CN108627507A - A kind of active detection method of dnmt rna - Google Patents
A kind of active detection method of dnmt rna Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 23
- 108010063593 DNA modification methylase SssI Proteins 0.000 claims abstract description 52
- 239000012488 sample solution Substances 0.000 claims abstract description 18
- 230000000694 effects Effects 0.000 claims abstract description 15
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- 238000007069 methylation reaction Methods 0.000 claims abstract description 7
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 5
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- 108010008532 Deoxyribonuclease I Proteins 0.000 claims abstract description 4
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- 230000011987 methylation Effects 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 24
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- 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 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
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- 238000006243 chemical reaction Methods 0.000 claims description 8
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- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical class O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 claims description 5
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- 238000004220 aggregation Methods 0.000 description 5
- XAUDJQYHKZQPEU-KVQBGUIXSA-N 5-aza-2'-deoxycytidine Chemical compound O=C1N=C(N)N=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 XAUDJQYHKZQPEU-KVQBGUIXSA-N 0.000 description 4
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- 230000008836 DNA modification Effects 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
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- VFNGKCDDZUSWLR-UHFFFAOYSA-L disulfate(2-) Chemical compound [O-]S(=O)(=O)OS([O-])(=O)=O VFNGKCDDZUSWLR-UHFFFAOYSA-L 0.000 description 1
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- 201000011549 stomach cancer Diseases 0.000 description 1
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- 238000003597 transferase activity assay Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
- G01N2021/786—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour with auxiliary heating for reaction
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Abstract
The invention discloses a kind of active detection methods of dnmt rna, include the following steps：1) it is that catalyst carries out methylation catalytic reaction to HP1 with CpG transmethylases (M.SssI)；2) M.SssI is inactivated；3) endonuclease HpaII is added in step 2 acquired solution；4) Au NPs HP2 are prepared；5) Au NPs HP2 and nickase Nt.AlwI are added in step 3 acquired solution；6) it adds graphene oxide into step 5 acquired solution, obtains GO Au NPs sample solutions；7) it uses ultraviolet-visible spectrophotometry to analyze GO Au NPs sample solutions, thus measures the activity of M.SssI.The present invention also provides the applications of the active detection method of dnmt rna.The detection method of the present invention is easily operated, has good sensitivity and selectivity, has a good application prospect in drug development and clinical diagnosis.
Description
Technical field
The invention belongs to optical bio field of sensing technologies, and in particular to one kind being based on graphene oxide (GO) and nanogold
The colorimetric analysis methyl transferase activity method of (Au NPs).
Background technology
DNA methylation refers to catalysis of the donor S-adenosyl methionine (SAM) in dnmt rna (DNA MTase)
The lower process that methyl is shifted to adenine or cytimidine.DNA MTase have been used as predicting various cancers (such as lung cancer, gastric cancer, knot
Intestinal cancer) biomarker in type and potential treatment target etc., it can be seen that detect the importance of methyl transferase activity.
Up to the present, the technology of various exploitations can be used for sensitive and selectively detection CpG transmethylases (M.SssI) are active
Mainly have Bergerat etc. (Bergerat, A., Guschlbauer, W., Fazakerley, G.V.,
1991.Proc.Natl.Acad.Sci.U.S.A.88,6394-6397.) using the work of radioisotope method detection M.SssI
Property, but the substrate of labelled with radioisotope is harmful to biological tissue；Cao and Zhang (Cao, A., Zhang, C.-Y.,
2012.Anal.Chem.84,6199-6205.) it needs Cytosines to be that urine is phonetic by disulfate processing rolling circle amplification
Pyridine, detection time are long；Lopez Torres etc. (Lopez Torres, A., YanezBarrientos, E., Wrobel, K.,
Wrobel, K., 2011.Anal.Chem.83,7999-8005.) using the activity of high performance liquid chromatography detection M.SssI, but
It is that this method is needed using expensive instrument high performance liquid chromatograph, therefore the detection method is unfavorable for promoting.Therefore, exploitation spirit
Quick and simple methyl transferase activity assay method is extremely urgent.
It is well known that nano material has specific physics and chemical characteristic, this makes it in test dna, Cancer Biology mark
Will object and virus aspect show many advantages.Nanogold (AuNPs) and graphene oxide (GO) are typical optical nano materials
Material, the operation for having many advantages, such as good sensing characteristics and being readily synthesized, therefore such material has broad application prospects.
Invention content
The purpose of the present invention is to provide a kind of colorimetric analysis being based on graphene oxide (GO) and nanogold (Au NPs)
Methyl transferase activity method, this method avoid relative complex DNA designs and cumbersome experimental implementations, and using simple
Instrument, this method are tested suitable for actual sample, have good sensitivity and selectivity.
The method that the present invention realizes is：Before measuring, HP2 passes through Au-S keys functional modification to Au nano particles
Surface on.As detection probe, HP1 hair clip substrates contain M.SssI and endonuclease (HpaII) unique identification sequence
5′-C↓CGG-3′.Downward arrow indicates the cleavage site of HpaII.It is cut at unmethylated HP1 corresponding sites, the mistake
Journey is irreversible.As a result, hairpin dna be cut into three it is single-stranded, wherein a single stranded DNA (S1 in Fig. 1) can be miscellaneous with HP2
The double of the sequence (- CCTAG-5 ' of 5 '-GGATCNNNN ↓ N-3 '/3 ') can be identified completely to be formed with nickase (Nt.AlwI) by handing over
Chain HP2/s1 compounds, down arrow indicate the cleavage site of Nt.AlwI.The HP2 of hydridization can be catalytically decomposed by Nt.AlwI into s2
With two segments of s3, then s1 discharged again to start removal process, to generate a large amount of s2 and s3.Meanwhile it thus generating
Au NPs-s3 and GO interact, and by the surfaces GO and be modified with Au nano particles DNA it is single-stranded between π-π heaps
Product interaction effectively has adjusted the Au NPs aggregations of compound.Due to the aggregation deposition containing bulky grain Au nano particles
Object, it is observed that limpid supernatant.On the contrary, in the presence of M.SssI, HPl can be methylated as 5 '-CmCGG-3 ',
HpaII shear histories are cut off, and so that HP1 is kept hair clip DNA structure and cannot be caused next reaction.Therefore, although then
It is added to GO, but the AuNPs of HP2 modifications does not assemble, and mixture keeps red.In this way, purple can be used
The activity of outer visible spectrophotometry detection M.SssI.
The active detection method of dnmt rna of the present invention, includes the following steps：1) using M.SssI as catalyst pair
HP1 carries out methylation catalytic reaction；2) M.SssI is inactivated；3) HpaII is added in step 2 acquired solution；4) AuNPs- is prepared
HP2；5) Au NPs-HP2 and Nt.AlwI are added in step 3 acquired solution；6) it adds graphene oxide into molten obtained by step 5
In liquid, GO-Au NPs sample solutions are obtained；7) ultraviolet-visible spectrophotometry is used to analyze GO-Au NPs sample solutions, thus
Measure the activity of M.SssI.
Preferably, the HP1 hair clips substrate contains 5 '-C of M.SssI and HpaII unique identification sequences ↓ CGG-3 '.
Preferably, methylation reaction described in step 1, reaction system are 50nM HP1,1 × NE buffer 2 and 0.1mM
S-adenosylmethionine and M.SssI；The NE buffer 2 include 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2、
1mM dithiothreitol (DTT)s, pH7.9, reaction condition are 37 DEG C of 3~5h of heat preservation.
Preferably, M.SssI is inactivated described in step 2, reaction condition is：Step 1 gained mixture is heated 20 at 65 DEG C
~30min.
Preferably, the preparation of AuNPs-HP2 includes the following steps in step 4：A) HP2 is restored with reducing agent, then uses color
It composes post separation and removes excessive reducing agent；B) acquired solution is mixed with Au NPs after detaching, overnight at 4 DEG C by gained mixture
It is spare；C) sodium chloride solution is added in the mixture obtained by step b, and is incubated 10~15h in dark surrounds, obtains Au
NPs-HP2。
Preferably, the reducing agent is dithiothreitol (DTT) or TCEP hydrochlorides.
Preferably, after completing step c, the mixture is washed with the pH Tris-HCl solution for being 7.4, is then centrifuged for, obtains
To upper layer and lower layer solution, containing Tris-HCl, sodium chloride, the AuNPs and HP2 not being coupled in upper solution, lower layer be containing
The solution of AuNPs-HP2.Upper solution is removed, the Tris-HCl solution that pH is 7.4 is then used to wash repeatedly lower layer's solution to obtain
To the Au NPs-HP2 solution of purifying, the Au NPs-HP2 of purifying are resuspended in standby in the Tris-HCl solution that pH is 7.4
With.
Preferably, in stepb, the grain size of the AuNPs is 13~15nm, and the AuNPs is with solid particle or colloidal sol
Form detach rear acquired solution with described and mix.
It is a further object of the present invention to provide the applications of the active detection method of the dnmt rna.
The solution have the advantages that：The present invention utilizes the conjugation crosslinking effect between the Au NPs and GO of double-stranded DNA modification
Cycle amplified signal should be carried out with by Nt.AlwI enzymes, developed for the active unmarked methods of colorimetric detection M.SssI.It should
Method is compared with prior art, easy to operate, is suitable for high-volume actual sample and tests, there is lower detection to limit (0.067U/
ML), there is good application prospect in drug development and clinical diagnosis.
Description of the drawings
Fig. 1 is the colorimetric analysis methyl transferase activity method schematic diagram based on graphene oxide (GO) and AuNPs.
Fig. 2 is the ultraviolet-visible absorption spectroscopy figure of different samples, wherein (a) is Au NPs-HP2 solution；(b) it is Au
The mixed solution of NPs-HP2 and GO；(c) it is the GO-Au NPs sample solutions containing 60U/mL M.SssI；(d) be without containing
The GO-Au NPs sample solutions of M.SssI.
(A) is the transmission electron microscope picture of the GO-Au NPs sample solutions containing 60U/mL M.SssI in Fig. 3；(B) be without
There is the transmission electron microscope picture of the GO-Au NPs sample solutions of M.SssI.
(A) is the absorption spectrum of the colorimetric analysis of the GO-AuNPs sample solutions containing various concentration M.SssI in Fig. 4；
(B) it is absorbance and M.SssI concentration relationship figures, interior illustration is the logarithmic relationship curve of absorbance and M.SssI concentration.
Fig. 5 is inhibitor concentration and M.SssI relative activity graph of relation：(A)5-Aza；(B)5-Aza-dC.
Specific implementation mode
The present invention is further elaborated in the following with reference to the drawings and specific embodiments, and the present invention is not limited thereto.
The detection method of the present invention is as follows：1) methylation reaction：By SAM (0.1mM), HP1 (50nM), 1 × NE
The mixing of the M.SssI of Buffer2 and various concentrations a series of incubates the reaction solution 3 hours at 37 DEG C；2) it inactivates：It will be anti-
Solution is answered to heat 20 minutes at 65 DEG C to inactivate M.SssI；3) HpaII (60U/mL) is added in step 2 acquired solution and 37
DEG C be incubated 2 hours；4) AuNPs-HP2 is prepared；5) AuNPs-HP2 (1 μM) and Nt.AlwI (20U/mL) are added to step 3 institute
It obtains in solution and is maintained at 37 DEG C 2 hours；6) 0.6mL GO solution (0.1mg/mL) is added to step 5 gained mixed solution
In, obtain GO-Au NPs sample solutions；7) it uses ultraviolet-visible spectrophotometry to analyze GO-Au NPs sample solutions, thus surveys
Determine the activity of M.SssI.
Wherein, the preparation process of AuNPs-HP2 is as follows：At room temperature by dithiothreitol (DTT) (DTT) by the HP2 of sulfhydrylation
Then activation is used Nap-5 post separations to remove DTT, acquired solution is mixed with Au NPs, by institute for 2.5 hours with Reduction of Disulfide
Mixture is obtained to be incubated overnight at 4 DEG C.After this step, sodium chloride solution is slowly added dropwise in three times in the resulting mixture,
Then it is placed on being incubated 12 hours in dark surrounds.The mixture is washed with the pH Tris-HCl solution for being 7.4, then
Centrifugation, obtains upper layer and lower layer solution, is containing Tris-HCl, sodium chloride, the AuNPs and HP2 not being coupled, lower layer in upper solution
Solution containing Au NPs-HP2.Upper solution is removed, the Tris-HCl solution that pH is 7.4 is then used to wash repeatedly lower layer's solution
With the Au NPs-HP2 solution purified, the Au NPs-HP2 of purifying are resuspended in the Tris-HCl solution that pH is 7.4
In it is spare.
From Figure 2 it can be seen that a-d sample solutions are to have maximum absorption band, wherein Au NPs-HP2 molten at 530nm in wavelength
The absorbance of liquid it is most strong (see curve a), AuNPs-HP2 and GO in Fig. 2 mixed solution and contain 60U/mL M.SssI's
The peak value of GO-Au NPs sample solutions is also stronger (see the curve b and c in Fig. 2).These experimental phenomenas show to be modified with Au
The HP2 of NPs is very stable in the solution of no M.SssI and HpaII, and the HP2 for being modified with Au NPs can be also stable in the presence of
M.SssI and HpaII is both in existing solution, this is because by the way that M.SssI, M.SssI catalysis are added in HP1 solution
The methylation reaction at the site places 5 '-CCGG-3 ' of identification forms the digestion process quilt of the HP1, HpaII and the Nt.AlwI that methylate
Forbid, therefore complete HP1 cannot cause the aggregation of Au NPs.However, there is no M.SssI, the solution of HP1
Single stranded DNA is processed by HpaII and Nt.AlwI and causes the aggregation of Au NPs that absorbance is caused to be substantially reduced (see the song in Fig. 2
Line d), this result are demonstrated conjugation cross-linked effect between the Au NPs and GO modified based on double-stranded DNA and by nickases
Nt.AlwI carries out cycle amplified signal, is used for the feasibility of the active methods of colorimetric detection M.SssI.
Morphology characterization is carried out to the sample solution of preparation using transmission electron microscope.By Fig. 3 A as it can be seen that containing 60U/
In the GO-Au NPs sample solutions of mL M.SssI, the surfaces GO are barely perceivable Au NPs.However, by Fig. 3 B as it can be seen that not
In the case of M.SssI, there are the Au NPs of aggregation on the surface of GO, this further demonstrate the present invention detection method can
Row.
In order to assess this method to the active analytical performances of M.SssI, this experiment is investigated under optimized experiment condition
Changing rule of the UV absorption intensity with M.SssI concentration.As shown in Figure 4 A, increase to from 0 with the concentration of M.SssI
60U/mL, Au NPs in sample solution are that absorbance value at 530nm gradually increases in wavelength.Au NPs absorbances with
The active logarithms of M.SssI linear correlation (Fig. 3 B) within the scope of 0.2 to 60U/mL.Related coefficient (R) is calculated as 0.9932,
Linear fit equation is as follows：
A=0.2491g [C]+0.214
Wherein A is absorbance, and C is M.SssI activity.Based on 36 regular (S/N=3), detection is limited to 0.067U/mL.
In order to study the validity of this method and screen M.SssI inhibitor, this experiment is in anticancer clinical test and many first
Two kinds of representative drugs 5-azacitidines (5-Aza) and 5-aza-2'-deoxycytidine (5-Aza- have been selected in baseization experiment
dC).The RA of M.SssI is reduced with the increase of two kinds of inhibitor concentrations as can be seen from Figure 5, this shows as inhibitor is dense
Its inhibiting effect of the increase of degree gradually increases.Calculate two kinds of drugs make M.SssI activity reduce by 50% 50 values of IC be respectively
4.58 ± 0.02 μM (5-Aza) and 48.82 ± 0.02 μM (5-Aza-dC).The experimental results showed that 5-Aza is active to M.SssI
Inhibiting effect ratio 5-Aza-dC is strong.Therefore, detection method of the invention, which can be used for screening, inhibits the active anticancer drugs of M.SssI,
It can be used as future drugs exploitation and the potential tool of clinical diagnosis.
The above is only a preferred embodiment of the present invention, it is noted that those skilled in the art, not
Under the premise of being detached from the technology of the present invention principle, several improvements and modifications can also be made, these improvements and modifications also should be regarded as this
The protection domain of invention.
Claims (9)
1. a kind of active detection method of dnmt rna, it is characterised in that：Include the following steps：1) it is to urge with M.SssI
Agent carries out methylation catalytic reaction to HP1；2) M.SssI is inactivated；3) endonuclease is added in step 2 acquired solution
HpaII；4) Au NPs-HP2 are prepared；5) Au NPs-HP2 and nickase Nt.AlwI are added in step 3 acquired solution；6) will
Graphene oxide is added in step 5 acquired solution, obtains GO-Au NPs sample solutions；7) ultraviolet-visible spectrophotometry is used
GO-Au NPs sample solutions are analyzed, the activity of M.SssI is thus measured.
2. according to the method described in claim 1, it is characterized in that：The HP1 hair clips substrate contains M.SssI and endonuclease
Enzyme spcificity identifies 5 '-C of sequence ↓ CGG-3 '.
3. according to the method described in claim 1, it is characterized in that：Methylation reaction described in step 1, reaction system 50nM
HP1,1 × NE buffer 2 and 0.1mM S-adenosylmethionines and M.SssI；The NE buffer 2 include 50mM
NaCl、10mM Tris-HCl、10mM MgCl2, 1mM dithiothreitol (DTT)s, pH7.9, reaction condition be 37 DEG C heat preservation 3~5h.
4. according to the method described in claim 1, it is characterized in that：M.SssI is inactivated described in step 2, reaction condition is：It will
Step 1 gained mixture heats 20~30min at 65 DEG C.
5. according to the method described in claim 1, it is characterized in that：The preparation of Au NPs-HP2 includes the following steps in step 4：
A) HP2 is restored with reducing agent, is then separated off excessive reducing agent with chromatographic column；B) acquired solution and Au NPs after detaching
Mixing, gained mixture is spare overnight at 4 DEG C；C) sodium chloride solution is added in the mixture obtained by step b, and in dark ring
It is incubated 10~15h in border, obtains Au NPs-HP2.
6. according to the method described in claim 5, it is characterized in that：The reducing agent is dithiothreitol (DTT) or TCEP hydrochlorides.
7. according to the method described in claim 5, it is characterized in that：After completing step c, the Tris-HCl solution for being 7.4 with pH
The mixture is washed, is then centrifuged for, upper solution is removed, the Tris-HCl solution that lower layer's solution is 7.4 with pH is repeated to wash
It washs, finally obtains the Au NPs-HP2 of purifying, the Au NPs-HP2 of purifying are resuspended in the Tris-HCl solution that pH is 7.4
In it is spare.
8. according to the method described in claim 5, it is characterized in that：In stepb, the grain size of the Au NPs be 13~
15nm, the Au NPs detach rear acquired solution mixing in the form of solid particle or colloidal sol with described.
9. the application of the active detection method of any one of the claim 1-8 dnmt rnas.
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