CN105018461A - Method for rapid screening of nucleic acid aptamer - Google Patents
Method for rapid screening of nucleic acid aptamer Download PDFInfo
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- CN105018461A CN105018461A CN201410178946.7A CN201410178946A CN105018461A CN 105018461 A CN105018461 A CN 105018461A CN 201410178946 A CN201410178946 A CN 201410178946A CN 105018461 A CN105018461 A CN 105018461A
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Abstract
The invention discloses a method for rapid screening of a nucleic acid aptamer. After to-be-screened target molecules are fixed on a processed inner wall of a micro pipeline, nucleic acid library molecules of DNAs or RNAs flow through the pipeline to be combined with the target molecules on the inner pipe of the pipeline, and then the library molecules which are not combined or weakly combined with the target molecules are washed away with buffer solution, so as to obtain aptamer molecules combined with a target. The aptamer molecules are amplified to obtain an enriched sublibrary. If the affinity does not meet the predetermined requirements, the process can be repeated many times until the required affinity is met. It is relatively simple and efficient to use the method to screen the nucleic acid aptamer, the method is low in cost and easy to implement, and automation is achieved easily. Meanwhile, the method can further be used for affinity detection.
Description
Technical field
The present invention relates to field of molecular biotechnology, particularly a kind of screening method of aptamer.
Background technology
Functional structure that aptamer is made up of one section of single stranded DNA or RNA, there is similar function with antibody, efficiently can be combined specifically with target molecules, can be applicable to medical diagnosis on disease and treatment, food inspection and safety, the fields such as environmental pollutant detection.Due to aptamers has can artificial Fast back-projection algorithm, can amplification in vitro, the active characteristic such as unaffected after experience thermally denature, overcome the antibody producing cycle long, poor stability, condition of storage requires high defect, thus makes it have more widespread use space than antibody.The technology of external acquisition aptamers is independently set up early than three laboratories of nineteen ninety by the U.S., and the application document of aptamers increases fast in recent years.
Although the characteristic with widespread use of aptamers has caused the attention of various countries scientist, the high quality aptamers quantity reported in document is very limited, and a large amount of literature research is only based on a few aptamers.If the aptamers of human thrombin is after report, by more than 700 sections of reference citations and for different application scenarios.And considerably less by screening the bibliographical information successfully obtaining high quality aptamers, the screening of high-quality nucleic acid aptamers, has become the bottleneck of its widespread use.Therefore, the triage techniques of the high-quality aptamers of quick obtaining is international study hotspot always.
The rapid screening method reported mainly contains following a few class: a class is the screening method based on capillary electrophoresis, and these class methods screening storage capacity is little, needs repeatedly to screen, and the screening cycle is long; Another kind of is the method for magnetic bead, be the most ripe method relatively, but screening efficiency is lower than capillary electrophoresis.The automatically screening method of current report is also based on paramagnetic particle method, these automatically screening methods, is only to replace by hand, method not breaking through with machine.Class methods are also had to be methods that solid phase surface screens.SPR-SELEX technology one is the plant and instrument of requirement costliness, and chip price used is expensive, causes screening cost higher; Secondly, due to long-pending very little for the die active surface screened in SPR-SELEX, reduce the probability that library molecule is combined with chip surface target molecule, a large amount of library molecule has no chance to be combined with the target molecules of chip surface just to have flowed away, and causes screening efficiency to improve further; Finally because the piping system in instrument easily introduces pollution, screening efficiency is caused to reduce.
Summary of the invention
The object of the invention is grow to solve the multi-turns screen cycle in existing aptamer screening method or need the defect of expensive instrument complicated operation, and propose a kind of fast and convenient screening method based on capillary tube inner wall coupling target molecules.Capillary tube inner wall as solid phase carrier, is overcome existing cDNA microarray technique table area not enough, the shortcoming that screening efficiency is low by the present invention; Relative to existing magnetic bead screening method, automatization is convenient in the present invention, and screening efficiency is higher.
The technical solution adopted in the present invention is for this reason:
A rapid screening method for aptamer, comprises the following steps:
(1) pre-treatment is carried out to quartz capillary, described pre-treatment refers to and described kapillary is cleaned up rear GTPS (3-(2,3-epoxy third oxygen) propyl trimethoxy silicane) (CAS:2530-83-8) (Chemical Abstracts Service's database: 2530-83-8) make described kapillary internal surface epoxy, then make described kapillary internal surface carboxylated with glycine;
(2) use NHS/EDC (N-hydroxy-succinamide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide mixing solutions) to make the activated carboxylic of described kapillary internal surface, then target proteins molecule is coupled on described kapillary internal surface;
(3) be pressed into after library molecule solution denaturing treatment in described kapillary, the target nucleic acid adaptor molecules in library molecule is combined with described target proteins molecule; Wash away the aptamer molecule do not combined with weak binding;
(4) pcr amplification is carried out to described target nucleic acid aptamers, then prepare strand aptamers by described amplified production; And the detection of SPR (surface plasma body resonant vibration) avidity is carried out to the strand aptamers obtained;
(3) and (4) (5) repeat above-mentioned steps, screening obtains the high strand aptamers of SPR avidity.
The rapid screening method of aptamer of the present invention, be further characterized in that: described step epoxy (1) refers to the described kapillary of press-in after analytical pure dry toluene and GTPS 4:1 mixing by volume, closed at both ends, in 50 DEG C ~ 60 DEG C reaction 12 ?16 hours, blow out the solution in described kapillary, cleaning, nitrogen dries up; The glycine solution of 10mg/mL to be pressed in described kapillary 34 DEG C ~ 40 DEG C reaction 12 ?and to blow out solution in described kapillary, cleaning after 16 hours by described carboxylated comprising, and nitrogen dries up; Described glycine solution is prepared by 3M ammoniumsulphate soln, and described ammoniumsulphate soln is by 0.1M phosphate buffered saline.
The coupling (2) of described step refers to and is pressed in described kapillary by the target proteins of the 10mM NaAc buffer solution with pH5.0, room temperature reaction 25 ~ 35 minutes, then closes rearmounted 4 DEG C of preservations with the 1M thanomin of 50mM Tris-Cl damping fluid or pH8.5.
Described step (3) library molecule solution is dissolved in by 1nmol library molecule in 100 μ L binding buffer liquid, described binding buffer liquid be pH7.4 by 20mM HEPES, 150mM NaCl, 2mM KCl, 2mM MgCl
2with 2mM CaCl
2the solution of composition.
Described step (2) target proteins molecule is selected from HER2 (proto-oncogene human epidermal growth factor acceptor), PA (prealbumin) or SA (Streptavidin).
(4) described step is prepared strand aptamers and is comprised the steps: that the solution got in centrifuge tube joins in denaturation buffer by centrifugal for described pcr amplification product super filter tube; With frozen water cooling after sex change, PAGE (polyacrylamide gel) electrophoresis, is separated and cuts glue; Boiling water boiling glue, gets supernatant liquor super filter tube centrifugal, repurity nucleic acid; Described denaturation buffer is the solution be made up of 178mMTris-HCl , 178mM Boricacid, 4mMEDTA, 7M Μ rea, 0.6mM Ficoll, 0.3mM Bromophenol blue and 0.74mMXylene Cyanole FF of pH8.0.
It is CM5 chip or self-control CM5 chip that described SPR avidity detects the chip used.Described self-control chip and preparation method thereof refers to " Chinese biological magazine, 2009,29 (1): 44-49 "
Technical scheme of the present invention has following beneficial effect:
1, the inventive method is utilized to carry out the screening of aptamer relatively simply efficiently, with low cost, easily realize, easily be automated.Screening method of the present invention decreases the multiplicity of screening compared with the capillary electrophoresis screening method of routine; Compared with the screening method utilizing surface plasma resonance instrument, do not need expensive chip consumptive material, reduce cost; Compared with utilizing the micro-fluidic method of carrying out screening of complexity, simple to operate, and fully used the automatic sampling of capillary electrophoresis apparatus and the function of cleaning, screening easily is automated.
2, in present method due to molecule is coupled on inside pipe wall, modifies compared with screening method that magnetic bead carries out with utilizing, can not because of magnetic bead operational difficulty, magnetic bead is lost and causes screen unsuccessfully, can tolerate high speed flushing simultaneously, make collection and cleaning process simple.
3, the ips that present method is used and length all can control, and compared with utilizing the screening method of surface plasma body resonant vibration instrument, improve target molecules and the effective bonded area of library molecule, substantially increase library molecule in conjunction with probability; Simultaneously owing to combining and cleaning without the need to additionally supporting pipeline, greatly reduce dead volume, decrease and introduce extra pollution.
As long as 4, target molecule can be coupled to tube wall in present method, compared with the capillary electrophoresis screening method of routine, without the need to carrying out the fine optimization of the separation condition such as ionic concn and pH selection, electrophoretic voltage of damping fluid.
Accompanying drawing explanation
Fig. 1 is screening principle schematic of the present invention.
In Fig. 1: pipe:
target molecules:
Difform adaptor molecules in nucleic acid library:
With target molecules in conjunction with strong adaptor molecules:
Fig. 2 is HER2 aptamers avidity detected result of the present invention.
Fig. 3 is SA aptamers avidity detected result of the present invention.
In Fig. 2 and 3: X-coordinate is the time that sample flows through chip surface, ordinate zou is associated value, and associated value represents the power that aptamers is combined with the target proteins of chip surface when flowing through chip surface, and the higher description taken in conjunction of associated value is more to the aptamers of chip surface.After aptamers flows through chip completely, just can observe by associated value the curve that aptamers dissociates from chip surface and target proteins.The degree that dissociation curve declines also shows the power of avidity.Decline sooner illustrate dissociate more, show that avidity is more weak.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described:
Fig. 1 is shown in screening principle signal of the present invention.
In following examples, without the correlation step of special declaration, all refer to be realized by this area routine techniques means.The reagent used in embodiment, instrument are commercially available.
Embodiment 1: preparation HER2 aptamers
One, the pre-treatment of kapillary:
1, getting internal diameter is 75 microns, and length is the quartz capillary 100mM NaOH wash tube inwall 30 minutes of 60 centimetres; By washed with de-ionized water 30 minutes; 30 minutes are cleaned with 100mM HCl; With washed with de-ionized water after 30 minutes nitrogen dry up;
2, analytical pure anhydrous toluene solution 800 μ L is got in 1.5mL centrifuge tube, add 200 μ L GTPS (CAS:2530-83-8) and mix rear taking-up, press-in kapillary, by closed at both ends (being inserted in little silica gel block at kapillary two ends), then 12-16 hour can be reacted at 50 DEG C ~ 60 DEG C (preferably 55 DEG C) after the other end mouth of pipe has solution to flow out; After reaction terminates, blowout liquid, by washed with de-ionized water 30 minutes, and dries up with nitrogen, obtains the undercoat kapillary that surface is epoxide group.
3, glycine is dissolved in the 3M ammonium sulfate with 0.1M phosphate buffered saline, final concentration 10mg/mL, be pressed into rear enclosed two ends in kapillary (being inserted in little silica gel block at kapillary two ends), 34 DEG C ~ 40 DEG C (preferably 37 DEG C) reaction 12-16 hour; Reaction terminates, and blowout liquid in pipe, with distilled water flushing 30 minutes, and nitrogen dried up, and obtains the kapillary that internal surface is carboxylated.
Two, the activation of kapillary internal surface carboxyl and the coupling of target proteins molecule
4, get the carboxyl 30 minutes that 400uL0.05M NHS/0.2M EDC is pressed into activation tube internal surface in kapillary, use subsequently damping fluid (containing 20mM HEPES, 150mM NaCl, 2mM KCl, 2mM MgCl
2, 2mM CaCl
2(pH7.4)) rinse 5 minutes and dry up; Press-in uses 10mM NaAc, HER2 (sigma company) target proteins of the buffer solution of pH5.0, after closed at both ends, and room temperature reaction 30 minutes.Be pressed into the group of the non-coupling of 50mMTris-Cl buffer blind inwall subsequently, also can close with the thanomin of 1M pH8.5; Close after with deionized water rinsing after 5 minutes nitrogen dry up.Kapillary after drying up can be put 4 DEG C and save backup.
Press-in in above-mentioned steps 2-4 and flushing are all adopt steel cylinder nitrogen pressure to carry out, and press-in pressure 40-50psi, sees and have solution to flow out from the pipe the other end, rinse and adopt same pressure maintenance solution to continue to flow out.。
Three, be pressed in kapillary after library molecule solution denaturing treatment and rinse
5, by the library molecule that 1nmol synthesizes, 100 μ L binding buffer liquid are dissolved in (containing 20mM HEPES, 150mM NaCl, 2mM KCl, 2mM MgCl2,2mM CaCl2 (pH7.4)) in, boiling water bath 5 minutes, is then slowly cooled to room temperature for subsequent use; By after above-mentioned denaturing treatment library molecule 5psi pressure press-in kapillary in and keep 1psi be pressed into pressure ensure be no less than 10 minutes in conjunction with actual.After library molecule all flows out kapillary, with binding buffer liquid (20mM HEPES, 150mM NaCl, 2mM KCl, 2mM MgCl2,2mM CaCl2 (pH7.4)) clean 20min under 10psi pressure and rinse capillary tube inner wall, wash away and do not combine and weak-bonded molecules, solution in blowout kapillary.
Four, target nucleic acid aptamers pcr amplification and prepare strand aptamers;
6, with the library molecule that 5 μ L50mM NaOH wash-out capillary tube inner walls combine, elutriant joins in 200 μ L PCRmix after taking out and carries out conventional amplification.Amplification condition is: 94 DEG C 10 seconds, 60 DEG C 10 seconds, 72 DEG C 30 seconds, coamplification 30 circulation.PCR instrument (model PTC-225, manufacturer MJ Research company).The operations such as the press-in of above-mentioned steps 5 and 6 Chinese library molecule, the flushing of binding buffer liquid and NaOH wash-out are all be placed in by kapillary in capillary electrophoresis apparatus (model: PA800, manufacturer: Beckman) to carry out.
7, above-mentioned PCR primer is amplified pcr amplification 2mL again, PCR primer 10K super filter tube is centrifugal to 90-110 μ L (preferential 100 μ L), the denaturation buffer (containing 178mMTris-HCl, 178mM Boric acid, 4mM EDTA, 7M Μ rea, 0.6mM Ficoll, 0.3mM Bromophenol blue and 0.74mM Xylene Cyanole FF, pH8.0) of 150 μ L is added after taking-up; Boiling water bath sex change is put into mixture of ice and water and is cooled 5 minutes fast after 10 minutes, utilize PAGE electrophoretic separation to cut glue; Boiling water boiling glue twice, is concentrated into 90-110 μ L (preferential 100 μ L) with the super filter tube of 3K after getting supernatant, purifies subsequently and obtain strand aptamers with Nucleic acid purification kits.This strand can be used for avidity and detects.
Five, the SPR avidity of strand aptamers detects
8, SPR is utilized to carry out the detection of avidity: 1) detection chip preparation: to get CM5 chip or make CM5 chip by oneself and put into Biacore3000, operate according to instrument working instructions.Flow velocity 2uL/min, 0.05M NHS/0.2M EDC is utilized to activate 1 and 2 passage 30 minutes, the 10ug/mL of selector channel 2 sample introduction coupling subsequently target proteins molecule 30 minutes, close unconjugated activation site in two passages with 1M pH8.5 thanomin subsequently, the chip this prepared 4 DEG C preservation is stand-by.2) detect: the above-mentioned chip prepared is put into Biacore3000, flow velocity 20uL/min, with damping fluid (containing 20mM HEPES, 150mM NaCl, 2mMKCl, 2mM MgCl
2, 2mM CaCl
2(pH7.4) after) cleaning chip, the aptamers solution 60uL of sample introduction concentration 100nM.3) avidity calculates: carry software by Biacore3000 and calculate avidity (avidity account form is existing known technology) according to aptamers concentration and associated value.If do not reach the avidity needed for aptamer of HER2, turn back to step 5 and can proceed screening, until reach expection avidity.Through three-wheel screening, the HER2 strand aptamers avidity of preparation reaches 120nM, is obviously better than conventional magnetic bead screening method.HER2 aptamers avidity detected result is shown in Fig. 2, the 60uL of sample introduction shown in figure, associated value 32RU.
Embodiment 2: preparation SA aptamers
Get the kapillary that internal diameter 25 μm of length are 30cm to process according to the method for step 1 ~ 4 described in above embodiment 1, its difference is only that described target proteins is SA, get the kapillary that inwall coupling has SA (the raw work in Shanghai) target proteins, by in the library molecule 10psi pressure press-in kapillary after above-mentioned denaturing treatment, after being no less than 10 minutes, use 50psi pressure wash 20min; PCRmix is directly pressed in kapillary, closed at both ends, 95 DEG C of heating 5min; PCRmix blowout in pipe is increased to 100 μ L PCR mix, and prepare strand aptamers by the method for embodiment 1 step 7, repeat to screen two-wheeled, strand library molecule is diluted to 100nM, utilize SPR technique to detect avidity, the SA strand aptamers avidity of preparation is about 500nM.SA aptamers avidity detected result is shown in Fig. 3, the 60uL of sample introduction shown in figure, associated value 19RU.
Embodiment 3
Get the kapillary that internal diameter 200 μm of length are 1m to process according to the method for step 1 ~ 4 described in above embodiment 1, its difference is only that described target proteins is PA, get the kapillary that inwall coupling has PA target proteins, library molecule binding buffer liquid is dissolved to 5uM, get 200uL with after above-mentioned condition denaturing treatment, in 2psi pressure press-in kapillary, after being no less than 10 minutes, use 10psi pressure wash 20min; PCRmix is directly pressed in kapillary, closed at both ends, 95 DEG C of heating 5min; PCRmix blowout in pipe is increased to 100 μ L PCR mix, and prepare strand aptamers by the method for embodiment 1 step 7, repeat to screen two-wheeled, strand library molecule is diluted to 100nM, utilize SPR technique to detect avidity, the PA strand aptamers avidity of preparation is about 212nM.
Claims (7)
1. a rapid screening method for aptamer, comprises the following steps:
(1) carry out pre-treatment to quartz capillary, described pre-treatment refers to that described kapillary is cleaned up rear GTPS makes described kapillary internal surface epoxy, then makes described kapillary internal surface carboxylated with glycine;
(2) make the activated carboxylic of described kapillary internal surface with NHS/EDC, then target proteins molecule is coupled on described kapillary internal surface;
(3) be pressed into after library molecule solution denaturing treatment in described kapillary, the target nucleic acid adaptor molecules in library molecule is combined with described target proteins molecule; Wash away the aptamer molecule do not combined with weak binding;
(4) pcr amplification is carried out to described target nucleic acid aptamers, then prepare strand aptamers by described amplified production; And the detection of SPR avidity is carried out to the strand aptamers obtained;
(3) and (4) (5) repeat above-mentioned steps, screening obtains the high strand aptamers of SPR avidity.
2. the rapid screening method of aptamer according to claim 1 and 2, it is characterized in that: described step epoxy (1) refers to the described kapillary of press-in after analytical pure dry toluene and GTPS 4:1 mixing by volume, closed at both ends, in 50 DEG C ~ 60 DEG C reaction 12-16 hour, blow out the solution in described kapillary, cleaning, nitrogen dries up; The glycine solution of 10mg/mL to be pressed in described kapillary 34 DEG C ~ 40 DEG C reactions and to blow out solution in described kapillary, cleaning after 12-16 hour by described carboxylated comprising, and nitrogen dries up; Described glycine solution is prepared by 3M ammoniumsulphate soln, and described ammoniumsulphate soln is by 0.1M phosphate buffered saline.
3. the rapid screening method of aptamer according to claim 1 and 2, it is characterized in that: the coupling (2) of described step refers to and is pressed in described kapillary by the target proteins of the 10mM NaAc buffer solution with pH5.0, room temperature reaction 25 ~ 35 minutes, then close rearmounted 4 DEG C of preservations with the 1M thanomin of 50mM Tris-Cl damping fluid or pH8.5.
4. the rapid screening method of aptamer according to claim 1 and 2, it is characterized in that: described step (3) library molecule solution is dissolved in by 1nmol library molecule in 100 μ L binding buffer liquid, described binding buffer liquid be pH7.4 by 20mM HEPES, 150mM NaCl, 2mM KCl, 2mM MgCl
2with 2mM CaCl
2the solution of composition.
5. the rapid screening method of aptamer according to claim 1 and 2, is characterized in that: described step (2) target proteins molecule is selected from HER2, PA or SA.
6. the rapid screening method of aptamer according to claim 1 and 2, it is characterized in that: (4) described step is prepared strand aptamers and comprised the steps: that the solution got in centrifuge tube joins in denaturation buffer by centrifugal for described pcr amplification product super filter tube; With frozen water cooling after sex change, PAGE electrophoretic separation, cuts glue; Boiling water boiling glue, gets supernatant liquor super filter tube centrifugal, repurity nucleic acid; Described denaturation buffer is the solution be made up of 178mM Tris-HCl, 178mM Boric acid, 4mM EDTA, 7M Μ rea, 0.6mMFicoll, 0.3mM Bromophenol blue and 0.74mM Xylene Cyanole FF of pH8.0.
7. the rapid screening method of aptamer according to claim 2, is characterized in that: it is CM5 chip or self-control CM5 chip that described SPR avidity detects the chip used.
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| CN113061610A (en) * | 2020-03-31 | 2021-07-02 | 中国科学技术大学 | Aptamer binding to novel coronavirus (SARS-CoV-2) spinous process protein S1 subunit and use thereof |
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| 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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| CN110577979A (en) * | 2018-06-08 | 2019-12-17 | 首都师范大学 | rapid screening method of aptamer based on crosslinking reaction and structure switch type aptamer obtained through screening |
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| 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 |
| CN109486824A (en) * | 2018-11-20 | 2019-03-19 | 安徽省昂普拓迈生物科技有限责任公司 | A kind of aptamer and its screening technique, application specifically binding Taq enzyme |
| CN113151282A (en) * | 2020-02-21 | 2021-07-23 | 中国科学技术大学 | Aptamer binding to novel coronavirus (SARS-CoV-2) nucleocapsid protein and use 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 |
| CN113061610A (en) * | 2020-03-31 | 2021-07-02 | 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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