CN109517877A - Screen m6A goes nucleotides substrate, kit and the method for modification enzyme inhibitor - Google Patents
Screen m6A goes nucleotides substrate, kit and the method for modification enzyme inhibitor Download PDFInfo
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- CN109517877A CN109517877A CN201811198076.4A CN201811198076A CN109517877A CN 109517877 A CN109517877 A CN 109517877A CN 201811198076 A CN201811198076 A CN 201811198076A CN 109517877 A CN109517877 A CN 109517877A
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Abstract
The present invention relates to screening m6A goes polynucleotide substrate, kit and the method for modification enzyme inhibitor.Presence or absence of m6In the case that A methylates, polynucleotide substrate disclosed by the invention, kit and method using the fluorescent marker and quenched label that fluorescence resonance energy transfer (FRET) can occur in restriction enzyme treated fluorescent value variation detection and evaluation small molecule compound to going the inhibiting effect of modificationization enzyme, and then filter out satisfactory inhibitor.
Description
Technical field
Modification enzyme is gone to inhibit this invention relates generally to RNA epigenetic modification, and more particularly to detection m6A is gone
Modification enzyme inhibitor effect and screening m6A goes nucleotides substrate, kit and the method for modification enzyme inhibitor.
Background technique
Epigenetics be study gene nucleotides sequence be listed in and do not change in the case where, gene expression it is heritable
Variation a science of heredity subdiscipline.Epigenetic modification include DNA methylation modification (5-methylcytosine, 5mC) and
Its relevant modifications (such as 5-hydroxymethyl cytosine, 5hmC), histone modification (such as methylation modification, acetylation modification) and are sent out recently
Existing RNA epigenetics modifies (such as N6Methyl adenine, m6A)。
m6A is modified as the mRNA of eukaryocyte abundance highest (0.1%-0.4% for accounting for adenine total amount in mRNA),
It is found that playing an important role in many normal biological piocesses, such as tissue development, stem cells self-renewal and differentiation, heat
Shock or DNA damage response etc..It is some research shows that m6A goes modification enzyme (such as FTO) and the generation of a variety of diseases closely related, because
And research and develop more m6A goes modification enzyme inhibitor to potentially contribute to effective therapeutic scheme that research and development are directed to various diseases, especially will
These inhibitor and other therapeutic agents are used in combination, and may treat at present to the drug resistant kinds of Diseases of active drug.
In the prior art, the detection of inhibitor effect is as shown in fig. 1, first progress m6A goes modification enzyme activity to react,
Then the principle of restriction enzyme enzyme spcificity cutting restriction enzyme site is utilized, that is, it can be with unmodified restricted
Restriction enzyme site and m cannot be cut through6The restriction enzyme site of A modification, passes through non-denaturing polyacrylamide (Native-PAGE)
Endonuclease bamhi size is analyzed in gel electrophoresis, to realize to m6A goes the detection and screening of modification enzyme inhibitor: depositing
In the case where inhibitor, DNA fragmentation has small one and large one two bands；And there are inhibitor, small DNA item
The color of band is shallower, and shoals with the raising of inhibitor concentration, it is possible thereby to judge the effect of inhibitor, and then carries out
The screening of inhibitor small molecule compound.However, there are the complicated for operation, few (flux of test sample number for the scheme of the prior art
It is low), clarity is low, is difficult to the disadvantages of carrying out accurate quantitative analysis and comparing.
Therefore, it is necessary to a kind of new detections that can overcome disadvantages mentioned above and screening technique.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of nucleotides substrate with fluorescent marker and quenched label,
Due to fluorescence resonance energy transfer (FRET), system fluorescent value minimizes above two label.But pass through restriction enzyme
After enzyme effect, the double-strand of modificationization is gone to disconnect, releases above-mentioned fluorescence resonance energy transfer (FRET), and then can detect very
High fluorescence；And the double-strand of modificationization is not gone to keep combining, continue that lower fluorescent value is presented.It can using fluorescent value size
To detect and evaluate small molecule compound to going the inhibiting effect of modificationization enzyme, and then filter out satisfactory inhibitor.
In a first aspect, containing at least one restriction enzyme the present invention provides a kind of double-stranded polynucleotide substrate
Cog region, the base containing at least one modification in the restriction enzyme cog region, and the presence of the modified base
Prevent shearing of the restriction enzyme corresponding with restriction enzyme cog region to nucleic acid primer；The double-stranded polynucleotide
Two chains of substrate are marked with fluorescent emission group and fluorescent quenching group respectively, and fluorescent emission group and fluorescent quenching group it
Between there are fluorescence resonance energy transfer.
Double-stranded polynucleotide substrate of the present invention, it is characterised in that the base of the modification is 5-methylcytosine, 5-
Hydroxymethyl cytosine, N6- methyl adenine etc..
Double-stranded polynucleotide substrate of the present invention, it is characterised in that the restriction enzyme cog region is GGATCC,
A therein is m6A, and the restriction enzyme is DpnII.
Double-stranded polynucleotide substrate of the present invention, it is characterised in that fluorescent emission group and fluorescent quenching group are positioned at double
One end of chain polynucleotide substrate, or it is located at the both ends of double-stranded polynucleotide substrate.
Double-stranded polynucleotide substrate of the present invention, it is characterised in that fluorescent emission group and fluorescent quenching group distinguish position
In the both ends of double-stranded polynucleotide substrate, the double-stranded polynucleotide substrate length < 20bp, preferably < 17bp.
Double-stranded polynucleotide substrate of the present invention, it is characterised in that fluorescent emission group and fluorescent quenching group are positioned at double
One end of chain polynucleotide substrate, between the fluorescent emission group and fluorescent quenching group label end and restriction enzyme site
Length, the length than double-stranded polynucleotide substrate is as short as few 6bp, preferably short 8bp, more preferable short 10bp.
Double-stranded polynucleotide substrate of the present invention, it is characterised in that the fluorescent emission group is TAMRA, the fluorescence
Quenching group is DABCYL.
Double-stranded polynucleotide substrate of the present invention, it is characterised in that two polynucleotide chains respectively include 5 '-CTTAAGG
(m6A) TCCGGTCAA-3 ' and 5 '-TTGACCGGATCCTTAAG-3 '.
Second aspect, the present invention provide a kind of nucleic acid and go modification enzyme activity detection method, comprising:
(1) modification enzyme activity determinand is gone to contact with double-stranded polynucleotide substrate of the present invention nucleic acid；
(2) utilization can identify the corresponding restriction enzyme of restriction enzyme cog region on double-stranded polynucleotide substrate
It is cut；
(3) the step of detecting fluorescent value.
Nucleic acid of the present invention goes modification enzyme activity detection method, it is characterised in that: the height and nucleic acid of the fluorescent value
Modification enzyme activity is gone to be positively correlated.
Nucleic acid of the present invention goes modification enzyme activity detection method, it is characterised in that step (3) is examined using fluorescence microplate reader
Survey fluorescent value.
The third aspect, the present invention provide a kind of nucleic acid and go modification inhibitor activity detection method, comprising:
(1) modification inhibitor activity determinand and nucleic acid is gone to remove modification enzyme, double-strand multicore glycosides of the present invention nucleic acid
Sour substrate reactions；
(2) restriction enzyme corresponding with restriction enzyme cog region on the double-stranded polynucleotide substrate is utilized
It is cut；
(3) the step of detecting fluorescent value.
Nucleic acid of the present invention goes modification enzyme activity detection method, it is characterised in that: the height and nucleic acid of the fluorescent value
Go modification inhibitor activity negatively correlated.
Nucleic acid of the present invention goes modification enzyme activity detection method, it is characterised in that reaction system is adjusted after step (2)
Temperature make it between the melting temperature of double-stranded polynucleotide substrate and the melting temperature of cleaved products.
Nucleic acid of the present invention goes modification enzyme activity detection method, it is characterised in that step (3) is examined using fluorescence microplate reader
Survey fluorescent value.
Compared with prior art, technical solution of the present invention has the advantage that
(1) easy to operate, high sensitivity.Present invention utilizes the fluorescence between fluorescent emission group and fluorescent quenching group
Resonance energy transfer (FRET) feature, compared with the existing technology in non-denaturing polyacrylamide (Native-PAGE) gel electricity
Not only easy to operate but also sensitivity of swimming is higher.
(2) quantitative analysis is carried out to enzyme activity.Direct testing result of the invention is fluorescent value, the size and core of fluorescence values
Acid goes the activity of modification enzyme is strong and weak to be positively correlated, to realize the quantitative analysis of enzyme activity according to fluorescent measurement.
(3) extensive, high-throughput detection.The present invention can detect the activity for removing modification enzyme by fluorescence microplate reader, high-throughput
Detect the Large-scale Screening for being especially suitable for candidate molecules.
(4) inventive substrate had both been suitble to short polynucleotide substrate, was also suitble to long polynucleotide substrate.For short more
Fluorescent emission group and fluorescent quenching group are marked on double-stranded polynucleotide both ends by nucleotides substrate respectively, when digestion occurs
When, both ends separately issue fluorescence；For long polynucleotide substrate, fluorescent emission group and fluorescent quenching group are marked on double
On two chains of chain polynucleotides one end, when digestion occurs, the length of double-strand shortens, melting temperature reduces, therefore system temperature
It is denatured double stranded separately to issue fluorescence when degree is higher than the melting temperature of digestion products.
Detailed description of the invention
By reading the detailed description of following preferred embodiment, various other advantage and benefit are for art technology
Personnel will be apparent.Attached drawing is not to be construed as limiting the invention merely for illustrating the purpose of preferred embodiment.?
In attached drawing:
The testing principle of Fig. 1 prior art and the schematic diagram of result.
Figure 1A: the nucleic acid demethyl enzyme inhibitor testing principle based on gel electrophoresis.
Figure 1B: the electrophoresis detection result (μM) of various concentration methylase FTO inhibitor Rhein.
Fig. 1 C: swimming lane 0 is blank control；Swimming lane 1-11 methylase FTO inhibitor to be measured；Swimming lane Rhein is methylation
Enzyme FTO inhibitor positive control Rhein.
The schematic diagram of Fig. 2 testing principle according to the preferred embodiment of the present invention and result.
Fig. 2A: fluorescence-based nucleic acid demethyl enzyme inhibitor testing principle.
Fig. 2 B: the fluorescence detection result of various concentration methylase FTO inhibitor Rhein.
Fig. 2 C: the fluorescence detection result of methylase FTO inhibitor compound 1 to be measured.
Specific embodiment
The illustrative embodiments that the present invention will be described in more detail below with reference to accompanying drawings.Although showing this hair in attached drawing
Bright illustrative embodiments, it should be understood that may be realized in various forms the present invention and should not be limited to the present invention and retouch
The embodiment stated.It is to be able to thoroughly understand the present invention on the contrary, providing these embodiments, and this can be sent out
Bright range communicates completely to those skilled in the art.
Embodiment 1
1, it recombinantly expresses and has isolated and purified before demethylase FTO Protein Assav and is spare.FTO substrate is the conjunction of external solid phase
At the single stranded DNA (ssDNA) containing 49 bases, comprising DpnII cleavage sequence GATC, the site m6A is contained in cleavage sequence.
SsDNA sequence is as follows:
5′
-TAGACATTGCCATTCTCGATAGG(m6A)TCCGGTCAAACCTAGACGAATTCCA-3′
Reaction system is 100 μ L, is incubated at room temperature 0.5h.95 DEG C of heating 5min of metal bath terminate reaction.
2, using DpnII as restriction enzyme, when cleavage sequence contains m6A, DpnII cannot play digestion function, when
After m6A methylation is removed by FTO, double-stranded DNA (dsDNA) can be cut into two sections by DpnII.
1) it anneals
Above-mentioned demethylation reaction system is transferred to PCR pipe, antisense ssDNA (50 μs complementary with substrate ssDNA are added
M) 2.2 μ l, wherein substrate ssDNA: antisense ssDNA is 1:1.1, is annealed in PCR instrument.
2) endonuclease reaction
2 μ l 10 × cutsmart buffer are added in PCR pipe in the product for taking 10 μ l to anneal, and 1 μ l DpnII is added,
Add MiliQ H2O to 20 μ l.It is placed in 37 DEG C of digestion 2h (taking-up when running glue loading) in PCR.
3, non-denaturing polyacrylamide (Native-PAGE) gel electrophoresis analysis
20%Native-PAGE glue is prepared, 4 μ l 6 × loading buffer are added in Xiang Shangshu digestion products, it mixes,
Loading 10uL, loading is twice side by side.150V runs 60min.Pre-dyed glue 60min, the ultraviolet transmission illumination in gel imaging system
It penetrates, it can be seen that blank group m6A can inhibit the enzymatic activity of DpnII, be primarily present the band of 49nt, and experimental group m6A can be by FTO
It is catalyzed demethylation, since enzyme activity efficiency part m6A is by demethyl, DpnII plays digestion effect, it can be observed that two bands,
49nt and 22&27nt respectively.
Figure 1B shows the electrophoresis detection of various concentration methylase FTO inhibitor Rhein as a result, according to electrophoretic band color
The depth inhibition of methylase FTO is correspondinglyd increase with the raising of Rhein concentration.Fig. 1 C shows 11 kinds of 100 μ of compound
To the inhibiting effect of FTO enzyme activity under M, in addition to compound 2 is able to suppress FTO enzyme activity, the inhibitory effect of other 10 kinds of compounds
It can not make and clearly judge, wherein No. 1 compound is displayed without inhibitory effect compared with positive control, with blank control group phase
Than being displayed without significant difference, band clarity is close.
Embodiment 2
1. nucleotides substrate designs and prepares
The nucleotides substrate used in this embodiment is containing 17 bases, including DpnII enzyme for external synthesis in solid state
The single stranded DNA (ssDNA) of sequence GATC is cut, contains m in cleavage sequence6The site A.The single stranded DNA --- that is, positive-sense strand --- with
The sequence difference of its antisense strand is as follows:
Sense strand sequence: 5 '-(TAMRA)-CTTAAGG (m6A)TCCGGTCAA-3'；
Antisense strand sequence: 5 '-TTGACCGGATCCTTAAG- (DABCYL) -3 '.
The external demethylase activity experiment of 2.FTO
It recombinantly expresses and has isolated and purified before demethylase FTO Protein Assav and is spare.Reaction system is 50 μ L, and room temperature is incubated
Educate 2h.95 DEG C of heating 5min of metal bath terminate reaction.
The system of table 1:FTO external demethylase activity experiment control group and experimental group
3. annealing
Above-mentioned demethylation reaction system is transferred to PCR pipe, antisense ssDNA (50 μs complementary with substrate ssDNA are added
M) 1.2 μ l, wherein substrate ssDNA: antisense ssDNA is 1:1.2, is annealed in PCR instrument.
4.DpnII digestion experiment
DpnII is commonly used for a kind of restriction enzyme of epigenetics research, when cleavage sequence contains m6When A,
DpnII cannot play digestion function, work as m6After A methylation is removed by FTO, double-stranded DNA (dsDNA) can be cut into two by DpnII
Section.
Table 2: endonuclease reaction system
| DnpII | 10 units |
| 10X NEBuffer | 10μl(10X) |
| H2O | Surplus |
| Overall reaction system | 50μl |
All hole endonuclease reaction system mix are configured, every hole needs 50 μ l, and test in next step is waited to use.
5. microplate reader fluorescence detection
Reaction solution is transferred to 96 orifice plate of black, microplate reader vibrates 30s, 37 DEG C of balance 5min (detection baseline excitation wavelengths
558nm and launch wavelength 580nm).
Instrument pops up 96 orifice plates, reserves time enough for the endonuclease reaction system volley of rifle fire and each 50 μ is added by 8 holes of each column
L, 37 DEG C are reacted and collect fluorescence signal 30min.
Table 3: microplate reader sample-adding signal
Blank is control group, and S is experimental group, and Positive is positive compound control group.
6. interpretation of result
As shown in Fig. 2 B, after the inhibitor of addition various concentration, microplate reader detects that different degrees of fluorescence is strong
Degree, and this fluorescence intensity is reduced with the increase of inhibitor concentration, illustrates that the present invention can quantitatively reflect inhibitor pair
Go the inhibiting effect of modificationization enzyme.Fig. 2 C shows that No. 1 compound differs greatly with blank control group fluorescent value, with positive control
It is not so good as positive control compared to inhibiting effect.
The above, the only preferred embodiments of the disclosure, but the scope of protection of the present invention is not limited thereto, appoints
What those familiar with the art in technical scope disclosed by the invention, can hundred million change or replacement being readily apparent that,
It should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the protection of the claim
Subject to range.
Sequence table
<110>Peking University
<120>screen m<sup>6</sup>a goes nucleotides substrate, kit and the method for modification enzyme inhibitor
<140> 201811198076.4
<141> 2018-10-15
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<223> n=m6a
<400> 1
cttaaggntc cggtcaa 17
<210> 2
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttgaccggat ccttaag 17
<210> 3
<211> 49
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<223> n=m6a
<400> 3
tagacattgc cattctcgat aggntccggt caaacctaga cgaattcca 49
Claims (10)
1. a kind of double-stranded polynucleotide substrate contains at least one restriction enzyme cog region, the restriction enzyme
In cog region containing at least one modification base, and the modified base there are preventions and restriction enzyme cog region
Shearing of the corresponding restriction enzyme to nucleic acid primer；Two chains of the double-stranded polynucleotide substrate are marked with fluorescence respectively
Emit group and fluorescent quenching group, and there are fluorescence resonance energy transfer between fluorescent emission group and fluorescent quenching group.
2. double-stranded polynucleotide substrate as described in claim 1, it is characterised in that the base of the modification is N6- methyl adenine
Deng.
3. double-stranded polynucleotide substrate as described in claim 1, it is characterised in that the restriction enzyme cog region is
GGATCC, A therein are m6A, and the restriction enzyme is DpnII.
4. double-stranded polynucleotide substrate as claimed in claim 3, it is characterised in that fluorescent emission group and fluorescent quenching group point
Not Wei Yu double-stranded polynucleotide substrate both ends, the double-stranded polynucleotide substrate length at least 17bp.
5. a kind of nucleic acid goes modification enzyme activity detection method, comprising:
(1) modification enzyme activity determinand is gone to contact with any double-stranded polynucleotide substrate of claim 1-7 nucleic acid；
(2) it is carried out using restriction enzyme corresponding with restriction enzyme cog region on the double-stranded polynucleotide substrate
Cutting；
(3) the step of detecting fluorescent value.
6. nucleic acid as claimed in claim 5 goes modification enzyme activity detection method, it is characterised in that: the height and core of the fluorescent value
Acid goes modification enzyme activity to be positively correlated.
7. a kind of nucleic acid goes modification inhibitor activity detection method, comprising:
(1) modification inhibitor activity determinand and nucleic acid is gone to go any double-strand of modification enzyme, claim 1-4 more nucleic acid
Nucleotides substrate reaction；
(2) it is carried out using restriction enzyme corresponding with restriction enzyme cog region on the double-stranded polynucleotide substrate
Cutting；
(3) the step of detecting fluorescent value.
8. nucleic acid as claimed in claim 7 goes modification enzyme activity detection method, it is characterised in that: the height and core of the fluorescent value
Acid goes modification inhibitor activity negatively correlated.
9. the detection method as described in claim 5-8 is any, it is characterised in that the temperature for adjusting reaction system after step (2) makes
It is between the melting temperature of double-stranded polynucleotide substrate and the melting temperature of cleaved products.
10. the detection method as described in claim 5-8 is any, it is characterised in that step (3) detects fluorescence using fluorescence microplate reader
Value.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110106231A (en) * | 2019-04-22 | 2019-08-09 | 武汉大学 | A method of utilizing N6 or N1 generation methylation modifications of adenine in dUTP or dTTP detection nucleic acid |
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|---|---|---|---|---|
| CN101855351A (en) * | 2007-09-14 | 2010-10-06 | 特拉纳探索公司 | Compositions and methods for the identification of inhibitors of retroviral infection |
| CN106755306A (en) * | 2016-11-21 | 2017-05-31 | 陕西师范大学 | T3DNA ligases and T4RNA ligases 2 are in detection N6Application in methyl adenine |
| CN107557430A (en) * | 2017-10-20 | 2018-01-09 | 郑州大学 | The method of high flux screening ALKBH5 micromolecular inhibitors |
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2018
- 2018-10-15 CN CN201811198076.4A patent/CN109517877B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101855351A (en) * | 2007-09-14 | 2010-10-06 | 特拉纳探索公司 | Compositions and methods for the identification of inhibitors of retroviral infection |
| CN106755306A (en) * | 2016-11-21 | 2017-05-31 | 陕西师范大学 | T3DNA ligases and T4RNA ligases 2 are in detection N6Application in methyl adenine |
| CN107557430A (en) * | 2017-10-20 | 2018-01-09 | 郑州大学 | The method of high flux screening ALKBH5 micromolecular inhibitors |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110106231A (en) * | 2019-04-22 | 2019-08-09 | 武汉大学 | A method of utilizing N6 or N1 generation methylation modifications of adenine in dUTP or dTTP detection nucleic acid |
| CN110106231B (en) * | 2019-04-22 | 2021-08-17 | 武汉大学 | Method for detecting methylation modification of adenine N6 or N1 bit in nucleic acid by using dUTP or dTTP |
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