CN110029153A - Nucleic acid polypeptide complex probe and its preparation method and application - Google Patents
Nucleic acid polypeptide complex probe and its preparation method and application Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
Nucleic acid polypeptide complex probe and its preparation method and application, structure is as follows:The R1 is DNA:5'-biotin-TCCATCATTACCCGGCAGTATTA-3'；R2 is substrate polypeptide: GDKAVLGVDPFR.The present invention is for the first time by the nuclease-mediated isothermal duplication strategy of double-stranded specific in conjunction with targeting protein omics technology, by double-stranded specific nuclease degradation selectivity DNA- polypeptide: the DNA chain in miRNA hybrid, the ability of the cyclic amplification of a large amount of nucleic acid polypeptide complex probe fragment and the high sensitivity of targeting protein omics technology examining report polypeptide are discharged with very small amount of miRNA, highly selective and wider dynamic range combines, a kind of quasi- targeting protein group method of completely new quantitative low abundance miRNA (such as miRNA in stem cell) is developed.
Description
Technical field
The invention belongs to biology and pharmaceutical technology fields, and in particular to a kind of nucleic acid polypeptide complex probe and its preparation side
Method and application.
Background technique
MicroRNAs (miRNAs) is a kind of single-stranded non-coding RNA with 18-25 length of nucleotides.They pass through
The translation of mRNA (mRNA) is inhibited to play important adjustment effect in many bioprocess.It is nearest the study found that
The unconventionality expression of miRNA and a series of cancers including breast cancer are closely related.Tumour is made of foreign cell, wherein doing
Cell has the characteristic of self-renewing and Multidirectional Differentiation ability, is the most important reason that tumour occurs, maintains, shifts and recur.
Therefore, we have reason to speculate that miRNAs is related with the key property of tumor stem cell (CSCs).Moreover, more and more evidences
Show that the unconventionality expression of miRNAs in tumor stem cell can be used as the potential source biomolecule marker and target of cancer diagnosis and treatment.
Regrettably, the research of tumor stem cell fails to find general, clear and informative gene expression profile so far.Cause
This determines that they are causing that people's is emerging as the potentiality of new marker and drug targets as indicated by nearest research
Interest.In order to preferably realize these targets, the expression of accurate quantitative analysis miRNAs is the prerequisite of its application.
Currently, since miRNAs has, length is short, similarity is high between family member, abundance is low etc. in total serum IgE sample
Unique property, detection miRNA are still challenging.Most of miRNA detection method is all indirect at present, including micro- battle array
Column, polymerase chain reaction (PCR) and next-generation sequencing technologies.These methods usually require to carry out chemistry/enzymatic to target miRNA
Modification or pre- amplification.Although such techniques offer a large amount of useful information, result obtained is generally difficult to interpret, and
Their indirect property may the special sexual deviation of calling sequence.The some Direct Inspection Technologies developed recently are (for example, based on capillary
The method of electrophoresis tube (CE), differential interference contrast (DIC) imaging and are based on the spectral detection that is assisted by double-stranded specific nuclease
The method of electrochemistry) it does not need that miRNA expand/modify.But allow they be applied to practice there is still a need for make much exerting
Power, because most of these technologies are in the early stage of exploitation.Moreover, these most of measurements can only provide it is limited qualitative
Data.Accordingly, it is difficult to be compared between different research/laboratories.With to the detection micro change of miRNA expression
Increase in demand, rather than just the presence or shortage for detecting specific miRNA, there is an urgent need to a kind of sensitive quantitative detections
The method of miRNA.Therefore, it is based on this demand, is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) we have developed a series of
Quasi- targeting protein group method quantify miRNA.
In recent years, since Modern Mass Spectrometry instrument has highly sensitive, highly selective and wide dynamic range excellent performance, base
Have become the indispensable tool of quantification of protein in the targeting protein group of LC-MS/MS.The key of this technology be
The testing goal albumen of peptide level specificity.Destination protein passes through the polypeptide of hydrolytic digestion generation as feature polypeptide, and
Pass through Selective reaction monitoring or multiple-reaction monitoring (SRM/MRM) Lai Dingliang.However, due to miRNA mass spectrum behavior complexity and not yet
It is parsed, directly with Mass Spectrometer Method miRNA and infeasible, this was discussed in research before this.In this context, we will
The concept of feature polypeptide be introduced into miRNA it is quantitative in, and a kind of method for developing quasi- targeting protein group.In the method
In, the signal of miRNA is converted into the mass signal of report peptide fragment by the nucleic acid polypeptide complex probe of designed, designed by we,
Then with LC-MS/MS come quantifying reporter peptide fragment.
It is well known that the amplitude of variation of miRNA is up to 4 orders of magnitude, count to from several copies of each cell more than 50,
000 copy number differs.For example, for tumor stem cell, inside every 1000 cells < 1.Therefore, miRNA quantity therein
It may be very low.For low-abundance miRNA, the sensitivity for the quasi- targeting proteins group detection method that we develop at present may
It is not enough to detect, especially if this method will be applied to clinical practice.
Summary of the invention
The technical issues of solution: the present invention provides a kind of nucleic acid polypeptide complex probe and its preparation method and application, tool
Body is to combine the nuclease-mediated isothermal of double-stranded specific for detecting probe of low-level miRNAs and preparation method thereof and its
Expand the application of strategy.
Technical solution: nucleic acid polypeptide complex probe, structure are as follows:
The R1 is DNA:5'-biotin-TCCATCATTACCCGGCAGTATTA-3'；R2 is substrate polypeptide:
GDKAVLGVDPFR
The preparation method of above-mentioned nucleic acid polypeptide complex probe, preparation step are as follows: be also with trichloroethyl phosphate in proportion
Former agent, the DNA:5'-biotin-TCCATCATTACCCGGCAGTATTA-3' that the end the 3' disulfide bond that 200 μ L concentration are 1 μM is modified
It is mixed with the TCEP of 20 μ L reduction pearl, shakes 2h in 37 DEG C of reactions；Then 1000 × g of sample is centrifuged 6min；Take above-mentioned preparation
The supernatant containing the DNA reduced, in supernatant be added same volume 20 μM of aminoterminal maleimide it is amine-modified
Substrate polypeptide: GDKAVLGVDPFR, 37 DEG C of oscillation 4h carry out conjugation reactions, are carried out using high performance liquid chromatography to reaction product
Purifying removes extra unbonded DNA and substrate polypeptide.
Application of the above-mentioned nucleic acid polypeptide complex probe in the nuclease-mediated isothermal duplication of joint double-stranded specific.
The specific steps of above-mentioned application are as follows: 100 μ L are contained into 1 × double-stranded specific nuclease buffer, are dissolved in 25mM
The 0.20U double-stranded specific nuclease of Tris-HCl, pH 8.0,50wt.% glycerol, 20U RNase inhibitor, 200nM nucleic acid
The target miRNA of polypeptide complex probe and various concentration reacts 1 hour for 60 DEG C in MJ Mini thermal cycler；Isothermal duplication
Afterwards, 100 μ L 10mM EDTA are added in said mixture to incubate 5 minutes at 60 DEG C, 20 μ L Streptavidin agar is then added
Sugared microballoon removes remaining biotinylated nucleic acid polypeptide complex probe, with 50mM NH4HCO3After washing pearl, collection contains
The supernatant of the nucleic acid polypeptide complex probe of digestion is in case subsequent digestion and Mass Spectrometer Method.
Application of the above-mentioned nucleic acid polypeptide complex probe in the kit that preparation detects low abundance miRNA level.
The kit for detecting low abundance miRNA level, contains above-mentioned nucleic acid polypeptide complex probe.
By the present invention in that with nucleic acid polypeptide complex probe, the thought of feature polypeptide can be by targeting protein group
It is effectively maintained.Nucleic acid polypeptide complex probe is to be combined to be formed by two kinds of molecule covalents of biotinylated DNA and polypeptide
, substrate polypeptide includes the positioned trypsin cleavage site before reporter polypeptide and reporter polypeptide.Target miRNA is mainly and biotin
The nucleic acid polypeptide complex probe of change hybridizes.MiRNA-DNA- polypeptide pair is cut by introducing double-stranded specific nucleic acid enzyme spcificity
Nucleic acid polypeptide complex probe chain in serobila discharges target miRNA integrally and miscellaneous with another nucleic acid polypeptide complex probe
It hands over, causes next round cutting, release and hybridization.One target miRNA wait mildly without fixing condition under be cut on thousands of
Ten thousand nucleic acid polypeptide complex probes, the process can recycle, so as to realize significant signal amplification.Finally, utilizing chain
Mould Avidin-Biotin interaction is removed excessive nucleic acid polypeptide complex probe by Streptavidin agarose microbeads
It removes, and digestion release is carried out to the report peptide fragment of supernatant and uses liquid chromatography mass spectrometric serial connection technology (LC-MS/MS) quantitative detection.
By this method, target miRNA signal can be converted into the level of reporter polypeptide.To nucleic acid polypeptide complex probe into
After row preparation and identification, the conditions of our isothermal duplication processes also nuclease-mediated to double-stranded specific (temperature of reaction with
Time, the concentration of double-stranded specific nuclease, the concentration of probe and reaction time) it optimizes.Finally, we are directed at targeting egg
White matter group method has carried out methodology validation, and this method is applied to the detection of low abundance miRNA, such as quantitatively mammary gland
Cancer stem cell (BCSCs) and horizontal from the miR-200c in the stem cell that tumor of breast separates, while by data of acquisition and logical
The data for crossing quantitative reverse transcription PCR (qRT-PCR) acquisition are compared.
The utility model has the advantages that 1) nucleic acid polypeptide complex probe of the present invention, can chemical synthesis, stability is good；2) this hair
It is bright that the nuclease-mediated isothermal duplication strategy of nucleic acid polypeptide complex probe joint double-stranded specific is applied to quantitative cream for the first time
Gland cancer stem cell and horizontal from the miR-200c in the stem cell that breast cancer tissue separates；3) nucleic acid polypeptide of the present invention is multiple
Closing physical prospecting needle, preparation method is simple, and what is utilized is that the terminal modified maleimide of polypeptide amino and DNA3' are end modified
Michael addition reaction occurs for sulfydryl, belongs to covalent linkage, and reaction rate is high, and time-consuming short, the purity and yield of product are higher, can
It is directly used in cell and tissue test；4) present invention for the first time that double-stranded specific is nuclease-mediated isothermal duplication strategy and target
It is combined to proteomic techniques, by double-stranded specific nuclease degradation selectivity DNA- polypeptide: the DNA in miRNA hybrid
Chain discharges the ability and targeting egg of the cyclic amplification of a large amount of nucleic acid polypeptide complex probe fragment with very small amount of miRNA
Highly sensitive, the highly selective and wider dynamic range of white matter omics technology examining report polypeptide combines, by quasi- target
Optimize to proteomics quantitative approach, has developed a kind of completely new quantitative low abundance miRNA (such as miRNA in stem cell)
Quasi- targeting protein group method；5) the nuclease-mediated amplification procedure of double-stranded specific of the present invention is an isothermal
Reaction process avoids the limitation of traditional miRNA amplification technique, such as high-precision requirement and thermal cycle to equipment well
Deng requirement.6) the nuclease-mediated amplification procedure of nucleic acid polypeptide complex probe joint double-stranded specific of the present invention is molten
Hybridization and double-stranded specific nucleic acid enzymatic hydrolysis are carried out in liquid, using the method for no immobilization probe, are avoided immobilization probe and are fixed
Change process space steric effect, the forfeiture of oligonucleotides configuration freedom degree, immobilization particle surface it is unstable the disadvantages of；7) originally
The isothermal duplication strategy that the invention double-stranded specific nuclease is mediated can be released largely by minimal amount of miRNA
Nucleic acid polypeptide complex probe, the sensitivity of detection is greatly improved；8) quasi- targeting protein group method of the present invention
Low abundance miRNA can be quantified on the nuclease-mediated isothermal duplication policy grounds of double-stranded specific.
Detailed description of the invention
Fig. 1 be amplifying nucleic acid polypeptide complex probe of the present invention preparation and its combine double-stranded specific it is nuclease-mediated etc.
The flow diagram of temperature amplification strategy detection miR-200c new method；
Fig. 2 is the map of reporter polypeptide AVLGVDPFR in the present invention, and wherein A is daughter ion figure, and B is LC-MS/MS chromatography
Figure, and the interior target chromatogram of corresponding isotope labelling；
Fig. 3 be the present invention in DNA and substrate polypeptide GDKAVLGVDPFR before the reaction after high-efficient liquid phase chromatogram, wherein
A:DNA and substrate polypeptide GDKAVLGVDPFR before the reaction with the efficient liquid after reaction at the UV absorption wavelength 260nm of DNA
Phase chromatogram；High performance liquid chromatography of the B:DNA and substrate polypeptide GDKAVLGVDPFR at the UV absorption wavelength 220nm of polypeptide
Figure；
Fig. 4 be in the present invention DNA- polypeptide complex and individual DNA with MALDI-TOF/TOF mass spectrum in cation mould
The mass spectrogram detected under formula, wherein A:DNA mass spectrogram, Theoretical Mass 7692.40；B:DNA- polypeptide complex mass spectrum
Figure, Theoretical Mass 9026.84；
Fig. 5 is high-efficient liquid phase chromatogram and liquid matter of the amplifying nucleic acid polypeptide complex probe of the present invention before digestion and after digestion
Coupled HPLC figure, wherein A: nucleic acid polypeptide complex probe is before digestion and after digestion at DNA UV absorption wavelength 260nm
High-efficient liquid phase chromatogram；B: nucleic acid polypeptide complex probe is before digestion and after digestion in the liquid chromatography mass on the channel MRM
Coupled HPLC figure；
Fig. 6 is to detect the hybridization of miR-200c and DNA- polypeptide probe and double-stranded specific nucleic acid by HPLC in the present invention
The product figure that digestion is cut.Wherein the retention time of miR-200c is 7.3min, and the retention time of nucleic acid polypeptide complex probe is
The retention time of 17.9min, miR-200c and the compound of nucleic acid polypeptide complex probe hybridization is 17.3min, miR-200c
For compound with the hybridization of nucleic acid polypeptide complex probe in the presence of double-stranded specific nuclease, enzymatic hydrolysis releases miR-200c；
Fig. 7 A is hybridization/double-stranded specific nuclease incubation temperature optimization figure in the present invention；
Fig. 7 B is the optimization figure of double center chain specific nucleic acid enzyme concentration of the present invention；
Fig. 7 C is the time dependence figure of various concentration miR-200c in the present invention；
Fig. 8 is that (i.e. 60 DEG C, 100pM miR-200c, 200nM nucleic acid polypeptide is multiple under identical experiment condition in the present invention
Close physical prospecting needle and 1h reaction time), with immobilization probe and the detection signal observed in the solution without immobilization probe；
Fig. 9 be double center chain specific nucleic acid enzyme of the present invention mediation isothermal duplication strategy miR-200c, SM-200c,
Selectivity on miR-200b, miR-141 and miR-21；
Figure 10 is the typical calibration curve (1fM to 100pM) figure of miR-200c standard in the present invention.By reporter polypeptide and stabilization
The relative peak area ratio with mutually homotactic internal standard peptide of isotope labelling is compared with concentration；
Figure 11 is the expression water for measuring miR-200c in MCF-7 cell and BCSCs in the present invention by qRT-PCR method
Flat figure；
The percentage example diagram that Figure 12 is BCSCs in 16 human breast carcinoma patients in the present invention；
Figure 13 is the miR-200c expression figure detected in 16 human breast carcinoma patients in the present invention.
Specific embodiment
The following examples can make those skilled in the art that the present invention be more fully understood, but not limit this in any way
Invention.Without departing from the spirit and substance of the case in the present invention, it modifies and replaces to made by the method for the present invention, step or condition
It changes, all belongs to the scope of the present invention.Unless otherwise specified, technological means used in embodiment is ripe for those skilled in the art
The conventional means known.
The preparation and identification of 1 nucleic acid polypeptide complex probe of embodiment
(1) preparation and purification of nucleic acid polypeptide complex probe
Using trichloroethyl phosphate as reducing agent, the DNA:5'-biotin- for the end the 3' disulfide bond modification that 200 μ L concentration are 1 μM
TCCATCATTACCCGGCAGTATTA-3'(Sheng Gong Bioisystech Co., Ltd, Shanghai) with the TCEP of 20 μ L restore pearl (Sai Mo
ThermoFisher Scientific Company, the U.S.) mixing, 2h is shaken in 37 DEG C of reactions；Then 1000 × g of sample is centrifuged 6min；Take above-mentioned system
The standby supernatant containing the DNA reduced, 20 μM of the aminoterminal maleimide that same volume is added in supernatant are amine-modified
Substrate polypeptide: GDKAVLGVDPFR (Jie Tai Biotechnology Co., Ltd, Shanghai), 37 DEG C of oscillation 4h carry out conjugation reactions, tightly
Followed by conjugation front and back retention time difference (retention time of DNA and DNA- polypeptide complex be respectively 10.4min and
18.2min, such as Fig. 3: A) reaction product is purified using semipreparative high performance liquid chromatography, remove extra unreacted
The purity of DNA and substrate polypeptide, product after purification carries out Purity by HPLC, and purity is 98% or more.In order to exclude by
It is total to the interference that appearance may cause in peptide fragment, remaining condition is constant, and the wavelength (220nm) with detection peptide fragment is compound to DNA- polypeptide
Object is detected, and as shown in Fig. 3: B, peptide fragment could not be eluted out from chromatographic column.
(2) Structural Identification of nucleic acid polypeptide complex probe
Newly synthesized DNA- polypeptide complex first passes around MALDI-TOF/TOF mass spectral analysis and is verified (such as Fig. 4),
The increased molecular weight of DNA and the molecular weight of substrate polypeptide match.Newly synthesized probe also by being characterized with tryptic digestion,
The nucleotide and polypeptide of digestion release are examined with high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometric combination (LC-MS/MS) respectively
It surveys.As expected, occur new chromatographic peak (such as Fig. 5) after digestion.It may be noted that explanation is new chromatographic peak
Retention time and for conjugation reaction DNA retention time and substrate polypeptide retention time it is not quite identical.Retain
The migration of time is loaded on DNA mainly since three amino acid residues fall off from substrate polypeptide.So practical
That we detect after upper digestion is DNA-GDK and AVLGVDPFR (reporter polypeptide).Finally measure nucleic acid polypeptide complex probe
Digesting efficiency be 95.4 ± 3.4%, compared with the efficiency (95.8 ± 2.6%) of the independent digestion of substrate polypeptide, difference is without statistics
Meaning is learned, shows that the nucleic acid moiety of probe will not have an impact the digestion of trypsase.
The nuclease-mediated isothermal duplication strategy of 2 double-stranded specific of embodiment
(1) condition optimizing of the nuclease-mediated amplification of double-stranded specific
The nuclease-mediated amplification of double-stranded specific is dependent on double-stranded specific nuclease to double-stranded DNA or DNA:RNA
The digestion selectivity that DNA chain is special in heteroduplex.In this study, by HPLC analysis confirm DNA- polypeptide complex and
The generation of miR-200c hybridization and the digestion of double-stranded specific nuclease.As shown in fig. 6, miR-200c and nucleic acid polypeptide complex are visited
The retention time of needle is respectively 7.3 and 17.9 minutes.After hybridizing with excessive miR-200c, nucleic acid polypeptide complex probe peak disappears
It loses, the hybrid product newly formed occurred at 17.3 minutes.After double-stranded specific nuclease is further processed, only observe by double
Chain specific nucleic acid enzymatic hydrolysis generate part DNA- polypeptide fragment and release miR-200c, and discharge miR-200c and its
Base level is identical.Importantly, the participation of hybridization and the digestion of double-stranded specific nuclease means that signal obtained exists
It is complementary during the two, so that experiment parameter optimizes some complexity.Therefore, we study and optimize the research
In all Key Experiment conditions (for example, concentration of reaction temperature and time and double-stranded specific nuclease and probe).
A) reaction temperature
For double-stranded specific nuclease, it loses activity under 70 DEG C or higher temperature, and 60 DEG C (about) are double-strand spies
The optimum temperature of specific nuclease activity.Other than the temperature requirement of double-stranded specific nuclease, hybridization is easier by temperature shadow
It rings, high temperature can promote the hybridization and subsequent dissociation of miRNA.Therefore, we have studied gradient temperature reactions.Work as reaction temperature
When between 30 DEG C and 60 DEG C, the significant increase of signal, but the significant reduction (Fig. 7 A) at 60 DEG C to 70 DEG C are detected.Meanwhile miR-
The melting temperature (Tm) of 200c is 66 DEG C.Therefore, signal reduces the denaturation that should be attributed to double-stranded helical.In following experiment, it selects
60 DEG C are selected as reaction temperature.
B) double-stranded specific nuclease concentration
Hybridization/cutting circulation can continue until the complete cutting of all DNA- peptide probes, this is because in this process
The consumption of double-stranded specific nuclease is seldom.In addition, the concentration of double-stranded specific nuclease can influence reaction rate.When double
When the concentration of chain specific nucleic acid enzyme is high, the enzymolysis speed of double-stranded specific nuclease quickly, and can be accordingly in short-term
It is interior to can achieve expected result.On the contrary, the time of enzymatic hydrolysis needs longer when the concentration of double-stranded specific nuclease is low.
It is consistent with speculating, the experimental results showed that reaction rate all increases sharply (Fig. 7 B) before concentration reaches 0.10U.
It is worth noting that, generating a major reason of linear relationship between miRNA concentration and detection signal is them
It is one-to-one dependency relationships.Since final signal is to digest co- controlling by hybridization and double-stranded specific nucleic acid, because
Only when through hybridization control probe release, signal is only the linear dependence of target miRNA concentration possible for this.Therefore, foot
Enough high double-stranded specific nuclease concentrations may insure that enzymatic lysis process will not be the step of determining rate.Based on these hairs
Existing, final we have selected double-stranded specific nuclease concentration for 0.20U.
C) concentration and probe concentration and reaction time
After determining double-stranded specific nuclease concentration and reaction temperature, it is thus necessary to determine that the concentration of probe and reaction time.
The double-stranded specific nuclease of 200nM nucleic acid polypeptide complex probe and 0.20U react at 60 DEG C, signal with miRNA and
Reaction time is scaled.As shown in fig. 7c, the up to miR-200c of 100pM has been observed good after being incubated for 1 hour
Good linear response.
(2) excessive DNA-polypeptide removal
It is previously most of to be related to the researchs or be fixed on probe or be fixed on target miRNA that miRNA is detected.
In view of immobilization process space steric effect, the forfeiture of oligonucleotides configuration freedom degree, immobilization particle surface it is unstable
The disadvantages of, hybridization and double-stranded specific nucleic acid enzymatic hydrolysis are carried out in the solution, and we have developed no immobilization strategies.Our result
Show to be compared under identical experiment condition, the initial reaction rate of nucleic acid in solution polypeptide complex probe is than fixing
It is high 1.8 times (Fig. 8) to change probe.
(3) the nuclease-mediated isothermal duplication of double-stranded specific
The nuclease-mediated isothermal duplication step of double-stranded specific: firstly, 100 μ L are contained 1 × double-stranded specific nucleic acid
Enzyme buffer liquid (50mM Tris-HCl, pH 8.0,5mM MgCl2, 1mM DTT), 0.20U double-stranded specific nuclease (is dissolved in
25mM Tris-HCl, pH 8.0,50wt.% glycerol), 20U RNase inhibitor, 200nM nucleic acid polypeptide complex probe and not
Target miRNA (1fM, 2.5fM, 10fM, 100fM, 1pM, 10pM and 100pM) with concentration is in MJ Mini thermal cycler (Bio-
Rad, Hercules, CA, USA) in 60 DEG C react 1 hour.After isothermal duplication signal, in order to inactivate double-stranded specific nuclease,
After 100 μ L 10mM EDTA are added, mixture is incubated 5 minutes at 60 DEG C.Then 20 μ L Streptavidin agaroses are added
Microballoon removes remaining biotinylated nucleic acid polypeptide complex probe, with 50mM NH4HCO 3After washing pearl, collection contains
The supernatant of the DNA- peptide probes of digestion is in case subsequent digestion and Mass Spectrometer Method.
Finally, we also have detected in this way to 5 kinds of miRNAs (i.e. miR-200c, SM-miR-200c (single bases
Mispairing), the ability of miR-200b (double alkali yl mispairing), miR-141 (four base mispairings) and base mispairing discrimination miR-21).
Base (100%) > single base mismatch (12.7%) > double alkali yl mispairing (7.3%) > tetra- base mispairing is matched as the result is shown
(3.5%) (Fig. 9).In addition, almost negligible signal peak can be observed in the presence of miR-21.In addition to hybrid process, this
The ability of the identification mispairing of kind method is also attributed to the fact that double-stranded specific nuclease, it can distinguish complementary and mispairing well
DNA-RNA double-strand.
Embodiment 3 quantifies the miR-200c in BCSC using the nuclease-mediated amplification joint LC-MS/MS of double-stranded specific
Using the method in embodiment 2, the content of the miR-200c in MCF-7 cell and breast carcinoma stem cell is determined,
Respectively (5.86 ± 1.40) × 103Copy/cell and (3.80 ± 0.66) × 102Copy/cell.In breast carcinoma stem cell
MiR-200c level is lower than cancer cell (p < 0.05), consistent with research report before.Before studies have shown that miR-200c can be with
Inhibit the expression of several target genes such as PDCD10 and BMI1, these genes participate in inhibiting self-renewing, differentiation and the tumour of BCSCs
Occur.Therefore, the reduction of miR-200c can be explained in BCSCs.Furthermore in order to compare with conventional method, we are also logical
It crosses qRT-PCR and has detected miR-200c (Figure 11) in above-mentioned cell.Compared with our method, miR- that qRT-PCR is detected
200c level is slightly lower, but there were significant differences without observation two methods result.
In addition, we also carry out to from the miR-200c in the stem cell of 16 human breast carcinoma tissue sample magnetic sortings
Quantitative analysis.The result shows that BCSCs accounts for 12.1 ± 4.3% (range: 3.0-21.5%) (Figure 12) of total cell in tumour；
The horizontal quantitative of miR-200c is (1.43 ± 0.96) × 103Copy/breast carcinoma stem cell (range: (0.50-3.13) × 103
Copy/breast carcinoma stem cell) (Figure 13).
The technical concepts and features of above example only to illustrate the invention, its object is to allow person skilled in the art to be
It cans understand the content of the present invention and implement it accordingly, it is not intended to limit the scope of the present invention.It is all smart according to the present invention
The equivalent transformation or modification that refreshing essence is done, should be covered by the protection scope of the present invention.
Sequence table
<110>Nanjing Medical University
<120>nucleic acid polypeptide complex probe and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tccatcatta cccggcagta tta 23
<210> 2
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Gly Asp Lys Ala Val Leu Gly Val Asp Pro Phe Arg
1 5 10
<210> 3
<211> 9
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Ala Val Leu Gly Val Asp Pro Phe Arg
1 5
Claims (7)
1. nucleic acid polypeptide complex probe, it is characterised in that structure is as follows:
The R1 is DNA:5'-biotin-TCCATCATTACCCGGCAGTATTA-3'；R2 is substrate polypeptide:
GDKAVLGVDPFR。
2. the preparation method of nucleic acid polypeptide complex probe described in claim 1, it is characterised in that preparation step are as follows: in proportion,
Using trichloroethyl phosphate as reducing agent, the DNA:5'-biotin- for the end the 3' disulfide bond modification that 200 μ L concentration are 1 μM
TCCATCATTACCCGGCAGTATTA-3' is mixed with the TCEP of 20 μ L reduction pearl, shakes 2h in 37 DEG C of reactions；Then by sample
1000 × g is centrifuged 6min；Same volume is added in the supernatant containing the DNA reduced for taking above-mentioned preparation in supernatant
The amine-modified substrate polypeptide of 20 μM of aminoterminal maleimide: GDKAVLGVDPFR, 37 DEG C of oscillation 4h carry out conjugation reaction, use
High performance liquid chromatography purifies reaction product, removes extra unbonded DNA and substrate polypeptide.
3. nucleic acid polypeptide complex probe described in claim 1 is in the nuclease-mediated isothermal duplication of joint double-stranded specific
Using.
4. application according to claim 3, it is characterised in that 100 μ L are contained into 1 × double-stranded specific nuclease buffer,
It is dissolved in 25mM Tris-HCl, pH 8.0, the 0.20U double-stranded specific nuclease of 50wt.% glycerol, 20U RNase inhibitor,
The target miRNA of 200nM nucleic acid polypeptide complex probe and various concentration reacts 1 hour for 60 DEG C in MJ Mini thermal cycler；
After isothermal duplication, 100 μ L 10mM EDTA are added in said mixture and are incubated 5 minutes at 60 DEG C, 20 μ L strepto- parents are then added
Remaining biotinylated nucleic acid polypeptide complex probe nucleic acid polypeptide complex probe is removed with avidin agarose microballoon, uses 50mM
NH4HCO3After washing pearl, the supernatant of the nucleic acid polypeptide complex probe containing digestion is collected in case subsequent digestion and mass spectrum are examined
It surveys.
5. application according to claim 4, it is characterised in that the temperature of isothermal amplification is 60 DEG C.
6. the answering in the kit that preparation detects low abundance miRNA level of nucleic acid polypeptide complex probe described in claim 1
With.
7. detecting the kit of low abundance miRNA level, it is characterised in that visited containing nucleic acid polypeptide complex described in claim 1
Needle.
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| CN110441527A (en) * | 2019-07-31 | 2019-11-12 | 南京医科大学 | Beta-lactamase responsive type nano-probe and its preparation method and application |
| CN113005179A (en) * | 2021-02-26 | 2021-06-22 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Mass spectrum method for quantifying nucleic acid based on DNA-polypeptide probe technology and application |
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2019
- 2019-03-12 CN CN201910184181.0A patent/CN110029153A/en active Pending
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| FEIFEI XU等: "A Combination of DNA-peptide Probes and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): A Quasi-Targeted Proteomics Approach for Multiplexed MicroRNA Quantification.", 《THERANOSTICS》 * |
| FEIFEI XU等: "Quantification of microRNA by DNA-Peptide Probe and Liquid Chromatography-Tandem Mass Spectrometry-Based Quasi-Targeted Proteomics.", 《ANALYTICAL CHEMISTRY》 * |
| 孙波等: "双链特异性核酸酶介导的放大策略联合液质联用的准定向蛋白质组学定量检测乳腺癌干细胞中的miRNA", 《2018年中国质谱学术大会（CMSC 2018）论文集》 * |
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| CN110441527A (en) * | 2019-07-31 | 2019-11-12 | 南京医科大学 | Beta-lactamase responsive type nano-probe and its preparation method and application |
| CN110441527B (en) * | 2019-07-31 | 2022-06-10 | 南京医科大学 | Beta-lactamase sensitive nanoprobe and preparation method and application thereof |
| CN113005179A (en) * | 2021-02-26 | 2021-06-22 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Mass spectrum method for quantifying nucleic acid based on DNA-polypeptide probe technology and application |
| CN113005179B (en) * | 2021-02-26 | 2021-11-16 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Mass spectrum method for quantifying nucleic acid based on DNA-polypeptide probe technology and application |
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Application publication date: 20190719 |