CN105985944A - Novel method of intracellular site-specific covalent RNA labeling - Google Patents
Novel method of intracellular site-specific covalent RNA labeling Download PDFInfo
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Abstract
The invention relates to a novel method of in-vitro or intracellular site-specific covalent RNA labeling. In the method, a tRNAlle2-agmatidine synthesis enzyme (Tias for short, wherein the nucleotide sequence is represented as the SEQ ID No.1, and the amino acid sequence is represented as the SEQ ID No.2), and achaeoglobus fulgidus tRNAlle2 (SEQ ID No. 3) or a derived nucleotide sequence thereof are introduced in in-vitro or intracellular manner to enable the Tias to be capable of specifically recognizing a special sequence of AftRNAlle2 in a transcriptome, so that sequence and site-specific combination between a small molecule, which contains an azide or an alkynyl functional group, and a target RNA containing a tRNAlle2 label, and further specific labeling and imaging on the target RNA are achieved through a chemical reaction between a fluorescent dye and the functional groups. The invention also relates to a kit for covalently labeling the target RNA in an intracellular site-specific manner, wherein the kit includes the tRNAlle2-agmatidine synthesis enzyme and the tRNAlle2-3-5.
Description
Technical field
The invention belongs to biochemical field.Specifically, the present invention provide a kind of in vitro or the new method of intracellular site specific covalent labeled rna.More specifically, this invention is by vitro or intracellular introducing tRNAIle2-agmatidine synzyme (tRNAIle2-agmatidine synthetase, is called for short Tias, and its nucleotide sequence is as shown in SEQ ID NO:1, and aminoacid sequence is as shown in SEQ ID NO:2) and the ancient green-ball bacterium of flicker (Archaeoglobus fulgidus is called for short Af) tRNAIle2(nucleotide sequence is as shown in SEQ ID NO:3), makes Af tRNA in Tias specific recognition transcript profileIle2Particular sequence, promote little molecule containing azide or alkynyl functionality with containing tRNAIle2Produce sequence between target RNA of label and locus specificity combines, and then realized specific marker and the imaging of target RNA by the chemical reaction of fluorescent dye Yu functional group.
Background technology
RNA is a kind of important biomacromolecule in organism, and it plays extremely important role in the biological process such as protein translation, gene expression regulation.Along with going deep into of scientific research, the function of RNA is more and more diversified, and its importance the most can match in excellence or beauty protein.
There is clear and definite Subcellular Localization intracellular in RNA, the different Subcellular Localization of RNA decide it and perform different biological function.Therefore, in exploitation cell, the biological function tool of we the accurate RNA of understanding is of great significance by specific mark and the imaging technique of RNA.
In order to disclose RNA dynamic transport in cell, position and interact, scientific research personnel has been developed over many new methods of non-covalent labeling RNA in mammalian cell, and has made some progress.The intracellular rna labelling developed at present and imaging technique are greatly promoted the functional study of RNA, have deepened our understanding to RNA function.But all there is deficiency in a way in existing intracellular rna labelling and imaging technique, which prevent the widely studied of RNA function.Therefore, developing new intracellular rna labelling and imaging technique will be greatly improved the level of RNA functional study, this also shoulders heavy responsibilities for vast researcher.
Compared to non-covalent approach, bio-orthogonal chemistry depends on the specificity of probe molecule, and covalently bound with target biomacromolecule.Therefore, the method has following several advantage: 1, probe molecule and target biomacromolecule have high affinity, therefore can limit the highly purified target biomacromolecule of elution requirement by strict；2, high-affinity makes low-abundance target biomacromolecule to visualize, and this is even more important for non-coding RNA；3, many probe molecules containing functional group (such as: fluorescence, nuclear magnetic resonance, NMR, infrared spectrum, electron paramagnetic resonance functional group etc.) may be incorporated on target biomacromolecule so that the method becomes the most flexible in application.Due to the function of these uniquenesses, bio-orthogonal chemistry has been successfully applied for the functional study of protein and polysaccharide, is mainly used to observe their expression in mammalian cell, position and interact.
At present, translation/post translational modification mechanism is combined with bio-orthogonal chemistry, has been able to carry out labelled protein with unprecedented precision and multifunctionality, but but do not have suitable method that RNA carries out in mammalian cell site and sequence-specific labelling.Therefore, the present invention intends finding suitable post translational modification component, it is combined with bio-orthogonal chemistry, develops the new method of RNA specific mark.
Summary of the invention
1, technical problem
In order to solve the problems referred to above, it is an object of the invention to provide a kind of in vitro or the new method of intracellular site specific covalent labeled rna.
The present invention provide a kind of in vitro or the new method of intracellular site specific covalent labeled rna.The present invention relates to by vitro or intracellular introducing tRNAIle2-agmatidine synzyme (tRNAIle2-agmatidine synthetase, is called for short Tias, and its nucleotide sequence is as shown in SEQ ID NO:1, and aminoacid sequence is as shown in SEQ ID NO:2) and the ancient green-ball bacterium of flicker (Archaeoglobus fulgidus is called for short Af) tRNAIle2(nucleotide sequence is as shown in SEQ ID NO:3), makes Af tRNA in Tias specific recognition transcript profileIle2Particular sequence, promote little molecule containing azide or alkynyl functionality with containing tRNAIle2Produce sequence between target RNA of label and locus specificity combines, and then realized specific marker and the imaging of target RNA by the chemical reaction of fluorescent dye Yu functional group.
2, technical scheme
In order to realize the specific marker of RNA, we are firstly the need of the unique ingredient found in a kind of post translational modification mechanism, in order to carry out bio-orthogonal chemical reaction.This component needs have two features: the first, it must be a kind of RNA modification enzyme, it is possible to identify the specific site in specific RNA sequence in transcript profile；The second, it allows for transferring to the little molecule containing unique function group (such as azide or alkynes functional group) specific part of RNA, covalent labeling is carried out subsequently by bio-orthogonal chemical reaction, realize locus specificity to combine, finally carry out fluorescence, nuclear magnetic resonance, NMR, electron paramagnetic resonance or infrared spectrum measurement.
In order to find this can in mammalian cell the post translational modification component of precise marking RNA, sight is turned to tRNA modification enzyme by the present inventor.Modify it is known that tRNA modification enzyme can be catalyzed more than 100 kind of tRNA.The loyal translation of genetic code depends on the chemical modification of tRNA, especially the 34th nucleotide of anticodon loop.Many acts on the tRNA modification enzyme in this site and is found to exist only in specific region, it means that they are probably independent evolution.
It should be noted that tRNA in archeobacteriaIle234 cytosine through tRNAIle2-agmatidine synzyme (tRNAIle2-agmatidine synthetase, is called for short Tias) catalysis can be by agmatine (agmatine, is called for short AGM, is referred to as compound 1 in the present invention) modification.According to former report, in the ancient green-ball bacterium of flicker (Archaeoglobus fulgidus is called for short Af), Tias identifies tRNAIle2Need seven nucleotide, respectively: G1, G2, C34, U36, A37, C71 and C72, and mammalian cell both there is no Tias, and the most do not contained the tRNA of these seven kinds of nucleotide sites.Therefore, inventors have contemplated that, if by Tias and the tRNA of archeobacteriaIle2Introduce in mammalian cell, and the Small-molecule probe containing nitrine or alkynyl functionality can be transferred in target RNA by Tias, perhaps can realize sequence and the site-specific labeling of RNA.
nullSuccessfully to realize RNA labelling and imaging，Except tRNA modification enzyme Tias，Also need to the Small-molecule probe containing functional group and corresponding fluorescent dye carries out bio-orthogonal chemical reaction，Therefore，The present inventor has bought/has synthesized three kinds of agmatine analog (structural formula sees Fig. 1): N-(4-aminobutyl)-2-azido acetamide (N-(4-aminobutyl)-2-azidoacetamide) and (has been called for short AGN，The present invention is referred to as compound 2)、Propargylamine (2-Propynylamine，The present invention is referred to as compound 3，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 is referred to as compound 4)；And three kinds of fluorescent dyes: BCN-FITC, Sulfo-Cy5-azide (purchased from Lumiprobe company), BCN-Cy5.Simultaneously, have purchased archeobacteria Archaeoglobus fulgidus (purchased from ATCC company, article No. 49558), clonal expression be purified into RNA modification enzyme Tias (, as shown in SEQ ID NO:1, aminoacid sequence is as shown in SEQ ID NO:2 for nucleotide sequence) from its genome.By design series of experiments, the most jointly hatch Tias, Af tRNAIle2Or the fusion nucleus nucleotide sequence of its derivative nucleotide sequence and target RNA, Small-molecule probe, fluorescent dye and other assist material, or at intracellular co expression Tias and Af tRNAIle2Or the fusion nucleus nucleotide sequence of its derivative nucleotide sequence and target RNA, and and Small-molecule probe, fluorescent dye and other auxiliary materials jointly hatch, be finally successfully realized specific marker and the fluorescence imaging of target RNA.Wherein said Small-molecule probe may be, but not limited to, selected from agmatine or the compound of agmatine analog.
The present invention provides following embodiment:
1. can include at a test kit for intracellular site specific covalent labelling target RNA, described test kit:
(1)tRNAIle2-agmatidine synzyme, its aminoacid sequence is SEQ ID NO:2,
(2)tRNAIle2Or its derivative nucleotide sequence, wherein said tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3,
(3) substrate, described substrate is agmatine or agmatine analog,
(4) reaction buffer；
(5) ATP liquid storage；
(6) DTT liquid storage；
(7) fluorescent dye, it is selected from BCN-FITC, BCN-Cy5 or Sulfo-Cy5-azide.
2. according to the 1st described test kit, wherein said tRNAIle2Derivative nucleotides sequence is classified as tRNAIle2-3-5, its nucleotides sequence is classified as SEQ ID NO:5, and it has the structure shown in Figure 11.
3., according to the 1st described test kit, wherein said agmatine analog is selected from N-(4-aminobutyl)-2-azido acetamide, propargylamine or butyl-3-alkynes-1-base (4-aminobutyl) t-butyl carbamate.
4., according to the 1st described test kit, wherein said reaction buffer is 100mM Tris-HCl, pH 8.0,10mM KCl, 5mM MgCl2。
5., according to the 1st described test kit, described test kit also includes making tRNAIle2Or the reagent that its derivative nucleotide sequence merges with target RNA.
6., in a method for intracellular site specific covalent labelling target RNA, described method comprises the steps:
(1) by tRNAIle2Or its derivative nucleotide sequence forms fusion rna, wherein said tRNA with described target RNA couplingIle2Nucleotides sequence be classified as SEQ ID NO:3；
(2) in described cell, co expression aminoacid sequence is the tRNA of SEQ ID NO:2Ile2The fusion rna that-agmatidine synzyme and step (1) obtain, containing selected from agmatine or the compound of agmatine analog in cell culture medium；
(3) cell lysis, extracts cell total rna, adds fluorescent dye mixing and hatches, is analyzed by agarose gel electrophoresis combined with fluorescent imaging, and wherein said fluorescent dye is selected from BCN-FITC, BCN-Cy5 or Sulfo-Cy5-azide.
6., in a method for intracellular site specific covalent labelling target RNA, described method comprises the steps:
(1) by tRNAIle2Or its derivative nucleotide sequence forms fusion rna, wherein said tRNA with described target RNA couplingIle2Nucleotides sequence be classified as SEQ ID NO:3；
(2) in described cell, co expression aminoacid sequence is the tRNA of SEQ ID NO:2Ile2The fusion rna that-agmatidine synzyme and step (1) obtain, containing selected from agmatine or the compound of agmatine analog in cell culture medium；
(3) adding fluorescent dye and the catalyst of catalyzed fluorogenic reaction in cell culture, hatch, analyzed by laser confocal microscope, wherein said fluorescent dye is selected from BCN-FITC, BCN-Cy5 or Sulfo-Cy5-azide.
8. a method for locus specificity covalent labeling target RNA in vitro, described method is included in vitro system and jointly hatches following material: Tias, tRNAIle2Or the fusion nucleus nucleotide sequence of its derivative nucleotide sequence and target RNA, Small-molecule probe, fluorescent dye and other assist material, wherein said tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3, described Small-molecule probe is selected from agmatine or the compound of agmatine analog.
9. according to the method according to any one of 6-8 item, wherein said tRNAIle2Derivative nucleotides sequence is classified as tRNAIle2-3-5, its nucleotides sequence is classified as SEQ ID NO:5, and it has the structure shown in Figure 11.
10., according to the method described in item according to any one of 6-8 item, wherein said agmatine analog is selected from N-(4-aminobutyl)-2-azido acetamide, propargylamine or butyl-3-alkynes-1-base (4-aminobutyl) t-butyl carbamate.
3, beneficial effect
At present, many modern RNA biologys and RNA biotechnology all refer to by chemical method site-specific integration modified nucleotide in RNA molecule, but, due to the restriction of integration efficiency, the length of target RNA molecule is typically smaller than 30-50 nucleotide.Our new method depends on the mechanism of transcribing and the sequence-specific of RNA modification enzyme Tias of RNA, therefore can integrate biophysics probe by high degree of specificity in any target RNA in mammalian cell.The method can apply to the RNA folding in external or mammalian cell, RNA-protein interaction, rna transport, RNA modify research and RNA nano material builds.
Compared to former non-covalent RNA formation method, such as Herba Spinaciae is fit, green fluorescence luminophore and Herba Spinaciae fit between noncovalent interaction be only capable of having affinity in micro-molar range；And our new method depends on Tias and Af tRNAIle2Selective recognition (both of which is not present in mammalian cell), again by introducing azido or alkynyl group, make to be formed between fluorophor and target RNA covalent bond, to realize the fluorescence imaging of low abundance RNA, therefore covalent labeling new method has higher specificity, affinity and versatility.The more important thing is, our new method is by introducing the little molecule containing difference in functionality group, after click chemistry reaction (click chemistry reaction), multiple method can be utilized, such as: fluorescence, infrared spectrum, nuclear magnetic resonance, NMR or electron paramagnetic resonance etc., carry out multi-functional locus specificity RNA labelling, without changing Tias albumen or Af tRNAIle2Sequence.
Accompanying drawing explanation
From detailed description below in conjunction with the accompanying drawings, features described above and the advantage of the present invention will be apparent from, wherein:
Fig. 1: the picture left above is the chemical structural formula of compound 1 (AGM), and top right plot is the chemical structural formula of compound 2 (AGN), and lower-left figure is the chemical structural formula 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 (owing to Figure of description page 2 could not show the synthetic route of complete BCN-FITC, therefore, continue display in Fig. 4 (Continued)) of BCN-FITC；
Fig. 5 is the synthetic route of BCN-Cy5；
Fig. 6 is enzyme used in the present invention and the nucleotide/aminoacid sequence of RNA；
Fig. 7 is the reaction schematic diagram that Tias modifies RNA；
Fig. 8: A figure is the polyacrylamide gel electrophoresis figure of the external modification of RNA, and B figure is the polyacrylamide gel electrophoresis figure of RNA decorating site checking, and C figure is non-modified tRNAIle2Nucleic acid fragment mass spectrum, D figure is the tRNA modified through compound 1Ile2Nucleic acid fragment mass spectrum, E figure is the tRNA modified through compound 2Ile2Nucleic acid fragment mass spectrum, F figure is the tRNA modified through compound 3Ile2Nucleic acid fragment mass spectrum；
Fig. 9 is the crystal structure figure that Tias combines 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 labelling and fluorescence imaging figure, B figure is the tRNA modified through compound 3Ile2In-vitro specificity labelling and fluorescence imaging figure；
Figure 11 is tRNAIle2-3-5 structure chart；
Figure 12 is tRNAIle2-3-5 In-vitro specificity labelling 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 at the intracellular specific marker of U2OS and fluorescence imaging figure, and wherein lastrow is not express tRNAIle2The micro-imaging figure of the U2OS cell of-5S fusion rna, next line is to express tRNAIle2The micro-imaging figure (left column is fluorescence imaging figure, and middle row are by DAPI passage microexamination picture, and right row are by DIC passage microexamination picture) of the U2OS cell of-5S fusion rna.
Detailed description of the invention
It is further elucidated with the present invention by the following examples.However, it should be understood that the purpose that described embodiment is merely illustrative, it is 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 used in following embodiment is the reagent of the analytical pure rank can buied by commercial sources.
Embodiment 1: the chemosynthesis of agmatine (AGM, compound 1) analog
1, the synthesis (Fig. 2) of compound 2 (N-(4-aminobutyl)-2-azidoacetamide, AGN):
Take 656mg BOC-1,4-butanediamine hydrochloride and 1.48g sodium carbonate is dissolved in 80mL, the ethyl acetate of 1: 1 (v/v) and aqueous mixtures jointly, 0 DEG C of ice bath.Dissolve the ethyl acetate solution of 702mg 2-acetyl bromide bromide toward interpolation 10ml in reactant liquor after 1 hour, be stirred at room temperature 2 hours.Take upper organic phase, Rotary Evaporators is utilized to be dried, then by silica gel chromatography at the upper purification of silica gel (being the ethyl acetate of 1: 1 containing volume ratio: petroleum ether), 320mg white intermediate product 1 is obtained: the tert-butyl group (4-(2-acetyl bromide amido) butyl) carbamate (Tert-butyl (4-(2-bromoacetamido) butyl) carbamate).
Take 230mg intermediate product 1 to be dissolved in 5mL acetone, 0 DEG C of ice bath.5mL Hydrazoic acid,sodium salt (250mg) aqueous solution, stirred overnight at room temperature (22 hours) is added after 30 minutes.Then reactant liquor is concentrated, and by silica gel chromatography at the upper purification of silica gel (being the ethyl acetate of 1: 1 containing volume ratio: petroleum ether), obtain 50mg intermediate product 2: the tert-butyl group (4-(2-nitrine acetamido) butyl) carbamate (Tert-butyl (4-(2-azidoacetamido) butyl) carbamate).
Take 44mg intermediate product 2 to be resuspended in 20ml hydrochloric acid/ethyl acetate (volume ratio is 1: 1) mixed liquor, stirred overnight at room temperature (12 hours), then rotary evaporation lyophilization, obtains 29mg end-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):
Take 140mg 3-butyne-1-ol and 424mg sodium carbonate is dissolved in 10mL tetrahydrofuran solution jointly, 0 DEG C of ice bath.Then in reactant liquor, add 198mg triphosgene, and stirring 12 hours at room temperature, obtain intermediate product.Take 380mg intermediate product to be dissolved in 50mL ethyl acetate/water (volume ratio ratio is 1: 1) solution, after being stirred at room temperature 12 hours, separate organic facies, be passed through HCL gas.Finally collect white fixing, obtain end-product.
Needed for above synthetic reaction, chemical reagent is if no special instructions, is purchased from Beijing Chemical Plant or AlfaAesar company, is the above rank of analytical pure.
Embodiment 2: the chemosynthesis of fluorescent dye
1, the synthesis (Fig. 4) of BCN-FITC:
Taking 15.0mL 1,5-cyclo-octadiene and the mixing of 281mg dimerization rhodium acetate to add in 10mL dichloromethane solution, be then added dropwise over 10mL in 3 hours and dissolve the dichloromethane solution of 15mmol ethyl diazoacetate, temperature is maintained at 0 DEG C simultaneously.After reacting 15 hours under room temperature condition, reaction system is replaced in ice-water bath and maintains the temperature at 0 DEG C, again added 10mL in 3 hours and dissolves the dichloromethane solution of 15mmol ethyl diazoacetate, continues reaction 21 hours at ambient temperature.Dichloromethane is removed subsequently by rotary evaporation, silicagel column separates (petroleum ether: ethyl acetate=20: 1, v/v) 2.52g intermediate product 1:(1R is obtained, 8S, 9r, Z)-ethyl bicycle [6.1.0] non-4-ene-9-carboxylate (endo product) and 2.08g intermediate product 2:(1R, 8S, 9s, Z)-ethyl bicycle [6.1.0] non-4-ene-9-carboxylate (exo product).
Take 370mg lithium aluminium hydride reduction and be dissolved in 30mL ether, be subsequently placed in 0 DEG C of ice-water bath, and toward suspension is added dropwise over 30mL dissolving the diethyl ether solution of 2.23g intermediate product 1.It is cooled to 0 DEG C after reacting 2 hours under room temperature condition, is added dropwise over deionized water subsequently until gray solid becomes white.Adding 8g sodium sulfate, solid is then filtered to remove, and fully washs with 100mL ether, the filtrate obtained removes ether by rotary evaporation.In the case of not being further purified, the 1.55g alcohol compound obtained is dissolved in 30mL dichloromethane and is placed in ice-water bath, be then added dropwise over 7.3mL and dissolve the dichloromethane solution of 649 μ L bromines, until solution keeps yellow constant.Reaction system 30mL, the hypo solution cancellation of 10%, and extract with 2 × 50mL dichloromethane.The organic facies anhydrous sodium sulfate obtained is dried, then removes dichloromethane by concentrating under reduced pressure, obtains 2.80g bis-bromo mixture.In the case of not being further purified, the 2.80g bis-bromo mixture obtained all is dissolved in 30mL oxolane and is placed in ice-water bath, in 10 minutes, be then added dropwise over 29.6mL catalyst KOtBu.Reaction system is cooled to room temperature 66 DEG C of backflows after 2 hours, with 100mL saturated ammonium chloride solution cancellation, then removes oxolane by rotary evaporation.The solution obtained 3 × 100mL dichloromethane extracts, after isolating organic layer, it is dried with anhydrous sodium sulfate, dichloromethane is removed subsequently by rotary evaporation, silicagel column separates (petroleum ether: ethyl acetate=4: 1, v/v) 724mg intermediate product 3 white solid is obtained: (1R, 8S, 9r)-Bicyclo [6.1.0] non-4-yn-9-ylmethanol.
Taking 222mg intermediate product 3 to be dissolved in 20mL dichloromethane solution, add 299 μ L pyridines and 368mg p-nitrophenyl chloro-formate, reaction system uses 35mL saturated ammonium chloride solution cancellation after being stirred at room temperature 40 minutes.Reactant liquor 2 × 50mL dichloromethane extracts, separate organic layer, then dichloromethane is removed by Rotary Evaporators, silicagel column separates (petroleum ether: ethyl acetate=5: 1, v/v) 459mg intermediate product 4 white solid is obtained: (1R, 8S, 9r)-bicyclo [6.1.0] non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate.
80 μ L tetra-methylenedimines are added dropwise in the methanol solution that 4mL dissolves 102mg Fluorescein isothiocyanate (being called for short FITC, purchased from sigma company), are stirred at room temperature 2 hours.After solution having red precipitate generate, filter precipitation and be dried, obtaining 100mg intermediate product 5:5-(3-(4-aminobutyl) thioureido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl) benzoicacid.
Take 83mg intermediate product 4 and 100mg intermediate product 5 to be jointly dissolved in 4mL DMF solution, add 111 μ L triethylamines, the most at ambient temperature stirring 3 hours.Silicagel column separates (dichloromethane: methanol=10: 1, v/v) 106mg end-product: 5-(3-(4-((((1R is obtained, 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 (being called for short BCN-FITC).
2, the synthesis (Fig. 5) of BCN-Cy5:
Taking 15g 4-pHydrazinebenzenesulfonic acid semihydrate and 25.2mL 3-methyl-2-butanone is dissolved in 45mL glacial acetic acid, then 110 DEG C are refluxed 3 hours.Remove acetic acid by Rotary Evaporators, the residue obtained is dissolved in methanol, and stirs in the isopropanol saturated solution containing potassium hydroxide, until solution becomes yellow, finally give 13.2g yellow intermediate product 1:Potassium2,3,3-trimethyl-3H-indole-5-sulfonate.
Take 1.39g intermediate product 1 and 1.42g 3-propantheline bromide hydrobromide and be jointly suspended in 10mL acetonitrile solution, then in tube sealing, keep 80 DEG C and reflux 24 hours.Reaction system is resuspended in ether after being concentrated by rotary evaporation.Filtering mixt obtains solid, and is redissolved in methanol, is again spin-dried for, and is subsequently adding 2.35g Bis(tert-butoxycarbonyl)oxide, the anhydrous chloroform of 30mL and 3.75mLN, N-diisopropylethylamine.After mixture is slowly warmed up reflux temperature 55 DEG C, continue stirring 4 hours.Separate (dichloromethane: methanol=4: 1) with silicagel column through concentration and obtain intermediate product 2:1-(3-((tert-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 react at 80 DEG C and within 24 hours, obtain crude product 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate.Take 535mg crude product and 570mg hydrochloric acid-N-(3-phenylamino-2-propylene subunit) aniline is dissolved in 10mL glacial acetic acid and 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.
Take 2.00mmol intermediate product 2,2.00mmol intermediate product 3 and 2.29g anhydrous sodium acetate is dissolved in 30mL ethanol, then refluxes 18 hours at 80 DEG C.(dichloromethane: methanol=4: 1 is separated with silicagel column through concentrating, v/v) 214mg product sodium1-(3-((tert-butoxycarbonyl) amino) propyl)-2-((1E is obtained, 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.Subsequently product is dissolved in 15mL methanol and 10mL, 37% hydrochloric acid, after 60 DEG C are reacted 6 hours, obtain Sodium 1-(3-aminopropyl)-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 (is called for short Cy5-NH2 HCl).
nullTake 99mg Cy5-NH2 HCl and 51mg (1R，8S，9r)-bicyclo [6.1.0] non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate is dissolved in 8mL N，Dinethylformamide，It is subsequently adding 86 μ L triethylamine solutions，Stir 6 hours under room temperature，Obtain end-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 (is called for short BCN-Cy5).
Needed for above synthetic reaction, chemical reagent is if no special instructions, is purchased from Beijing Chemical Plant or AlfaAesar company, is the above rank of analytical pure.
Embodiment 3:RNA modification enzyme Tias's is recombinant expressed
The present inventor have purchased archeobacteria Archaeoglobus fulgidus (purchased from ATCC company, registration number 49558), clonal expression be purified into activated RNA modification enzyme Tias (, as shown in SEQ ID NO:1, aminoacid sequence is as shown in SEQ ID NO:2 for nucleotide sequence) from its genome.
Concrete operation method is: by pET22b Af Tias plasmid transformation escherichia coli BL21 (DE3), and picking monoclonal is in the LB fluid medium containing ammonia benzyl antibiotic, and 37 DEG C are shaken bacterium overnight；Again bacterium solution is transferred in the LB fluid medium containing ammonia benzyl antibiotic according to 1: 100 volume ratio, 37 DEG C are shaken bacterium to OD600=0.6, add 1mM IPTG, induce 4 hours for 37 DEG C, centrifugal receipts bacterium, after abandoning culture medium, with containing 50mM Tris, 500mM NaCl, 5% glycerol, 10mM imidazoles, the solution of pH8.0 is resuspended, ultrasonic disruption, it is purified with His label purification column (purchased from Nanjing Jin Sirui company) again, albumen continuation sephadex G200 (purchased from GE company) after purification is further purified, the albumen obtained is active RNA modification enzyme Tias, save it in 100mMTris-HCl, pH 8.0, in 10mM KCl solution.
Embodiment 4:Af tRNAIle2Preparation
Af tRNAIle2T7 Ribomax Express Large Scale RNA production System (P1320) test kit of the preparation Promega company of (SEQ ID NO:3) carries out in vitro transcription, 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, put 37 DEG C of water-baths and react 30 minutes, then add 1 μ L RQ1 RNase-Free DNase and enter reaction system, puts 37 DEG C of water-baths and reacts 15 minutes.
The external modification of embodiment 5:RNA and checking
At reaction buffer (100mM Tris-HCl, pH 8.0,10mM KCl, 5mM MgCl2The Tias (SEQ ID NO:2), the Af tRNA of 20pmol in vitro transcription of 8 μ g embodiment 3 purification is added in)Ile2(SEQ ID NO:3), 1mMATP, 5mM DTT, finally it is separately added into 1mM compound 1,2,3 or 4.After 37 DEG C hatch 2 hours, separate modification RNA and non-modified RNA by acetic acid-Urea Polyacrylamide gel (acid-urea PAGE gel) electrophoresis.As shown in Figure 8 A, tRNA is worked asIle2After being modified, the translational 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 all can covalent modification tRNAIle2, cause its translational speed in acetic acid-Urea Polyacrylamide gel slack-off.But the tRNA that by contrast, compound 3 is modifiedIle2The tRNA modified than compound 2Ile2Having shown higher mobility, this is likely due to the reason that the molecular weight of compound 3 is relatively low.And tRNA after compound 4 modificationIle2Present two master tapes, show tRNAIle2Only partly being modified, this may is that because bigger carbamate and alkynes functional group have impact on Tias and the recognition efficiency of RNA result in not exclusively modification.Therefore, the present inventor will select compound 2 and compound 3 to carry out follow-up research.
It follows that the present inventor constructs tRNAIle2Whether C34U (SEQ ID NO:4) mutant, be Af tRNA in order to verify that Tias is catalyzedIle2The specificity of the 34th cytosine is modified.As shown in Figure 8 B, Tias can be covalently bound to tRNA by success catalytic cpd 1 and 2 to resultIle2On, but can not catalytic cpd 1/2 and tRNAIle2The combination of C34U mutant.This result fully demonstrates Tias and is possible not only to be catalyzed natural substrate compound 1, it is also possible to is catalyzed non-natural substrate analogue and is specifically bound to Af tRNAIle2The 34th cytosine on.
In order to verify that compound 2 and 3 can actually accurately combine Af tRNA furtherIle2C34, the present inventor uses RNase T1 to Af tRNAIle2Digest, then contained the nucleic acid fragment of anticodon loop by mass spectral analysis.Result as shown in Figure 8 C, non-modified tRNAIle2Fragments molecules amount is 3184.08Da, 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), the most closely 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 is differed with Theoretical Calculation molecular weight 3337.5Da, the present inventor analyzes, during its reason is possibly due to the irradiation of mass spectrographic laser, nucleic acid fragment lost two nitrogen-atoms, add two hydrogen atoms, the Mass Spectrometer Method that this situation is former the most once occurred.In a word, our result is still able to illustrate that Tias can be catalyzed the specific binding Af tRNA of AGM (compound 1) analog compounds 2 and 3 containing azide/alkynes functional groupIle234 cytosine.
Embodiment 6:Tias crystallographic structural analysis
Identify the molecular basis of AGN (compound 2) to untie Tias (SEQ ID NO:2), the present inventor has resolved Tias and has combined the crystal structure (Fig. 9) of AGN.The backbone carbonyl of Asp193 and the side chain of Asn194 azido with AGN respectively defines two hydrogen bonds, and the guanidine radicals of Arg217 also carbonyl with AGN defines a hydrogen bond, and meanwhile, the side chain of Val203 can stablize the carbochain of agmatine by hydrophobic interaction.According to this crystal structure, the present inventor is it is inferred that similar hydrophobic interaction also is able to promote the Tias identification to propargylamine (2-Propynylamine, compound 3).
Embodiment 7:RNA In-vitro specificity labelling and imaging
The present inventor then tests whether Tias can realize RNA fluorescent labeling by bio-orthogonal chemical method？The present inventor adds, toward mixing in buffer (20mM Tris, pH 7.4,150mM NaCl), the tRNA that 100 μMs of BCN-FITC and 5pmol modify through compound 2Ile2, simultaneously to add the most adorned tRNAIle2As comparison, incubated at room 30 minutes, after 6.5% polyacrylamide gel (containing 40mM Tris, pH8.0,1mM EDTA, 8M carbamide) electrophoretic analysis, carry out fluorescence imaging (488nm excites, and 520nm launches).From Figure 10 A, through the tRNA that compound 2 is modifiedIle2Can react with BCN-FITC, generation fluorescence, and the most modified tRNAIle2Any reaction is not produced with BCN-FITC.This result demonstrate BCN-FITC can specifically with modify after tRNAIle2Azido group reaction.
The present inventor under conditions of 1mM cuprous ion makees catalyst, the tRNA modified through compound 3 with same measurement of test method againIle2React (633nm excites, and 670nm launches) with the fluorescence imaging of Sulfo-Cy5-azide, have also been obtained identical reaction result (Figure 10 B), i.e. Sulfo-Cy5-azide can only be with the tRNA after modifyingIle2React and produce fluorescence.
Although present inventors have demonstrated that Tias can be catalyzed azide or alkynyl functionality's locus specificity combines tRNAIle2, but the present inventor also wonders, can the method be used for other targets RNA of labelling？In order to realize this target, the present inventor passes through in vitro transcription, has synthesized the tRNA containing 3 ' and 5 ' elongated endsIle2(referred to as tRNAIle2-3-5, structure is shown in Figure 11, and 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 mix, and simultaneously to be added without the mixed liquor of AGN as comparison, hatches 2 hours for 37 DEG C.Then remove the AGN of excess, add 100 μMs of BCN-FITC, incubated at room 30 minutes.Reactant liquor is analyzed by polyacrylamide gel electrophoresis.As shown in figure 12, only adding AGN, BCN-FITC could optionally labelling tRNAIle2-3-5, this illustrates at tRNA the most simultaneouslyIle2Two ends are respectively plus after 3 ' and 5 ' elongated ends, and Tias still can be catalyzed the specific bond of itself and AGN.
Embodiment 8: intracellular target RNA specific marker and imaging
Owing to above method is RNA labeled in vitro, then the present inventor's continual exploitation method of specific mark RNA in mammalian cell.We select 293T cell (purchased from ATCC company), will be simultaneous with tRNAIle2The pCMV Myc 5S Tias plasmid transfection 293T cell of-5S fusion gene and Tias gene, obtaining a strain can be with co expression Tias (SEQ ID NO:1) and tRNAIle2The cell strain of-5S fusion rna (SEQ ID NO:6), simultaneously by only with the pCMV MycTias plasmid transfection 293T cell of Tias gene, obtaining another strain can only express Tias and the cell strain of the fusion rna that is beyond expression.Both cells are incubated in the DMEM culture medium containing 1mM compound 2 by we, carry out cell lysis by TRIZOL detergent subsequently, extract cell total rna.Take 50pmol cell total rna and after 100 μMs of BCN-Cy5 mixing hatches 30 minutes, be analyzed by agarose gel electrophoresis combined with fluorescent imaging.Result as shown in figure 13, when cell does not has tRNAIle2When-5S fusion rna is expressed, BCN-Cy5 cannot any RNA in labelling 293T cell transcription group, and normal expression tRNA in cellIle2During-5S fusion rna, BCN-Cy5 can be with pass flag this subject fusion RNA.
In order to verify that the new method of the present invention carries out the effect of RNA imaging in mammalian cell further, the present inventor uses U2OS cell (purchased from ATCC company, registration number: HTB-96) instead and compound 3 repeats above-mentioned experiment.In two strain U2OS cells, one can express Tias and tRNA simultaneouslyIle2-5S fusion rna (SEQ ID NO:6), another kind can only express Tias and the fusion rna that is beyond expression, cultivate by the DMEM culture medium containing 1mM compound 3, then cell is hatched with Sulfo-Cy5-azide, be simultaneously introduced cuprous ion as catalyst.Utilize laser confocal microscope that cell is observed.The present inventor has obtained identical experimental result: only in the cell of normal expression fusion rna, and RNA can be by pass flag imaging (Figure 14).
We are marked by 293T cell extraction RNA, and to be because the transfection efficiency of 293T cell high, causes tRNA in the total serum IgE that we extractIle2The ratio of-5S fusion rna is high, and this is easy to the fluorochrome label that we are follow-up, because our detection method is use Fluorescence Scanner imaging after sepharose electrophoresis, its sensitivity is relatively low, so the most abundant albumen of needs.The most this method is it is observed that be labeled the relative molecular weight position of RNA, it is simple to we verify tRNAIle2The labelling of-5S fusion rna.
And we carry out cell imaging with U2OS cell and are because relative to 293T cell, U2OS cell attachment ratio is stronger, and extensibility is bigger, it is simple to be used for carrying out laser confocal imaging.But the transfection efficiency ratio of U2OS cell is relatively low, but for laser confocal imaging, it is only necessary to the positive cell of minority just can observe, and sensitivity is higher.
The operational difference of the intracellular experiment of above-mentioned two is the difference of research purpose: observe the size of labeled RNA in 293T cell, observes the labeled RNA distribution at intracellular locations in U2OS cell, and experiment purpose is different.
Should be appreciated that, although with reference to the embodiment that it is exemplary, the present invention carried out particularly shown and described, but it will be apparent to an ordinarily skilled person in the art that, under conditions of without departing substantially from by the spirit and scope of the present invention defined in appended claims, the change of various forms and details can be carried out wherein, the combination in any of various embodiment can be carried out.
Claims (10)
1. can be described at a test kit for intracellular site specific covalent labelling target RNA
Test kit includes:
(1)tRNAIle2-agmatidine synzyme, its aminoacid sequence is SEQ ID NO:2,
(2)tRNAIle2Or its derivative nucleotide sequence, wherein said tRNAIle2Nucleotide sequence
For SEQ ID NO:3,
(3) substrate, described substrate is agmatine or agmatine analog,
(4) reaction buffer；
(5) ATP liquid storage；
(6) DTT liquid storage；
(7) fluorescent dye, it is selected from BCN-FITC, BCN-Cy5 or Sulfo-Cy5-azide.
Test kit the most according to claim 1, wherein said tRNAIle2Derivative nucleotides sequence
It is classified as tRNAIle2-3-5, its nucleotides sequence is classified as SEQ ID NO:5, and it has a structure in which
Test kit the most according to claim 1, wherein said agmatine analog is selected from N-(4-
Aminobutyl)-2-azido acetamide, propargylamine or butyl-3-alkynes-1-base (4-aminobutyl) amino first
Tert-butyl acrylate.
Test kit the most according to claim 1, wherein said reaction buffer is 100mM
Tris-HCl, pH 8.0,10mM KCl, 5mM MgCl2。
Test kit the most according to claim 1, described test kit also includes making tRNAIle2Or its
The reagent that derivative nucleotide sequence merges with target RNA.
6. in a method for intracellular site specific covalent labelling target RNA, described method bag
Include following step:
(1) by tRNAIle2Or its derivative nucleotide sequence is formed with described target RNA coupling and merges
RNA, wherein said tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3；
(2) in described cell, co expression aminoacid sequence is SEQ ID NO:2
tRNAIle2The fusion rna that-agmatidine synzyme and step (1) obtain, contains in cell culture medium
Selected from agmatine or the compound of agmatine analog；
(3) cell lysis, extracts cell total rna, adds fluorescent dye mixing and hatches, passes through agar
Sugar gel electrophoresis combined with fluorescent imaging be analyzed, wherein said fluorescent dye selected from BCN-FITC,
BCN-Cy5 or Sulfo-Cy5-azide.
7. in a method for intracellular site specific covalent labelling target RNA, described method bag
Include following step:
(1) by tRNAIle2Or its derivative nucleotide sequence is formed with described target RNA coupling and merges
RNA, wherein said tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3；
(2) in described cell, co expression aminoacid sequence is SEQ ID NO:2
tRNAIle2The fusion rna that-agmatidine synzyme and step (1) obtain, contains in cell culture medium
Selected from agmatine or the compound of agmatine analog；
(3) in cell culture, add fluorescent dye and the catalyst of catalyzed fluorogenic reaction, incubate
Educate, analyzed by laser confocal microscope, wherein said fluorescent dye selected from BCN-FITC,
BCN-Cy5 or Sulfo-Cy5-azide.
8. a method for locus specificity covalent labeling target RNA in vitro, described method includes
System hatches following material in vitro jointly: Tias, tRNAIle2Or its derivative nucleotide sequence with
The fusion nucleus nucleotide sequence of target RNA, Small-molecule probe, fluorescent dye and other auxiliary materials,
Wherein said tRNAIle2Nucleotides sequence be classified as SEQ ID NO:3, described Small-molecule probe is for being selected from
Agmatine or the compound of agmatine analog.
9. according to the method according to any one of claim 6-8, wherein said tRNAIle2Derive
Nucleotides sequence is classified as tRNAIle2-3-5, its nucleotides sequence is classified as SEQ ID NO:5, and it has following
Structure:
10. according to the method according to any one of claim 6-8, wherein said agmatine analog
Selected from N-(4-aminobutyl)-2-azido acetamide, propargylamine or butyl-3-alkynes-1-base (4-aminobutyl)
T-butyl carbamate.
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