CN106990084A - A kind of being used for based on single quantum dot detects the nano-sensor of dnmt rna - Google Patents
A kind of being used for based on single quantum dot detects the nano-sensor of dnmt rna Download PDFInfo
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Abstract
The nano-sensor of dnmt rna is detected the invention discloses being used for based on single quantum dot.The nano-sensor only relates to single detection probe, it is to avoid complicated detection design and cumbersome signal amplification procedure, while have the high selectivity and high sensitivity to dnmt rna.The nano-sensor includes：The hair clip detection probe of 3' terminal biotins, the detection probe 5' end marks fluorogen flower cyanines 5 (Cy5), 3' ends are also marked with quencher Black Hole Quencher (BHQ2), and stem area includes the recognition sequence 5'GATC 3' of target DNA transmethylase；The quantum dot of pan coating Streptavidin；The probe is interacted by Streptavidin biotin to be connected on the quantum dot surface that Streptavidin is modified, and forms the Black Hole Quencher compounds of quantum dot/probe/Hua Jing 5/；The nano-sensor also includes methylation-specific endonuclease (DpnI).
Description
Technical field
The invention belongs to bioassay technique field, and in particular to a kind of being used for based on single quantum dot detects DNA first
The nano-sensor of based transferase.
Background technology
DNA methylation is main epigenetic modification in cell, is played a crucial role during various important cells, such as
Gene regulation, chromatin Structure is maintained and cell development.DNA methylation is catalyzed by dnmt rna (MTases) family.It is logical
Cross by methyl from S-adenosylmethionine (SAM) be transferred to specific dna sequence in adenine or cytosine residues, DNA methyl
Certain genomic methylation pattern can be set up and be maintained to transferase.Abnormal dnmt rna activity may interfere with
Normal methylation patterns, cause the generation of many diseases including cancer.For example, dnmt rna (1,3a and
Overexpression and the tumour of retinoblastoma 3b) occurs closely related.The activity of cytosine dna transmethylase is in colon
Cancer increases during being in progress.The expression rise of dnmt rna -1 in non-small cell lung cancer.In addition, passing through specificity suppression
Agent suppresses dnmt rna activity and provides new chance for the treatment of human cancer.Therefore, the standard of dnmt rna
Really detection and the screening of inhibitor are most important for medical diagnosis on disease and treatment.
At present, dnmt rna detection standard method be based on its co-factor radioactive label ([methyl-3
Hydrogen]-S-adenosylmethionine), but the participation of radioactive substance greatly limit the extensive use of this method.In order to avoid putting
Radioactive pollution, has developed on-radiation high performance liquid chromatography (HPLC) determination method, but this method process is cumbersome, takes
It is many.Recently, various new methods, including colorimetric method, chemoluminescence method, fluorescence method, electrochemical process etc. are established.Although these methods
Effective platform is provided for the quantitative detection of dnmt rna, but they are still not enough to sensitively detect very low
The target of abundance.In order to improve the sensitivity of detection, having been introduced into several signal amplification methods is used for dnmt rna
Detection, the target circulation of such as exonuclease III auxiliary, the amplification of endonuclease auxiliary, double-stranded specific nuclease auxiliary
Amplification, rolling circle amplification (RCA) and strand displacement amplification (SDA).In these methods, the test limit of dnmt rna is significantly
Improve, however, they need cumbersome multiple probe with amplification template to complete amplification of signal step, and due to non-specificity
Amplification often produces high background signal.Compared with traditional fluorescin and organic dyestuff, semiconductor-quantum-point has light stable
Property it is good, quantum yield is high, and wide to excite, the unique optical properties of narrow transmitting have been widely used for bio-imaging and bio-sensing
Field.But so far, rarely have is used to detect dnmt rna by integrating single molecule fluorescence detection and technology of quantum dots
Report.
The content of the invention
For above-mentioned the deficiencies in the prior art, the present invention proposes a kind of nano-sensor based on single quantum dot, described to receive
Rice sensor only relates to single detection probe, it is to avoid complicated detection design and cumbersome signal amplification procedure, has simultaneously
To the high selectivity and high sensitivity of dnmt rna.
Specifically, the present invention relates to following technical scheme：
First, the present invention provides a kind of nano-sensor based on single quantum dot, and the nano-sensor includes：
The hair clip detection probe of 3' terminal biotins, the detection probe 5' end marks fluorogen flower cyanines 5 (Cy5),
3' ends are also marked with quencher Black Hole Quencher (BHQ2), and stem area includes the recognition sequence 5'- of target DNA transmethylase
GATC-3'；
The quantum dot of pan coating Streptavidin；
The probe is connected to the quantum dot surface that Streptavidin is modified by Streptavidin-biotin interaction
On, form the Black Hole Quencher compounds of quantum dot/probe/Hua Jing 5/；
The nano-sensor also includes methylation-specific endonuclease (DpnI).
Nano-sensor of the present invention, by Cy5 in the Black Hole Quencher compounds of quantum dot/probe/Hua Jing 5/ with
The FRET of quantum dot, for fluorescence molecule Cy5 selection, although prior art is a variety of can be with quantum dot
Occur the luminescent dye molecule of FRET, such as TAMRA/Cy3/Texas Red/ rhodamines, but inventor logical
Cross comparative experimental research discovery, FRET (FRET) of this pair of the combinations of QD/Cy5 in the method for the invention
It is more efficient.
It is preferred that, the hair clip detection probe length is 36nt, and the base sequence of the hair clip detection probe is：5'-
black hole quencher 2(BHQ2)-ACA GTG ATC ATT GTT TTC AAT GAT CAC TGT-Cy5-TTT
TTT-Biotin-3'；
It is preferred that, quantum dot of the present invention is 605QDs；605QDs of the present invention refers to from company Invitrogen
The Qdot 605ITK that Corporation (California, U.S.A.) is commercially available, it is a kind of streptavidin-
Coated CdSe/ZnS QDs, maximum emission wavelength is 605nm.
It is preferred that, the nano-sensor also includes S-adenosylmethionine and methylation-specific endonuclease enzyme reaction
Cushioning liquid；
It is further preferred that the methylation-specific endonuclease enzyme reaction cushioning liquid composition is：50 mMs every
The potassium acetate risen, 20 mMs every liter of trishydroxymethylaminomethane-acetic acid, 10 mMs every liter of magnesium acetate, 100 micrograms are every
The bovine serum albumin(BSA) of milliliter, pH 7.9.
The invention also discloses the method that the nano-sensor is used to detect dnmt rna, specific method is as follows：
(1) testing sample is added and reacted in the biotinylated hair clip detection probe solution, after reaction terminates
Carry out high temperature deactivation；
(2) quantum dot that pan coating Streptavidin is added into step (1) carries out incubation reaction；
(3) Cy5 fluorescence signal is examined using the single molecular imaging system of full interior angle reflected fluorescent light technology (TIRF)
Survey, with the dnmt rna in quantitative detection testing sample.
It is preferred that, reaction temperature is 30-40 DEG C (most preferably 37 DEG C) in the step (1), and the reaction time is 1h；High temperature loses
Reaction temperature living is 70-90 DEG C (most preferably 80 DEG C), and the reaction time is 20 minutes；
It is preferred that, incubation reaction temperature is room temperature (20 DEG C) in the step (2), and the reaction time is 10min.
The invention also discloses above-mentioned nano-sensor and/or detection method in detection dnmt rna activity and/or
Application in screening DNA methyltransferase inhibitors.
The principle of nano-sensor detection method of the present invention is：Technical solution of the present invention devises a biology first
The hair clip detection probe of elementization, its 5' end marks fluorogen flower cyanines 5 (Cy5), 3' end mark quencher Black Hole Quenchers
(BHQ2), stem area includes the recognition sequence 5'-GATC-3' of target DNA transmethylase.Probe passes through Streptavidin-biotin
Interaction is connected on the quantum dot surface of Streptavidin modification, is formed quantum dot/Black Hole Quenchers of probe/Hua Jing 5/ and is answered
Compound.In the compound, because quantum dot and the insignificant transmitting crosstalks of Hua Jing 5 and the transmitting of quantum dot and flower cyanines 5 are sharp
Significant spectra overlapping between hair, quantum dot is used as the donor of FRET, and flower cyanines 5 are used as fluorescence resonance energy
The acceptor of transfer.In theory, the distance between adjacent base is about 0.34nm in DNA, the quantum dot of Streptavidin modification
Maximum radius is 7.5nm.Quantum dot in the Black Hole Quencher compounds of quantum dot/probe/Hua Jing 5/ is with spending the distance between cyanines 5
9.2nm is calculated as, and (for quantum dot/Hua Jing 5,2R in the range of FRET0=14nm).Therefore, measure
May occur effective fluorescence resonance between quantum dot and Hua Jing 5 in the sub- Black Hole Quencher nanostructureds of point/probe/Hua Jing 5/
Energy transfer, produces obvious flower cyanines 5 and launches, however, the transmitting of flower cyanines 5 is quenched completely by neighbouring Black Hole Quencher.Work as target
In the presence of dnmt rna, dnmt rna will be catalyzed methylating for recognition sequence 5'-GATC-3', and generation methylates
Product 5'-GAmTC-3'.Then, restriction endonuclease (DpnI) can cut the probe methylated, cause black hole to be quenched
Agent part discharges from the Black Hole Quencher compounds of quantum dot/probe/Hua Jing 5/.Therefore, Hua Jing 5 transmitting recovers.Finally, lead to
The photon for calculating the flower cyanines 5 being imaged based on total internal reflection fluorescent is crossed, detection target DNA transmethylase can be quantified.
Beneficial effects of the present invention：
1. the nano-sensor that the present invention is prepared is used to detect dnmt rna, when target DNA methylase
During Dam presence, detection probe is then methylated restriction endonuclease DpnI cuttings, caused by specific methylation
Black Hole Quencher BHQ2 discharges from detection probe.Therefore, the transmitting of the Cy5 from FRET recovers, and
It can be detected by the monomolecular counting being imaged based on total internal reflection fluorescent (TIRF).Whole process only relates to single detection and visited
Pin, it is to avoid complicated detection design and cumbersome signal amplification procedure, at the same it is short using molecule detection analysis time,
The few advantage of sample consumption, improves the simplicity of detection, and effectively reduces the generation of false positive signal；
2. the present invention takes full advantage of the high s/n ratio of Single Molecule Detection and the fluorescence resonance energy based on single quantum dot
The low-down background level of the nano-sensor of transfer is measured, by combining monomolecular counting technology and based on the glimmering of single quantum dot
Photoresonance energy transfer, realizes fabulous selectivity and very high sensitivity, experiment proves that, calculate test limit
For 0.002U/mL, the test limit of technology is low than ever, or even is surveyed than the target circulation fluorescence of the restriction endonuclease mediation without amplification step
The test limit of amount is low 5 times, and this method can be further used in screening and the complex biological sample of methylase inhibitors
Accurate detection, the value of great popularization and application.
Brief description of the drawings
Fig. 1：Nano-sensor based on single quantum dot sensitively detects that the principle of dnmt rna illustrates schematic diagram.
Fig. 2：Detection probe methylate and cleaved products polyacrylamide gel electrophoresis (PAGE) analysis.Wherein, swim
Road 1, the Characterization of The Products during probe of every liter of the nanomole of individualism 96；The probe and 10 units of every liter of the nanomole of swimming lane 2,96
Methylation-specific endonuclease (DpnI) in the presence of Characterization of The Products；The probe of every liter of the nanomole of swimming lane 3,96 and 20
Characterization of The Products in the presence of the dnmt rna of unit；The probe of every liter of the nanomole of swimming lane 4,96 and the methyl of 10 units
Change the Characterization of The Products in the presence of the dnmt rna of specific endonucleases (DpnI) and 20 units.
Fig. 3：(A) target DNA transmethylase (control) is not present glimmering with total internal reflection when there is target DNA transmethylase
Photoimaging (TIRF) analysis chart.(B) target DNA transmethylase (control) is not present and cyanines 5 are spent when there is target DNA transmethylase
Counting, the consumption of target DNA transmethylase is 40 units per mls.What error bar was represented is the standard of three repetition experiments
Deviation.
Fig. 4：The counting figure of the corresponding colored cyanines 5 of dnmt rna of various concentrations.Accompanying drawing display flower cyanines 5 are counted and DNA
Linear relationship between the logarithmic form of transmethylase concentration.What error bar was represented is the standard deviation of three repetition experiments.
Fig. 5：Haemophilus haemolyticus transmethylase (HhaIMTase) (40 units per mls), CpG transmethylases
(M.SssIMTase) (40 units per mls), dnmt rna (40 units per mls), bovine serum albumin(BSA) (BSA)
The count measurement value of flower cyanines 5 in the presence of (100 every liter of nanomole), using the sample without any ferment treatment as a control group
(Control).What error bar was represented is the standard deviation of three repetition experiments.
Fig. 6：Gentamicin suppresses the analysis of dnmt rna activity, and what error bar was represented is three repetition experiments
Standard deviation.
Embodiment
It is noted that described further below is all exemplary, it is intended to provide further instruction to the application.Unless another
Indicate, all technologies used herein and scientific terminology are with usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation, device, component and/or combinations thereof.
As background technology is introduced, in the prior art to detecting that the method generally existing of dnmt rna is detected
Sensitivity is low, radioactive pollution, it is time-consuming longer the shortcomings of.
In view of this, there is provided a kind of nano-sensor based on single quantum dot in an exemplary embodiment of the invention,
The nano-sensor includes：
The hair clip detection probe of 3' terminal biotins, the detection probe 5' end marks fluorogen flower cyanines 5 (Cy5),
3' end mark quencher Black Hole Quenchers (BHQ2), stem area includes the recognition sequence 5'-GATC- of target DNA transmethylase
3'；
The quantum dot of pan coating Streptavidin；
The probe is connected to the quantum dot surface that Streptavidin is modified by Streptavidin-biotin interaction
On, form the Black Hole Quencher compounds of quantum dot/probe/Hua Jing 5/；
The nano-sensor also includes methylation-specific endonuclease DpnI.
In the another exemplary embodiment of the present invention, the hair clip detection probe length is 36nt, and the hair clip detection is visited
The base sequence of pin is：5'-black hole quencher 2(BHQ2)-ACA GTG ATC ATT GTT TTC AAT GAT
CAC TGT-Cy5-TTT TTT-Biotin-3'；
In the another exemplary embodiment of the present invention, the quantum dot is 605QDs；605QDs of the present invention refer to from
The Qdot 605ITK that company Invitrogen Corporation (California, U.S.A.) is commercially available, it is a kind of
Streptavidin-coated CdSe/ZnS QDs, maximum emission wavelength is 605nm.
In the another exemplary embodiment of the present invention, the nano-sensor also includes S-adenosylmethionine and methylated
Specific endonucleases react cushioning liquid；
In the another exemplary embodiment of the present invention, the methylation-specific endonuclease enzyme reaction cushioning liquid composition
For：50 mMs every liter of potassium acetate, 20 mMs every liter of trishydroxymethylaminomethane-acetic acid, 10 mMs every liter of vinegar
Sour magnesium, the bovine serum albumin(BSA) of 100 micrograms per millilitres, pH 7.9.
There is provided the nano-sensor for detecting dnmt rna in the another exemplary embodiment of the present invention
Method, specific method is as follows：
(1) testing sample is added and reacted in the biotinylated hair clip detection probe solution, after reaction terminates
Carry out high temperature deactivation；
(2) quantum dot that pan coating Streptavidin is added into step (1) carries out incubation reaction；
(3) Cy5 fluorescence signal is examined using the single molecular imaging system of full interior angle reflected fluorescent light technology (TIRF)
Survey, with the dnmt rna in quantitative detection testing sample.
In the another exemplary embodiment of the present invention, the biotinylated hair clip detection probe solution includes biotin
The hair clip detection probe of change, S-adenosylmethionine and the restriction endonuclease (Dpn I) that methylates, the hair clip detection are visited
Pin 5' end marks fluorogen flower cyanines 5 (Cy5), 3' end mark quencher Black Hole Quenchers (BHQ2), stem area includes target DNA
The recognition sequence 5'-GATC-3' of transmethylase；
The quantum dot of pan coating Streptavidin；
The probe is connected to the quantum dot surface that Streptavidin is modified by Streptavidin-biotin interaction
On, form the Black Hole Quencher compounds of quantum dot/probe/Hua Jing 5/；
The nano-sensor also includes methylation-specific endonuclease DpnI.
In the another exemplary embodiment of the present invention, the hair clip detection probe length is 36nt, and the hair clip detection is visited
The base sequence of pin is：5'-black hole quencher 2(BHQ2)-ACA GTG ATC ATT GTT TTC AAT GAT
CAC TGT-Cy5-TTT TTT-Biotin-3'；
In the another exemplary embodiment of the present invention, the quantum dot is 605QDs；605QDs of the present invention refer to from
The Qdot 605ITK that company Invitrogen Corporation (California, U.S.A.) is commercially available, it is a kind of
Streptavidin-coated CdSe/ZnS QDs, maximum emission wavelength is 605nm.
In the another exemplary embodiment of the present invention, the nano-sensor also includes S-adenosylmethionine and methylated
Specific endonucleases react cushioning liquid；
In the another exemplary embodiment of the present invention, the methylation-specific endonuclease enzyme reaction cushioning liquid composition
For：50 mMs every liter of potassium acetate, 20 mMs every liter of trishydroxymethylaminomethane-acetic acid, 10 mMs every liter of vinegar
Sour magnesium, the bovine serum albumin(BSA) of 100 micrograms per millilitres, pH 7.9.
In the another exemplary embodiment of the present invention, reaction temperature is 30-40 DEG C (most preferably 37 in the step (1)
DEG C), the reaction time is 1h；High temperature deactivation reaction temperature is 70-90 DEG C (most preferably 80 DEG C), and the reaction time is 20 minutes；
In the another exemplary embodiment of the present invention, incubation reaction temperature is room temperature (20 DEG C), reaction in the step (2)
Time is 10min.
In the another exemplary embodiment of the present invention, above-mentioned nano-sensor and/or detection method are disclosed in detection DNA
Application in methyl transferase activity and/or screening DNA methyltransferase inhibitors.
It is further elaborated with reference to embodiment.
Embodiment 1
The detection of dnmt rna and the formation of the Black Hole Quencher nanostructureds of quantum dot/probe/Hua Jing 5/：DNA first
The detection of based transferase includes three continuous steps：First, all oligonucleotides 10 × trishydroxymethylaminomethane-second two
Amine tetraacethyl (Tris-EDTA) buffer solution dilution prepares stock solution.The hairpin probe of 1 every liter of micromole is slow in annealing in 95 DEG C
Rush solution (50 mMs every liter of sodium chloride, 5 mMs every liter of pH 8.0 trishydroxymethylaminomethane-hydrochloric acid (Tris-
HCl reaction 4 minutes, are then slowly dropped to room temperature, the hairpin probe substrate of formation is stored in 4 DEG C and used in)).Second, hair clip is visited
Methylating for pin is carried out with cleavage reaction in 50 microlitres of reaction system, includes the hairpin probe of 96 every liter of nanomoles, different
The dnmt rna of concentration, 1 × transmethylase reaction cushioning liquid (50 mMs every liter of trishydroxymethylaminomethane-
Hydrochloric acid (Tris-HCl), 10 mMs every liter of ethylenediamine tetra-acetic acid, 5 mMs every liter of 2 mercapto ethanol, pH 7.5), 160
The S-adenosylmethionine of every liter of micromole, the restriction endonuclease that methylates (Dpn I) of 6 units and 1 × limitation
Property endonuclease enzyme reaction cushioning liquid (50 mMs every liter of potassium acetate, 20 mMs every liter of trishydroxymethylaminomethane-
Acetic acid, 10 mMs every liter of magnesium acetate, the bovine serum albumin(BSA) of 100 micrograms per millilitres, pH 7.9).Reaction is anti-at 37 DEG C
Answer 1 hour, after at 80 DEG C inactivate 20 minutes.3rd, the reaction product and the quantum dot of 2 every liter of nanomoles that 50 microlitre of the above
(605QD) is in 100 microlitres of incubation cushioning liquid (3 mMs every liter of magnesium chloride, the three of 100 mMs every liter of pH 8.0
Hydroxymethyl aminomethane-hydrochloric acid (Tris-HCl), 10 mMs every liter of ammonium sulfate) in room temperature reaction 10 minutes, formed quantum
The Black Hole Quencher nanostructureds of point/probe/Hua Jing 5/.
Gel electrophoresis：Before being reacted with quantum dot, methylate with the product of cleavage reaction with 12% non denatured polypropylene
Acrylamide gel electrophoresis is analyzed.
Single Molecule Detection and data analysis：Before Single Molecule Detection, first by reaction product imaging cushioning liquid (1 milli
Gram every milliliter of glucose oxidase, 0.4% (w/v) D-Glucose, 0.04% milligram every milliliter of catalase, 50 is micro-
Gram every milliliter of bovine serum albumin(BSA), 67 mMs every liter of glycine-potassium hydroxide, 1 milligram every milliliter of water soluble vitamin life
Plain E, 2.5 mMs every liter of magnesium chloride, pH is 9.4) to dilute 10 times.It is for total internal reflection fluorescent imaging, 10 μ L samples are straight
Connect and be drawn on cover glass.Use 488 nanometers laser (50 milliwatts, Coherent, USA) excitation quantum point of sapphire
(605QD).Quantum dot and Hua Jing 5 photon are collected by 100 × object lens (Olympus, Japan), and passes through AndorIxon
DU897EMEMCD is with 500ns exposure time imaging.Image-region (200 × 400 is selected using image J (image J) software
Pixel) it is used to spend the counting of cyanines 5 (Cy5) molecule.
Specificity and inhibitor analysis：The CpG transmethylases (M.SssIMTase) and 5'-CG- of 40 units per mls
The bovine serum albumin(BSA) (BSA) of 3' transmethylases (HhaIMTase) and 100 every liter of nanomoles is used for the spy for probing into this method
The opposite sex.In order to further probe into the hair clip of the inhibitory action of gentamicin, the gentamicin of various concentrations and 96 every liter of nanomoles
Probe reacts 15 minutes in 1 × methylation reaction cushioning liquid in 37 DEG C.Then, by the DNA methyl of 40 units per mls
Transferase (MTase), the methylation-specific endonuclease (DpnI) of 6 units and the S- adenosine first of 160 every liter of micromoles
Methyllanthionine is added in solution, is reacted 1 hour at 37 DEG C, is inactivated 20 minutes after 80 DEG C.Experiment turns with above-mentioned detection DNA methyl
The detection mode for moving enzyme is identical.
The detection of dnmt rna in blood serum sample：One total reaction volume is 50 microlitres of mixture, including adds
5% human serum of various concentrations dnmt rna, the hairpin probe of 96 every liter of nanomoles, the S- glands of 160 every liter of micromoles
Glycosides methionine, the methylation-specific endonuclease (DpnI) of 6 units, 1 × specific endonucleases reaction buffering
Solution, 37 DEG C react 1 hour, after 80 DEG C inactivate 20 minutes.Test the detection mode phase with above-mentioned detection DNA methylation enzyme
Together.
Embodiment 2
The experimental verification of 2.1 principles
Technical solution of the present invention principle：Technical solution of the present invention devises a biotinylated hair clip detection and visited first
Pin, its 5' end marks fluorogen flower cyanines 5 (Cy5), 3' end mark quencher Black Hole Quenchers (BHQ2), stem area includes target
The recognition sequence 5'-GATC-3' of dnmt rna.Probe is connected to strepto- by Streptavidin-biotin interaction
On the quantum dot surface of Avidin modification, the Black Hole Quencher compounds of quantum dot/probe/Hua Jing 5/ are formed.In the compound,
Significant spectrum weight between being excited due to quantum dot and the insignificant transmitting crosstalks of Hua Jing 5 and the transmitting of quantum dot and flower cyanines 5
Folded, quantum dot is used as the donor of FRET, and flower cyanines 5 are used as the acceptor of FRET.In theory,
The distance between adjacent base is about 0.34nm in DNA, and the maximum radius of the quantum dot of Streptavidin modification is 7.5nm.Amount
The distance between quantum dot and flower cyanines 5 in the sub- Black Hole Quencher compounds of point/probe/Hua Jing 5/ are calculated as 9.2nm, and glimmering
(for quantum dot/Hua Jing 5,2R in the range of photoresonance energy transfer0=14nm).Therefore, quantum dot/probe/Hua Jing 5/ is black
Effective FRET may occur between quantum dot and Hua Jing 5 in the quencher nanostructured of hole, produce obvious
Flower cyanines 5 launch, however, flower cyanines 5 transmitting be quenched completely by neighbouring Black Hole Quencher.When target DNA transmethylase is present
When, dnmt rna will be catalyzed methylating for recognition sequence 5'-GATC-3', produce methylate 5'-GAmTC-3'.With
Afterwards, restriction endonuclease (DpnI) can cut the probe methylated, cause Black Hole Quencher part from quantum dot/spy
Discharged in the Black Hole Quencher compounds of pin/Hua Jing 5/.Therefore, Hua Jing 5 transmitting recovers.Finally, by calculating based on complete interior anti-
The photon of the flower cyanines 5 of fluorescence imaging is penetrated, detection target DNA transmethylase can be quantified.
In order to verify methylating and subsequent cleavage reaction for probe, we handle the colored black hole of cyanines 5/ at different conditions
Quencher dual labelled probe, by directly exciting Hua Jing 5, is analyzed with 12% native polyacrylamide gel electrophoresis (PAGE)
Product.As shown in Fig. 2 when only existing probe, it is impossible to it was observed that the characteristic bands (swimming lane 1) that flower cyanines 5 are excited, illustrate in probe
Black Hole Quencher colored cyanines 5 have been quenched completely.By contrast, when the restriction endonuclease that methylates (DpnI) and DNA methyl
Transferase simultaneously in the presence of, the characteristic bands (swimming lane 4) that Hua Jing 5 is excited can be clearly observed, show probe be cut, it is black
Hole quencher therefrom discharges, and the fluorescence of flower cyanines 5 recovers.In addition, in no dnmt rna or the restriction nuclease that methylates
In the case of restriction endonuclease (DpnI), Hua Jing 5 characteristic bands (swimming lane 2 and swimming lane 3) are not observed equally, show the cutting of probe
Can only occur in two kinds of enzymes in the presence of simultaneously.These results clearly show that detection probe can be by target DNA transmethylase first
Base, is then methylated restriction endonuclease (DpnI) specificity cutting.
For the feasibility that validating DNA transmethylase is detected, pass through the list being imaged based on total internal reflection fluorescent (TIRF)
Numerator counts detect the activity of dnmt rna.As shown in Figure 3A, the swashing in wavelength 488nm with quantum dot (605QD)
Hair, in the presence of target DNA transmethylase, can detect obvious flower cyanines 5 (Cy5) signal.On the contrary, there is no DNA first
In the presence of based transferase (Control), then Hua Jing 5 (Cy5) signal can not be detected.In addition, the counting by flower cyanines 5 also may be used
With the activity of quantitative measurment dnmt rna.As shown in Figure 3 B, it can be detected in the presence of dnmt rna aobvious
High flower cyanines 5 are write to count, and in the case of no dnmt rna (Control groups), then not it is observed that obvious flower
Cyanines 5 are counted.In summary, the technical program is feasible.
2.2 sensitivity technique
In order to determine the sensitivity that the technical program detects dnmt rna, we turn in the DNA methyl of various concentrations
Move under enzyme and measure the counting of flower cyanines 5.As shown in figure 4, as the concentration of dnmt rna from 0 increases to 80U/mL, Hua Jing 5
Counting increases successively.It is worth noting that, on logarithmic scale, Hua Jing 5 counting and the concentration of dnmt rna from
Linear correlation (Fig. 4) is presented in the range of 0.004 to 2U/mL.
Its regression equation is N=57.02+16.98log10C, calculates detection and is limited to 0.002U/mL, than ever technology
Test limit it is low, or even than without amplification step restriction endonuclease mediation target circulation fluorescence measurement test limit it is low 5 times.Improve
Sensitivity is mainly due to following reason：Multiple detection probes are connected on (1) single quantum dot, make FRET
Efficiency is sufficiently high；(2) Single Molecule Detection has sufficiently high signal to noise ratio；(3) FRET based on single quantum dot
Nano-sensor can be produced.To sum up illustrate that the sensitivity of the technical scheme is sufficiently high.
2.3 specific detection
In order to assess the specificity of the technical program, we use CpG transmethylases (M.SssIMTase), and hemolytic is thermophilic
Blood bacillus transmethylase (HhaIMTase) and bovine serum albumin(BSA) (BSA) carry out specificity experiments as negative control.CpG
Cytosine residues in transmethylase (M.SssIMTase) catalytic sequence 5'-GCGC-3' methylate, the bloodthirsty bar of hemolytic
Cytosine residues methylate in fungus beetle based transferase (HhaIMTase) catalytic sequence 5'-CG-3', bovine serum albumin(BSA) (BSA)
It is used as incoherent protein control.In theory, these non-specific targets can not all be catalyzed methylating for detection probe, to produce
The 5'-GA of methylation-specific endonuclease (DpnI)mTC-3' recognition sites, thus Black Hole Quencher can not from quantum dot/
Discharged in the Black Hole Quencher compounds of probe/Hua Jing 5/, and then can't detect Hua Jing 5 signal.As shown in figure 5, in target
In the presence of dnmt rna, it is observed that the signal for the flower cyanines 5 being remarkably reinforced.By contrast, CpG transmethylases
(M.SssIMTase), in the presence of Haemophilus haemolyticus transmethylase (HhaIMTase) and bovine serum albumin(BSA) (BSA),
Compared with the control group of not any enzyme, all without the significantly colored signal intensity of cyanines 5.It is indicated above that the technical program has height
Specificity, can distinguish target DNA transmethylase and non-target dnmt rna and other uncorrelated albumen, can be used in
Target DNA transmethylase is detected in complicated biotic environment.
The inhibitor analysis of 2.4 dnmt rnas
Can the inhibitor for detecting dnmt rna to detect the technical program be used for, we be all with a kind of many institutes
Broad-spectrum antibiotic-the gentamicin known, is used as model inhibitor.As shown in fig. 6, as gentamicin concentration from 0 increases to 50
Every liter of micromole, the relative activity of dnmt rna is significantly reduced.Calculate the 503nhibiting concentration (IC for obtaining gentamicin50,
I.e. by the inhibitor concentration needed for the activity reduction by 50% of dnmt rna) it is 11.25 every liter of micromoles, this is with being reported
503nhibiting concentration (IC50) it is that every liter of 10.0 micromole is consistent.These results clearly show that, the detection that we are proposed
Method can be successfully applied to the screening of dnmt rna inhibitor, have very big potentiality to the discovery of antibiotic.
The preferred embodiment of the application is the foregoing is only, the application is not limited to, for the skill of this area
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair
Change, equivalent substitution, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. a kind of nano-sensor based on single quantum dot, it is characterised in that the nano-sensor includes：
The hair clip detection probe of 3' terminal biotins, the detection probe 5' end marks fluorogen flower cyanines 5 (Cy5), 3' ends
End is also marked with quencher Black Hole Quencher (BHQ2), and stem area includes the recognition sequence 5'-GATC- of target DNA transmethylase
3'；
The quantum dot of pan coating Streptavidin；
The probe is connected on the quantum dot surface that Streptavidin is modified by Streptavidin-biotin interaction,
Form the Black Hole Quencher compounds of quantum dot/probe/Hua Jing 5/；
The nano-sensor also includes methylation-specific endonuclease DpnI.
2. the nano-sensor as claimed in claim 1 based on single quantum dot, it is characterised in that the hair clip detection probe is long
Spend for 36nt, the base sequence of the hair clip detection probe is：5'-black hole quencher 2(BHQ2)-ACA GTG
ATC ATT GTT TTC AAT GAT CAC TGT-Cy5-TTT TTT-Biotin-3'。
3. the nano-sensor based on single quantum dot as claimed in claim 1, it is characterised in that the quantum dot is
605QDs。
4. the nano-sensor as claimed in claim 1 based on single quantum dot, it is characterised in that the nano-sensor is also wrapped
Include S-adenosylmethionine and methylation-specific endonuclease enzyme reaction cushioning liquid.
5. the nano-sensor as claimed in claim 4 based on single quantum dot, it is characterised in that the methylation-specific core
Sour inscribe enzyme reaction cushioning liquid is constituted：50 mMs every liter of potassium acetate, 20 mMs every liter of trihydroxy methyl amino first
Alkane-acetic acid, 10 mMs every liter of magnesium acetate, the bovine serum albumin(BSA) of 100 micrograms per millilitres, pH 7.9.
6. any one of the claim 1-5 nano-sensors are used for the method for detecting dnmt rna, it is characterised in that
Method is as follows：
(1) testing sample is added and reacted in the biotinylated hair clip detection probe solution, reaction is carried out after terminating
High temperature deactivation；
(2) quantum dot that pan coating Streptavidin is added into step (1) carries out incubation reaction；
(3) Cy5 fluorescence signal is detected using the single molecular imaging system of full interior angle reflected fluorescent light technology (TIRF), with
Quantitatively detect the dnmt rna in testing sample.
7. detection method as claimed in claim 6, it is characterised in that reaction temperature is 30-40 DEG C in the step (1), excellent
Elect 37 DEG C as, the reaction time is 1h；High temperature deactivation reaction temperature is 70-90 DEG C (most preferably 80 DEG C), and the reaction time is 20 minutes.
8. detection method as claimed in claim 7, it is characterised in that reaction temperature is 37 DEG C in the step (1)；High temperature loses
Reaction temperature living is 80 DEG C.
9. detection method as claimed in claim 6, it is characterised in that incubation reaction temperature is room temperature in the step (2), instead
It is 10min between seasonable.
10. any one of any one of the claim 1-5 nano-sensors and/or claim 6-9 detection method is in inspection
The application surveyed in dnmt rna activity and/or screening DNA methyltransferase inhibitors.
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