CN105420239B - Luminous point can occur and hit microRNA of chemical reaction and its preparation method and application - Google Patents

Luminous point can occur and hit microRNA of chemical reaction and its preparation method and application Download PDF

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CN105420239B
CN105420239B CN201510728437.1A CN201510728437A CN105420239B CN 105420239 B CN105420239 B CN 105420239B CN 201510728437 A CN201510728437 A CN 201510728437A CN 105420239 B CN105420239 B CN 105420239B
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sirna
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张艳
张辰宇
李金波
黄磊
肖潇
周正权
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Nanjing University
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Abstract

Luminous point can occur and hit microRNA of chemical reaction and its preparation method and application, structure is as follows：Present invention combination luminous point hits chemical reaction realization to fluorescent marker, molecular modification, target spot the separation identification of siRNA and miRNA, the separation identification of other interaction biomolecule and with this assessment to toxic side effect in siRNA and miRNA medicinal applications.

Description

Luminous point can occur and hit microRNA of chemical reaction and its preparation method and application
Technical field
The invention belongs to luminous points to hit technical field of chemical reactions, and in particular to the small molecule that luminous point hits chemical reaction can occur RNA and its preparation method and application.
Background technology
In mammalian cell, the RNA interference (RNA interference, RNAi) is a kind of extensively and basic Bioprocess, and conclusive effect is played in pathological processes.During RNAi, small molecule non-coding RNA, such as Endogenic miRNA (microRNA, miRNA) and exogenous small interference ribonucleic acid (small Interfering RNA, siRNA) their target messenger RNA (mRNA) quilt can be made by the principle of base pair complementarity It degrades or inhibits their translation, it is this widely to the close phase of occurrence and development of the regulation process of expression of target gene and disease It closes.Thus, siRNA and miRNA have been considered as to realize the novel gateway drug of one kind of therapeutic purposes by RNAi in recent years Object.However, the mechanism of drug action of siRNA and miRNA includes transport in vivo, distribution, action target spot, interaction in vivo Other biological molecule, possible toxic side effect etc. are still within the unknown stage, more than a kind of novel method solution of development Unknown problem in siRNA and miRNA medicinal applications.
It is a kind of novel bio-orthogonal reaction, it can be achieved that the modification to large biological molecule and fluorescence that luminous point, which hits chemical reaction, Label.In the present invention, inventor is found that hit to chemically react using luminous point and realizes the fluorescent marker to siRNA and miRNA, divides Son modification, target spot detach identification, the separation identification of other interaction biomolecule and with this to siRNA and miRNA drugs The assessment of toxic side effect in.
Invention content
The technical issues of solution：Present invention offer can occur luminous point and hit microRNA of chemical reaction and preparation method thereof And application, in conjunction with luminous point hit chemical reaction realize to the fluorescent marker, molecular modification, target spot of siRNA and miRNA separation identification, The separation of other interaction biomolecule is identified and with this assessment to toxic side effect in siRNA and miRNA medicinal applications.
Technical solution：The microRNA that luminous point hits chemical reaction can occur, structure is as follows：
Wherein RNA represents arbitrary siRNA or miRNA, and X is disulfide bond, amido bond, ester bond or ehter bond, and X is connected to The positive-sense strand of siRNA or miRNA or 3 ' ends of antisense strand or 5 ' ends, Y are the alkyl containing one to ten carbon, oligomeric or poly second two Alcohol, Z is disulfide bond, amido bond, ester bond, ehter bond, the alkyl containing one to ten carbon, any one in oligomeric or poly ethylene glycol Kind, Tet is that the tetrazole chemical small molecule that luminous point hits chemical reaction can occur, and chemical constitution is as follows：
Wherein Ar1And Ar2For phenyl ring or naphthalene nucleus, R1-R14Respectively hydrogen, nitrogen, oxygen, sulphur, the alkyl containing one to ten carbon, Oligomeric or poly ethylene glycol, allyl, propargyl, nitrine, acyl group or ester group, 3- allyloxys or 4- alkene butoxy, Z are connected to Ar1Or Ar2Any position on.
The RNA is miR-122.
The Tet is
The structural formula that the microRNA that luminous point hits chemical reaction occurs is：
The preparation method that luminous point occurs and hits the microRNA of chemical reaction, process route are：
The above method is preferably, using the carboxyl of succinimide activated micromolecular compound, then be modified with amino RNA is condensed, and reaction system is the DMSO/PBS of pH=7.4.
Application of the above-mentioned microRNA in label, imaging.
Application of the above-mentioned microRNA in the separation identification of its target messenger RNA.
Application of the above-mentioned microRNA in preparing the RNA interference drug.
Application of the above-mentioned microRNA in the different biological molecules that identification interacts therewith.
The present invention is small numerator modified using modern molecular biology and chemicobiological technique study tetrazole Applications of the siRNA and miRNA in the RNA interference.
(1) synthesis of tetrazole chemical small molecule
Tetrazole chemical small molecule synthesis synthetic route be：
(2) modification of siRNA and miRNA
(3) label of siRNA and miRNA
Luminous point between the functional molecular modified by tetrazole chemical small molecule and double bond hits chemical reaction realization pair Functional modifications different with miRNA siRNA.
(4) luciferase assay
The siRNA and miRNA modified by tetrazole chemical small molecule stimulates intracellular luciferase signal to change Judge that its bioactivity, detailed process are as follows：
(1) luciferase plasmids containing siRNA or miRNA binding sites are built；
(2) in siRNA the and miRNA cotransfections to cell for modifying plasmid and tetrazole chemical small molecule；
(3) luciferase signal is measured.
(5) experiment is pulled down
Chemical reaction is hit in conjunction with luminous point, biotin is connected on siRNA and miRNA, and utilizes biotin and streptomysin Between strong affinity interaction to being detached into the cell with the siRNA and miRNA biomolecule acted on, and reflected accordingly to it It is fixed.Detailed process is as follows：
(1) siRNA or miRNA that tetrazole chemical small molecule is modified are transfected into cell；
(2) in cell cracking, chemical reaction is hit by luminous point, biotin is connected on siRNA or miRNA；
(3) obtained siRNA or miRNA and the magnetic bead with streptomysin are acted on, makes siRNA or miRNA and therewith phase The biomolecule fixation of interaction is to magnetic bead surfaces；
(4) biomolecule on magnetic bead is detached；
(5) siRNA or miRNA of separation analyze using quantitative fluorescent PCR quantitative；
(6) the target messenger RNA of separation to interact with siRNA or miRNA is carried out using quantitative fluorescent PCR Separation identification；
(7) pair it is separated by electrophoresis with the siRNA or miRNA albumen to interact, western blot analysis or silver staining analysis, Protein spectrum is analyzed.
(6) fluorescence imaging of siRNA and miRNA
By luminous point hit chemical reaction in living cells siRNA and miRNA carry out fluorescence imaging, detailed process is as follows：
(1) siRNA or miRNA that tetrazole chemical small molecule is modified are transfected into cell；
(2) chemical reaction is hit with the intramolecular luminous point of two-photon excitation tetrazole；
(3) fluorescence imaging is carried out to siRNA or miRNA.
(7) Apoptosis detects
Cell viability after hitting siRNA and the miRNA stimulation for chemically reacting label by luminous point is measured, detailed process is as follows：
(1) being transfected into the siRNA of label and miRNA into the cell stimulates cell；
(2) Apoptosis is measured using flow cytometer.
Advantageous effect：Present invention offer can occur luminous point and hit microRNA of chemical reaction and its preparation method and application, In conjunction with luminous point hit chemical reaction realize to the fluorescent marker, molecular modification, target spot of siRNA and miRNA separation identification, other mutually Act on the separation identification of biomolecule and with this assessment to toxic side effect in siRNA and miRNA medicinal applications.
Description of the drawings
Fig. 1 is the nucleus magnetic hydrogen spectrum of tetrazole compound 1；
Fig. 2 is that the nuclear-magnetism carbon of tetrazole compound 1 is composed；
Fig. 3 is the nucleus magnetic hydrogen spectrum of tetrazole compound 2；
Fig. 4 is that the nuclear-magnetism carbon of tetrazole compound 2 is composed；
Fig. 5 is the nucleus magnetic hydrogen spectrum of tetrazole compound 3；
Fig. 6 is that the nuclear-magnetism carbon of tetrazole compound 3 is composed；
Fig. 7 is the luciferase assay result that tetrazole compound 1 modifies miR-122；
MiR-122 analysis results in the drop-down experiment that Fig. 8 is the modification of tetrazole compound 1 miR-122；
Fig. 9 is the said target mrna analysis result for pulling down miR-122 in experiment that tetrazole compound 1 modifies miR-122；
Figure 10 is the interaction protein for pulling down miR-122 in experiment that tetrazole compound 1 modifies miR-122 (AGO2) analysis result；
Figure 11 is the fluorescence imaging result that tetrazole compound 2 and 3 modifies miR-122.
Specific implementation mode
The synthesis of embodiment 1, tetrazole chemical small molecule 1
(1) compound 1a (1.5g, 10mmol) and methanol (50mL) is dissolved in Methyl benzenesulfonyl hydrazine (1.86g, 10mmol) And in water (50mL), it is stirred at room temperature 5 minutes, a large amount of white solids is precipitated, filters to obtain 1b 3g (yield 93%).
(2) diazonium salt of aniline is by being poured into sodium nitrite solution (5mmol) cold 2mL containing 1.3mL at 5 DEG C or less It is prepared in the 50vt.% ethanol waters of the aniline (5mmol) of concentrated hydrochloric acid (37.5wt.%).Diazol is added drop-wise to 30mL is dissolved in the pyridine solution of 1b (1.6g, 5mmol), and controlling reaction temperature is at -10 DEG C to -15 DEG C.It is added dropwise completely subsequent continuous After reaction 3 hours, with dichloromethane and water extraction reaction, organic layer is washed after merging with dilute hydrochloric acid (mass concentration is no more than 20%) It washs and is dried with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain yellow powder 1c 0.7g (productions by column chromatography Rate 53%).
(3) 1c (0.6g, 2.5mmol) is dissolved in Dioxane (10mL), is heated to reflux (80 DEG C) Na2S (3g, Aqueous solution (5mL) 7.5mmol) is added drop-wise in Dioxane.Continue to be heated to reflux 2 hours, dichloromethane and water extraction reaction, Organic layer is dried after merging with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain yellow powder 1d by column chromatography 0.5g (yield 80%).
(4) 1d (100mg, 0.4mmol) is dissolved in dichloromethane (5mL), and 100 μ L of triethylamine are added, and fat is then added Acyl chlorides (113mg, 0.6mmol) is stirred at room temperature 2 hours.With dichloromethane and water extraction reaction, organic layer uses dilute hydrochloric acid after merging It washs and is dried with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain white powder 1e 160mg by column chromatography (yield 99%).
(5) 1e (160mg, 0.4mmol) is dissolved in 50vt.% tetrahydrofuran aqueous solutions, and LiOHH is added2O 177mg (4.2mmol).It is stirred at room temperature 1 hour, with hydrochloric acid solution tune pH to acidity, ethyl acetate is added to extract, with dilute after organic layer merging Salt acid elution is simultaneously dried with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain white powder 1 by column chromatography 100mg (yield 65%).The nucleus magnetic hydrogen spectrum of compound 1 is as shown in Fig. 1, and nuclear-magnetism hydrogen carbon spectrum is as shown in Fig. 2.
The synthesis of embodiment 2, tetrazole chemical small molecule 2
(1) diazol 2a is dense containing 1.3mL by being poured at 5 DEG C or less sodium nitrite solution (5mmol) cold 2mL It is prepared in the 50vt.% ethanol waters of the naphthylamines (0.7g, 5mmol) of hydrochloric acid (37.5wt.%).
(2) diazol 2b is added drop-wise to 30mL dissolved in the pyridine solution of 1b (1.6g, 5mmol), controlling reaction temperature- 10 DEG C to -15 DEG C.After the complete subsequent continuous reaction of dropwise addition 3 hours, reacted with dichloromethane and water extraction, with dilute after organic layer merging Salt acid elution is simultaneously dried with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain yellow powder 2c by column chromatography 0.8g (yield 50%).
(3) 2c (0.8g, 2.5mmol) is dissolved in Dioxane (10mL), is heated to reflux (80 DEG C) Na2S (3g, Aqueous solution (5mL) 7.5mmol) is added drop-wise in Dioxane.Continue to be heated to reflux 2 hours, dichloromethane and water extraction reaction, Organic layer is dried after merging with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain yellow powder 2d by column chromatography 0.6g (yield 84%).
(4) 2d (100mg, 0.35mmol) is dissolved in dichloromethane (5mL), and 100 μ L of triethylamine are added, and fat is then added Acyl chlorides (100mg, 0.525mmol) is stirred at room temperature 2 hours.It is reacted with dichloromethane and water extraction, with dilute salt after organic layer merging Acid elution is simultaneously dried with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain white powder 2e by column chromatography 150mg (yield 99%).
(5) 2e (150mg, 0.35mmol) is dissolved in 50vt.% tetrahydrofuran aqueous solutions, and LiOHH is added2O 147mg(3.5mmol).It is stirred at room temperature 1 hour, with hydrochloric acid solution tune pH to acidity, ethyl acetate is added to extract, after organic layer merges It is washed with dilute hydrochloric acid and is dried with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain white powder 2 by column chromatography 80mg (yield 55%).The nucleus magnetic hydrogen spectrum of compound 2 is as shown in Fig. 3, and nuclear-magnetism hydrogen carbon spectrum is as shown in Fig. 4.
The synthesis of embodiment 3, tetrazole chemical small molecule 3
(1) compound 3a (1.9g, 10mmol) is dissolved in 100mL acetone, is heated to reflux, is separately added into potassium carbonate (13.8g, 10mol) and allyl bromide, bromoallylene (11.9g, 10mol).Continue to be heated to reflux 24 hours, be extracted with dichloromethane and water anti- It answers, is dried after organic layer merges and with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain yellow powder by column chromatography Last 3b 2g (yield 87%).
(2) 3b (2g, 8.7mmol) is dissolved in Dioxane (50mL), is heated to reflux (80 DEG C) Na2S (17g, Aqueous solution (5mL) 7.5mmol) is added drop-wise in Dioxane.Continue to be heated to reflux 2 hours, dichloromethane and water extraction reaction, Organic layer is dried after merging with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain yellow powder 3c by column chromatography 1.5g (yield 86%).
(3) diazol 3d is dense containing 1.3mL by being poured at 5 DEG C or less sodium nitrite solution (5mmol) cold 2mL It is prepared in the 50vt.% ethanol waters of the 3c (1g, 5mmol) of hydrochloric acid (37.5wt.%).
(4) diazol 3d is added drop-wise to 30mL dissolved in the pyridine solution of 1b (1.6g, 5mmol), controlling reaction temperature- 10 DEG C to -15 DEG C.After the complete subsequent continuous reaction of dropwise addition 3 hours, reacted with dichloromethane and water extraction, with dilute after organic layer merging Salt acid elution is simultaneously dried with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain yellow powder 3e 1g by column chromatography (yield 53%).
(5) 3e (0.9g, 2.5mmol) is dissolved in Dioxane (10mL), is heated to reflux (80 DEG C) Na2S (3g, Aqueous solution (5mL) 7.5mmol) is added drop-wise in Dioxane.Continue to be heated to reflux 2 hours, dichloromethane and water extraction reaction, Organic layer is dried after merging with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain yellow powder 3f by column chromatography 0.8g (yield 93%).
(6) 3f (100mg, 0.3mmol) is dissolved in dichloromethane (5mL), and 100 μ L of triethylamine are added, and fat is then added Acyl chlorides (78mg, 0.45mmol) is stirred at room temperature 2 hours.With dichloromethane and water extraction reaction, organic layer uses dilute hydrochloric acid after merging It washs and is dried with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain white powder 3g 140mg by column chromatography (yield 99%).
(7) 3g (140mg, 0.3mmol) is dissolved in 50vt.% tetrahydrofuran aqueous solutions, and LiOHH is added2O (126mg, 3mmol).It is stirred at room temperature 1 hour, with hydrochloric acid solution tune pH to acidity, ethyl acetate is added to extract, after organic layer merges It is washed with dilute hydrochloric acid and is dried with anhydrous sodium sulfate.After vacuum distillation removal solvent, propose to obtain white powder 3 by column chromatography 90mg (yield 63%).The nucleus magnetic hydrogen spectrum of compound 3 is as shown in Fig. 5, and nuclear-magnetism hydrogen carbon spectrum is as shown in Fig. 6.
Embodiment 4, tetrazole chemical small molecule 1 modify the synthesis of miR-122 (TetI-miR-122)
Compound 1 (160mg, 0.44mmol) is dissolved in 10mL tetrahydrofurans and dichloromethane (volume ratio 1:1) in solution, point It Jia Ru not DCC (108mg, 0.52mmol) and NHS (60mg, 0.52mmol).It is stirred overnight at room temperature, white solid, filtering is precipitated Organic phase is obtained, obtain white solid after vacuum distillation removal solvent is made into 5mM storing solutions without being further purified with anhydrous DMSO. Amido modified miRNA PBS (pH=7.4) dissolve (50 μM), and the DMSO that same volume is added is made into 5mM storing solutions (100eq). Overnight, HPLC purifying freezes dry white powder solid for room temperature reaction.
Embodiment 5, tetrazole chemical small molecule 2 modify the synthesis of miR-122 (TetII-miR-122)
Compound 2 (100mg, 0.24mmol) is dissolved in 10mL tetrahydrofurans and dichloromethane (volume ratio 1:1) in solution, point It Jia Ru not DCC (54mg, 0.29mmol) and NHS (30mg, 0.29mmol).It is stirred overnight at room temperature, white solid is precipitated, filters Organic phase, vacuum distillation removal solvent after white solid be made into 5mM storing solutions with anhydrous DMSO without being further purified.Ammonia The miRNA of base modification dissolves (50 μM) with PBS (pH=7.4), and the DMSO that same volume is added is made into 5mM storing solutions (100eq).Room Overnight, HPLC purifies ice and does to obtain white powder solid for temperature reaction.
Embodiment 6, tetrazole chemical small molecule 3 modify the synthesis of miR-122 (TetIII-miR-122)
Compound 3 (80mg, 0.17mmol) is dissolved in 10mL tetrahydrofurans and dichloromethane (volume ratio 1:1) in solution, point It Jia Ru not DCC (42mg, 0.2mmol) and NHS (24mg, 0.2mmol).It is stirred overnight at room temperature, white solid is precipitated, filters to have Machine phase, vacuum distillation removal solvent after white solid be made into 5mM storing solutions with anhydrous DMSO without being further purified.Amino The miRNA of modification PBS (pH=7.4) dissolve (50 μM), and the DMSO that same volume is added is made into 5mM storing solutions (100eq).Room temperature Overnight, HPLC is purified and white powder solid is lyophilized to obtain for reaction.
Embodiment 7, luciferase assay
(1) structure of reporter plasmid
The 3 ' of Luciferase reporter plasmids (Promega) is sheared using HindIII and Spel (I Takara) restriction endonuclease Obtained segment is carried out electrophoretic separation purifying by the ends UTR.The complementary series of miR-122 passes through DNA ligase, T4 respectively Ligase (Takara) is inserted into the luciferase reporter plasmids that purifying obtains, and is transformed into competent E.coli, It screens to obtain possible positive plasmid by the screening flat board containing ammonia benzyl again.This report plasmid is finally subjected to sequencing and confirms it Correctness (Invitrogen).
(2) luciferase assay
Reporter plasmid after identification is entered by transfection in HepG2 cells.First by cell kind to 24 orifice plates, work as cell When density reaches 70%-80%, 0.25 μ g miR-122 reporter plasmids and 0.15 μ g β-galactosidase internal reference plasmids are used 2000 reagents of Lipofectamine are transfected into cell, and transfection procedure follows the Invitrogen descriptions of product.In transfection 4 hours Afterwards, the miR-122 (TetI-miR-122) that 0.25 μ g compounds 1 are modified is transfected with 2000 reagents of Lipofectamine into thin Born of the same parents.After 5-6 hours, cell training liquid is changed into containing 2vt.%FBS, the DMEM in high glucose culture solution of 1vt.%PS continues culture 48 hours Afterwards, luciferase and β-galactosidase internal reference signals are measured.The result shows that after the modification of tetrazole chemical small molecule, MiR-122 still has the activity (Fig. 7) for inhibiting luciferase expression.
Embodiment 8, drop-down experiment
The control (TetI-NC) of 30 μ g tetrazoles chemical small molecules 1 modification is used respectively with TetI-miR-122 The transfections of Lipofectamine 2000 enter 1.5 × 107HepG2 is intracellular.After 24 hours, by cell respectively with 150 μ L 0.1vt.%Triton PBS cracking, by the way that supernatant is collected by centrifugation.The biotin molecule of 100 μM of double bonds modification is added in supernatant, and With 302nm ultraviolet lights 10 minutes.Next, cell pyrolysis liquid super filter tube and PBS are washed 4 times.The supernatant of collection with Streptomysin magnetic-particle (New England Biolabs) is after being incubated at room temperature 2 hours, by magnetic-particle washing buffer It washs 4 times and 1 time respectively with low salt buffer, the biomolecule acted on magnetic-particle is eluted with elution buffer finally and is received Collection carries out reversing and quantitative PCR or western blot analysis respectively.Quantitative PCR experiment finds that treated with TetI-miR-122 There are the enrichment (Fig. 8) of apparent miR-122, target gene that can also realize enrichment and identification (Fig. 9) in sample.
The measurement of embodiment 9, miRNA expression quantity
The RNA progress reverse transcription reactions for taking out 5 μ L prepare corresponding cDNA samples.10 μ L systems of specific reverse transcription reaction For：
5x AMV buffer(Takara) 2μL
AMV(Takara) 0.5μL
RT primer(ABI) 0.5μL
dNTP mixture(Takara) 1μL
DEPC H2O 1μL
RNA 5μL
Response procedures are：16 DEG C of 30min, 42 DEG C of 30min, 85 DEG C of 5min, 4 DEG C of preservations.The cDNA prepared uses probe again Method (Taqman) PCR carries out real-time quantitative, 20 μ L to miR-122；
Taqman PCR reaction systems are：
10x buffer(Takara) 2μL
MgCl2(25mM)(Takara) 1.2μL
Taq(Takara) 0.3μL
dNTP mixture(10mM) 0.4μL
Probe(ABI) 0.33μL
cDNA 1μL
Distilled H2O 14.77μL
PCR response procedures are 95 DEG C of 5min, and 60 DEG C of 1min40 cyclic amplifications after 95 DEG C of 15s, real-time fluorescent signals exist The 60 DEG C of acquisitions each recycled.
Embodiment 10, the measurement of mrna expression amount
The RNA progress reverse transcription reactions for taking out 5 μ L prepare corresponding cDNA samples.20 μ L systems of specific reverse transcription reaction For：
5x AMV buffer(Takara) 4μL
AMV(Takara) 1μL
OligodT(Takara) 1μL
dNTP mixture(Takara) 2μL
RRI(Takara) 0.5μL
DEPC H2O 6.5μL
RNA 5μL
Response procedures are：42 DEG C of 60min, 85 DEG C of 5min, 4 DEG C of preservations.The cDNA prepared uses dye method PCR to mRNA again Carry out real-time quantitative, 20 μ L；
Taqman PCR reaction systems are：
10x buffer(Takara) 2μL
MgCl2(25mM)(Takara) 1.6μL
Taq(Takara) 0.2μL
dNTP mixture(10mM) 0.4μL
SYBR Green 1μL
Forward+Reverse primers 0.4μL
cDNA 1μL
Distilled H2O 13.4μL
PCR response procedures are 95 DEG C of 5min, 95 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 30s, 40 cyclic amplifications, real-time fluorescence letter Number in 72 DEG C of acquisitions of each cycle.
Embodiment 11, western blot analysis
After protein lysate and 1 × SDS loading buffer mixings, various concentration is selected according to the size of albumen (SDS-PAGE of general 10%) carries out Protein Separation to sample.After albumen is successfully separated, it is transferred to albumen is wet on pvdf membrane, Carry out 1 hour sealer with the TBST/Tween-20 buffer solutions containing 5wt.% skimmed milk powers again, then with primary antibody 4 DEG C incubate overnight It educates, TBST/Tween-20 10min are washed four times, and secondary antibody is incubated at room temperature 1 hour, then is washed with TBST/Tween-20 10min Four times, finally use ECL reagents development protein band.It as can see from Figure 10, can be in the cell of TetI-miR-122 processing It is clearly visible the enrichment of AGO2 albumen.
The real-time fluorescence imaging of miR-122 in embodiment 12, living cells
Take the miR-122 of 5 ' terminal modified Cy3 as precursor modification tetrazole chemical small molecule 2 and 3.Then by 2 μ g MiR-122-Cy3, TetII-miR-122-Cy3 and TetIII-miR-122-Cy3 use Lipofectamine 2000 to transfect respectively Into 1 × 105HepG2 is intracellular.After 4 hours, remove culture solution and by cells rinsed with PBS 3 times, after it is aobvious in aggregation fluorescence It is acquired after being excited one minute with 700nm light under micro mirror green (excitation wavelength 405nm emits detection range 450-550nm) and red Color (excitation wavelength 543nm emits detection range 580-650nm) fluorescence.In the cell of TetIII-miR-122-Cy3 processing It can clearly be seen that green fluorescence is opened, and other two groups are not observed similar phenomenon, and green fluorescence has with red fluorescence Preferable common location effect (Figure 11) shows the fluorescence imaging that miRNA in living cells is realized using light click chemistry.
The above-mentioned specific implementation mode technical solution that the invention is not limited in any way, it is every to use equivalent replacement or wait The technical solution that the mode of effect transformation is obtained all falls within protection scope of the present invention.

Claims (2)

1. the microRNA that luminous point hits chemical reaction can occur, it is characterised in that the structural formula is：
2. application of the microRNA described in claim 1 in preparing label, imaging agents.
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