleic acid, as to the cutting of nucleic acid chains be connected etc.And be requisite process in the nucleic acid operating process to the cutting of nucleic acid chains.At present, the kind in order to the nucleic acid cleavage agent of nucleic acid chains cutting has isolating various natural acid enzymes or the simulation nuclease of synthetic and the chemical reagent of other kinds from organism.At this, the reagent of the nucleic acid chains that can rupture by any way is referred to as nucleic acid cleavage agent.Outline as follows:
(1) natural acid enzyme, promptly naturally occurring enzyme with hydrolysed nucleic acid phosphodiester bond function.
Wherein restriction enzyme is the hydrolase nucleic acid that a class can be discerned specific base sequence in the double-stranded DNA, and its recognition sequence is generally the palindrome of 4 or 6 base pairs, and the fragment that cutting produces is shorter, is difficult to obtain complete gene fragment.The nucleic acid fragment that other endonuclease and excision enzyme cutting produce is then shorter.Therefore, existing natural acid enzyme can not satisfy research and need fix a point during gene to cut to obtain the requirement of big fragment gene bunch.
(2) metal and complex compound thereof
A, with the cutting agent of free radical mechanism cutting nucleic acid: comprising EDTA-Fe (II), Cu (II)-mercaptan, 1,10-phenanthroline-Cu (I), xitix-Cu (II), Cu (II)-Gly-Gly-His, CNBr-methyl thioether, Mn (II)-methyl hydrazine, etc.This class cutting agent is by producing the free radical nucleic acid chains that ruptures, can produce living radical, and itself will cause the injury to organism, can not be used for gene therapy.
B, with the cutting agent of transesterification mechanism cutting nucleic acid: comprising Zn (II), Cu (II), Co (III) and trivalent lanthanide metal ion, i.e. (La, Ce, Eu, Gd, Tb, amine complex compound Lu).Though simple Ln
A+Can effectively promote the cutting of RNA, still, must select suitable part just can obtain the metal complex of catalytic activity meticulously.On the other hand, part must be thermodynamically stable, and requiring again on kinetics is inert, to prevent the release of metal ion.In addition, metal complex need just can reach the purpose of fixed point cutting equally with the recognition system coupling.Therefore, metal ion can be combined on the aglucon that links to each other with suitable identifier, remain a key.As if the treatment that metal complex is used under the physiological condition, just more strict.
C. cut the cutting agent of nucleic acid with hydrolysis phosphodiester bond mechanism: comprising Co (III), Ln (III) etc.At present report all needs higher temperature.
(3) contain the compound of imidazolyl
This class cutting agent mainly is to utilize imidazolyl agent reaches the purpose of hydrolysis RNA as general acid-base catalysis.But be not suitable for cutting DNA.
The objective of the invention is existing seryl Histidine, phosphorylated Serine, phosphorylated Threonine are used for the nucleic acid cutting.The limited amount of natural acid enzyme own, the fragment that the effect back produces is too little; And there are all deficiencies in above-described chemical chop agent on using, the present invention is according to the cutting mechanism of natural acid enzyme active center to nucleic acid, the active group that utilizes natural amino acid, little peptide and derivative thereof to have, with hydrolysis mechanism cutting nucleic acid, thereby obtain the hydrolysis end identical, provide feasibility for being applied to molecular biology with the natural enzyme hydrolysis products.In addition, in order to be applied to gene therapy, antiviral, antitumor etc., natural amino acid, little peptide and derivative thereof that the present invention uses can not bring toxic side effect.
Content of the present invention is the structure according to the natural enzyme active centre, designs and synthesizes the compound of tool DNA (thymus nucleic acid), RNA (Yeast Nucleic Acid) cutting function, belongs to biological high-technology and explores the field.The present invention adopts seryl Histidine (Ser-His dipeptides), the Histidine solution cutting DNA and the RAN of phosphorylated Serine, phosphorylated Threonine.
1, Ser-His cutting DNA and RNA
Ser-His is dissolved in aseptic high purity water, wiring solution-forming.This solution is mixed with the cutting substrate, and it is double-stranded linear DNA (selecting λ DNA among the present invention for use), super spirial plasmid DNA (selecting pUC8 and pBR322 plasmid DNA among the present invention for use), RNA (selecting poly and UpolyA among the present invention for use) that the nucleic acid that is cut promptly cuts substrate.Bloomsbury with 20mM (mmole/liter) is smooth-and Robison (Britton-Robison) (be made up of phosphoric acid, acetate, boric acid, its concentration is respectively 0.04M) damping fluid is as buffer system, with the scope of sodium hydroxide adjust pH at 4.0-9.0.The final concentration of Ser-His is 1-200 mmole/liter (mM), and the final concentration of substrate is 0.05-50mM (concentration by single base is calculated).The mixture of Ser-His and substrate is placed 10-80 ℃ of insulation down, and insulation 0-96h takes out, and promptly finishes the cutting to substrate.DNA after the cutting is carried out agarose gel electrophoresis detect, RNA adopts ultraviolet spectrophotometry to detect, and its result is as follows: not obvious in the phenomenon of cutting below 30 ℃; In the time of 37 ℃, the Ser-His of 5mM promptly has tangible cutting phenomenon behind insulation 24h.Along with the raising of temperature raising or Ser-His concentration, cutting speed in feet per minute is accelerated; At 50 ℃ of insulation 4h, tangible cutting phenomenon is just arranged.In above-mentioned pH value scope, Ser-His has nicking activity.When the pH value was 7 left and right sides, cutting speed in feet per minute was the fastest.
2, phosphorylated Serine, phosphorylated Threonine cutting DNA and RNA
Phosphorylated Serine of the present invention is N-O, O-di-isopropyl phosphorylated Serine (DIPP-Ser), N-O, O-di-n-butyl phosphorylated Serine (DBP-Ser) and N-O, O-solutions of dimethyl phosphoryl Serine (DMP-Ser).Phosphorylated Threonine of the present invention is N-O, O-di-isopropyl phosphorylated Threonine (DIPP-Thr), N-O, O-di-n-butyl phosphorylated Threonine (DBP-Thr).With aseptic high purity water compound concentration is the saturated Histidine solution of 280mM, is that phosphorylated Serine, phosphorylated Threonine are dissolved in wherein wiring solution-forming with cutting agent.This solution and the nucleic acid that is cut are promptly cut substrate to be mixed, the cutting substrate is double-stranded wire NDA (selecting λ DNA among the present invention for use), super spirial plasmid DNA (selecting the pUC8 plasmid DNA among the present invention for use), RNA (selecting yeast tRNA among the present invention for use), with Histidine as buffer system, the pH value is adjusted at about 7.5, and the concentration range of histidine buffering liquid is 10-280mM.The final concentration of cutting agent is 0.5-200mM, and the final concentration of substrate is 0.05-50mM (concentration by single base is calculated).The mixture of cutting agent, Histidine and substrate is placed 10-80 ℃ of insulation down, and insulation 0-96h takes out and promptly finishes the substrate cutting.To the solution after the cutting, carry out agarose gel electrophoresis and detect, its result is as follows: at 37 ℃ of insulation 24h, the DIPP-Ser of 0.5mM promptly has tangible cutting phenomenon to λ DNA; Super coiled DNA can be cut into wire; TRNA cuts into pieces with yeast.Along with temperature improves or the increase of phosphorylated serine concentration, cutting speed in feet per minute is accelerated.
The present invention has found Ser-His, phosphoryl amino acid can cutting DNA and RNA.It is the new nucleic acid cutting reagent of a class.With itself and the coupling of nucleic acid recognizing system, can become nucleic acid fixed point cutting reagent, as the artificial toolenzyme and the gene therapy medicament of molecular biology research.Cutting agent of the present invention can cut nucleic acid in the temperature of broad and pH scope, range of application is wider.But owing to phosphoryl amino acid, little peptide autoactivation, transesterify on the multiple imitation biochemistry reaction-phosphorus takes place, have peptide of one's own, become ester, phosphoryl N → O transposition etc.Its reactive behavior is regulated and control by the precision of side chain functionalities, and reaction type changes with the variation of phosphoryl, amino acid and reaction medium, temperature, pH value.Thereby they have the enzyme feature.
Embodiment:
1, cut the wire double-stranded DNA with Ser-His (seryl Histidine) in the aqueous solution: the working concentration of Ser-His is 5mM, 7.5mM, 15mM, 20mM; The effect substrate is λ DNA, and concentration of substrate is 0.15mM (in a base pair), and temperature of reaction is 37 ℃, and the pH value of reaction system is 7.0, and the reaction times is 0-96h.Cutting is the result detected by agarose gel electrophoresis.Behind the insulation 48h, all visible significantly cutting of 4 kinds of concentration phenomenons, and with the increase or the holding time prolonging of concentration, the cutting degree increases gradually.
The relation of nicking activity and pH value: the pH value that experiment is adopted is pH4.98, pH6.05, and pH6.3, pH6.7, pH7.0, pH8.0, soaking time is 50h.About pH7, cutting speed in feet per minute is the fastest.
The relation of nicking activity and temperature: the temperature that experiment is adopted is-20 ℃, 10 ℃, and 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃.Be incubated 45h in the Britton-Robison of pH6.7 damping fluid, cutting is the result detected by agarose gel electrophoresis.Temperature raises, and cutting speed in feet per minute is accelerated.Be incubated 45h below 30 ℃, cut not obvious.40 ℃ of insulation 45h have tangible cutting phenomenon.In the time of 50 ℃, as seen insulation 4h promptly cuts phenomenon; Insulation 8h, the cutting phenomenon is obvious.
2, the working concentration that cuts super spirial plasmid DNA:Ser-His with Ser-His in the aqueous solution is 10mM, 20mM, the effect substrate is pBR322 plasmid DNA, pUC8 plasmid DNA, concentration of substrate is 0.20mM (in a base pair), temperature of reaction is 37 ℃, the pH value of reaction system is 7.0, and the reaction times is 0-96h.Cutting is the result detected by agarose gel electrophoresis, for pBR322DNA, and insulation 24h, promptly visible significantly cutting phenomenon; For pUC8DNA, most of superhelix form has been cut into the wire form behind the 24h, all is cut into wire behind the 48h, behind the 72h as seen by the wire section of cutting into pieces.Ser-His concentration increases, and cutting speed in feet per minute is accelerated.
1,2 as seen by, Ser-His concentration increases, and cutting speed in feet per minute is accelerated; Temperature of reaction improves, and cutting speed in feet per minute is accelerated; Ser-His has cutting action to double-stranded DNA in certain pH range.Except testing the condition in 1 and 2, Ser-His also might (such as change buffer system under same pH value) have nicking activity to DNA and RNA under conditions of similarity.
3, the working concentration that cuts RNA:Ser-His with Ser-His in the aqueous solution is 10mM, and the effect substrate is polyA and polyU, and concentration of substrate is 0.06mM (in a base), and temperature of reaction is 37 ℃, the pH7.0 of reaction system, and the reaction times is 0-60h.Can increase because nucleic acid is cut the back uv-absorbing, cutting is the result detected by ultraviolet spectrophotometry.Absorbancy is mapped to soaking time, can get a near linear, increased value is 3.6 * 10
-3/ h.
4, with N-DIPP-Ser (N-O; O-di-isopropyl phosphorylated Serine) Histidine solution cutting wire double-stranded DNA: working concentration 0.1mM, the 0.2mM of N-DIPP-Ser, 0.6mM, 0.9mM, 1.3mM; the working concentration of Histidine is 200mM-280mM; the effect substrate is λ DNA; the concentration of substrate is 0.2mM (in base pair); temperature of reaction is 37 ℃, reaction pH7.5, and the reaction times is 24h.Cutting is the result detected by agarose gel electrophoresis, and the N-DIPP-Ser of each concentration has the cutting phenomenon, and along with the increase of concentration, the cutting degree obviously increases, and the fragment that cutting produces reduces gradually.The same DIPP-Ser of cutting condition of DBP-Ser, DMP-Ser.The nicking activity size order is DIPP-Ser＜DBP-Ser＜DMP-Ser when same concentration.
5, with N-DIPP-Ser (N-O; O-di-isopropyl phosphorylated Serine) the working concentration 1.1mM of Histidine solution cutting super spirial plasmid DNA:N-DIPP-Ser; 3.3mM; the working concentration of Histidine is 200mM-280mM; the effect substrate is the pUC8 plasmid DNA, and concentration of substrate is 0.2mM (in a base pair), and temperature of reaction is 37 ℃; reaction pH7.0, the reaction times is 24-48h.Cutting is the result detected by agarose gel electrophoresis, and the DIPP-Ser of 3.3mM all is cut into the wire form with the DNA of superhelix form behind the 48h.The same DIPP-Ser of cutting condition of DBP-Ser, DMP-Ser.The nicking activity size order is DIPP-Ser＜DBP-Ser＜DMP-Ser when same concentration.
6, using the working concentration of the Histidine solution cutting RNA:N-DIPP-Ser of N-DIPP-Ser (N-O, O-di-isopropyl phosphorylated Serine) is 0.5mM, 1.0mM, 1.5mM, 2.0mM, 2.5mM; The working concentration 280mM of Histidine, the effect substrate is a yeast rna, and concentration of substrate is 0.5mM (in a base), and temperature of reaction is 37 ℃, the pH7.5 of reaction system, the reaction times is 17.5h.Cutting is the result detected by agarose gel electrophoresis, compared with the control, under five kinds of concentration cutting arranged all, and along with the increase of DIPP-Ser concentration, the cutting degree strengthens gradually.The same DIPP-Ser of cutting condition of DBP-Ser, DMP-Ser.The nicking activity size order is DIPP-Ser＜DBP-Ser＜DMP-Ser when same concentration.
4,5,6 as seen by, DIPP-Ser, DBP-Ser, DMP-Ser can cutting double-stranded DNA and RNA in Histidine solution.And with the increase of concentration, cutting speed in feet per minute is accelerated; Improve temperature, nicking activity improves.
7, be 0.5mM with the Histidine solution cutting super spirial plasmid DNA of N-DIPP-Thr and the working concentration of wire double-stranded DNA: N-DIPP-Thr, 1mM, 3mM, 5mM, 10mM, the working concentration of Histidine is 20mM-280mM, the effect substrate is pUC8 plasmid DNA, λ DNA, and concentration of substrate is 0.2mM (in a base pair), and temperature of reaction is 37 ℃, reaction pH7.5, the reaction times is 24h.Cutting is the result detected by agarose gel electrophoresis, and the DIPP-Thr of 10mM all is cut into the wire form with the DNA of superhelix form behind the 24h.And along with the increase of DIPP-Thr concentration, cutting speed in feet per minute is accelerated.For the λ DNA of wire, visible significantly cutting phenomenon behind the insulation 24h.The same DIPP-Thr of cutting condition of DBP-Thr.The nicking activity size order is DIPP-Thr＜DBP-Thr when same concentration.And along with the increase of phosphorylated Threonine concentration, cutting speed in feet per minute is accelerated; Improve temperature, cutting speed in feet per minute is accelerated.
Claims (2)
1, a kind of with the purposes of seryl Histidine as nucleic acid cleavage agent, it is characterized in that the nucleic acid that is cut is DNA or RNA, its cutting process is: the seryl Histidine is dissolved in aseptic high purity water, wiring solution-forming, this solution and the nucleic acid that is cut are promptly cut substrate to be mixed, the cutting substrate is double-stranded linear DNA, super spirial plasmid DNA and RNA, Bloomsbury with 20mM (mmole/liter) is smooth-Robison (Britton-Robison), by phosphoric acid, acetate, boric acid is formed, its concentration is respectively the 0.04M damping fluid as buffer system, with the scope of sodium hydroxide adjust pH at 4.0-9.0, the final concentration of seryl Histidine is-200 mmoles/liter (mM), the final concentration of substrate is 0.05-50mM (concentration by single base is calculated), the mixture of seryl Histidine and substrate is placed 10-80 ℃ of insulation down, insulation 0-96h takes out, and promptly finishes the cutting to substrate.
2; a kind of with the phosphorylated Serine; the phosphorylated Threonine is as the purposes of nucleic acid cleavage agent; it is characterized in that the nucleic acid that is cut is DNA territory RNA; cutting agent phosphorylated Serine is N-O; O-di-isopropyl phosphorylated Serine (DIPP-Ser); N-O; O-di-n-butyl phosphorylated Serine (DBP-Ser) and N-O; O-solutions of dimethyl phosphoryl Serine (DMP-Ser); cutting agent phosphorylated Threonine is N-O; O-di-isopropyl phosphorylated Threonine (DIPP-Thr) and N-O; O-di-n-butyl phosphorylated Threonine (DBP-Thr); its cutting process is: be the saturated Histidine solution of 280mM with aseptic high purity water compound concentration; with cutting agent is the phosphorylated Serine; the phosphorylated Threonine is dissolved in wherein; wiring solution-forming; then this solution and the nucleic acid that is cut promptly being cut substrate mixes; the cutting substrate is double-stranded linear DNA; super spirial plasmid DNA and RNA; with Histidine as buffer system; the pH value is adjusted at about 7.5; the concentration range of histidine buffering liquid is 10-280mM; the final concentration of cutting agent is 0.5-200mM; the final concentration of substrate is 0.05-50mM (concentration by single base is calculated); at last with cutting agent; the mixture of Histidine and substrate places 10-80 ℃ of insulation down, and insulation 0-96h takes out and promptly finishes the substrate cutting.
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| CN96114313A CN1045448C (en) | 1996-12-13 | 1996-12-13 | Using seryl histidine, phosphorylated serine, phosphorylated threonine as nucleic acid chipping agent |
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| CN96114313A CN1045448C (en) | 1996-12-13 | 1996-12-13 | Using seryl histidine, phosphorylated serine, phosphorylated threonine as nucleic acid chipping agent |
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| CN106046047B (en) * | 2016-05-26 | 2018-10-02 | 清华大学 | The method for preparing phosphorylation serine phosphonic acids analogies |
| CN108484754B (en) * | 2018-03-26 | 2021-02-19 | 南华大学 | Preparation and application of artificial metallohydrolase based on metal ion-myoglobin mutant compound |
| CN111229327B (en) * | 2020-03-10 | 2022-11-29 | 南华大学 | Artificial metalloenzyme and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN87100610A (en) * | 1986-02-05 | 1987-09-09 | 瓦克劳·西巴尔斯基 | General retriction endonuclease |
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| CN87100610A (en) * | 1986-02-05 | 1987-09-09 | 瓦克劳·西巴尔斯基 | General retriction endonuclease |
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