CN105985944B - A kind of new method of intracellular site specific covalent labeled RNA - Google Patents
A kind of new method of intracellular site specific covalent labeled RNA Download PDFInfo
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- CN105985944B CN105985944B CN201510046658.0A CN201510046658A CN105985944B CN 105985944 B CN105985944 B CN 105985944B CN 201510046658 A CN201510046658 A CN 201510046658A CN 105985944 B CN105985944 B CN 105985944B
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- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- HVTICUPFWKNHNG-UHFFFAOYSA-N iodoethane Chemical compound CCI HVTICUPFWKNHNG-UHFFFAOYSA-N 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000012280 lithium aluminium hydride Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- LPNYRYFBWFDTMA-UHFFFAOYSA-N potassium tert-butoxide Chemical compound [K+].CC(C)(C)[O-] LPNYRYFBWFDTMA-UHFFFAOYSA-N 0.000 description 1
- ZWIOSPCYIJAHKM-UHFFFAOYSA-M potassium;2,3,3-trimethylindole-5-sulfonate Chemical compound [K+].C1=C(S([O-])(=O)=O)C=C2C(C)(C)C(C)=NC2=C1 ZWIOSPCYIJAHKM-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 229960005439 propantheline bromide Drugs 0.000 description 1
- WGYKZJWCGVVSQN-UHFFFAOYSA-N propylamine Chemical group CCCN WGYKZJWCGVVSQN-UHFFFAOYSA-N 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- SVOOVMQUISJERI-UHFFFAOYSA-K rhodium(3+);triacetate Chemical compound [Rh+3].CC([O-])=O.CC([O-])=O.CC([O-])=O SVOOVMQUISJERI-UHFFFAOYSA-K 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to it is a kind of in vitro or the new method of intracellular site specific covalent labeled RNA.This method by introducing tRNA in vitro or into the celllle2- agmatidine synzyme (abbreviation Tias, nucleotide sequence is as shown in SEQ ID NO:1, and amino acid sequence is as shown in SEQ ID NO:2) and the ancient green-ball bacterium tRNA of flashinglle2(SEQ ID NO:3) or its derivatized nucleotide sequence, make AftRNA in Tias specific recognition transcript profilelle2Particular sequence, promote the small molecule containing azide or alkynyl functionality and to contain tRNAlle2Sequence is generated between the target RNA of label and locus specificity combines, and then specific marker and the imaging of target RNA are realized by the chemical reaction of fluorescent dye and functional group.The invention further relates to the kits of locus specificity covalent labeling target RNA in the cell, and it includes tRNAlle2- agmatidine synzyme and tRNAlle2‑3‑5。
Description
Technical field
The invention belongs to biochemical fields.Specifically, the present invention provides one kind in vitro or intracellular site is specific
The new method of covalent labeling RNA.More specifically, the invention by introducing tRNA in vitro or into the cellIle2- agmatidine is closed
At enzyme (tRNAIle2- agmatidine synthetase, abbreviation Tias, nucleotide sequence is as shown in SEQ ID NO:1, ammonia
Base acid sequence is as shown in SEQ ID NO:2) and the ancient green-ball bacterium (Archaeoglobus fulgidus, abbreviation Af) of flashing
tRNAIle2(nucleotide sequence is as shown in SEQ ID NO:3), makes Af tRNA in Tias specific recognition transcript profileIle2Specific sequence
Column promote the small molecule containing azide or alkynyl functionality and contain tRNAIle2Sequence is generated between the target RNA of label
And locus specificity combines, and then the specific marker of target RNA is realized by the chemical reaction of fluorescent dye and functional group
And imaging.
Background technique
RNA is a kind of important large biological molecule in organism, in biology such as protein translation, gene expression regulations
It plays an extremely important role in the process.With going deep into for scientific research, the function of RNA is more and more diversified, importance
Can match in excellence or beauty protein at present.
There is specific subcellular localization in RNA, the different subcellular localizations of RNA decide that it executes different lifes in the cell
Object function.Therefore, the biological function that the specific mark of RNA and imaging technique in cell accurately understand us RNA is developed
It has a very important significance.
In order to disclose dynamic transport, positioning and interaction of the RNA in cell, scientific research personnel has developed many and has existed
The new method of non-covalent labeling RNA in mammalian cell, and have made some progress.Current developed intracellular rna
Label and imaging technique are greatly promoted the functional study of RNA, have deepened our understanding to RNA function.But it is existing
All there is deficiency in a way in intracellular rna label and imaging technique, which prevent the extensive researchs of RNA function.Therefore,
Develop the level that RNA functional study will be greatly improved in new intracellular rna label and imaging technique, this is to vast research work
It is also to shoulder heavy responsibilities for person.
Compared to non-covalent approach, bio-orthogonal chemistry depends on the specificity of probe molecule, and raw with target
The covalent linkage of object macromolecular.Therefore, this method has following several advantages: 1, probe molecule and target biomacromolecule have height
Compatibility is spent, therefore can be by limiting elution requirement strictly come highly purified target biomacromolecule；2, high-affinity makes low
The target biomacromolecule of abundance can visualize, and this is even more important for non-coding RNA；3, many contains functional group
The probe molecule of (such as: fluorescence, nuclear magnetic resonance, infrared spectroscopy, electron paramagnetic resonance functional group etc.) may be incorporated into target
On large biological molecule, so that this method above becomes very flexibly in application.Due to these unique functions, bio-orthogonal chemistry is
Be successfully applied to the functional study of protein and polysaccharide, be mainly used to observe in mammalian cells their expression,
Positioning and interaction.
Currently, translation/posttranslational modification mechanism is combined with bio-orthogonal chemistry, have been able to unprecedented essence
Degree and the multifunctionality method carrying out labelled protein, but be not suitable in mammalian cells to RNA carry out site and
Sequence-specific label.Therefore, the present invention is quasi- finds suitable posttranslational modification component, it is mutually tied with bio-orthogonal chemistry
It closes, the new method of Lai Kaifa RNA specific mark.
Summary of the invention
1, technical problem
To solve the above-mentioned problems, the purpose of the present invention is to provide it is a kind of in vitro or intracellular site specific covalent
The new method of labeled RNA.
The present invention provide it is a kind of in vitro or the new method of intracellular site specific covalent labeled RNA.The present invention relates to
By introducing tRNA in vitro or into the cellIle2- agmatidine synzyme (tRNAIle2- agmatidine synthetase,
Abbreviation Tias, nucleotide sequence is as shown in SEQ ID NO:1, and amino acid sequence is as shown in SEQ ID NO:2) and the ancient life of flashing
Coccus (Archaeoglobus fulgidus, abbreviation Af) tRNAIle2(nucleotide sequence is as shown in SEQ ID NO:3), makes
Af tRNA in Tias specific recognition transcript profileIle2Particular sequence, promote the small molecule containing azide or alkynyl functionality
With contain tRNAIle2Sequence is generated between the target RNA of label and locus specificity combines, and then passes through fluorescent dye and function
Specific marker and the imaging of group chemically reacted to realize target RNA.
2, technical solution
In order to realize the specific marker of RNA, we are firstly the need of unique group found in a kind of posttranslational modification mechanism
Point, to carry out bio-orthogonal chemical reaction.The component needs tool, and there are two features: the first, it must be a kind of RNA modification
Enzyme can identify the specific site in specific RNA sequence in transcript profile；The second, it allows for that unique function base will be contained
The small molecule of group (such as azide or alkynes functional group) is transferred to the privileged site of RNA, then passes through bio-orthogonal chemistry
Reaction carries out covalent labeling, realizes that locus specificity combines, finally carries out fluorescence, nuclear magnetic resonance, electron paramagnetic resonance or red
External spectrum measurement.
In order to find it is this can precise marking RNA in mammalian cells posttranslational modification component, the present inventor
Sight is turned into tRNA modification enzyme.It is well known that tRNA modification enzyme can be catalyzed more than 100 kinds of tRNA modifications.The loyalty of genetic code
Real translation depends on the chemical modification of tRNA, especially the 34th of anticodon loop nucleotide.Many acts on the position
The tRNA modification enzyme of point is found to exist only in specific region, it means that they may be independently to evolve.
It is worth noting that, tRNA in archeobacteriaIle234 cytimidines through tRNAIle2- agmatidine synzyme
(tRNAIle2- agmatidine synthetase, abbreviation Tias) catalysis can be by agmatine (agmatine, abbreviation AGM, this hair
Compound 1 is referred to as in bright) modification.According to pervious report, flash ancient green-ball bacterium (Archaeoglobus fulgidus,
Abbreviation Af) in, Tias identifies tRNAIle2Seven nucleotide are needed, are respectively: G1, G2, C34, U36, A37, C71 and C72, and
Both without Tias in mammalian cell, the also tRNA not containing this seven kinds of nucleotide sites.Therefore, inventors have contemplated that,
If by the tRNA of Tias and archeobacteriaIle2It is introduced into mammalian cell, and Tias can will contain nitrine or alkynyl function
The Small-molecule probe of group is transferred on target RNA, perhaps can realize sequence and the site-specific labeling of RNA.
RNA label and imaging are successfully realized, in addition to tRNA modification enzyme Tias, it is also necessary to the small molecule containing functional group
Probe and corresponding fluorescent dye carry out bio-orthogonal chemical reaction, therefore, the present inventor's purchase/synthesized three kinds of agmatine classes
Like object (structural formula is referring to Fig. 1): N- (4- aminobutyl) -2- nitrine yl acetamide (N- (4-aminobutyl) -2-
Azidoacetamide) (abbreviation AGN, the present invention in referred to as compound 2), propargylamine (2-Propynylamine, the present invention
In referred to as compound 3, be purchased from AlfaAesar company), butyl- 3- alkynes -1- base (4- aminobutyl) t-butyl carbamate (but-
3-yn-1-yl (4-aminobutyl) carbamate, the present invention in referred to as compound 4)；And three kinds of fluorescent dyes: BCN-
FITC, Sulfo-Cy5-azide (being purchased from Lumiprobe company), BCN-Cy5.Meanwhile having purchased archeobacteria
Archaeoglobus fulgidus (be purchased from ATCC company, article No. 49558) clonal expression and is purified into from its genome
RNA modification enzyme Tias (for nucleotide sequence as shown in SEQ ID NO:1, amino acid sequence is as shown in SEQ ID NO:2).By setting
A series of experiments is counted, is incubated for Tias, Af tRNA jointly in vitroIle2Or its derivative nucleotide sequence and target RNA merge
Nucleotide sequence, Small-molecule probe, fluorescent dye and other auxiliary materials, or Tias and Af is co-expressed in the cell
tRNAIle2Or the nucleotide sequence that merges of its derivative nucleotide sequence and target RNA, and and Small-molecule probe, fluorescent dye
It is incubated for jointly with other auxiliary materials, is finally successfully realized the specific marker and fluorescence imaging of target RNA.It is wherein described small
Molecular probe may be, but not limited to, the compound selected from agmatine or agmatine analog.
The present invention provides following embodiments:
1. one kind is capable of the kit of locus specificity covalent labeling target RNA in the cell, the kit includes:
(1)tRNAIle2- agmatidine synzyme, amino acid sequence are SEQ ID NO:2,
(2)tRNAIle2Or its derivative nucleotide sequence, wherein the tRNAIle2Nucleotides sequence be classified as SEQ ID NO:
3,
(3) substrate, the substrate be agmatine or agmatine analog,
(4) reaction buffer；
(5) ATP liquid storage；
(6) DTT liquid storage；
(7) fluorescent dye is selected from BCN-FITC, BCN-Cy5 or Sulfo-Cy5-azide.
2. the kit according to the 1st, wherein the tRNAIle2Derivative nucleotides sequence is classified as tRNAIle2- 3-5,
Its nucleotides sequence is classified as SEQ ID NO:5, with structure shown in Figure 11.
3. the kit according to the 1st, wherein the agmatine analog is selected from N- (4- aminobutyl) -2- nitrine
Yl acetamide, propargylamine or butyl- 3- alkynes -1- base (4- aminobutyl) t-butyl carbamate.
4. the kit according to the 1st, wherein the reaction buffer is 100mM Tris-HCl, pH 8.0,
10mM KCl, 5mM MgCl2。
5. the kit according to the 1st, the kit further includes making tRNAIle2Or its derivative nucleotide sequence
The reagent merged with target RNA.
6. a kind of method of locus specificity covalent labeling target RNA in the cell, the method includes the following steps:
(1) by tRNAIle2Or its derivative nucleotide sequence and the target RNA are coupled to form fusion rna, wherein described
tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3；
(2) tRNA that amino acid sequence is SEQ ID NO:2 is co-expressed in the cellIle2- agmatidine is closed
At the fusion rna that enzyme and step (1) obtain, contain the compound selected from agmatine or agmatine analog in cell culture medium；
(3) lytic cell extracts cell total rna, and fluorescent dye mixing is added and is incubated for, is combined by agarose gel electrophoresis
Fluorescence imaging is analyzed, wherein the fluorescent dye is selected from BCN-FITC, BCN-Cy5 or Sulfo-Cy5-azide.
6. a kind of method of locus specificity covalent labeling target RNA in the cell, the method includes the following steps:
(1) by tRNAIle2Or its derivative nucleotide sequence and the target RNA are coupled to form fusion rna, wherein described
tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3；
(2) tRNA that amino acid sequence is SEQ ID NO:2 is co-expressed in the cellIle2- agmatidine is closed
At the fusion rna that enzyme and step (1) obtain, contain the compound selected from agmatine or agmatine analog in cell culture medium；
(3) catalyst of fluorescent dye and catalyzed fluorogenic reaction is added into cell culture, is incubated for, passes through laser
Laser Scanning Confocal Microscope analysis, wherein the fluorescent dye is selected from BCN-FITC, BCN-Cy5 or Sulfo-Cy5-azide.
8. a kind of method of locus specificity covalent labeling target RNA in vitro, the method includes in system in vitro
It is incubated for following substance: Tias, tRNA jointlyIle2Or the nucleotide sequence that merges of its derivative nucleotide sequence and target RNA, it is small
Molecular probe, fluorescent dye and other auxiliary materials, wherein the tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3, it is described
Small-molecule probe is the compound selected from agmatine or agmatine analog.
9. the method according to any one of 6-8, wherein the tRNAIle2Derivative nucleotides sequence is classified as
tRNAIle2- 3-5, nucleotides sequence is classified as SEQ ID NO:5, with structure shown in Figure 11.
10. method described in the item according to any one of 6-8, wherein the agmatine analog is selected from N- (4-
Aminobutyl) -2- nitrine yl acetamide, propargylamine or butyl- 3- alkynes -1- base (4- aminobutyl) t-butyl carbamate.
3, beneficial effect
Currently, many modern times RNA biology and RNA biotechnology all refer to through chemical method the site in RNA molecule
Specific integration modified nucleoside acid, however, the length of target RNA molecule is usually less than 30-50 due to the limitation of integration efficiency
Nucleotide.Our new method depends on the transcription mechanism of RNA and the sequence-specific of RNA modification enzyme Tias, therefore can
Biophysics probe is integrated with high degree of specificity on any target RNA in mammalian cells.This method can be applied to
The external or RNA folding in mammalian cell, the interaction of RNA- protein, rna transport, RNA modification research and RNA receive
Rice material building.
Compared to pervious non-covalent RNA imaging method, such as spinach aptamer, green fluorescence luminophore and spinach aptamer
Between noncovalent interaction be only capable of in the micromolar range have affinity；And our new method depends on Tias
With Af tRNAIle2Selective recognition (both of which is not present in mammalian cell), then by introducing azido or alkynes
Base group makes to form covalent bond between fluorophor and target RNA, to realize the fluorescence imaging of low abundance RNA, therefore covalent labeling
New method has higher specificity, compatibility and versatility.Importantly, our new method is by introducing containing difference
The small molecule of functional group can use a variety of methods after click chemistry reacts (click chemistry reaction),
Such as: fluorescence, infrared spectroscopy, nuclear magnetic resonance or electron paramagnetic resonance etc. carry out multi-functional locus specificity RNA label, without
Need to change Tias albumen or Af tRNAIle2Sequence.
Detailed description of the invention
From detailed description with reference to the accompanying drawing, features described above of the invention and advantage be will be apparent from, in which:
Fig. 1: the picture left above is the chemical structural formula of compound 1 (AGM), and top right plot is the chemical structure of compound 2 (AGN)
Formula, lower-left figure are the chemical structural formulas of compound 3, and bottom-right graph is the chemical structural formula of compound 4；
Fig. 2 is the synthetic route of compound 2；
Fig. 3 is the synthetic route of compound 4；
Fig. 4 and Fig. 4 (Continued) is the synthetic route of BCN-FITC (since Figure of description page 2 could not show completely
Therefore the synthetic route of BCN-FITC continues to show in Fig. 4 (Continued))；
Fig. 5 is the synthetic route of BCN-Cy5；
Fig. 6 is enzyme used in the present invention and nucleotide/amino acid sequence of RNA；
Fig. 7 is the reaction schematic diagram of Tias modification RNA；
Fig. 8: A figure is the polyacrylamide gel electrophoresis figure that RNA is modified in vitro, and B figure is poly- the third of RNA decorating site verifying
Acrylamide gel electrophoresis figure, C figure are non-modified tRNAIle2Nucleic acid fragment mass spectrogram, D figure are the tRNA modified through compound 1Ile2
Nucleic acid fragment mass spectrogram, E figure are the tRNA modified through compound 2Ile2Nucleic acid fragment mass spectrogram, F figure are modified through compound 3
tRNAIle2Nucleic acid fragment mass spectrogram；
Fig. 9 is the crystal structure figure of Tias combination AGN, and wherein A figure is overall structure figure, and B figure is local structural graph；
Figure 10: A figure is the tRNA modified through compound 2Ile2In-vitro specificity label and fluorescence imaging figure, B figure are through changing
Close the tRNA that object 3 is modifiedIle2In-vitro specificity label and fluorescence imaging figure；
Figure 11 is tRNAIle2- 3-5 structure chart；
Figure 12 is tRNAIle2- 3-5 In-vitro specificity label and fluorescence imaging figure；
Figure 13 is tRNAIle2- 5S fusion rna specific marker and fluorescence imaging figure；
Figure 14 is tRNAIle2- 5S fusion rna is in the intracellular specific marker of U2OS and fluorescence imaging figure, wherein lastrow
It is not express tRNAIle2The micro-imaging figure of the U2OS cell of -5S fusion rna, next line are expression tRNAIle2- 5S fusion rna
The micro-imaging figure of U2OS cell (left column is fluorescence imaging figure, and middle column are by the channel DAPI microexamination picture, and right column are
Pass through the channel DIC microexamination picture).
Specific embodiment
It is next by the following examples that the present invention is furture elucidated.However, it should be understood that the embodiment is merely illustrative
Purpose, be not intended to limit scope and spirit of the present invention.
It should be appreciated by those skilled in the art that unless stated otherwise, chemical reagent as used in the following examples is can
Pass through the reagent for the pure rank of analysis that commercial sources are bought.
Embodiment 1: the chemical synthesis of agmatine (AGM, compound 1) analog
1, the synthesis (Fig. 2) of compound 2 (N- (4-aminobutyl) -2-azidoacetamide, AGN):
656mg BOC-1 is taken, 4- butanediamine hydrochloride and 1.48g sodium carbonate are dissolved in the acetic acid of 80mL, 1: 1 (v/v) jointly
In ethyl ester and aqueous mixtures, 0 DEG C of ice bath.The second of 10ml dissolution 702mg 2- acetyl bromide bromide is added after 1 hour into reaction solution
Acetate solution is stirred at room temperature 2 hours.Upper organic phase is taken, it is dry using Rotary Evaporators, then existed by silica gel chromatography
It is purified on silica gel (ethyl acetate for being 1: 1 containing volume ratio: petroleum ether), obtains 320mg white intermediate product 1: tert-butyl (4-
(2- acetyl bromide amido) butyl) carbamate (Tert-butyl (4- (2-bromoacetamido) butyl)
carbamate)。
230mg intermediate product 1 is taken to be dissolved in 5mL acetone, 0 DEG C of ice bath.5mL sodium azide is added after 30 minutes
(250mg) aqueous solution, be stirred overnight at room temperature (22 hours).Then reaction solution is concentrated, and (is contained by silica gel chromatography in silica gel
The ethyl acetate that volume ratio is 1: 1: petroleum ether) on purify, obtain 50mg intermediate product 2: tert-butyl (4- (2- nitrine acetyl
Amido) butyl) carbamate (Tert-butyl (4- (2-azidoacetamido) butyl) carbamate).
It takes 44mg intermediate product 2 to be resuspended in 20ml hydrochloric acid/ethyl acetate (volume ratio 1: 1) mixed liquor, is stirred at room temperature
Overnight (12 hours), it then rotary evaporation and is freeze-dried, obtains 29mg final product N- (4- aminobutyl) -2- nitrine acetamide
Hydrochloride (N- (4-aminobutyl) -2-azidoacetamide hydrochlorate).
2, the synthesis (Fig. 3) of compound 4 (but-3-yn-1-yl (4-aminobutyl) carbamate):
140mg 3- butyne-1-ol and 424mg sodium carbonate is taken to be dissolved in 10mL tetrahydrofuran solution jointly, 0 DEG C of ice bath.So
198mg triphosgene is added in backward reaction solution, and stirs 12 hours at room temperature, obtains intermediate product.Take 380mg intermediate product
It is dissolved in 50mL ethyl acetate/water (volume ratio ratio is 1: 1) solution, after being stirred at room temperature 12 hours, separates organic phase, be passed through HCL
Gas.It is fixed finally to collect white, obtains final product.
Chemical reagent needed for the above synthetic reaction unless otherwise instructed, is purchased from Beijing Chemical Plant or AlfaAesar company,
It is the pure above rank of analysis.
Embodiment 2: the chemical synthesis of fluorescent dye
1, the synthesis (Fig. 4) of BCN-FITC:
15.0mL 1 is taken, 5- cyclo-octadiene and the mixing of 281mg dimerization rhodium acetate are added in 10mL dichloromethane solution, then
The dichloromethane solution of 10mL dissolution 15mmol ethyl diazoacetate is added dropwise in 3 hours, while temperature is maintained at 0 DEG C.
After reacting 15 hours under room temperature, reaction system, which is replaced in ice-water bath, maintains the temperature at 0 DEG C, adds in 3 hours again
The dichloromethane solution for entering 10mL dissolution 15mmol ethyl diazoacetate continues to react 21 hours at room temperature.It is then logical
It crosses rotary evaporation and removes methylene chloride, silica gel post separation (petroleum ether: ethyl acetate=20: 1, v/v) obtains 2.52g intermediate product
1:(1R, 8S, 9r, Z) in-ethyl bicycle [6.1.0] non-4-ene-9-carboxylate (endo product) and 2.08g
Between product 2:(1R, 8S, 9s, Z)-ethyl bicycle [6.1.0] non-4-ene-9-carboxylate (exo product).
It takes 370mg lithium aluminium hydride reduction to be dissolved in 30mL ether, is subsequently placed in 0 DEG C of ice-water bath, and be added dropwise into suspension
The diethyl ether solution of 30mL dissolution 2.23g intermediate product 1.It is cooled to 0 DEG C after reacting 2 hours under room temperature, is then added dropwise
Deionized water is until gray solid becomes white.8g sodium sulphate is added, is then removed by filtration solid, and with 100mL second
Ether sufficiently washs, and obtained filtrate removes ether by rotary evaporation.In the case where not being further purified, by what is obtained
1.55g alcohol compound is dissolved in 30mL methylene chloride and is placed in ice-water bath, and 7.3mL is then added dropwise and dissolves 649 μ L bromines
Dichloromethane solution, until solution keeps yellow constant.Reaction system is quenched with the hypo solution of 30mL, 10%, and
It is extracted with 2 × 50mL methylene chloride.After the anhydrous sodium sulfate drying of obtained organic phase, then by the way that removing dichloromethane is concentrated under reduced pressure
Alkane obtains bis- bromo mixture of 2.80g.It is in the case where not being further purified, obtained bis- bromo mixture of 2.80g is complete
Portion is dissolved in 30mL tetrahydrofuran and is placed in ice-water bath, and 29.6mL catalyst KOtBu is then added dropwise in 10 minutes.Reaction
System is cooled to room temperature after flowing back 2 hours at 66 DEG C, is quenched with 100mL saturated ammonium chloride solution, then is removed by rotary evaporation
Tetrahydrofuran.Obtained solution is extracted with 3 × 100mL methylene chloride, dry with anhydrous sodium sulfate after isolating organic layer, with
Methylene chloride is removed by rotary evaporation afterwards, silica gel post separation (petroleum ether: ethyl acetate=4: 1, v/v) obtains among 724mg
3 white solid of product: (1R, 8S, 9r)-Bicyclo [6.1.0] non-4-yn-9-ylmethanol.
It takes 222mg intermediate product 3 to be dissolved in 20mL dichloromethane solution, 299 μ L pyridines and 368mg p-nitrophenyl is added
Chloro-formate, reaction system is stirred at room temperature to be quenched with 35mL saturated ammonium chloride solution after forty minutes.Reaction solution 2 × 50mL bis-
Chloromethanes extraction, separates organic layer, then removes methylene chloride, silica gel post separation (petroleum ether: acetic acid second by Rotary Evaporators
Ester=5: 4 white solid of 459mg intermediate product: (1R, 8S, 9r)-bicyclo [6.1.0] non-4-yn-9- 1, v/v) is obtained
ylmethyl(4-nitrophenyl)carbonate。
80 μ L tetra-methylenedimines are added dropwise to 4mL dissolution 102mg fluorescein isothiocynate, and (abbreviation FITC, is purchased from
Sigma company) methanol solution in, be stirred at room temperature 2 hours.After having red precipitate generation in solution, filtering is precipitated and is done
It is dry, obtain 100mg intermediate product 5:5- (3- (4-aminobutyl) thioureido) -2- (6-hydroxy-3-oxo-3H-
xanthen-9-yl)benzoicacid。
It takes 83mg intermediate product 4 and 100mg intermediate product 5 to be dissolved in 4mL n,N-Dimethylformamide solution jointly, adds
Enter 111 μ L triethylamines, then stirs 3 hours at room temperature.Silica gel post separation (methylene chloride: methanol=10: 1, v/v)
To 106mg final product: 5- (3- (4- ((((1R, 8S, 9r)-bicyclo [6.1.0] non-4-yn-9-ylmethoxy)
carbonyl)amino)butyl)thioureido)-2-(6-hydroxy-3-oxo-3H 3Hxanthen-9-yl)benzoic
Acid (abbreviation BCN-FITC).
2, the synthesis (Fig. 5) of BCN-Cy5:
15g 4- pHydrazinebenzenesulfonic acid semihydrate and 25.2mL 3- methyl -2- butanone is taken to be dissolved in 45mL glacial acetic acid, then
110 DEG C are flowed back 3 hours.Acetic acid is removed by Rotary Evaporators, obtained residue is dissolved in methanol, and containing hydroxide
It is stirred in the isopropanol saturated solution of potassium, until solution becomes yellow, finally obtains 13.2g yellow intermediate product 1:
Potassium2,3,3-trimethyl-3H-indole-5-sulfonate.
It takes 1.39g intermediate product 1 and 1.42g 3- propantheline bromide hydrobromide to be suspended in 10mL acetonitrile solution jointly, then exists
80 DEG C are kept in tube sealing to flow back 24 hours.Reaction system is resuspended in ether after being concentrated by rotary evaporation.Filtering
Mixture obtains solid, and is redissolved in methanol, is spin-dried for again, and 2.35g di-tert-butyl dicarbonate, 30mL anhydrous three is then added
Chloromethanes and 3.75mLN, N- diisopropylethylamine.After mixture is slowly warmed up 55 DEG C of reflux temperature, continue stirring 4 hours.
(methylene chloride: methanol=4: intermediate product 2:1- (3- ((tert- 1) is obtained by concentration and silica gel post separation
Butoxycarbonyl) amino) propyl) -2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate.
Contain addition 0.828mL iodoethane in the acetonitrile solution of 1.39g intermediate product 1 to 10mL, be placed in a tube sealing,
Then it is reacted at 80 DEG C and obtains within 24 hours crude product 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium-5-
sulfonate.535mg crude product and 570mg hydrochloric acid-N- (3- phenylamino -2- propylene subunit) aniline is taken to be dissolved in 10mL glacial acetic acid
In 10mL acetic anhydride, 110 DEG C are reacted 4 hours, obtain intermediate product 3:1-ethyl-3,3-dimethyl-2- ((1E, 3E)-
4- (N-phenylacetamido) buta-1,3-dien-1-yl) -3H-indol-1-ium-5-sulfonate.
2.00mmol intermediate product 2,2.00mmol intermediate product 3 and 2.29g anhydrous sodium acetate is taken to be dissolved in 30mL ethyl alcohol,
Then it flows back 18 hours at 80 DEG C.By concentration and silica gel post separation (methylene chloride: methanol=4: 1, v/v) obtain 214mg produce
Object sodium1- (3- ((tert-butoxycarbonyl) amino) propyl) -2- ((1E, 3E, 5Z) -5- (1-ethyl-3,
3-dimethyl-5-sulfonatoindolin-2-ylidene) penta-1,3-dien-1-yl) -3,3-dimethyl-3H-
indol-1-ium-5-sulfonate.Then product is dissolved in 15mL methanol and 10mL, 37% hydrochloric acid, 60 DEG C are reacted 6 hours
Afterwards, Sodium 1- (3-aminopropyl) -2- ((1E, 3E, 5Z) -5- (1-ethyl-3,3-dimethyl-5- are obtained
Sulfonatoindolin-2-ylidene) penta-1,3-dien-1-yl) -3,3-dimethyl-3H-indol-1-ium-
5-sulfonate (abbreviation Cy5-NH2HCl).
Take 99mg Cy5-NH2HCl and 51mg (1R, 8S, 9r)-bicyclo [6.1.0] non-4-yn-9-ylmethyl
(4-nitrophenyl) carbonate is dissolved in 8mL n,N-Dimethylformamide, and 86 μ L triethylamine solutions, room temperature is then added
Lower stirring 6 hours, obtains final product Sodium 1- (3- ((((1R, 8S, 9r)-bicyclo [6.1.0] non-4-yn-9-
Ylmethoxy) carbonyl) amino) propyl) -2- ((1E, 3E, 5Z) -5- (1-ethyl-3,3-dimethyl-5-
Sulfonatoindolin-2-ylidene) penta-1,3-dien-1-yl) -3,3-dimethyl-3H-indol-1-ium-
5-sulfonate (abbreviation BCN-Cy5).
Chemical reagent needed for the above synthetic reaction unless otherwise instructed, is purchased from Beijing Chemical Plant or AlfaAesar company,
It is the pure above rank of analysis.
The recombinant expression of embodiment 3:RNA modification enzyme Tias
The present inventor has purchased archeobacteria Archaeoglobus fulgidus (purchased from ATCC company, registration number 49558),
Clonal expression and active RNA modification enzyme Tias (nucleotide sequence such as SEQ ID NO:1 institute is purified into from its genome
Show, amino acid sequence is as shown in SEQ ID NO:2).
Concrete operation method are as follows: by pET22b Af Tias plasmid convert e. coli bl21 (DE3), picking monoclonal in
In the LB liquid medium of the antibiotic of benzyl containing ammonia, 37 DEG C are shaken bacterium and stayed overnight；Bacterium solution is transferred according to 1: 100 volume ratio in containing ammonia again
In the LB liquid medium of benzyl antibiotic, 37 DEG C are shaken bacterium to OD600=0.6,1mM IPTG are added, 37 DEG C induce 4 hours, centrifugation
Bacterium is received, after abandoning culture medium, is resuspended, is surpassed with the solution of Tris containing 50mM, 500mM NaCl, 5% glycerol, 10mM imidazoles, pH8.0
Sonication, then purified with His label purification column (being purchased from Nanjing Jin Sirui company), albumen after purification continues to use
Sephadex G200 (being purchased from GE company) is further purified, and obtained albumen is active RNA modification enzyme Tias, will
It is stored in 100mMTris-HCl, pH 8.0, in 10mM KCl solution.
Embodiment 4:Af tRNAIle2Preparation
Af tRNAIle2The preparation of (SEQ ID NO:3) the T7 Ribomax Express Large of Promega company
Scale RNA production System (P1320) kit is transcribed in vitro, and reaction system is 20 μ L, includes 10 μ L
RiboMAXTM Express T7 2X Buffer、3μL T7Af tRNAIle2Transcription templates, 2 μ L T7 Express Enzyme
Mix, 3 μ L RNase inhibitor set 37 DEG C of water-baths and react 30 minutes, then plus 1 μ L RQ1 RNase-Free DNase enter
Reaction system is set 37 DEG C of water-baths and is reacted 15 minutes.
Embodiment 5:RNA is modified and is verified in vitro
In reaction buffer (100mM Tris-HCl, pH 8.0,10mM KCl, 5mM MgCl2) 8 μ g embodiments of middle addition
The Tias (SEQ ID NO:2) of 3 purifying, the Af tRNA that 20pmol is transcribed in vitroIle2(SEQ ID NO:3), 1mMATP, 5mM
DTT is finally separately added into 1mM compound 1,2,3 or 4.After 37 DEG C are incubated for 2 hours, pass through acetic acid-Urea Polyacrylamide gel
(acid-urea PAGE gel) electrophoresis modifies RNA and non-modified RNA to separate.As shown in Figure 8 A, work as tRNAIle2It is modified
Afterwards, the movement speed in gel can be slack-off.The present inventor sees very happily, the analog of three kinds of compounds 1: compound 2,
3 and 4 can covalent modification tRNAIle2, cause its movement speed in acetic acid-Urea Polyacrylamide gel slack-off.But it compares
Under, the tRNA of the modification of compound 3Ile2The tRNA modified than compound 2Ile2Shown higher mobility, this may be by
In the relatively low reason of the molecular weight of compound 3.And tRNA after the modification of compound 4Ile2Two master tapes are presented, are shown
tRNAIle2It is only partially modified, this may is that because biggish carbamate and alkynes functional group affect Tias to RNA's
Recognition efficiency and result in incomplete modification.Therefore, the present inventor carries out subsequent grind for compound 2 and compound 3 is selected
Study carefully.
Next, the present inventor constructs tRNAIle2C34U (SEQ ID NO:4) mutant, to verify Tias catalysis
Whether be Af tRNAIle2The special sex modification of 34th cytimidine.As a result as shown in Figure 8 B, Tias being capable of successful catalysis
It closes object 1 and 2 and is covalently bound to tRNAIle2On, but it is unable to catalytic cpd 1/2 and tRNAIle2The combination of C34U mutant.This
As a result natural substrate compound 1 can be not only catalyzed by sufficiently demonstrating Tias, and it is special can also to be catalyzed non-natural substrate analogue
The opposite sex is integrated to Af tRNAIle2The 34th cytimidine on.
Af tRNA can actually be accurately combined in order to further verify compound 2 and 3Ile2C34, the present inventor use
RNase T1 is to Af tRNAIle2It is digested, the nucleic acid fragment of anticodon loop is then contained by mass spectral analysis.As a result such as
Shown in Fig. 8 C, non-modified tRNAIle2Fragments molecules amount is 3184.08Da, is connect very much with theoretical calculation molecular weight 3184.4Da
Closely.The tRNA that compound 1 and compound 3 are modifiedIle2Fragments molecules amount is respectively 3295.29 and 3220.26Da (Fig. 8 D and F),
Also all very close theoretical calculation molecular weight 3295.5 and 3220.3Da.But the tRNA that compound 2 is modifiedIle2Fragments molecules amount is
3311.15Da (Fig. 8 E), 26.3Da, the present inventor's analysis are differed with theoretical calculation molecular weight 3337.5Da, and reason may be
Because during mass spectrographic laser irradiation, nucleic acid fragment is lost two nitrogen-atoms, increase two hydrogen atoms, such case with
Also once occurred in preceding Mass Spectrometer Method.In short, our result is still able to illustrate that Tias can be catalyzed containing Azide
Object/alkynes functional group AGM (compound 1) analog --- compound 2 and 3 specifically binds Af tRNAIle234 born of the same parents it is phonetic
Pyridine.
Embodiment 6:Tias crystallographic structural analysis
In order to unlock the molecular basis of Tias (SEQ ID NO:2) identification AGN (compound 2), the present inventor is parsed
The crystal structure (Fig. 9) of Tias combination AGN.The side chain of the backbone carbonyl of Asp193 and Asn194 respectively with the azido shape of AGN
At two hydrogen bonds, the guanidine radicals of Arg217 also forms a hydrogen bond with the carbonyl of AGN, meanwhile, the side chain of Val203 can lead to
Cross the carbochain that hydrophobic effect stablizes agmatine.According to the crystal structure, the present inventor is it is inferred that similar hydrophobic effect also can
Enough promote identification of the Tias to propargylamine (2-Propynylamine, compound 3).
Embodiment 7:RNA In-vitro specificity label and imaging
Can the present inventor then tests Tias realize RNA fluorescent marker by bio-orthogonal chemical method? this
100 μM of BCN-FITC and 5pmol warp is added toward mixing in buffer (20mM Tris, pH 7.4,150mM NaCl) in inventor
The tRNA that compound 2 is modifiedIle2, while the tRNA not being modified is addedIle2It as control, is incubated at room temperature 30 minutes, warp
After 6.5% polyacrylamide gel (Tris containing 40mM, pH8.0,1mM EDTA, 8M urea) electrophoretic analysis, fluorescence imaging is carried out
(488nm excitation, 520nm transmitting).As can be seen from fig. 10A the tRNA modified through compound 2Ile2It can be carried out with BCN-FITC anti-
It answers, generation fluorescence, and unmodified tRNAIle2Any react is not generated with BCN-FITC.The result demonstrates BCN-
FITC can specifically with tRNA after modificationIle2Azido group reaction.
The present inventor tests under conditions of 1mM cuprous ion makees catalyst, and with same test method through chemical combination
The tRNA that object 3 is modifiedIle2With the fluorescence imaging reaction (633nm excitation, 670nm transmitting) of Sulfo-Cy5-azide, also obtain
Identical reaction result (Figure 10 B), i.e. Sulfo-Cy5-azide can only be with the tRNA after modificationIle2It reacts and generates fluorescence.
Although present inventors have demonstrated that Tias can be catalyzed azide or alkynyl functionality locus specificity combines
tRNAIle2, but the present inventor also wonders, can this method be used to mark other targets RNA? in order to achieve this goal, originally
Inventor has synthesized the tRNA for containing 3 ' and 5 ' elongated ends by being transcribed in vitroIle2(referred to as tRNAIle2- 3-5, structure are shown in figure
11, nucleotide sequence is as shown in SEQ ID NO:5).The present inventor is by 5pmol tRNAIle2- 3-5,8 μ g Tias, 5mMDTT,
1mM ATP and 1mM AGN mixing, while the mixed liquor to be added without AGN, as control, 37 DEG C are incubated for 2 hours.Then it removed
The AGN of amount adds 100 μM of BCN-FITC, is incubated at room temperature 30 minutes.Reaction solution is carried out by polyacrylamide gel electrophoresis
Analysis.As shown in figure 12, AGN is only added, BCN-FITC could selectively mark tRNAIle2- 3-5, this also illustrates simultaneously
In tRNAIle2After both ends add 3 ' and 5 ' elongated ends respectively, Tias can still be catalyzed the specific bond of itself and AGN.
Embodiment 8: intracellular target RNA specific marker and imaging
Since above method is RNA labeled in vitro, then the present inventor's continual exploitation is special in mammalian cell
The method of labeled RNA.We select 293T cell (purchased from ATCC company), will be simultaneous with tRNAIle2- 5S fusion and
The pCMV Myc 5S Tias plasmid transfection 293T cell of Tias gene, Tias (SEQ ID can be co-expressed by obtaining one plant
) and tRNA NO:1Ile2The cell strain of -5S fusion rna (SEQ ID NO:6), while the pCMV that will only have Tias gene
MycTias plasmid transfection 293T cell, Tias can only be expressed and the cell strain for the fusion rna that is beyond expression by obtaining another strain.We
By both cell culture in the DMEM culture medium containing 1mM compound 2, then cracked by TRIZOL detergent thin
Born of the same parents extract cell total rna.After taking 50pmol cell total rna to mix incubation with 100 μM of BCN-Cy5 30 minutes, pass through agarose
Gel electrophoresis combination fluorescence imaging is analyzed.As a result as shown in figure 13, when there is no tRNA in cellIle2The expression of -5S fusion rna
When, BCN-Cy5 can not mark any RNA in 293T cell transcription group, and normal expression tRNA in cellIle2- 5S fusion rna
When, BCN-Cy5 can be with pass flag subject fusion RNA.
In order to further verify the effect that new method of the invention carries out RNA imaging in mammalian cells, the present invention
People uses U2OS cell (purchased from ATCC company, registration number: HTB-96) instead and compound 3 repeats above-mentioned experiment.Two plants of U2OS cells
In, one kind can express Tias and tRNA simultaneouslyIle2- 5S fusion rna (SEQ ID NO:6), another kind can only express Tias and
Be beyond expression fusion rna, is cultivated with the DMEM culture medium containing 1mM compound 3, then by cell and Sulfo-Cy5-
Azide is incubated for, while cuprous ion is added as catalyst.Cell is observed using laser confocal microscope.This hair
Bright people has obtained identical experimental result: only in the cell of normal expression fusion rna, RNA can by pass flag and at
As (Figure 14).
It is to cause us to extract because the transfection efficiency of 293T cell is high that we are marked with 293T cell extraction RNA
Total serum IgE in tRNAIle2The ratio of -5S fusion rna is high, this is convenient for our subsequent fluorochrome labels, because of our inspection
Survey method is imaged after agarose electrophoresis with Fluorescence Scanner, its sensitivity is relatively low, so needing opposite high abundance
Albumen.This method is observed that the relative molecular weight position of labeled RNA simultaneously, verifies tRNA convenient for usIle2- 5S melts
Close the label of RNA.
And it is because relative to 293T cell that we, which carry out cell imaging with U2OS cell, the adherent comparison of U2OS cell is firm
Gu extensibility is larger, convenient for being used to carry out laser confocal imaging.But the transfection efficiency of U2OS cell is relatively low, but it is right
For laser confocal imaging, it is only necessary to which a small number of positive cells can observe, and sensitivity is higher.
Above-mentioned two operational difference tested into the cell is the difference of research purpose: observing quilt in 293T cell
The size of labeled RNA, observes the distribution of the position in the cell labeled RNA in U2OS cell, and experiment purpose is different.
It should be understood that although carrying out particularly shown and description to the present invention with reference to its illustrative embodiment,
It should be understood by those skilled in the art that without departing substantially from spirit of the invention as defined in appended claims
Under conditions of range, any of various embodiments can be carried out in the variation for wherein carrying out various forms and details
Combination.
Claims (6)
1. one kind is capable of the kit of locus specificity covalent labeling target RNA in the cell, the kit includes:
(1)tRNAIle2- agmatidine synzyme, amino acid sequence are SEQ ID NO:2,
(2)tRNAIle2Or its derivative nucleotide sequence, wherein the tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3,
(3) substrate, the substrate is agmatine or agmatine analog, wherein the agmatine analog is selected from N- (4- amino
Butyl) -2- nitrine yl acetamide, propargylamine or butyl- 3- alkynes -1- base (4- aminobutyl) t-butyl carbamate,
(4) reaction buffer；
(5) ATP liquid storage；
(6) DTT liquid storage；
(7) fluorescent dye is selected from BCN-FITC, BCN-Cy5 or Sulfo-Cy5-azide,
The wherein tRNAIle2Derivative nucleotides sequence is classified as tRNAIle2- 3-5, nucleotides sequence are classified as SEQ ID NO:5,
Structure is shown in formula I:
2. kit according to claim 1, wherein the reaction buffer is 100mM Tris-HCl, pH8.0,10mM
KCl, 5mM MgCl2。
3. kit according to claim 1, the kit further includes making tRNAIle2Or its derivative nucleotide sequence
The reagent merged with target RNA.
4. a kind of method of locus specificity covalent labeling target RNA in the cell, the method includes the following steps:
(1) by tRNAIle2Or its derivative nucleotide sequence and the target RNA are coupled to form fusion rna, wherein described
tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3, the tRNAIle2The derivative entitled tRNA of nucleotide sequenceIle2- 3-5,
Its nucleotides sequence is classified as SEQ ID NO:5, and structure is as shown in Formulas I in claim 1；
(2) tRNA that amino acid sequence is SEQ ID NO:2 is co-expressed in the cellIle2- agmatidine synzyme and
The fusion rna that step (1) obtains, containing the compound selected from agmatine or agmatine analog in cell culture medium, wherein institute
It states agmatine analog and is selected from N- (4- aminobutyl) -2- nitrine yl acetamide, propargylamine or butyl- 3- alkynes -1- base (4- amino fourth
Base) t-butyl carbamate；
(3) lytic cell extracts cell total rna, and fluorescent dye mixing is added and is incubated for, passes through agarose gel electrophoresis combination fluorescence
Imaging is analyzed, wherein the fluorescent dye is selected from BCN-FITC, BCN-Cy5 or Sulfo-Cy5-azide.
5. a kind of method of locus specificity covalent labeling target RNA in the cell, the method includes the following steps:
(1) by tRNAIle2Or its derivative nucleotide sequence and the target RNA are coupled to form fusion rna, wherein described
tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3, the tRNAIle2The derivative entitled tRNA of nucleotide sequenceIle2- 3-5,
Its nucleotides sequence is classified as SEQ ID NO:5, and structure is as shown in Formulas I in claim 1；
(2) tRNA that amino acid sequence is SEQ ID NO:2 is co-expressed in the cellIle2- agmatidine synzyme and
The fusion rna that step (1) obtains, containing the compound selected from agmatine or agmatine analog in cell culture medium, wherein institute
It states agmatine analog and is selected from N- (4- aminobutyl) -2- nitrine yl acetamide, propargylamine or butyl- 3- alkynes -1- base (4- amino fourth
Base) t-butyl carbamate；
(3) catalyst of fluorescent dye and catalyzed fluorogenic reaction is added into cell culture, is incubated for, is copolymerized by laser
Focusing microscope analysis, wherein the fluorescent dye is selected from BCN-FITC, BCN-Cy5 or Sulfo-Cy5-azide.
6. a kind of method of locus specificity covalent labeling target RNA in vitro, the method includes common in system in vitro
It is incubated for following substance: tRNAIle2- agmatidine synzyme, tRNAIle2Or its derivative nucleotide sequence and target RNA are melted
Synkaryon nucleotide sequence, Small-molecule probe, fluorescent dye and other auxiliary materials, wherein the tRNAIle2- agmatidine synthesis
The nucleotides sequence of enzyme is classified as SEQ ID NO:1, the tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3, the tRNAIle2Spread out
The entitled tRNA of raw nucleotide sequenceIle2- 3-5, nucleotides sequence are classified as SEQ ID NO:5, in structure such as claim 1
Shown in Formulas I, the Small-molecule probe is the compound selected from agmatine or agmatine analog, wherein the agmatine analog
Selected from N- (4- aminobutyl) -2- nitrine yl acetamide, propargylamine or butyl- 3- alkynes -1- base (4- aminobutyl) carbamic acid uncle
Butyl ester.
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