CN105018461B - A kind of rapid screening method of aptamer - Google Patents
A kind of rapid screening method of aptamer Download PDFInfo
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Abstract
The invention discloses a kind of rapid screening method of aptamer.After target molecules to be screened are fixed on treated microtubule inner wall, the nucleic acid library molecular flow piping of DNA or RNA is combined with the target molecules of its inner wall again, then fallen with wash buffer and be not associated with target molecules and with reference to weak library molecule, obtain the adaptor molecules combined with target.This adaptor molecules is expanded, the word bank after being enriched with.If affinity is not up to pre-provisioning request, above-mentioned process can be repeated several times, untill reaching the affinity of requirement.It is relatively easy efficiently that the screening of aptamer is carried out using the method for the present invention, it is of low cost, it is easy to implement, it is easy to accomplish automation.At the same time it can also be detected for affinity.
Description
Technical field
The present invention relates to field of molecular biotechnology, particularly a kind of screening technique of aptamer.
Background technology
Functional structure that aptamer is made of one section of single stranded DNA or RNA, has similar work(with antibody
Can, it specifically can efficiently be combined with target molecules, can be applied to medical diagnosis on disease and treatment, food inspection and safety, environment
The fields such as pollutant monitoring.Due to aptamers have can artificial Fast back-projection algorithm, can amplification in vitro, after thermal denaturation undergo it is active not
The characteristic such as impacted, overcomes antibody producing cycle length, and stability is poor, and condition of storage requires the defects of high, so that it is than anti-
Body has more extensively using space.Three laboratory independences of the external technology for obtaining aptamers most earlier than nineteen ninety by the U.S.
Establish, the application document of aptamers quickly increases in recent years.
Although the attention for having caused scientists from all over the world with widely applied characteristic of aptamers, reported in the literature
High quality aptamers quantity is very limited, and substantial amounts of literature research is based only upon a few aptamers.Such as the adaptation of human thrombin
Body by more than 700 reference citations and is used for different application scenarios from after reporting.And high quality is successfully obtained by screening and is adapted to
The document report of body is considerably less, the screening of high-quality nucleic acid aptamers, it has also become its widely applied bottleneck.Therefore, quickly
The screening technique for obtaining the aptamers of high quality is always international research hotspot.
It has been reported that rapid screening method mainly have following a few classes：One kind is the screening technique based on Capillary Electrophoresis,
This kind of method screening storage capacity is small, it is necessary to repeatedly screen, screening cycle length；Another kind of is the method for magnetic bead, and opposite is most ripe
Method, but screening efficiency is less than capillary electrophoresis.The automatically screening method reported at present is also based on paramagnetic particle method, these are certainly
Dynamicization screening technique, only replaces by hand, not breaking through in method with machine.Also a kind of method is solid phase surface screening
Method.SPR-SELEX technologies one are the instrument and equipments of requirement costliness, and chip price used is expensive, cause screening cost compared with
It is high；Secondly as being used for the die active surface product very little screened in SPR-SELEX, library molecule and chip surface are reduced
The probability that target molecule combines, substantial amounts of library molecule have no chance to be combined with the target molecules of chip surface and just flow away, cause
Screening efficiency can not further improve；Finally since the pipe-line system in instrument is easily introduced pollution, screening efficiency is caused to reduce.
The content of the invention
The purpose of the present invention is to solve multi-turns screen cycle length in existing aptamer screening technique or need expensive
The defects of weight instrumentation is complicated, and propose a kind of fast and convenient screening technique based on capillary tube inner wall coupling target molecules.
The present invention overcomes existing cDNA microarray technical face product deficiency, screening efficiency is low using capillary tube inner wall as solid phase carrier
The shortcomings that；For existing magnetic bead screening technique, the present invention is easy to automation, screening efficiency higher.
The technical solution adopted in the present invention is for this：
A kind of rapid screening method of aptamer, comprises the following steps：
(1) quartz capillary is pre-processed, the pretreatment refers to use GTPS after the capillary is cleaned up
(3- (the third oxygen of 2,3- epoxies) propyl trimethoxy silicane) (CAS：2530-83-8) (Chemical Abstract Service database：2530-83-8)
Make the capillary inner surface epoxy, then make the capillary inner surface carboxylated with glycine；
(2) NHS/EDC (n-hydroxysuccinimide and 1- ethyls -3- (3- dimethylaminopropyls)-carbodiimides are used
Mixed solution) make the activated carboxylic of the capillary inner surface, then by target proteins molecule coupling labeled in the capillary inner surface
On；
(3) will be pressed into after library molecule solution denaturation treatment in the capillary so that the target nucleic acid in library molecule is fitted
Ligand molecular is combined with the target proteins molecule；Wash away uncombined and weak binding aptamer molecule；
(4) PCR amplification is carried out to the target nucleic acid aptamers, then single-stranded aptamers are prepared by the amplified production；
And the detection of SPR (surface plasma body resonant vibration) affinity is carried out to the single-stranded aptamers of acquisition；
(3) and (4) (5) repeat the above steps, screening obtains the high single-stranded aptamers of SPR affinity.
The rapid screening method of the aptamer of the present invention, is further characterized in that：The epoxy of the step (1) is
Finger will analyze pure dry toluene and GTPS by volume 4：The capillary is pressed into after 1 mixing, it is closed at both ends, in 50 DEG C~60 DEG C
When reaction 12-16 is small, the solution in the capillary is blown out, is cleaned, nitrogen drying；The carboxylated is included 10mg/mL's
Glycine solution blows out the solution in the capillary after being pressed into when 34 DEG C~40 DEG C reaction 12-16 are small in the capillary, clearly
Wash, nitrogen drying；The glycine solution is prepared by 3M ammonium sulfates, and the ammonium sulfate is delayed by 0.1M phosphate
Fliud flushing is prepared.
The coupling of the step (2) refers to described in the press-in of the target proteins of the 10mM NaAc buffer solutions of pH5.0
In capillary, react at room temperature 25~35 minutes, then postposition is closed with the 1M monoethanolamines of 50mM Tris-Cl buffer solutions or pH8.5
4 DEG C of preservations.
(3) library molecule solution is that 1nmol library molecules are dissolved in 100 μ L combination buffers to the step, the knot
Conjunction buffer solution is pH7.4 by 20mM HEPES, 150mM NaCl, 2mM KCl, 2mM MgCl2With 2mM CaCl2What is formed is molten
Liquid.
(2) target proteins molecule is selected from HER2 (proto-oncogene human epidermal growth factor acceptor) to the step, and PA is (preceding white
Albumen) or SA (Streptavidin).
(4) the step prepares single-stranded aptamers and includes the following steps：The pcr amplification product is centrifuged with super filter tube, is taken
Solution in centrifuge tube is added in denaturation buffer；Cooled down after denaturation with frozen water, PAGE (polyacrylamide gel) electrophoresis, point
From cutting glue；Boiling water boiling glue, takes supernatant super filter tube to centrifuge, repurity nucleic acid；The denaturation buffer is pH8.0 by 178mM
Tris-HCl 、178mM Boric acid、4mM EDTA、7M Μrea、0.6mM Ficoll、0.3mM Bromophenol
The solution of blue and 0.74mM Xylene Cyanole FF compositions.
The chip that the SPR affinity detection uses is CM5 chips or self-control CM5 chips.The self-control chip and its system
Preparation Method refers to " Chinese biological magazine, 2009,29 (1)：44-49 "
Technical scheme has the advantages that：
1st, it is relatively easy efficiently that the screening of aptamer is carried out using the method for the present invention, it is of low cost, it is easy to implement, easily
Automated in realizing.The screening technique of the present invention reduces the repetition time of screening compared with conventional Capillary Electrophoresis screening technique
Number；Compared with the screening technique using surface plasma resonance instrument, it is not necessary to which expensive chip consumptive material, reduces cost；With profit
Compared with the complicated micro-fluidic method screened, it is easy to operate, and fully used capillary electrophoresis it is automatic into
Sample and the function of cleaning so that screening is easy to automate.
2nd, in this method due to molecule coupling labeled on inside pipe wall, with using modify magnetic bead progress screening technique compared with,
It will not lose because of magnetic bead operating difficulties, magnetic bead and cause screening failure, while can be resistant to and rinse at a high speed so that collect and clear
It is simple to wash journey.
3rd, the tube inner diameter used in this method and length can control, with the screening using surface plasma body resonant vibration instrument
Method is compared, and improves target molecules and the effective bonded area of library molecule, substantially increases the combination probability of library molecule；Together
When due to combining and cleaning without extra supporting pipeline, greatly reduce dead volume, reduce and introduce extra pollution.
As long as the 4, target molecule can be coupled to tube wall in this method, with conventional Capillary Electrophoresis screening technique
Compare, the fine optimization of the separation condition such as ion concentration and pH selections, electrophoretic voltage without carrying out buffer solution.
Brief description of the drawings
Fig. 1 is the screening principle schematic of the present invention.
In Fig. 1：Pipe：Target molecules：
Adaptor molecules of different shapes in nucleic acid library：
Strong adaptor molecules are combined with target molecules：
Fig. 2 is the HER2 aptamers affinity testing results of the present invention.
Fig. 3 is the SA aptamers affinity testing results of the present invention.
In Fig. 2 and 3：Abscissa flows through the time of chip surface for sample, and ordinate is associated value, and associated value represents adaptation
The power combined when body flows through chip surface with the target proteins of chip surface, the higher explanation of associated value are attached to chip surface
Aptamers are more.After aptamers flow completely through chip, by associated value aptamers can be observed from chip surface and target
Mark the curve of albumen dissociation.The degree that dissociation curve declines also indicates that the power of affinity.Decline it is faster explanation dissociation more
It is more, show that affinity is weaker.
Embodiment
The invention will be further described with specific embodiment below in conjunction with the accompanying drawings：
Fig. 1 is shown in the screening principle signal of the present invention.
In following embodiments, the correlation step of no special declaration, refers both to realize by this area conventional technical means.Implement
Reagent, instrument used in example are commercially available.
Embodiment 1：Prepare HER2 aptamers
First, the pretreatment of capillary：
1st, it is 75 microns to take internal diameter, and length is that 60 centimetres of quartz capillary cleans inside pipe wall 30 with 100mM NaOH and divides
Clock；Cleaned 30 minutes with deionized water；Cleaned 30 minutes with 100mM HCl；Nitrogen dries up after being cleaned 30 minutes with deionized water；
2nd, the pure 800 μ L of anhydrous toluene solution of analysis are taken to add 200 μ L GTPS (CAS in 1.5mL centrifuge tubes：2530-
83-8) take out after mixing, be pressed into capillary, can be by closed at both ends (by capillary after the other end mouth of pipe has solution outflow
Pipe both ends are inserted into small silica gel block), then when 50 DEG C~60 DEG C (preferably 55 DEG C) reaction 12-16 is small；After reaction, blow out
Liquid, is cleaned 30 minutes with deionized water, and is dried up with nitrogen, obtains the undercoating capillary that surface is epoxide group.
3rd, glycine is dissolved in the 3M ammonium sulfate with 0.1M phosphate buffered salines, final concentration 10mg/mL, by it
It is pressed into rear enclosed both ends in capillary (capillary both ends are inserted into small silica gel block), 34 DEG C~40 DEG C (preferably 37 DEG C) reaction
When 12-16 is small；Reaction terminates, and blows out liquid in pipe, and with distilled water flushing 30 minutes, and nitrogen dried up, and obtains inner surface carboxyl
The capillary of change.
2nd, the activation of capillary inner surface carboxyl and the coupling of target proteins molecule
4th, the carboxyl of activation pipe internal surface in 400uL0.05M NHS/0.2M EDC press-in capillaries is taken 30 minutes, then
(contain 20mM HEPES, 150mM NaCl, 2mM KCl, 2mM MgCl with buffer solution2,2mM CaCl2(pH7.4)) 5 points are rinsed
Clock simultaneously dries up；HER2 (sigma companies) target proteins of the press-in buffer solution of 10mM NaAc, pH5.0, it is closed at both ends
Afterwards, react at room temperature 30 minutes.The group that 50mMTris-Cl buffer blind inner walls are not coupled is then pressed into, 1M can also be used
The monoethanolamine of pH8.5 is closed；With deionized water rinsing, nitrogen dries up after five minutes after closing.Capillary after drying can be with
4 DEG C are put to save backup.
Press-in and flushing in above-mentioned steps 2-4 are carried out using steel cylinder nitrogen pressure, are pressed into pressure 40-50psi,
Seeing has solution to be flowed out from the pipe other end, rinses and keeps solution persistently to flow out using same pressure..
3rd, it is pressed into capillary and rinses after library molecule solution denaturation treatment
5th, the library molecule for synthesizing 1nmol, is dissolved in 100 μ L combination buffers (containing 20mM HEPES, 150mM
NaCl, 2mM KCl, 2mM MgCl2,2mM CaCl2 (pH7.4)) in, boiling water bath 5 minutes, it is standby to be then slowly cooled to room temperature
With；Library molecule 5psi pressure after above-mentioned denaturation treatment is pressed into capillary and keeps 1psi press-in pressure to ensure to combine in fact
Border is no less than 10 minutes.When library molecule all outflow capillary after, with combination buffer (20mM HEPES, 150mM NaCl,
2mM KCl, 2mM MgCl2,2mM CaCl2 (pH7.4)) 20min is cleaned under 10psi pressure rinse capillary tube inner wall, wash away not
With reference to and weak-bonded molecules, blow out capillary in solution.
4th, target nucleic acid aptamers PCR amplification and single-stranded aptamers are prepared；
6th, the library molecule combined with 5 μ L50mM NaOH elution capillary tube inner walls, eluent are added to 200 μ L after taking out
Conventional amplification is carried out in PCRmix.Amplification condition is:94 DEG C 10 seconds, 60 DEG C 10 seconds, 72 DEG C 30 seconds, the circulation of coamplification 30.PCR
Instrument (model PTC-225, manufacturer MJ Research company).The press-in of 5 and 6 Chinese library molecule of above-mentioned steps, combination buffer punching
Wash and the operations such as NaOH is eluted are that capillary is placed in capillary electrophoresis (model：PA800, manufacturer：Beckman) in
Carry out.
7th, above-mentioned PCR product is amplified into PCR amplification 2mL again, PCR product is centrifuged (excellent to 90-110 μ L with 10K super filter tubes
First 100 μ L), the denaturation buffer of 150 μ L is added after taking-up (containing 178mM Tris-HCl, 178mM Boric acid, 4mM
EDTA, 7M Μ rea, 0.6mM Ficoll, 0.3mM Bromophenol blue and 0.74mM Xylene Cyanole FF,
pH8.0)；Boiling water bath denaturation is put into mixture of ice and water quick cooling 5 minutes after ten minutes, and glue is cut using PAGE electrophoretic separation；
Boiling water boiling glue twice, takes and is concentrated into 90-110 μ L (preferential 100 μ L) with the super filter tube of 3K after supernatant, then tried with nucleic acid purification
Agent box is purified up to single-stranded aptamers.This is single-stranded available for affinity detection.
5th, the SPR affinity detection of single-stranded aptamers
8th, the detection of affinity is carried out using SPR：1) prepared by detection chip：CM5 chips or self-control CM5 chips is taken to be put into
In Biacore3000, operated according to instrument operation instructions.Flow velocity 2uL/min, is lived using 0.05M NHS/0.2M EDC
Change 1 and 2 passage 30 minutes, subsequent 2 sample introduction of selector channel coupling 10ug/mL target proteins molecule 30 minutes, then uses 1M
PH8.5 monoethanolamines close the activation site being not associated with two passages, and 4 DEG C of preservations of chip that this is prepared are stand-by.2) detect：
The above-mentioned chip prepared is put into Biacore3000, flow velocity 20uL/min, with buffer solution (containing 20mM HEPES,
150mM NaCl,2mM KCl,2mM MgCl2,2mM CaCl2(pH7.4)) after cleaning chip, the aptamers of sample introduction concentration 100nM
Solution 60uL.3) affinity calculates：Software is carried by Biacore3000 affinity is calculated according to adaptation bulk concentration and associated value
(affinity calculation is existing known technology).If the affinity not up to needed for the aptamer of HER2, returns to
Step 5 can continue to screen, until reaching expected affinity.Screened by three-wheel, the single-stranded aptamers parents of HER2 of preparation
Reach 120nM with power, hence it is evident that better than conventional magnetic bead screening technique.HER2 aptamers affinity testing results are shown in Fig. 2, show in figure
Go out sample introduction 60uL, associated value 32RU.
Embodiment 2：Prepare SA aptamers
Take at method of the capillary according to step 1~4 described in above example 1 that 25 μm of length of internal diameter are 30cm
Reason, it is SA that it, which differs only in the target proteins, takes inner wall coupling to have the capillary of SA (Shanghai life work) target proteins, will be upper
State in the press-in capillary of the library molecule 10psi pressure after denaturation treatment, with reference to no less than using 50psi pressure wash after ten minutes
20min；PCRmix is pressed directly into capillary, closed at both ends, 95 DEG C of heating 5min；PCRmix in pipe is blown out to 100 μ
Expanded in L PCR mix, and single-stranded aptamers are prepared by the method for 1 step 7 of embodiment, repeat to screen two-wheeled, will be single-stranded
Library molecule is diluted to 100nM, detects affinity using SPR technique, the single-stranded aptamers affinity of SA of preparation is about 500nM.
SA aptamers affinity testing results are shown in Fig. 3, and sample introduction 60uL, associated value 19RU are shown in figure.
Embodiment 3
Take at method of the capillary according to step 1~4 described in above example 1 that 200 μm of length of internal diameter are 1m
Reason, it is PA that it, which differs only in the target proteins, takes inner wall coupling to have the capillary of PA target proteins, library molecule is tied
Buffer solution is closed to 5uM, after taking 200uL with above-mentioned condition denaturation treatment, in 2psi pressure press-in capillary, with reference to no less than
10psi pressure wash 20min is used after ten minutes；PCRmix is pressed directly into capillary, closed at both ends, 95 DEG C of heating 5min；
PCRmix in pipe is blown out into 100 μ L PCR mix and is expanded, and single-stranded fit is prepared by the method for 1 step 7 of embodiment
Ligand, repeats to screen two-wheeled, single-stranded library molecule is diluted to 100nM, detects affinity using SPR technique, the PA of preparation is mono-
Chain aptamers affinity is about 212nM.
Claims (6)
1. a kind of rapid screening method of aptamer, comprises the following steps：
(1) quartz capillary is pre-processed, the pretreatment refers to after the capillary is cleaned up to be made with GPS described
Capillary inner surface epoxy, then make the capillary inner surface carboxylated with glycine；
(2) the activated carboxylic of the capillary inner surface is made with NHS/EDC, then by target proteins molecule coupling labeled in the capillary
On inner surface；
(3) will be pressed into after library molecule solution denaturation treatment in the capillary so that the target nucleic acid aptamers in library molecule
Molecule is combined with the target proteins molecule；Wash away uncombined and weak binding aptamer molecule；
(4) PCR amplification is carried out to the target nucleic acid aptamers, then single-stranded aptamers are prepared by the amplified production；It is and right
The single-stranded aptamers obtained carry out SPR affinity detections；
(3) and (4) (5) repeat the above steps, screening obtains the high single-stranded aptamers of SPR affinity；
The epoxy of the step (1) refers to analyze pure dry toluene and GPS by volume 4：The hair is pressed into after 1 mixing
Tubule, it is closed at both ends, when 50 DEG C~60 DEG C reaction 12-16 are small, the solution in the capillary is blown out, is cleaned, nitrogen drying；
After the carboxylated is included when 34 DEG C~40 DEG C reaction 12-16 are small in the glycine solution press-in capillary by 10mg/mL
The solution in the capillary is blown out, is cleaned, nitrogen drying；The glycine solution is prepared by 3M ammonium sulfates, described
Ammonium sulfate is by 0.1M phosphate buffered salines.
2. the rapid screening method of aptamer according to claim 1, it is characterised in that：The coupling of the step (2)
Refer to be pressed into the capillary with the target proteins of the 10mM NaAc buffer solutions of pH5.0, react at room temperature 25~35 points
Clock, then close 4 DEG C of preservations of postposition with the 1M monoethanolamines of 50mM Tris-Cl buffer solutions or pH8.5.
3. the rapid screening method of aptamer according to claim 1, it is characterised in that：The step (3) divide by library
Sub- solution is that 1nmol library molecules are dissolved in 100 μ L combination buffers, and the combination buffer is pH7.4 by 20mM
HEPES、150mM NaCl、2mM KCl、2mM MgCl2With 2mM CaCl2The solution of composition.
4. the rapid screening method of aptamer according to claim 1, it is characterised in that：The step (2) target egg
White molecule is selected from HER2, PA or SA.
5. the rapid screening method of aptamer according to claim 1, it is characterised in that：(4) the step prepares list
Chain aptamers include the following steps：The pcr amplification product is centrifuged with super filter tube, takes the solution in centrifuge tube to be added to denaturation
In buffer solution；Cooled down after denaturation with frozen water, PAGE electrophoretic separation, cuts glue；Boiling water boiling glue, takes supernatant to be centrifuged with super filter tube, then
Purification of nucleic acid；The denaturation buffer is pH8.0 by 178mM Tris-HCl, 178mM Boric acid, 4mM EDTA, 7M
Μrea、0.6mM Ficoll、
The solution of 0.3mM Bromophenol blue and 0.74mM Xylene Cyanole FF compositions.
6. the rapid screening method of aptamer according to claim 1, it is characterised in that：The SPR affinity inspection
It is CM5 chips or self-control CM5 chips to survey the chip used.
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| EP3406721B1 (en) * | 2016-01-22 | 2023-10-04 | The University Of Tokyo | Method for screening nucleic acid aptamer |
| CN105678112B (en) * | 2016-02-03 | 2018-08-03 | 中国农业科学院北京畜牧兽医研究所 | A kind of implementation method of computer-aided screening micromolecular compound target aptamers |
| CN107064485B (en) * | 2017-01-13 | 2020-05-19 | 南京大学 | Application of decahedral nano silver probe in screening aptamer by using surface plasmon resonance imaging technology |
| CN110577979B (en) * | 2018-06-08 | 2022-09-27 | 首都师范大学 | Rapid screening method of aptamer based on crosslinking reaction and structure switch type aptamer obtained through screening |
| CN109486824B (en) * | 2018-11-20 | 2021-11-16 | 安徽省昂普拓迈生物科技有限责任公司 | Aptamer specifically combined with Taq enzyme, and screening method and application thereof |
| CN113151282B (en) * | 2020-02-21 | 2022-04-19 | 中国科学技术大学 | Aptamer binding to novel coronavirus (SARS-CoV-2) nucleocapsid protein and use thereof |
| CN113061610B (en) * | 2020-03-31 | 2022-04-19 | 中国科学技术大学 | Aptamer binding to novel coronavirus (SARS-CoV-2) spinous process protein S1 subunit and use thereof |
| CN113495111A (en) * | 2020-04-02 | 2021-10-12 | 北京化工大学 | Method for representing binding affinity of aptamer and target molecule and application |
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| WO2009011855A2 (en) * | 2007-07-16 | 2009-01-22 | California Institute Of Technology | Selection of nucleic acid-based sensor domains within nucleic acid switch platform |
| CN101358963A (en) * | 2007-08-01 | 2009-02-04 | 中国计量学院 | Method for rapidly detecting malachite green in aquatic products by Aptamer |
| CN103525820A (en) * | 2013-10-29 | 2014-01-22 | 中国科学技术大学 | Affinity nucleic acid molecule screening method based on sectioned collection |
| CN103540672A (en) * | 2013-10-29 | 2014-01-29 | 中国科学技术大学 | Quick identification and separation method of affine nucleic acid molecule |
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| WO2009011855A2 (en) * | 2007-07-16 | 2009-01-22 | California Institute Of Technology | Selection of nucleic acid-based sensor domains within nucleic acid switch platform |
| CN101358963A (en) * | 2007-08-01 | 2009-02-04 | 中国计量学院 | Method for rapidly detecting malachite green in aquatic products by Aptamer |
| CN103525820A (en) * | 2013-10-29 | 2014-01-22 | 中国科学技术大学 | Affinity nucleic acid molecule screening method based on sectioned collection |
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