CN104611318B - A kind of multienzyme complex and method of regulation and control nucleotide sequence selectivity - Google Patents
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Abstract
The invention discloses a kind of multienzyme complex and method of regulation and control nucleotide sequence selectivity, establish a kind of brand-new chemical regulation means, make to obtain sequence selectivity without the nuclease (DNase and RNase) of sequence-specific originally, and can be according to the sequence selectivity for needing flexible modulation enzyme of required cutting sequence.So as to provide strong new tool for molecular biology research, drug development and technique for gene engineering exploitation etc..
Description
Technical field
The invention belongs to technical field of bioengineering, is related to the field that artificial regulatory is carried out to the property of enzyme, and in particular to
A kind of multienzyme complex and method of regulation and control nucleotide sequence selectivity.
Background technology
The substrate selective and catalytic activity of artificial regulatory enzyme all have in terms of life science and application of biological engineering
There is important meaning.On the one hand, enzyme is as natural biocatalyst, and its catalytic performance is powerful, especially in catalysis macromolecular
In reaction, catalyst type artificial synthesized at present is also considerably less, and enzyme has irreplaceable extensive use.On the other hand, enzyme
Long preparation period, yield is small, the limitednumber of existing enzyme, far can not also meet the needs of practical application.Therefore, to existing
Enzyme transformed, so as to obtain new property, can greatly widen the application of enzyme, for correlative study and application provide
Strong technological means.The method of artificial regulatory enzymatic property the most frequently used at present is mainly to be changed by the means of molecular biology
The amino acid sequence of enzyme, and the rare report of method modified after being carried out to enzyme.
Nuclease (nuclease) is the class of enzymes hydrolyzed using nucleic acid as substrate, catalytic phosphatase diester linkage.Nuclease includes
Deoxyribonuclease (DNase) and ribalgilase (RNase).The sequence selectivity of nuclease has emphatically in genetic engineering
The application wanted, the DNA or RNA of certain sequence are specifically cut, the more convenient neatly shearing of people, assembly can be helped or gone
Except specific DNA or RNA fragments, a variety of applications are realized.Restriction enzyme is the sequence selection being most widely used at present
Property nickase, they can identify that particular sequence is cut, and be toolenzyme important in laboratory research and genetic engineering.But
It is that the restriction enzyme having been found that only has kind more than 300, every kind of restriction enzyme can only identify a kind of specific sequence, because
This sequence that can be identified and selectively cut also only has kind more than 300.Many research groups are directed to by being mutated amino always
Acid (Bloom, J.et al.Evolving strategies for enzyme engineering.Current Opinion
In Structural Biology, 2005,15,447-452) or protein fusion technology (Koide, S.Generation of
new protein functions by nonhomologous combinations and rearrangements of
Domains and modules.2009, Curr Opin Biotech 20,398-404) it is next artificial reconstructed existing restricted
Restriction endonuclease, it is desirable to obtain new recognition sequence.But the workload of these methods is big, and does not possess versatility.Therefore need to open
The more simple and effective control measures of hair, make nuclease obtain more rich and varied sequence selectivity, so that in actual applications,
Different target cutting sequences can be directed to, realizes corresponding selective regulation.
The content of the invention
It is an object of the invention to provide a kind of multienzyme complex and method of regulation and control nucleotide sequence selectivity, establish a kind of complete
New chemical regulation means, make to obtain sequence selectivity without the nuclease (DNase and RNase) of sequence-specific originally, and
Can be according to the sequence selectivity for needing flexible modulation enzyme of required cutting sequence (following to be referred to as " target sequence ").
During the present invention is established, applicants have discovered that an important phenomenon：
As shown in figure 1, by the complete thio DNA single-stranded (being represented with S-DNA) of a phosphate backbones with DNase I according to certain
Ratio mixing, 1h is reacted at 37 DEG C, the DNase I@S-DNA obtained after ultrafiltration purification can only cut the complementation of the S-DNA
Chain, and almost no longer cut the DNA of the unrelated single stranded DNA of other sequences, double-stranded DNA and hair clip type structure.It is this by wide spectrum sequence
Row cleavage activity shows that S-DNA is acted on DNase I template-mediated, i.e., to the change of properties only cut to particular sequence
S-DNA and DNase I form stable compound, and cuttings of the DNase I to independent object chain is inhibited in structure, and only
Stronger cleavage activity is shown to the complementary strand of the S-DNA template strands.Test and also found, the combination of DNase I and S-DNA chains
Act on it is highly stable, room temperature place one day after, DNase I@S-DNA still show sequence selectivity.
In order to compare, applicant use without any processing normal DNase I act on above-mentioned unrelated DNA and
S-DNA complementary strand, find to show stronger cleavage activity to two kinds of chains.Applicant further applies this method to
On RNaseA, RNaseA and complete thio RNA chains (S-RNA) are blended in one hour of incubation reaction at 37 DEG C by a certain percentage
Afterwards, ultrafiltration removes unnecessary S-RNA, and the RNaseA@S-RNA compounds of acquisition still have very high to S-RNA complementary strand
Cleavage activity, but the cleavage activity of other unrelated RNA chains is suppressed by very big, show the S-RNA combined with RNaseA
Acted on template-mediated.
The important discovery of applicant can be summarized as：Thio DNA is single-stranded entirely can form closely with DNase I under certain condition
With reference to DNase I@S-DNA compounds, suppress DNase I and cut the activity of other DNAs, but repressed DNase I exist
When running into the complementary dna chain of the complete thio chain, activity can restart, the fly-cutting chain.The method is also applied for RNase A,
Other unrelated RNA sequences are no longer cut with reference to the RNase A S-RNA that complete thio RNA chains are formed, and only to the mutual of the S-RNA chains
Mend chain and show cleavage activity.The above-mentioned single-stranded and complete thio RNA of complete thio DNA being combined respectively with DNase I and RNaseA
Referred to as complete thio template strand below chain.
According to above-mentioned important discovery, the present invention provides a kind of regulation and control nuclease (DNase I/RNaseA) sequence selectivity
Multienzyme complex, the multienzyme complex is the compound of nuclease and complete thio template strand, available for selectivity cutting and the full sulphur
For the complementary target sequence of template strand.The mol ratio of the nuclease and the complete thio template strand is 1：1~1：10.
Further, the length of the complete thio template strand is between 15-50 nucleotides.
Further, it is non-covalent between the nuclease and the complete thio template strand or is covalently attached.
Further, the target sequence and the base number of complete thio template strand complementation are between 15-30, target sequence
The total length of row is unlimited.
A kind of method of regulation and control nucleotide sequence selectivity, comprises the following steps：
1) the design synthesis complete thio template strand complementary with target sequence.
2) by nuclease and complete thio template strand hybrid reaction in pH7.0-8.0 cushioning liquid, 1 is incubated at 37 DEG C
Hour.
3) the full sulphur to dissociate in above-mentioned reaction system is removed using milipore filter ultrafiltration of the cutoff between 20-40kD
For chain, nuclease@thio chain cpds entirely are obtained.
4) using nuclease@, thio chain cpd cuts target sequence entirely.
The sequence of the complete thio template strand used in the present invention is directly obtained by chemical method synthesis.Wherein complete thio mesh
Be to prevent the template strand by nuclease hydrolysis.Complete thio sequence is unsuitable long, can be shorter than target sequence to be cut.In order to
It is enough that free complete thio chain is effectively removed in ultra-filtration process, retain the compound of nuclease and complete thio chain, complete thio chain length
Degree control is more suitable below 50 nucleotides.The dosage of nuclease is 10 μ g magnitudes.
The reaction condition of nuclease and complete thio template strand is：Nuclease concentration 0.1mg/mL, the concentration 15- of complete thio chain
The mol ratio of 30 μM (preferably 29 μM), nuclease and complete thio chain is 1：1~1：10.Reaction temperature is 29-37 DEG C (preferably 37
DEG C), reaction time 45min-1h (preferably 1h).Reaction solution is neutral buffer (pH scopes：7.0-8.0 preferably 0.05M
Phosphate buffer, sodium chloride containing 0.15M, pH 7.2).Under these conditions, DNase I and complete thio DNA it is single-stranded and
RNaseA can form non-covalent stable bond thing, i.e. DNase I@S-DNA compounds and RNaseA@S- with complete thio RNA chains
RNA。
On this basis, further nuclease and complete thio template strand can also be covalently attached, make nuclease with
The combination of complete thio template strand is more firm.The covalent reaction type that can be used has：Complete thio template strand it is end modified-
SH, utilize SPDP (Chinese names：3- (2- pyridines dimercapto) propionic acid N-hydroxy-succinamide ester) or SMCC (Chinese names：Amber
Amber acid imide -4- thiacyclohexane -1- carbonic esters) it is catalyzed-the SH and amino on albumen reaction；Or repaiied in complete thio template chain end
Decorations-COOH, utilize EDC/NHS (Chinese names：1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides/N- hydroxysuccinimidyls
Acid imide) reaction of amino etc. on catalysis-COOH and albumen.Non-covalent linking or covalent attachment can obtain stabilization, have
The nuclease@of sequence selectivity thio chain cpds entirely, Comparatively speaking, non-covalent linking method are simpler easy.
The beneficial effects of the present invention are：We, which have developed, a kind of simple and easy makes nuclease obtain sequence selectivity
Method.Compound is formed by using different complete thio template strands and enzyme, different target sequences can be optionally cut, be
Molecular biology research, drug development and technique for gene engineering exploitation etc. provide strong new tool.
Brief description of the drawings
Fig. 1 is the original of complete thio DNA single-stranded (S-DNA) regulation and control deoxyribonuclease enzyme I (DNase I) sequence selectivity
Reason figure.
Fig. 2 (a) is that embodiment 1 uses S-DNA and after DNase I Non-covalent bindings, the DNase I@S-DNA of generation are cut
The target dna chain of fluorescence labeling and the real-time fluorescence curves figure of unrelated DNA.
Fig. 2 (b) is after DNase I@S-DNA cut unmarked target dna chain and unrelated DNA in embodiment 1, to production
Thing carries out the image of gel electrophoresis.
Fig. 3 (a) is after cutting target RNA chains using RNaseA@S-RNA in embodiment 2, to product
Carry out the image of gel electrophoresis.
Fig. 3 (b) is after cutting unrelated RNA chains using RNaseA@S-RNA in embodiment 2, and gel electrophoresis is carried out to product
Image.
Fig. 4 is after using S-DNA and DNase I covalent bonds in embodiment 3, and the DNase I@S-DNA of generation are covalently multiple
The target dna chain of compound cutting fluorescence labeling and the speed ratio (numbering 2 in figure) of the unrelated DNA of cutting in embodiment 2 with obtaining
Non-covalent complex cutting fluorescence labeling target dna chain and cut unrelated DNA speed ratio (numbering 1 in figure) control
Figure.
Embodiment
Below in conjunction with the accompanying drawings, the present invention is expanded on further by specific embodiment.Those skilled in the art should be understood
These embodiments are merely to illustrate the present invention and not limited the scope of the invention.
Embodiment 1<Regulate and control DNase I sequence selectivity>
In this embodiment, target dna chain-ordering is：
5'-TAT CTG CAC TAGATG CAC CTT-3'；
Unrelated DNA sequence is：
5'-CGGAAGACAGATGCUAAGTGCTTGACCTTCCG-3'；
The sequence of the complete thio DNA profiling chain (S-DNA) of the target dna sequence of synthesis is：
5'-AAAAAAAAAAAGGTGCATCTAGTGCAGATA-3'；
Above-mentioned S-DNA and DNase I combine to form the principle of DNase I@S-DNA and sequence selectivity measure referring to Fig. 1.
In the present invention, in order to effectively indicate DNase I/RNaseA to intending cutting target dna/RNA sequence and other nothings
The cutting situation of sequence is closed, applicant has used two methods：
1) fluorescence probe finger-length measurement.As shown in fig. 1, intend cutting target sequence and unrelated sequences marked fluorophor
And quencher, efficient FRET can occur between two kinds of groups.Common includes FAM and TAMRA；FAM and BHQ1；TET
With BHQ2；Cy3 and Cy5 etc..When above ditag sequence keeps complete, the fluorescence that fluorophor is sent after being excited is quenched
Group absorptions, so as to can't detect fluorescence.And after these sequences are cut by DNase I/RNaseA, quencher is away from fluorescence
Group, fluorescence can be detected caused by solution, so as to effectively instruction add sequence whether by with entirely thio chain combination
DNase I/RNaseA cutting and cleavage reaction speed and carry out degree.
2) agarose gel electrophoresis method.By the DNase I@S-DNA compounds or RNaseA@S- with complete thio chain combination
RNA compounds are with after unmarked target sequence and unrelated sequences reaction, taking out a small amount of reaction mixture (9 μ L) solution and carrying out agar
Sugared gel electrophoresis experiment.
Specific implementation step is as follows：
1) by 1 μ L 5mg/mL DNase I and 1.45nmol, thio DNA is single-stranded entirely is mixed in 50 μ L buffer solutions, buffers
Liquid forms：Solution is placed at 37 DEG C and incubated 1 hour by 50mM sodium phosphates, 150mM sodium chloride, pH 7.2..
2) the use of cutoff is 30kD, the super filter tube ultrafiltration 1 that processing volume is 500 μ L) in obtained solution.Ultrafiltration
Buffer solution used is the same as the buffer solution used in 1).Rotating speed used in first time ultrafiltration is 5000g, ultrafiltration time 15min.Second super
Filter rotating speed is 3000g, time 15min.
3) 3 μ L 2 are taken) in 50 μM of obtained DNase I@S-DNA and 1 μ L target dna chain or unrelated DNA mix
Afterwards, measure fluorescent value in real-time fluorescence PCR instrument (Rotor gene 6500) is put at once.
The target dna chain-ordering of fluorescence labeling is：
5'FAM-TAT CTG CAC TAGATG CAC CTT-3'BHQ-1
The unrelated DNA sequence of fluorescence labeling is：
5'FAM-CGGAAGACAGATGCUAAGTGCTTGACCTTCCG-3'Dabcyl。
4) the DNase I obtained by further being proved 3) using the experiment of 2.5% agarose gel electrophoresis possess sequence choosing
Selecting property.
Experimental result：
As shown in Fig. 2 (a), DNase I@S-DNA can relatively rapid cut target dna sequence, can not cut unrelated
DNA sequence dna.Common undressed DNase I can fly-cutting target dna sequence, the unrelated DNA sequences of and can fly-cutting
Row.
Six group of 2.5% agarose gel electrophoresis experiment is designed, wherein swimming lane 1-3 corresponds to the testing result of target sequence, because
Target sequence is single stranded DNA, is observed after being dyed with Gelsafe nucleic acid dyes.For strengthen fluorescent dyeing effect, three parts
The complementary strand that target sequence is added in reaction product solution is allowed to be hybridized to double-strand.Swimming lane 4-6 corresponds to the detection of unrelated sequences
As a result, because unrelated sequences itself are the hair clip type structure containing 6 complementary bases, easily it is colored, therefore need not it is possible to additionally incorporate
Complementary strand：
Swimming lane 1：Common DNase I cutting target dna sequences.
Swimming lane 2：DNase I@S-DNA compounds cut target dna sequence.
Swimming lane 3：Target dna sequence.
By the visual target DNA sequence dna of swimming lane 3 in itself with the band after complementary strand thereof and the band of unnecessary complementary strand.Swimming lane
1 and 2 displays, common DNase I and DNase I@S-DNA compounds can effectively cut target sequence, target sequence and complementation
The band of chain hybridization is almost disappeared, and the band of the complementary strand of addition is can only see in swimming lane.
Swimming lane 4：Common DNase I cutting extraneous DNA sequences.
Swimming lane 5：DNase I@S-DNA compounds cut extraneous DNA sequences.
Swimming lane 6：Extraneous DNA sequences.
From swimming lane 4, band has disappeared after extraneous DNA sequences are cut by common DNase, and swimming lane 5 shows DNase
I@S-DNA compounds hardly cut extraneous DNA sequences, and the band corresponding to it has almost no change.
Embodiment 2<Regulate and control RNaseA sequence selectivity>
In this embodiment,
Target RNA chain-orderings are：5'-UAUCUGCACUAGAUGCACCUU-3'；
Unrelated RNA chain-orderings are：5'-UUGUACUACACAAAAGUACUG-3'；
Synthesis complete thio RNA sequence be：
5'-AUACAGCUAAGGUGCAUCUAGUGCAGAUA-3'。
Specific implementation step is as follows：
1) by 1 μ L 2mg/mL RNaseA and 1.60nmol, thio RNA chains are mixed in 50 μ L buffer solutions entirely, buffer solution
Form and be：Solution is placed at 37 DEG C and incubated 1 hour by 50mM sodium phosphates, 150mM sodium chloride, pH 7.2..
2) the use of cutoff is 30kD, the super filter tube ultrafiltration 1 that processing volume is 500 μ L) in obtained solution.Ultrafiltration
Buffer solution used is the same as the buffer solution used in 1).Rotating speed used in first time ultrafiltration is 5000g, ultrafiltration time 15min.Second super
Filter rotating speed is 3000g, time 15min.
3) after 50 μM of the μ L of RNase A 3 obtained in taking 2) and 1 μ L target RNA chains or the mixing of unrelated RNA chains, at once
It is put into measure fluorescent value in real-time fluorescence PCR instrument (Rotor gene 6500).The target RNA chain-orderings of fluorescence labeling are：
5'FAM-UAUCUGCACUAGAUGCACCUU-3'BHQ-1；
The unrelated RNA chain-orderings of fluorescence labeling are：
5'-UUGUACUACACAAAAGUACUG-3'。
4) RNaseA obtained by further being proved 3) using the experiment of 2.5% agarose gel electrophoresis possesses sequence selection
Property.
Design six group of 2.5% agarose gel electrophoresis experiment：
In Fig. 3 (a)：RNA object chains used marked fluorophor and quencher, therefore use 535nm
Green fluorescence sense channel：
Swimming lane 1：RNaseA@S-RNA compounds cut target RNA sequence.
Swimming lane 2：Common RNaseA cuttings target RNA sequence.
Swimming lane 3：Target RNA sequence.In Fig. 3 (b)：Unrelated RNA sequence used does not carry out fluorescence labeling,
With using 590nm fluorescence detection channels after Gelsafe nucleic acid staining dyes：
Swimming lane 1：Unrelated RNA sequence.
Swimming lane 2：RNaseA@S-RNA compounds cut unrelated RNA sequence.
Swimming lane 3：Common RNaseA cuts unrelated RNA sequence.
Experimental result：
As shown in Fig. 3 (a)：Swimming lane 3 shows that FAM and BHQ-1 forms FRET in complete target RNA sequence, and fluorescence is in sudden
Go out state, therefore can't see the band of target sequence；Swimming lane 1 and the display target RNA sequence of swimming lane 2 are released by corresponding cleavage
Release intense fluorescence.
As shown in Fig. 3 (b)：Swimming lane 1 shows the pillar location of unrelated RNA sequence；Swimming lane 2 shows that unrelated RNA sequence can not
Cut by RNaseA@S-RNA compounds；Swimming lane 3 shows that common RNaseA can cut unrelated RNA sequence.
Embodiment 3
Applicant once carried out covalent coupling reaction using SPDP methods after DNase I are combined with S-DNA, and reactions steps are such as
Under：
1. utilize the DNase that 0.05M sodium phosphate buffers (sodium chloride containing 0.15M, pH 7.2) compound concentration is 10mg/mL
I solution.In the 1mL DNase I solution, it is that 6.2mg/mL is dissolved in SPDP solution in DMSO to add 25 μ L concentration, at room temperature
Stand overnight.
2. ultrafiltration removes unnecessary SPDP, the buffer solution composition used in ultrafiltration is：0.05M sodium phosphates, 0.15M sodium chloride,
0.01M EDTA, pH 7.2.
3.-SH S-DNA and the DNase I solution that is obtained in step 2 in molar ratio 1 by end mark：1 ratio is mixed
Close, room temperature left overnight.
6. ultrafiltration removes free DNase I and S-DNA.
DNase I@S-DNA covalent complexes also show the specificity to S-DNA complementary strands as made from above-mentioned steps
Cutting.Fig. 4 compared for the target dna chain of DNase I@S-DNA covalent complexes cutting fluorescence labeling and cut unrelated DNA
Speed ratio (numbering 2 in figure) and the target of the DNase I@S-DNA non-covalent complexes cutting fluorescence labeling obtained in embodiment 2
DNA and the speed ratio (numbering 1 in figure) for cutting unrelated DNA, it is seen that the ability of the two selectively cutting target sequence is basic
Unanimously.
Claims (6)
1. a kind of multienzyme complex of regulation and control nucleotide sequence selectivity, the multienzyme complex are nuclease and complete thio template strand
Compound, the target sequence complementary with the complete thio template strand, the nuclease and the complete thio mould are cut for selectivity
It is prepared by the following method between plate chain for non-covalent linking, the multienzyme complex：
It is by nuclease and complete thio template strand hybrid reaction, reaction condition in pH7.0-8.0 cushioning liquid first：Nuclease
The mol ratio of concentration 0.1mg/mL, 15-30 μM of the concentration of complete thio template strand, nuclease and complete thio chain is 1：1~1：10, instead
It is 29-37 DEG C to answer temperature, reaction time 45min-1h；Then milipore filter ultrafiltration of the cutoff between 20-40kD is used
The complete thio template strand to dissociate in above-mentioned reaction system is removed, obtains nuclease thio template chain cpd entirely；
The nuclease is DNase I or RNase A, and when the nuclease is DNase I, the complete thio template strand is full sulphur
Single-stranded for DNA, the multienzyme complex is used for selectivity cutting and the single-stranded target DNA sequence dna of the complete thio DNA single-stranded complementaries；
When the nuclease is RNase A, the complete thio template strand is complete thio RNA chains, and the multienzyme complex is used for selectivity and cut
Cut the target RNA sequence complementary with the complete thio RNA chains.
2. the multienzyme complex of regulation and control nucleotide sequence selectivity as claimed in claim 1, it is characterised in that the complete thio mould
The length of plate chain is between 15-50 nucleotides.
3. the multienzyme complex of regulation and control nucleotide sequence selectivity as claimed in claim 1, it is characterised in that the target sequence
Complementary base number is between 15-30 with complete thio template strand.
4. a kind of method of regulation and control nucleotide sequence selectivity, the nuclease is DNase I or RNase A, including following step
Suddenly：
1) the design synthesis complete thio template strand complementary with target sequence, when the nuclease is DNase I, the complete thio mould
Plate chain is that the complete thio DNA complementary with single-stranded target DNA sequence dna is single-stranded, when the nuclease is RNase A, the complete thio mould
Plate chain is the complete thio RNA chain complementary with target RNA sequence；
2) it is by nuclease and complete thio template strand hybrid reaction in pH7.0-8.0 cushioning liquid, reaction condition：Nuclease
The mol ratio of concentration 0.1mg/mL, 15-30 μM of the concentration of complete thio template strand, nuclease and complete thio chain is 1：1~1：10, instead
It is 29-37 DEG C to answer temperature, reaction time 45min-1h；
3) the complete thio mould to dissociate in above-mentioned reaction system is removed using milipore filter ultrafiltration of the cutoff between 20-40kD
Plate chain, obtains nuclease thio template chain cpd entirely, and the nuclease is non-covalent linking with complete thio template strand；
4) using nuclease, thio template chain cpd cuts target sequence entirely.
5. the method for regulation and control nucleotide sequence selectivity as claimed in claim 4, it is characterised in that the complete thio template chain length
Degree is below 50 nucleotides.
6. as claimed in claim 4 regulation and control nucleotide sequence selectivity method, it is characterised in that the cushioning liquid be containing
0.15M sodium chloride, pH 7.2 0.05M phosphate buffers.
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| Generation of artificial sequence-specific nucleases via a preassembled inert-template;Xianjin Xiao et al.;《Chemical Science》;20151207;第6卷;2051-2057 * |
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