CN1912135B - RNA affinity media and preparation method thereof - Google Patents
RNA affinity media and preparation method thereof Download PDFInfo
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- CN1912135B CN1912135B CN2005100286457A CN200510028645A CN1912135B CN 1912135 B CN1912135 B CN 1912135B CN 2005100286457 A CN2005100286457 A CN 2005100286457A CN 200510028645 A CN200510028645 A CN 200510028645A CN 1912135 B CN1912135 B CN 1912135B
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Abstract
The invention discloses RNA compatible medium and its manufacturing method used to separate and identify RNA singleness conjugated protein. It includes the following steps: fixing one end of DNA fragment of the plasmid pSP65 on glass dust of amino silane to form double chain DNA-glass dust; cloning target RNA gene or cDNA into another reciprocal clone site plasmid Psp64 to gain recombinant plasmid; gaining DNA fragment with extended nucleotide sequence by enzyme cutting; transcript RNA target sequence with RNA complementation fragment which is complemented with the plasmid pSP65 DNA fragment; heating the double chain DNA-glass dust and RNA target sequence together then cooling to make the RNA target sequence and signal chain DNA hybridize to form new RNA compatible medium which has the advantage of easy preparation, strong selectivity, repeatable utilization, etc.
Description
Technical field
The invention belongs to biology field, specifically, is about a kind of single-minded protein-bonded RNA affinity media of isolation identification RNA and preparation method thereof that is used for.
Background technology
In molecular biology research work, single-minded conjugated protein of isolation identification RNA is the important means of research genetic expression and regulation and control, also is simultaneously the important method that the molecular mechanism of development takes place study of disease.
At present, the single-minded protein-bonded method of isolation identification RNA is a lot, its principle all is an analog cell microenvironment in vitro, in the binding buffer liquid of artificial preparation, the affinity media that has target RNA is contacted with cell protein solution, so that target RNA conjugated protein mutually combine single-minded with it elutes and identified conjugated protein then.In these methods, the core is the RNA affinity media.
The simplest affinity media is that the oligomerization ribonucleotide is covalently bound on the polymer dextrane gel, such affinity media has merchandise sales, such as the series product of Amersham Biosciences (branch of PE Healthcare company) except that be used to purify mRNA and cDNA, still the special protein of minority that can be used to purify, conjugated protein as polyA.Because this oligomerization ribonucleotide is lacked (being generally tens nucleotide residues), and can not guarantee it to be to be connected with dextrane gel, so its purposes and specificity are all more limited with terminal.
The RNA affinity media of using morely at present, be with the surface hydroxyl activation of activator (as cyanogen bromide etc.) with carrier (as dextrane gel etc.), make itself and covalently bound (the Kumel G. of RNA again, Daus H., Mauch H.Improvedmethod for the cyanogen bromide activation of agarose beads.J Chromatogr.1979; 172:221-6).The purposes of the affinity media that this method is made is wider, and various rna binding proteins can be used to purify.But its shortcoming remains that any possible position all can form covalent linkage on the RNA molecule, therefore can not guarantee that all parts of RNA are free state, thereby makes its protein-bonded specificity and the separation efficiency all can be influenced.In addition, cyanogen bromide is a highly toxic substance, and potential safety hazard is arranged in the experimental implementation.
Also have a class affinity media to be to use bifunctional reagent with chain molecule structure, RNA and carrier are coupled together (Liu Dinggan, Li Zaiping is with the molecular hybridization method of right-diazobenzene sulfone ethyl paper Covalent Immobilization nucleic acid, Science Bulletin 1982; 27 (16): 1011-1014).Though RNA has bigger freedom of movement in this class medium,, might change the sterie configuration of RNA molecule, so RNA effect protein-bonded with it still can be influenced because bifunctional reagent still can combine with it in all possible position of RNA molecule.
Summary of the invention
Purpose of the present invention just is to overcome the above-mentioned shortcoming and defect of existing RNA affinity media, thereby a kind of novel easy to prepare, strong, the reusable RNA affinity media of bio-identification ability are provided.
Another object of the present invention is to provide the preparation method of described RNA affinity media.
RNA affinity media of the present invention comprises: the solid phase carrier that is used for fixing DNA; The covalently bound line style single stranded DNA fragment on described solid phase carrier of one end; Subsidiary have one section target RNA with the part or all of nucleotide sequence complementary RNA complementary sequence of described single stranded DNA, and wherein said RNA complementary sequence is hybridized with described single stranded DNA and linked to each other.
In the above-mentioned RNA affinity media, the line style single stranded DNA fragment of a plasmid in a pair of reciprocal cloning site plasmid that the preferred multiple clone site direction of described DNA is opposite, all the other nucleotide sequences are identical; Described a pair of preferred pSP64 of reciprocal cloning site plasmid and pSP65.
In the above-mentioned RNA affinity media, described solid phase carrier preferably adopts the solid phase carrier that contains amino group, preferably the glass granules of aminosilaneization (glass powder).
The preparation method of RNA affinity media of the present invention comprises the steps:
(a) end with the line style double chain DNA fragment of a plasmid in a pair of reciprocal cloning site plasmid covalently bind on the solid phase carrier, obtains double-stranded DNA-solid phase carrier;
(b) gene or the cDNA with described target RNA is cloned into another reciprocal cloning site plasmid, forms recombinant plasmid;
(c) recombinant plasmid is carried out enzyme and cut, obtain having extended the dna sequence dna of the dna fragmentation of one section institute's cloned plasmids at the end of its gene or cDNA;
(d) step (c) gained dna sequence dna is carried out in-vitro transcription, obtain the subsidiary one section target RNA with the part or all of sequence complementary of the described DNA of step (a) RNA complementary sequence that has;
(e) step (a) gained double-stranded DNA-solid phase carrier is joined in the renaturation buffered soln that contains target RNA, it is single stranded DNA that heating makes the DNA sex change, and by lowering the temperature gradually target RNA is connected with the single stranded DNA renaturation.
When RNA affinity media of the present invention is used for isolation identification albumen, the sequence of target RNA is complete freely to be suspended in the solution, make the protein molecular in RNA target sequence and the solution approach identification and combination mutually under the natural condition, promptly can be used for identifying eluting then with the single-minded bonded albumen of RNA.
Compare existing RNA affinity media, RNA affinity media of the present invention has advantages such as selectivity is strong, preparation is easy, can reuse.
Description of drawings
Fig. 1 is the hit schema of transcribing of RNA of affinity media of the present invention, and wherein, recombinant plasmid pSP64/0.28 is cut into line style with PvuII, is transcribed into the subsidiary target RNA that a section " tail " arranged then.Have the P representation DNA of circle or the phosphate group of RNA chain 5 ' end among the figure.
Fig. 2 is the covalently bound synoptic diagram of glass powder of plasmid DNA and aminosilaneization in the affinity media of the present invention, and plasmid pSP65 is cut into line style with EcoRI, and is connected with the amino covalence on glass powder surface, cuts the back with the HindIII enzyme and forms double-stranded DNA-glass powder; Heating makes the DNA sex change, obtains single stranded DNA-glass powder.Among the figure, the parantheses of restriction enzyme site represents that this site does not exist, and this moment, DNA was a strand.
Fig. 3 is target RNA and the plasmid DNA connection diagram that is fixed on the glass powder surface, and the DNA renaturation on 3 ' of target RNA-end incidental " tail " and the single stranded DNA-glass powder forms the heterozygosis two strands.
Fig. 4 is the proteic SDS-polyacrylamide gel electrophoresis of affine bonded figure, and A is among the figure: in conjunction with preceding cell protein liquid; B is: the cell protein liquid after the combination; C is: with RNA-DNA-glass powder bonded albumen (shown in the arrow); D is: with single stranded DNA-glass powder bonded albumen (few, as can not to dye with coomassie brilliant blue staining); MW is: protein molecular weight standard is followed successively by from top to bottom: 94kDa, 66kDa, 44kDa, 29kDa.
Fig. 5 for the RNA affinity media reuse 2 times after proteic SDS-polyacrylamide gel electrophoresis figure, MW is among the figure: protein molecular weight standard is followed successively by 94kDa, 66 kDa, 44kDa under going up certainly; Wash-out is: the albumen of wash-out from the RNA-DNA-glass powder; Be not adsorbed as: through the cell protein liquid behind the association reaction; Stoste is: the cell protein liquid before the association reaction.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.It is pointed out that the protection domain of requirement of the present invention is not limited to the specific form of following embodiment.
As everyone knows, it is opposite to exist its multiple clone site direction in biology field, and the identical reciprocal cloning site plasmid of all the other nucleotide sequences, such as pSP64 and pSP65, the GenBank sequence library of the visible National Library of Medicine of its complete sequence, network address: www.ncbi.nlm.nih.gov/entrez.Utilize its structural characteristics, can on a plasmid, clone and the identical dna fragmentation of another reciprocal cloning site plasmid DNA partial sequence, and then accurately transcribe out and reciprocal cloning site plasmid DNA partial sequence complementary RNA.
RNA can be connected by the hydrogen bond between the pairing base with single stranded DNA, DNA can be connected with some solid phase carrier by the covalent attachment mode, these methods all are technology well known in the art, utilize the annexation between them can construct a kind of novel RNA affinity media, make the RNA molecule be in free state fully, to hinder the interactional factor of protein and RNA to minimize, thereby make this affinity media can extract albumen efficientlyly and single-mindedly.
Among the present invention, described " reciprocal cloning site plasmid " is meant so a pair of plasmid, and the difference between them only is that the direction of its multiple clone site is opposite, and all the other nucleotide sequences are identical.Structure such as pSP64 and pSP65 just has such characteristics.
Among the present invention, described " target RNA " be meant can with certain protein specificity bonded RNA sequence.
Among the present invention, described " tail " is meant the incidental one section RNA sequence of target RNA, is used for being connected with the segmental hybridization of single stranded DNA (heterozygosis), is also referred to as " RNA complementary section " or " RNA complementary sequence ".
Among the present invention, described " DNA-solid phase carrier " is meant the solid-phase media that double-stranded DNA or single stranded DNA one end are connected with solid phase carrier and form.
Among the present invention, described " RNA-DNA-glass powder " is meant the RNA affinity media that the glass granules of target RNA, single stranded DNA and aminosilaneization is connected and forms.
Among the present invention, described " the sequence amount of comprising " is meant: hit into micromolecular fragment when protein is done mass spectroscopy, the molecular weight and the sequence of these fragments are monitored and are analyzed by mass spectrograph immediately.With the molecular weight and the sequence of these small molecules fragments, compare with the known protein matter sequence that stores in the Computer Database, fragment can overlap with the corresponding section of a certain known array.The fragment that overlaps is the sequence amount of comprising with the ratio of whole known arrays.The sequence amount of comprising is big more, and the result is just sure more.
The present invention is target RNA with 3 '-non-translational region of the mRNA of nuclear factor C/EBP β, with pSP64 and pSP65 is reciprocal cloning site plasmid, and be solid phase carrier with the glass granules of aminosilaneization, prepare a kind of RNA affinity media, from cell protein liquid, isolate the single-minded conjugated protein of target RNA and identify.
Embodiment 1, have the preparation of the target RNA of " tail "
1.1, the length of 3 '-non-translational region of the mRNA of nuclear factor C/EBP β is the cDNA of 0.28kb, concrete nucleotide sequence is seen that Liu Ding does and is waited (Liu Dinggan, Zhu Lihua, Chen Zhenzhen, wild Tian Liang, Guo Lihe, Li Zaiping, nucleotide sequence with the active cDNA clone of antioncogene, Acta Biochimica et Biophysica Sinica 1991; 23 (3): document 246-250) is cloned into its forward the multiple clone site of plasmid pSP64 (Promega company).Clone's concrete operations see Liu Ding do to wait (Liu Dinggan examines good quiet man, Tadamitsu Kishimoto, Li Zaiping, answer be among the RR a kind of with reply relevant protein expression enhancing, Chinese science B collects 1995; 25 (4): 372-378), obtain recombinant plasmid pSP64/0.28.As shown in Figure 1, in the downstream of plasmid multiple clone site,, obtain including the linear DNA of pSP promotor and 0.28kb cDNA with PvuII linearization for enzyme restriction recombinant plasmid pSP64/0.28.
1.2, with test kit (RiboMax large scale RNA production kit, Promega company) above-mentioned linearization plasmid DNA is carried out extensive in-vitro transcription.By the RNA that the appended specification sheets of kit is purified and transcribed, obtain in the subsidiary RNA target sequence (Fig. 1) that the 0.28kb of a section " tail " is arranged of 3 '-end, the dna sequence dna complementation in the linear DNA of this " tail " and plasmid pSP64 between EcoRI and the PvuII.
The processing and the aminosilaneization of embodiment 2, glass powder carrier
Simple glass powder (50～100 order) with chloroform flush away greasy dirt after, use 5N NaOH and 5N HCl thorough washing successively, again with a large amount of distilled water flushings to remove residual HCl, dry for standby.
Clean glass powder is immersed in 50% acetone soln of 3-aminopropyltriethoxywerene werene (available from Sigma company), soaks half an hour under the room temperature, again with the residual 3-aminopropyltriethoxywerene werene of the abundant flush away of acetone, 50 ℃ of dry for standby.
Embodiment 3, at aminosilane glass powder carrier surface fixed dna
3.1, as shown in Figure 2, plasmid pSP65 (Promega) uses the EcoRI linearization for enzyme restriction, actual conditions is as follows:
Plasmid pSP65 20mg 2mL
10 times of concentration EcoRI damping fluid 250 μ L
0.1% bovine serum albumin, 250 μ L
EcoRI 1000 units (100 μ L)
37 ℃ of incubated overnight (12～16 hours).
Next day, get whether fully enzymolysis of 2 μ L electrophoretic examinationss; Incomplete as enzymolysis, then add the 1000 EcoR I of unit again, continue insulation 4 hours, electrophoretic examinations confirms that enzyme cuts entirely.
3.2, enzyme cuts completely reaction solution (v/v1: 1) extracting once with the saturated phenol/chloroform of equal-volume Tris-HCl (pH8), the chloroform extracting is once taken out water, adds the long-pending dehydrated alcohol of diploid, centrifugal 20 minutes of desk centrifuge 12000rpm, precipitation pSP65 plasmid DNA.
The line style pSP65 DNA that obtains contains the HindIII restriction enzyme site, the dna sequence dna identical (Fig. 1) in the dna sequence dna between HindIII and PvuII site and the linear DNA of plasmid pSP64 between EcoRI and the PvuII, and with " tail " complementation of target RNA.
3.3, in 10mg (20mg/mL) plasmid DNA solution, add 7mg1-ethyl-3-(3-dimethylamino) propyl group carbodiimide (EDC) crystallization and 10mg N-hydroxy-succinamide (NHS) crystallization, under room temperature, be incubated 30min after the stirring and dissolving, with the long-pending dehydrated alcohol deposit D NA of diploid.Precipitation is dissolved in 300 μ L TE (10mmol/LTris-HCl (pH8.0), 1mmol/L EDTA) solution, adds 0.5g aminosilane glass powder carrier, room temperature jolting 2hr.Stop jolting, glass powder is precipitated voluntarily, remove supernatant liquor, wash glass powder 3 times, each 1mL with TE.
3.4, carefully exhaust liquid, add the 1 * HindIII enzyme buffer liquid that contains 200 unit limit enzyme HindIII that just floods glass powder, 37 ℃ of insulation 2hr.Clean glass powder with TE, be suspended among the TE, standby-20 ℃ of preservations.
3.5, the joint efficiency of DNA and glass powder identifies with radioisotope method: get the pSP65DNA of 1 μ L (1 μ g) line style, with 5 ' [γ-
32P] ATP and T4 polynucleotide kinase mark 5 ' end (write referring to " molecular cloning experiment guide " (second edition) .Sambrook (U.S.), Jin Dongyan, Li Mengfeng translate. Science Press, Beijing, 1996).In the pSP65 of above-mentioned line style DNA, mix about 10
5The mark pSP65 DNA of cpm fixes according to the method for embodiment 3.3, and the glass powder after fixing is washed 5 times with TE, and each 1mL measures the radioactivity of glass powder then.Calculate the amount of the pSP65 DNA that is connected with glass powder with following formula:
The amount (mg) of the pSP65 DNA that is connected with glass powder=and the amount (mg) of glass powder blended pSP65 DNA * (radioactivity amount on the glass powder (cpm)/and the amount of pSP65 DNA blended radioactivity pSP65 DNA) (cpm).
Through measuring, the radioactivity amount in the present embodiment on the glass powder is 1.6 * 10
4The amount of cpm and pSP65 DNA blended radioactivity pSP65 DNA is 1 * 10
5Cpm is so the amount of the pSP65 DNA that glass powder connects is a 3.2mg/g glass powder.
The renaturation of embodiment 4, target RNA and DNA is connected
4.1,3 ' the non-translational region RNA of the mRNA of 1mg C/EBP β is dissolved in 250 μ LRNA renaturation buffers (20mM HEPES pH7.8,2mM EDTA, 90mM NaCI), add double-stranded DNA-glass powder 0.6 gram, be sealed in the centrifuge tube, at 500mL H
2Be heated to 90 ℃ among the O, insulation 2min unwinds the double-stranded DNA sex change and becomes single stranded DNA (as shown in Figure 2); In former water-bath, naturally cool to 37 ℃ and be incubated 1hr then, make target RNA be connected (as shown in Figure 3), naturally cool to 4 ℃ again with the single stranded DNA renaturation.Wash 5 times with the RNA renaturation buffer of ice-cold (0～4 ℃), each 1mL removes liquid, preserves down in 4 ℃.
The RNA affinity media that the glass granules that obtains target RNA, single stranded DNA and aminosilaneization is connected and forms abbreviates RNA-DNA-glass powder as.
4.2, the joint efficiency of target RNA and DNA identifies with radioisotope method: with 5 ' [α-
32P] UTP and Riboprobe test kit (Promega company) mark 5 ' end (marking method is seen the specification sheets that this test kit is appended).In above-mentioned RNA, mix about 5 * 10
4The labeled rna of cpm is fixed according to the method for embodiment 4.1, and the glass powder after fixing is washed 5 times with the RNA renaturation buffer of ice-cold (0～4 ℃), and 1mL at every turn measures the radioactivity of glass powder then.Calculate the amount of the RNA that is connected with glass powder with following formula:
The amount (μ g) of the amount of the RNA that is connected with glass powder (μ g)=fixing employed RNA * (radioactivity amount on the glass powder (cpm)/and the amount (cpm) of RNA blended radioactivity RNA).
Through measuring, the radioactivity amount in the present embodiment on the glass powder is 1.5 * 10
3The amount of cpm and RNA blended radioactivity RNA is 5 * 10
4Cpm is so the amount of the RNA that single stranded DNA-glass powder connects is 50 μ g/g glass powder.
The extracting of embodiment 5, cell soluble proteins and combine with the single-minded of target RNA
5.1, all operations of present embodiment all carries out under 4 ℃.10
7Individual SMMC-7721 human liver cancer cell (available from Chinese Academy of Sciences's cell bank) adds the solution of 1mL 250mM sucrose, 1mg/mL digitonin, stir evenly rapidly, go up the centrifugal 3min of 12000rpm at Eppendorf5417R high speed freezing centrifuge (U.S. Eppendorf company) immediately.Supernatant is cell protein liquid.
5.2, in 1mL cell protein liquid, add 0.5g RNA-DNA-glass powder, add 0.2mL 5 * binding buffer liquid (200mM KCl, 15mM MgCl again
2, 50mM HEPES pH7.8,5mM DTT, 15%v/v glycerine, 50mg/mL heparin sodium, 200 μ g/mL yeast tRNA), be incubated 4 hours to spending the night 0～4 ℃ of vibration.Single stranded DNA-glass powder with no target RNA is contrast, and wherein single stranded DNA-glass powder is that RNA-DNA-glass powder is heated to 95 ℃ in TE, removes the solid-phase media that obtains behind the RNA.
Embodiment 6, the proteic wash-out of single-minded bonded, electrophoretic separation and evaluation
6.1, be combined with proteic RNA-DNA-glass powder and single stranded DNA-glass powder and wash 5 times with 1 * binding buffer liquid down, 1mL at every turn at 0～4 ℃.Exhaustion liquid adds 1 * SDS protein electrophoresis sample solution of 250 μ L, 100 ℃ of insulation 2min.Sucking-off liquid suitably concentrates the back and (continues insulation 2～5min), last 8%SDS polyacrylamide gel electrophoresis at 80 ℃.With Xylene Brilliant Cyanine G R-250 dyeing, downcut zone of protein, carry out mass spectroscopy.
6.2, proteic electrophorogram sees Fig. 4.As shown in Figure 4, in the about 50kDa scope of molecular weight, show 3 tangible protein bands behind the elutriant electrophoresis of RNA-DNA-glass powder; The elutriant of single stranded DNA-glass powder is not then significantly distinguished band in same scope.This just shows, these 3 district's bands (i.e. 3 protein) are to be incorporated into 0.28kb RNA (target RNA) single-mindedly, while single stranded DNA-glass powder to these 3 proteic separation efficiencies far below the RNA affinity media.
6.3,3 protein that electrophoresis is obtained deliver NUS's protein and mass spectroscopy is carried out at the protein group center, analytical results is:
Protein 1 vimentin [people] gi|4507895; Mark 601; Nominal molecular weight (M
r) 53710; Calculate pI value 5.06; Classification Homo sapiens; The sequence amount of comprising 54%.
Protein 2 cytokeratin 8gi|181573; Mark 452; Nominal molecular weight (M
r) 53529; Calculate pI value 5.52; Classification Homo sapiens; The sequence amount of comprising 42%.
Protein 3 cytokeratin 18 (424AA) [people] gi|30311; Mark 877; Nominal molecular weight (M
r) 47305; Calculate pI value 5.27; Classification Homo sapiens; The sequence amount of comprising 53%.
6.4 mass spectrometry results proves: these albumen are respectively vimentin (vimentin), cytokeratin 8 and 18 (cytokeratins 8 and 18).These 3 albumen are the albumen that is incorporated into 0.28kb RNA in the cell protein liquid single-mindedly.
The reusability of embodiment 7, RNA affinity media
After the above-mentioned protein separation, RNA-DNA-glass powder is heated to 95 ℃ in TE, and keeps 1 minute, to remove RNA.Discard liquid, cool off in frozen water immediately, the single stranded DNA-glass powder that obtains is in 4 ℃ of preservations.
Get single stranded DNA-glass powder that 0.3g handled, substituting double-stranded DNA-glass powder is connected by the method renaturation of embodiment 4 with 0.5mg 0.28kb RNA, the RNA-DNA-glass powder that obtains carries out association reaction, wash-out and electrophoretic analysis with cell protein liquid by the method for embodiment 5 and 6 again, and the result as shown in Figure 5.
As seen from Figure 5, the electrophoresis result of the conjugated protein reaction first of the electrophoresis result of elutriant and RNA affinity media shown in Figure 4 does not have obvious difference in the 2nd repeated experiments.Above-mentioned experiment shows that the RNA affinity media can reuse, and proves that once more vimentin, cytokeratin 8 and 18 are the single-minded conjugated protein of 0.28kb RNA simultaneously.
Though below only with pSP64 and pSP65 as reciprocal cloning site plasmid, be target RNA and be that solid phase carrier is an example with the glass granules of aminosilaneization with 3 '-non-translational region of the mRNA of nuclear factor C/EBP β, technical scheme of the present invention is verified, but according to of the present invention open, adopt reciprocal cloning site plasmid of other kinds and solid phase carrier can prepare the RNA affinity media easily equally, this is conspicuous for a person skilled in the art.Therefore, adopt the preparation of reciprocal cloning site plasmid of other kinds and solid phase carrier to be used for the single-minded protein-bonded RNA affinity media of isolation identification RNA, should belong to scope of the present invention equally.
In sum, RNA affinity media preparation method of the present invention and uncomplicated in single-minded conjugated protein of isolation identification RNA, shows higher bio-identification ability, and selectivity is strong, and can reuse.Therefore, RNA affinity media of the present invention can be widely used in biology field and drug development.
Claims (8)
1. a RNA affinity media is characterized in that, described RNA affinity media is by the solid phase carrier that is used for fixing DNA; The covalently bound line style single stranded DNA on described solid phase carrier of one end; Attach and form by one section target RNA with the part or all of nucleotide sequence complementary RNA complementary sequence of described DNA, described DNA is the line style single stranded DNA fragment of a plasmid in a pair of reciprocal cloning site plasmid that the multiple clone site direction is opposite, all the other nucleotide sequences are identical, and described RNA complementary sequence links to each other with described single stranded DNA hybridization.
2. RNA affinity media as claimed in claim 1 is characterized in that, described solid phase carrier is the solid phase carrier that contains amino group.
3. RNA affinity media as claimed in claim 1 is characterized in that, described solid phase carrier is the aminosilane glass granules.
4. RNA affinity media as claimed in claim 1 is characterized in that, described a pair of reciprocal cloning site plasmid is pSP65 and pSP64.
5. the preparation method of a RNA affinity media is characterized in that comprising the steps:
(a) end with the line style double chain DNA fragment of a plasmid in a pair of reciprocal cloning site plasmid covalently bind on the solid phase carrier, obtains double-stranded DNA-solid phase carrier;
(b) gene or the cDNA with target RNA is cloned into another reciprocal cloning site plasmid, forms recombinant plasmid;
(c) recombinant plasmid is carried out enzyme and cut, obtain having extended the dna sequence dna of the dna fragmentation of one section institute's cloned plasmids at the end of its gene or cDNA;
(d), obtain the subsidiary one section target RNA with the part or all of sequence complementary of the line style double-stranded DNA RNA complementary sequence of the described plasmid of step (a) that has with step (c) gained dna sequence dna in-vitro transcription;
(e) step (a) gained double-stranded DNA-solid phase carrier is joined in the renaturation buffered soln that contains target RNA, it is single stranded DNA that heating makes the DNA sex change, and by cooling target RNA is connected with the single stranded DNA renaturation.
6. preparation method as claimed in claim 5 is characterized in that, described a pair of reciprocal cloning site plasmid is pSP65 and pSP64.
7. preparation method as claimed in claim 5 is characterized in that, described solid phase carrier is the solid phase carrier that contains amino group.
8. preparation method as claimed in claim 5 is characterized in that, described solid phase carrier is the aminosilane glass granules.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6238867B1 (en) * | 1998-02-23 | 2001-05-29 | Invitro Diagnostics Inc | Compositions, methods and kits for identifying naturally occurring RNA sequences having affinity for RNA-binding proteins |
| WO2002097049A2 (en) * | 2001-05-31 | 2002-12-05 | Q-Rna | Compositions and methods for binding agglomeration proteins |
| US6927031B2 (en) * | 2002-04-12 | 2005-08-09 | Rigel Pharmaceuticals, Incorporated | Methods for identifying polypeptide factors interacting with RNA |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6238867B1 (en) * | 1998-02-23 | 2001-05-29 | Invitro Diagnostics Inc | Compositions, methods and kits for identifying naturally occurring RNA sequences having affinity for RNA-binding proteins |
| WO2002097049A2 (en) * | 2001-05-31 | 2002-12-05 | Q-Rna | Compositions and methods for binding agglomeration proteins |
| US6927031B2 (en) * | 2002-04-12 | 2005-08-09 | Rigel Pharmaceuticals, Incorporated | Methods for identifying polypeptide factors interacting with RNA |
Non-Patent Citations (5)
| Title |
|---|
| Harry R. Burrell et al.Affinity binding of Escherichia coli ribosomal proteins toimmobilized RNA.FEBS Letters49 3.1975,49(3),306-309. |
| Harry R. Burrell et al.Affinity binding of Escherichia coli ribosomal proteins toimmobilized RNA.FEBS Letters49 3.1975,49(3),306-309. * |
| Jeffrey O. Langland et al.Nucleic acid affinity chromatography: preparation andcharacterization of double-stranded RNA agarose.Protein Expression and Purification6.1995,625-32. * |
| Stuart M. Heywood et al.Messenger RNA affinity column fractionation of eukaryoticinitiation factor and the translation of myosin messenger RNA.Archives of Biochemistry and Biophysics192 1.1979,192(1),270-281. |
| Stuart M. Heywood et al.Messenger RNA affinity column fractionation of eukaryoticinitiation factor and the translation of myosin messenger RNA.Archives of Biochemistry and Biophysics192 1.1979,192(1),270-281. * |
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