CN114990109A - Ribonucleic acid purification partner and application thereof - Google Patents
Ribonucleic acid purification partner and application thereof Download PDFInfo
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- CN114990109A CN114990109A CN202210704287.0A CN202210704287A CN114990109A CN 114990109 A CN114990109 A CN 114990109A CN 202210704287 A CN202210704287 A CN 202210704287A CN 114990109 A CN114990109 A CN 114990109A
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- 229920002477 rna polymer Polymers 0.000 title claims abstract description 142
- 238000000746 purification Methods 0.000 title claims abstract description 47
- 239000002608 ionic liquid Substances 0.000 claims abstract description 87
- 238000010828 elution Methods 0.000 claims abstract description 75
- 238000000034 method Methods 0.000 claims abstract description 30
- 239000012501 chromatography medium Substances 0.000 claims abstract description 17
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 9
- 150000001768 cations Chemical class 0.000 claims abstract description 8
- 150000001450 anions Chemical class 0.000 claims abstract description 5
- 239000003480 eluent Substances 0.000 claims description 58
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- 108020004999 messenger RNA Proteins 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 31
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 29
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 27
- 235000002639 sodium chloride Nutrition 0.000 claims description 20
- 239000011780 sodium chloride Substances 0.000 claims description 18
- 239000007853 buffer solution Substances 0.000 claims description 15
- 239000000872 buffer Substances 0.000 claims description 14
- 239000008363 phosphate buffer Substances 0.000 claims description 10
- 238000005571 anion exchange chromatography Methods 0.000 claims description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 8
- -1 rRNA Proteins 0.000 claims description 8
- 108020004414 DNA Proteins 0.000 claims description 6
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 6
- 238000013518 transcription Methods 0.000 claims description 5
- 230000035897 transcription Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 4
- 239000007995 HEPES buffer Substances 0.000 claims description 4
- 239000008351 acetate buffer Substances 0.000 claims description 4
- 238000000338 in vitro Methods 0.000 claims description 4
- 239000003446 ligand Substances 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 2
- 102000053642 Catalytic RNA Human genes 0.000 claims description 2
- 108090000994 Catalytic RNA Proteins 0.000 claims description 2
- 108020005004 Guide RNA Proteins 0.000 claims description 2
- 108020004688 Small Nuclear RNA Proteins 0.000 claims description 2
- 102000039471 Small Nuclear RNA Human genes 0.000 claims description 2
- 108020003224 Small Nucleolar RNA Proteins 0.000 claims description 2
- 102000042773 Small Nucleolar RNA Human genes 0.000 claims description 2
- 108020004459 Small interfering RNA Proteins 0.000 claims description 2
- 108091028113 Trans-activating crRNA Proteins 0.000 claims description 2
- 108091032917 Transfer-messenger RNA Proteins 0.000 claims description 2
- 241000726445 Viroids Species 0.000 claims description 2
- 239000006167 equilibration buffer Substances 0.000 claims description 2
- 238000010829 isocratic elution Methods 0.000 claims description 2
- 108091070501 miRNA Proteins 0.000 claims description 2
- 239000002679 microRNA Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 108091092562 ribozyme Proteins 0.000 claims description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims 1
- 108091029810 SaRNA Proteins 0.000 claims 1
- 229940078677 sarna Drugs 0.000 claims 1
- 108010083644 Ribonucleases Proteins 0.000 abstract description 17
- 102000006382 Ribonucleases Human genes 0.000 abstract description 17
- 239000003381 stabilizer Substances 0.000 abstract description 4
- 238000001042 affinity chromatography Methods 0.000 abstract description 3
- 230000006872 improvement Effects 0.000 abstract description 3
- 238000004255 ion exchange chromatography Methods 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 230000006641 stabilisation Effects 0.000 abstract description 2
- 238000011105 stabilization Methods 0.000 abstract description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 30
- 230000000052 comparative effect Effects 0.000 description 23
- 230000000694 effects Effects 0.000 description 23
- 238000012360 testing method Methods 0.000 description 17
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 15
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 13
- 229960001231 choline Drugs 0.000 description 13
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 7
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 7
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 4
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 4
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 239000005090 green fluorescent protein Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
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- 238000006467 substitution reaction Methods 0.000 description 4
- 230000002195 synergetic effect Effects 0.000 description 4
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- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- KAIPKTYOBMEXRR-UHFFFAOYSA-N 1-butyl-3-methyl-2h-imidazole Chemical compound CCCCN1CN(C)C=C1 KAIPKTYOBMEXRR-UHFFFAOYSA-N 0.000 description 1
- IBZJNLWLRUHZIX-UHFFFAOYSA-N 1-ethyl-3-methyl-2h-imidazole Chemical compound CCN1CN(C)C=C1 IBZJNLWLRUHZIX-UHFFFAOYSA-N 0.000 description 1
- WGVGZVWOOMIJRK-UHFFFAOYSA-N 1-hexyl-3-methyl-2h-imidazole Chemical compound CCCCCCN1CN(C)C=C1 WGVGZVWOOMIJRK-UHFFFAOYSA-N 0.000 description 1
- KTUWFYALZIAAGE-UHFFFAOYSA-N 1-methyl-3-octyl-2h-imidazole Chemical compound CCCCCCCCN1CN(C)C=C1 KTUWFYALZIAAGE-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- 102000010638 Kinesin Human genes 0.000 description 1
- 108010063296 Kinesin Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 239000012564 Q sepharose fast flow resin Substances 0.000 description 1
- 108010046983 Ribonuclease T1 Proteins 0.000 description 1
- 108091026924 Sar RNA Proteins 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940006460 bromide ion Drugs 0.000 description 1
- 238000002983 circular dichroism Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- JXTHNDFMNIQAHM-UHFFFAOYSA-M dichloroacetate Chemical compound [O-]C(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-M 0.000 description 1
- 238000000113 differential scanning calorimetry Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 238000003505 heat denaturation Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000004190 ion pair chromatography Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- JZMJDSHXVKJFKW-UHFFFAOYSA-M methyl sulfate(1-) Chemical compound COS([O-])(=O)=O JZMJDSHXVKJFKW-UHFFFAOYSA-M 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
Abstract
The invention relates to a ribonucleic acid purification partner and application thereof, wherein the ribonucleic acid purification partner is an ionic liquid consisting of cations and anions, the purification method is a chromatography method, and the purification partner is used for simultaneously improving the elution efficiency of ribonucleic acid on a chromatography medium and improving the stability of the ribonucleic acid. Such as ion exchange chromatography, affinity chromatography, and the like. The improvement of the stability of the ribonucleic acid refers to the simultaneous stabilization of the secondary structure of the ribonucleic acid and the inhibition of the enzymolysis of the ribonucleic acid by the ribonuclease. The ribonucleic acid purification partners of the invention can also be used alone as stabilizers for ribonucleic acids.
Description
Technical Field
The invention belongs to the technical field of separation and purification of ribonucleic acid, and relates to a ribonucleic acid purification mate and application thereof.
Background
Ribonucleic acid (RNA) is a long-chain polymer formed by condensing ribonucleotide through a phosphodiester bond, and is widely applied to the fields of biology, medicine, nanotechnology and the like. In organisms, RNA is key in the central rule for kinesin synthesis. RNA related research such as structural, biochemical and vaccinology research requires high purity RNA. Purification of the sample is therefore a critical step in RNA research.
Currently, a variety of liquid chromatography techniques have been used to purify RNA in vivo or obtained by in vitro transcription, including: affinity chromatography, size exclusion chromatography, ion exchange chromatography, reverse phase ion pair chromatography, and the like. The anion exchange chromatography adsorbs the biomolecules by electrostatic attraction between positively charged ligands and negatively charged biomolecules on a chromatographic medium, and then desorbs and elutes the biomolecules adsorbed on the medium by increasing the salt concentration in the eluent and shielding the charge effect. The presence of a large number of negatively charged phosphate groups on the RNA strand makes it negatively charged over a wide pH range, suitable for purification using anion exchange chromatography. Although anion exchange chromatography can be suitable for RNA purification, conventional ion exchange high salt elution conditions may not completely dissociate RNA from positively charged ligands due to the strong negative charge of RNA, resulting in low recovery of the elution. In response to similar problems, there are reports in the relevant literature of optimized methods to facilitate elution of DNA from solid phase adsorption surface materials.
However, these methods for optimizing the elution of DNA are not suitable for RNA because RNA is more unstable than protein or DNA, its structure is more susceptible to degradation by temperature, pH and ribonuclease, and thus it is difficult to store for a long period of time, and the structural integrity of RNA is crucial for its biological activity. Therefore, how to maintain the stability of purified RNA is an important issue to be focused on in RNA research. At present, in addition to preventing RNA degradation by low-temperature storage (-80 ℃ to-20 ℃), improving the stability of the RNA self-structure and inhibiting the degradation of RNA by ribonuclease are two main strategies for stabilizing RNA at present. For example, the mRNA itself may be structurally stabilized by capping the 5 'end of the mRNA, pseudouracil modifying the coding region, or adding poly A to its 3' end. Or using cations such as Mg 2+ 、K + 、Na + And the like shields the negatively charged phosphate group and the chelated 2' hydroxyl group in the RNA skeleton to improve the structural stability of the RNA. By using diethyl pyrocarbonate (DEPC), vanadyl riboside complexesNuclease inhibitors such as compound (RVC), guanidinium isothiocyanate or RNase inhibit the activity of ribonuclease in liquid chromatography buffers or RNA stocks. However, the existing RNA stabilizers have only a single function, and cannot simultaneously stabilize the RNA structure and inhibit the activity of ribonuclease.
Therefore, how to achieve both improvement of elution efficiency in RNA purification and maintenance of RNA stability after purification has become an urgent problem to be solved by those skilled in the art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a ribonucleic acid purification mate and application thereof. The ribonucleic acid purification partners of the invention can also be used alone as stabilizers for ribonucleic acids.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a ribonucleic acid purification partner, which is an ionic liquid comprising a cation and an anion, and which is purified by chromatography, for simultaneously increasing the efficiency of elution of ribonucleic acid from a chromatography medium and increasing the stability of the ribonucleic acid.
Such as ion exchange chromatography, affinity chromatography, and the like.
The elution efficiency is the recovery rate after ribonucleic acid elution, and the elution efficiency is the amount of ribonucleic acid after elution/the amount of ribonucleic acid before elution x 100%.
The improvement of the stability of the ribonucleic acid refers to the simultaneous stabilization of the secondary structure of the ribonucleic acid and the inhibition of the enzymolysis of the ribonucleic acid by the ribonuclease.
The secondary structure refers to a single-stranded region structure, a stem-loop structure or a double-stranded structure formed by complementary base pairing in a ribonucleic acid single strand.
The ribonuclease refers to an enzyme that hydrolyzes RNA phosphodiester bonds, including ribonuclease a, ribonuclease T1, or ribonuclease H.
Preferably, the ribonucleic acid is a long-chain molecule formed by condensing ribonucleotides through phosphodiester bonds, and comprises any one or a combination of at least two of mRNA, tRNA, rRNA, miRNA, snRNA, snoRNA, siRNA, sarRNA, tmRNA, crRNA, tracrRNA, gRNA, ribozyme, viroid or telase RNA.
Preferably, the ribonucleic acid is extracted from an animal, plant or microorganism, or is obtained by in vitro transcription of DNA.
Preferably, the microorganism comprises yeast.
Preferably, the ionic liquid comprises an imidazole-type ionic liquid and/or a choline-type ionic liquid.
Preferably, the cation in the imidazole type ionic liquid is [ C ] n mim] + (1-alkyl-3-methylimidazole) n ═ 2-8(2, 3, 4, 5, 6, 7 or 8), for example, [ C 2 mim] + (1-ethyl-3-methylimidazole), [ C 4 mim] + (1-butyl-3-methylimidazole), [ C 6 mim] + (1-hexyl-3-methylimidazole), [ C 8 mim] + (1-octyl-3-methylimidazole), and the like.
Preferably, the cation in the choline-type ionic liquid is [ Cho] + (Choline).
Preferably, the anion in the ionic liquid comprises [ Tf 2 N] - (bis (trifluoromethanesulfonylimide) ion), [ SCN [ ]] - (thiocyanate ion), [ N (CN) 2 ] - (dicyanimide ion), [ TfO ]] - (trifluoromethylsulfonate Ionic), [ MeOSO ] 3 ] - (methyl sulfate ion), [ Br] - (Bromide ion), [ Cl ]] - (chloride ion), [ BF ] 4 ] - (tetrafluoroborate ion), [ EtOSO ] 3 ] - (ethyl sulfate ion) or [ dca] - (dichloroacetate ion) or a combination of at least two thereof.
Combinations of said at least two, e.g. [ Tf 2 N] - And [ EtOSO 3 ] - Combination of [ A ] and [ Cl ]] - And [ SCN] - Combination of [ dca ]] - And [ TfO ]] - And the like, and any other combination may be used.
Preferably, the chromatography medium comprises an anion exchange chromatography medium.
Preferably, the ligand in the anion exchange chromatography medium comprises TMAM (-CH) 2 N + (CH 3 ) 3 )、TEAE(—(CH 2 ) 2 N + (C 2 H 5 ) 3 )、HEDMAM(—CH 2 N + (CH 3 ) 2 C 2 H 4 OH)、QAE(—CH 2 N + (C 2 H 5 ) 2 —CH 2 CH(OH)CH 3 )、DEAE(—(CH 2 ) 2 N(C 2 H 5 ) 2 )、MP(—C 5 H 5 NCH 3 )、GE(—C 2 H 4 C(NH 2 )＝N + H 2 )、DEAHP(—CH 2 CH(OH)CH 2 NH(C 2 H 5 ) 2 )、AE(—C 2 H 4 N + H 3 ) Or PEI (- (C)) 2 H 4 NH) n C 2 H 4 N + H 3 ) Any one of them.
Preferably, when the ribonucleic acid is extracted from yeast, the ionic liquid comprises [ Cho][EtOSO 3 ]、[C 8 mim][MeOSO 3 ]、[C 6 mim][Tf 2 N]、[C 4 mim][dca]、[Cho][Cl]、[Cho][BF 4 ]Or [ C 4 mim][Cl]Any one or a combination of at least two of them.
Combinations of said at least two, e.g. [ Cho][EtOSO 3 ]And [ C 8 mim][MeOSO 3 ]Combination of [ C ] 6 mim][Tf 2 N]And [ C 4 mim][dca]Combination of [ Cho ]][BF 4 ]And [ C 4 mim][Cl]Combinations of (3) and (3), and any other combination.
Preferably, when the ribonucleic acid is mRNA (preferably transcribed in vitro), the ionic liquid comprises [ Cho][Cl]、[Cho][Br]、[C 4 mim][Cl]、[C 2 mim][Br]、[C 6 mim][Tf 2 N]Or [ C 4 mim][dca]Any one or a combination of at least two of them.
Preferably, the ionic liquid comprises [ Cho][BF 4 ]And/or [ C 4 mim][Cl]。
Preferably, said [ Cho][BF 4 ]And [ C 4 mim][Cl]The concentration ratio of (1) to (4) is (3-6) to (7).
Specific numerical values in (3-6) above are, for example, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.
Specific numerical values in the above (4-7) are, for example, 4, 4.5, 5, 5.5, 6, 6.5, 7 and the like.
In a second aspect, the invention provides the use of a ribonucleic acid purification partner according to the first aspect in the preparation of a chromatography eluent for the purification of ribonucleic acid.
In a third aspect, the invention provides a method for purifying ribonucleic acid, comprising feeding ribonucleic acid to be purified to a chromatography column filled with a chromatography medium, eluting with an eluent comprising a ribonucleic acid purification partner of the first aspect, collecting the eluted fractions, and completing the purification.
Preferably, the eluent is obtained by mixing water or a buffer solution with the ribonucleic acid purification partner, wherein the buffer solution comprises any one of acetate buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer or carbonate buffer or a combination of at least two of the above.
Preferably, the buffer solution has a concentration of 5-200mM, e.g., 5mM, 10mM, 20mM, 50mM, 100mM, 150mM, 200mM, etc.
Preferably, the pH of the buffer solution is 6.0-8.5, such as 6.0, 6.2, 6.4, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.5, and the like.
Preferably, the concentration of the ribonucleic acid purification partner in the buffer solution is 0.05M-2M, such as 0.05M, 0.08M, 0.1M, 0.2M, 0.4M, 0.6M, 1M, 1.2M, 1.4M, 1.6M, 1.8M, 2.0M, and the like.
Preferably, the eluent further contains an inorganic salt, wherein the inorganic salt comprises any one or a combination of at least two of sodium chloride, potassium chloride or ammonium sulfate, for example, a combination of sodium chloride and ammonium sulfate, a combination of potassium chloride and ammonium sulfate, sodium chloride and potassium chloride, and the like, and any other combination can be adopted.
Preferably, the concentration of the inorganic salt in the eluent is 0.5-2M, such as 0.5M, 0.6M, 0.8M, 1M, 1.2M, 1.4M, 1.6M, 1.8M, 2.0M, etc.
Preferably, the manner of elution comprises isocratic elution or gradient elution, which comprises linear gradient elution or step gradient elution.
Preferably, the elution further comprises rinsing the chromatography medium with an equilibration buffer comprising any one or a combination of at least two of an acetate buffer, a phosphate buffer, a Tris-HCl buffer, a HEPES buffer, or a carbonate buffer.
The numerical ranges set forth herein include not only the points recited above, but also any points between the numerical ranges not recited above, and are not exhaustive of the particular points included in the ranges for reasons of brevity and clarity.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively provides an ionic liquid as a ribonucleic acid purification partner: has the following advantages:
(1) the method can improve the elution efficiency of RNA on anion exchange chromatography and can also improve the stability of RNA in an eluted component obtained after elution;
(2) the RNA structure is stabilized through the synergistic effect of simultaneously stabilizing the RNA molecular structure and inhibiting the activity of ribonuclease, which cannot be realized by the existing RNA stabilizer;
(3) can be used as stable solution of RNA from various sources, has good solubility and simple use, and has wide application prospect.
In addition, the effect of different ionic liquids is different, and in the aspect of improving the stability of the yeast RNA structure, [ Cho][EtOSO 3 ]Better effect, [ C ] in reducing ribonuclease A stability 8 mim][MeOSO 3 ]、[C 6 mim][Tf 2 N]And [ C 4 mim][dca]Has better effect, and can improve the elution efficiency to more than 99 percent (by using the prior ionic liquid) in the aspect of improving the elution efficiency of the yeast RNAThe conventional eluent in the technology can only reach 71-77% of elution efficiency). In terms of improving stability after yeast RNA elution, [ Cho][Cl]The effect of (2) is better. In terms of improving the efficiency of mRNA elution, [ C ] 4 mim][Cl]Has better effect, and in the aspect of improving the stability of mRNA after elution, [ Cho][BF 4 ]And [ C 4 mim][Cl]The effect of (2) is better.
The present inventors have also surprisingly found that [ C ] is selected in the case of concentrations of 1M 4 mim][Cl]And [ Cho][BF 4 ]The combination of [ C ] is better than that of the ionic liquid with any single component, and the result shows that 4 mim][Cl]And [ Cho][BF 4 ]The mutual cooperation has unexpected synergistic effect in improving mRNA elution efficiency and elution stability.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
In the following examples, unless otherwise specified, reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise indicated, all experimental methods and technical means used are those conventional in the art.
Yeast RNA (CAS number 63231-63-0) referred to in the following examples and comparative examples was purchased from Sigma, cat #: and R6625.
Example 1
This example provides a yeast RNA solution containing choline-type ionic liquid, which is prepared as follows:
1) adding a certain amount of choline type ionic liquid [ Cho][EtOSO 3 ]Dissolving in 20mM Tris-HCl buffer, pH7.5, adjusting pH to pH7.5 with 4mol/L NaOH or 2mol/L HCl to obtain [ Cho ] 0.4M][EtOSO 3 ]A solution;
2) to 1mL of a 1mg/mL yeast RNA solution (pH 7.5), 1mL of the [ Cho ] solution described above was added][EtOSO 3 ]Solution to give [ Cho ] supplemented with 0.2M][EtOSO 3 ]The yeast RNA solution of (1).
Example 2
This example provides a yeast RNA solution containing a choline-type ionic liquid, which is prepared by a method different from that of example 1 only in that [ Cho][EtOSO 3 ]Replacement by equal concentrations of [ Cho][dca]Other conditions were as in example 1.
Example 3
This example provides a yeast RNA solution containing imidazole-type ionic liquid, which is prepared as follows:
1) adding a certain amount of choline type ionic liquid [ C ] 4 mim][Tf 2 N]Dissolving in 20mM Tris-HCl buffer solution, pH7.5, adjusting pH to pH7.5 with 4mol/L NaOH or 2mol/L HCl, and preparing [ C ] with concentration of 0.2M 4 mim][Tf 2 N]A solution;
2) 1mL of the above [ C ] was added to 1mL of a 1mg/mL yeast RNA solution (pH 7.5) 4 mim][Tf 2 N]Solution to obtain [ C ] with 0.1M addition 4 mim][Tf 2 N]The yeast RNA solution of (1).
Example 4
This example provides an imidazole-type ionic liquid-containing yeast RNA solution, which is prepared by a method different from that of example 3 only in that [ C ] is added 4 mim][Tf 2 N]By substitution with isoconcentrated [ C 6 mim][SCN]Other conditions were as in example 3.
Example 5
This example provides an imidazole-type ionic liquid-containing yeast RNA solution, which is prepared by a method different from that of example 3 only in that [ C ] is added 4 mim][Tf 2 N]Replacement by isoconcentration of [ C 8 mim][MeOSO 3 ]Other conditions were as in example 3.
Example 6
This example provides an eluent containing choline-type ionic liquid, which is prepared as follows:
an eluate containing 1M [ Cho ] [ Cl ] was prepared by dissolving a certain amount of choline-type ionic liquid [ Cho ] [ Cl ] in 50mM, pH6.5, phosphate buffer containing 2mM EDTA and adjusting its pH to pH6.5 with 4mol/L NaOH or 2mol/L HCl.
Example 7
This example provides an eluent containing choline-type ionic liquid, which was prepared in a manner different from that of example 6 only in that the concentration of [ Cho ] [ Cl ] in the eluent was changed to 2M, and the other conditions were as in example 6.
Example 8
This example provides an eluent containing choline-type ionic liquid, which was prepared according to the method different from that of example 6 except that the concentration of [ Cho ] [ Cl ] in the eluent was changed to 1.5M, and the other conditions were as described in example 6.
Example 9
This example provides an eluent containing a choline-type ionic liquid, which was prepared by the method different from that of example 6 except that [ Cho ] [ Cl ] was replaced with [ Cho ] [ Br ] at an equal concentration, and the other conditions were as described in example 6.
Example 10
This example provides an eluent containing a choline-type ionic liquid, which was prepared by a method different from that of example 6 only in that [ Cho][Cl]Substitution to isoconcentrated [ Cho][BF 4 ]Other conditions were as in example 6.
Example 11
This example provides an eluent containing imidazole-type ionic liquid, which is prepared as follows:
adding a certain amount of choline type ionic liquid [ C ] 4 mim][Cl]Dissolved in 50mM, pH6.5, phosphate buffer containing 2mM EDTA and adjusted to pH6.5 with 4mol/L NaOH or 2mol/L HCl to prepare a solution containing 1M [ C ] 4 mim][Cl]The eluent of (4).
Example 12
This example provides an eluent containing an imidazole-type ionic liquid, which was prepared in a manner different from that of example 11 only in that [ C ] in the eluent was used 4 mim][Cl]The concentration was changed to 2M, and other conditions were as in example 11.
Example 13
This example provides an eluent containing an imidazole-type ionic liquid, which was prepared in a manner different from that of example 11 only in that [ C ] was included in the eluent 4 mim][Cl]The concentration was changed to 1.5M, other conditions were referred toExample 11.
Example 14
This example provides an eluent containing an imidazole-type ionic liquid, which was prepared by a method different from that of example 11 only in that [ C ] was used 4 mim][Cl]Replacement by isoconcentration of [ C 2 mim][Br]Other conditions were as in example 11.
Example 15
This example provides an eluent containing an imidazole-type ionic liquid, which was prepared by a method different from that of example 11 only in that [ C ] was used 4 mim][Cl]By substitution with isoconcentrated [ C 6 mim][Tf 2 N]Other conditions were as in example 11.
Example 16
This example provides an eluent containing an imidazole-type ionic liquid, which was prepared by a method different from that of example 11 only in that [ C ] was used 4 mim][Cl]Replacement by isoconcentration of [ C 4 mim][dca]Other conditions were as in example 11.
Example 17
This example provides a composition containing 0.7M [ C ] in combination 4 mim][Cl]And 0.3M [ Cho ]][BF 4 ]The preparation method of the eluent comprises the following steps:
a certain amount of imidazole type ionic liquid [ C ] 4 mim][Cl]And a certain amount of choline type ionic liquid [ Cho][BF 4 ]Dissolved in 50mM, pH6.5, 2mM EDTA-containing phosphate buffer and adjusted to pH6.5 with 4mol/L NaOH or 2mol/L HCl to prepare a composition containing 0.6M of [ C ] C 4 mim][Cl]And 0.4M [ Cho ]][BF 4 ]The eluent of (4).
Example 18
This example provides a composition containing 0.5M C 4 mim][Cl]And 0.5M [ Cho ]][BF 4 ]The eluent according to example 17 was prepared.
Example 19
This example provides a composition containing 0.4M [ C ] in combination 4 mim][Cl]And 0.6M [ Cho ]][BF 4 ]The eluent according to example 17 was prepared.
Example 20
This example provides an eluate containing a choline-type ionic liquid and 1M NaCl, prepared as follows:
a certain amount of choline-type ionic liquid [ Cho ] [ Br ] was dissolved in 50mM, pH6.5, phosphate buffer containing 1M NaCl and 2mM EDTA, and the pH was adjusted to pH6.5 with 4mol/L NaOH or 2mol/L HCl to prepare an eluent containing 0.2M [ Cho ] [ Cl ] and 1M NaCl.
Example 21
This example is an eluent containing imidazole type ionic liquid and 1M ammonium sulfate, and the preparation method is as follows:
adding a certain amount of choline type ionic liquid [ C ] 2 mim][dca]Dissolved in 50mM, pH6.5, phosphate buffer containing 1M ammonium sulfate and 2mM EDTA and adjusted to pH6.5 with 4mol/L NaOH or 2mol/L HCl to prepare a solution containing 0.2M of [ C ] 2 mim][Cl]And 1M ammonium sulfate.
Example 22
This example provides an eluent containing only imidazole-type ionic liquid, which was prepared as follows:
adding a certain amount of choline type ionic liquid [ C ] 2 mim][Br]Dissolving in water to obtain a solution containing 0.05M of C 2 mim][Br]The eluent of (4).
Comparative example 1
This comparative example provides a yeast RNA solution which differs from example 1 only in that the solution does not contain an ionic liquid, and is specifically prepared as follows:
to 1mL of a 1mg/mL yeast RNA solution (pH 7.5), 1mL of a 20mM Tris-HCl buffer solution (pH 7.5) was added to obtain a yeast RNA solution.
Comparative example 2
This comparative example provides a conventional eluent which differs from example 6 only in that the eluent contains no ionic liquid but 1M NaCl and is prepared as follows:
an eluent containing 1M NaCl was prepared by dissolving an amount of NaCl in a phosphate buffer solution containing 2mM EDTA at 50mM, pH6.5, and adjusting the pH to pH6.5 with 4mol/L NaOH or 2mol/L HCl.
Comparative example 3
This comparative example provides a conventional eluent which differs from example 6 only in that the eluent contains no ionic liquid but 1.5M NaCl and is prepared as follows:
an eluent containing 1.5M NaCl was prepared by dissolving a certain amount of NaCl in 50mM, pH6.5, phosphate buffer containing 2mM EDTA and adjusting the pH to pH6.5 with 4mol/L NaOH or 2mol/L HCl.
Comparative example 4
This comparative example provides a conventional eluent which differs from example 6 only in that the eluent contains no ionic liquid but 2M NaCl and is prepared as follows:
an eluent containing 2M NaCl was prepared by dissolving an amount of NaCl in a phosphate buffer solution containing 2mM EDTA at 50mM, pH6.5, and adjusting the pH to pH6.5 with 4mol/L NaOH or 2mol/L HCl.
Test example 1 Yeast RNA stability test
T of RNA in Yeast RNA solutions of examples 1 to 5 and comparative example 1 m Values were determined to reflect the stability of RNA structure, T, in each set of solutions m The value is defined as the temperature at which half of the secondary structure of the RNA is lost, a higher temperature indicating a more stable secondary structure of the RNA.
The specific method comprises the following steps: firstly, removing ionic liquid which is not combined with RNA in each group of solution by a G25 desalting column, measuring the change condition of RNA secondary structure spectrogram at 210nm-320nm under the condition of gradient temperature rise (2 ℃/min) at 20-90 ℃ by using circular dichroism, analyzing a temperature rise curve by using Global 3 software, and calculating the T of the RNA m The value is obtained. The results are shown in Table 1.
TABLE 1
| Group of | T m Value (. degree. C.) | Group of | T m Value (. degree. C.) |
| Example 1 | 55.6 | Example 4 | 51.7 |
| Example 2 | 54.0 | Example 5 | 50.2 |
| Example 3 | 52.3 | Comparative example 1 | 42.5 |
The results show that: as is clear from the results of comparison between examples 1 to 5 and comparative example 1, both of the choline-type and imidazole-type ionic liquids can increase the T of yeast RNA m The value is beneficial to keeping the stability of the secondary structure of RNA, and under the condition of consistent concentration, the choline ionic liquid has better effect of improving the stability of the RNA structure than the imidazole ionic liquid, wherein the choline ionic liquid [ Cho][EtOSO 3 ]The best effect (example 1).
Test example 2 RNase stability test
T for ribonucleases using Differential Scanning Calorimetry (DSC) m The values were measured. After 0.5mg/mL RNase A was added to each of the solutions of examples 1-5, 6, 9-10, 11, 14-16, 1 and 3, respectively, the samples were placed in a MicrolCal TM In the VP-DSC instrument, the temperature rise range of the instrument is 20 ℃ to 90 ℃ (1 ℃/min), and under the condition, the T of the ribonuclease in each group of solution is measured m The value (heat denaturation temperature), higher value indicates the RNase stability higher, the results are shown in Table 2.
TABLE 2
| Group of | T m Value (. degree. C.) | Group of | T m Value (. degree. C.) |
| Example 1 | 60.4 | Example 6 | 61.3 |
| Example 2 | 58.2 | Example 9 | 58.7 |
| Example 3 | 54.3 | Example 10 | 58.9 |
| Example 4 | 55.1 | Example 11 | 57.2 |
| Example 5 | 52.9 | Example 14 | 58.6 |
| Comparative example 1 | 63.5 | Example 15 | 52.1 |
| Comparative example 2 | 64.0 | Example 16 | 51.0 |
The results show that: from the results of comparison of the yeast RNA solutions (examples 1-5 and comparative example 1), it can be seen that the choline and imidazole type ionic liquids can reduce the T of RNase A m The RNase A is more unstable and inactivated in the ionic liquid environment, and the choline and imidazole type ionic liquid can simultaneously achieve the effects of improving the stability of RNA and reducing the stability of RNase according to the results of test example 1. Wherein, under the condition of consistent concentration, the imidazole ionic liquid has better effect of reducing the stability of ribonuclease than choline ionic liquid, wherein [ C 8 mim][MeOSO 3 ]The best effect (example 5).
The same conclusions were drawn from the comparison of the various sets of eluents (examples 6, 9 to 11, 14 to 16 and comparative example 3): reduction of both choline and imidazole type ionic liquidsT of ribonuclease A m Value, make ribonuclease A in the ionic liquid environment is more unstable and inactive. Wherein, under the condition of consistent concentration, when the ionic liquid in the eluent is [ C ] 6 mim][Tf 2 N]Or [ C 4 mim][dca]In this case, the effect of reducing the stability of ribonuclease was the best (examples 15 and 16).
Test example 3 Yeast RNA elution efficiency test
After equilibrating the column with 50mM PB +0.2mM EDTA and pH6.5 buffer (POROS dee as chromatography medium) at 25 ℃, 0.9mg/mL of yeast RNA was injected into the column, after completion of the injection, the column was eluted with 50mM PB +0.2mM EDTA and pH6.5 buffer, and then eluted with the eluents of examples 6 to 16 and comparative examples 2 to 4, respectively, and the elution fractions were collected and the elution efficiency, i.e., the total amount of RNA in the elution fraction/total amount of RNA injected into the column × 100%, was calculated, and the results are shown in table 3.
TABLE 3
| Group of | Elution efficiency (%) | Group of | Elution efficiency (%) |
| Example 6 | >99 | Example 13 | >99 |
| Example 7 | >99 | Example 14 | >99 |
| Example 8 | >99 | Example 15 | >99 |
| Example 9 | >99 | Example 16 | >99 |
| Example 10 | >99 | Comparative example 2 | 77 |
| Example 11 | >99 | Comparative example 3 | 75 |
| Example 12 | >99 | Comparative example 4 | 71 |
The results show that: when the traditional NaCl is used as an eluent, the elution efficiency of the yeast RNA on an anion exchange medium is low and is only 71% -77%, and when the ionic liquid is used as a purification partner for elution, the elution efficiency of the RNA can be remarkably improved to more than 99%.
Test example 4 determination of stability of Yeast RNA after elution
Yeast RNA was eluted with each elution set according to the method of test example 3Line elution, after elution, the fractions were collected and the T of RNA in each fraction was determined according to the method of test example 1 m The value is obtained. The results are shown in Table 4.
TABLE 4
The results show that: eluting with eluent containing ionic liquid as purification partner to obtain elution fraction containing RNA T m The value is obviously higher than the result of elution by using the conventional eluent without the ionic liquid, which shows that the ionic liquid can stabilize the secondary structure of RNA in the elution process and obviously improve the stability of the RNA after elution.
From the comparison results of examples 6, 9, 10, 11, 14, 15, and 16, it is clear that [ Cho ] [ Cl ] has a higher effect of improving RNA elution stability than other ionic liquids at a uniform concentration.
From the comparison results of examples 6 to 8 and the comparison results of examples 11 to 13, it is understood that the concentration of the ionic liquid in the eluate has a certain influence on the RNA elution stability, and the effect is best when the concentration is 2M.
Test example 5 mRNA elution efficiency test
After the column was equilibrated with 50mM PB +0.2mM EDTA and pH6.5 buffer solution (DEAE Sepharose FF as chromatography medium) at 25 deg.C, 142. mu.g/mL of mRNA expressing green fluorescent protein (obtained by transcription from green fluorescent protein DNA, the transcription method being a method conventional in the art) was injected into the column, and after the injection was completed, the column was eluted with 50mM PB +0.2mM EDTA and pH6.5 buffer solution, and then eluted with the eluents of examples 6-7, examples 9-12, examples 17-19, and comparative examples 2 and 4, respectively, to collect the eluted fractions, and the elution efficiency was calculated, and the results are shown in Table 5.
TABLE 5
The results show that: when the ionic liquid is used as a purification partner for elution, the elution efficiency of mRNA can be obviously improved. Wherein when the ionic liquid is [ C ] 4 mim][Cl]The elution efficiency was highest.
Furthermore, from the comparison results of examples 11 to 12, it is understood that the concentration of the ionic liquid in the eluate greatly affects the mRNA elution efficiency, and the effect is best when the concentration is 2M.
As is clear from the comparison results of examples 10, 11 and 17, [ C ] was selected when the concentrations were all 1M 4 mim][Cl]And [ Cho][BF 4 ]The combination of [ C ] is better than that of the ionic liquid with any single component, and the result shows that 4 mim][Cl]And [ Cho][BF 4 ]The two components are matched with each other, and have unexpected synergistic effect in the aspect of improving the mRNA elution efficiency.
Test example 6 determination of mRNA stability after elution
After mRNA was eluted with different eluents according to the method described in test example 5, the fractions were collected and left at 4 ℃ for 72 hours, and then the content of mRNA in the fractions was measured by the change in the gradation of agarose electrophoresis, and the remaining percentage (%) of mRNA was calculated to reflect the degradation of mRNA. And T of mRNA in the eluted fractions was determined according to the method described in test example 1 m The values reflect the structural stability of the mRNA and the results are shown in Table 6.
TABLE 6
The results show that: after the eluent containing the ionic liquid as the purification partner is used for elution, the stability of mRNA in the obtained elution component is obviously higher than that of the eluent which does not contain the ionic liquid.
From the comparison results of examples 6, 9, 10 and 11, it is understood that [ Cho ] is observed when the concentrations are the same][BF 4 ]And [ C 4 mim][Cl]The effect of improving the mRNA elution stability is better than that of other ionic liquids.
The concentration of the ionic liquid in the eluate had a certain influence on the mRNA elution stability, and the comparison between examples 6 to 7 and examples 11 to 12 revealed that the effect was better at 2M.
As is clear from the comparison results of examples 10, 11 and 17, [ C ] was selected when the concentrations were all 1M 4 mim][Cl]And [ Cho][BF 4 ]The combination of [ C ] is better than that of the ionic liquid with any single component, and the result shows that 4 mim][Cl]And [ Cho][BF 4 ]The mutual matching has unexpected synergistic effect in improving the elution stability of mRNA.
Test example 7 mRNA elution efficiency and post-elution stability test
Equilibrating the column with 50mM PB +0.2mM EDTA, pH6.5 buffer solution (Q Sepharose FF as chromatographic medium) at 25 deg.C, then feeding 142. mu.g/mL mRNA expressing green fluorescent protein into the column, rinsing the column with 50mM PB +0.2mM EDTA, pH6.5 buffer solution after feeding, then eluting with the eluents of examples 20 and 21 and comparative example 2, respectively, collecting the eluted fractions, calculating the elution efficiency, and determining the T of mRNA in the eluted fractions according to the method described in test example 1 m The value is obtained. The results are shown in Table 7.
TABLE 7
| Group of | Elution efficiency (%) | T m Value (. degree. C.) |
| Example 20 | 44 | 43.1 |
| Example 21 | 58 | 46.2 |
| Comparative example 2 | 41 | 41.5 |
The results show that: the ionic liquid is used as a purification partner, so that the secondary structure of RNA can be stabilized, the activity of ribonuclease can be inhibited, the elution efficiency of RNA on anion exchange chromatography can be improved, and the ionic liquid has great practical value.
Test example 8 mRNA stability test after elution (other chromatography)
Equilibrating the chromatographic column (oligo (dT)) with 20mM Tris-HCl +1.0M NaCl and pH7.5 buffer at 25 ℃, injecting 180. mu.g/mL mRNA expressing green fluorescent protein into the chromatographic column, eluting the chromatographic column with 50mM PB +0.2mM EDTA and pH6.5 buffer after injection, eluting with the eluent of example 22 and deionized water respectively, collecting the eluted components, and determining the T of the mRNA in the eluted components according to the method described in test example 1 m The value is obtained.
The results show that: t of mRNA eluted with the eluent of example 22 m The value is 42.1 ℃ and the T of the mRNA after elution with water as eluent m The value was 39.5 ℃ indicating that the use of ionic liquids as purification partners for other chromatographies also improved the stability of the RNA after elution.
The applicant states that the present invention is described by the above examples to describe a ribonucleic acid purification partner and applications thereof, but the present invention is not limited to the above examples, i.e., it is not meant to be construed that the present invention is necessarily dependent on the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A ribonucleic acid purification partner, wherein the ribonucleic acid purification partner is an ionic liquid consisting of a cation and an anion, and the purification method is a chromatography method, and the purification partner is used for simultaneously improving the elution efficiency of ribonucleic acid on a chromatography medium and improving the stability of the ribonucleic acid.
2. The ribonucleic acid purification partner of claim 1, wherein the ribonucleic acid comprises any one of mRNA, tRNA, rRNA, miRNA, snRNA, snoRNA, siRNA, saRNA, tmRNA, crRNA, tracrRNA, gRNA, ribozyme, viroid, or telase RNA, or a combination of at least two thereof;
preferably, the ribonucleic acid is extracted from an animal, plant, or microorganism, or is obtained by in vitro transcription of DNA;
preferably, the microorganism comprises yeast.
3. The ribonucleic acid purification partner according to claim 1 or 2, characterised in that the ionic liquid comprises an imidazole-type ionic liquid and/or a choline-type ionic liquid;
preferably, the cation in the imidazole type ionic liquid is [ C ] n mim] + ，n＝2-8；
Preferably, the cation in the choline-type ionic liquid is [ Cho] + 。
4. The ribonucleic acid purification partner of any one of claims 1 to 3, characterised in that the anion in the ionic liquid comprises [ Tf [ 2 N] - 、[SCN] - 、[N(CN) 2 ] - 、[TfO] - 、[MeOSO 3 ] - 、[Br] - 、[Cl] - 、[BF 4 ] - 、[EtOSO 3 ] - Or [ dca] - Any one or a combination of at least two of them.
5. The ribonucleic acid purification partner according to any of claims 1 to 4, characterised in that the chromatography medium comprises an anion exchange chromatography medium;
preferably, the ligand in the anion exchange chromatography medium comprises any one of TMAM, TEAE, hedam, QAE, DEAE, MP, GE, DEAHP, AE or PEI.
6. The ribonucleic acid purification partner according to any of claims 1 to 5, characterised in that the ionic liquid comprises [ Cho ] when the ribonucleic acid is extracted from yeast][EtOSO 3 ]、[C 8 mim][MeOSO 3 ]、[C 6 mim][Tf 2 N]、[C 4 mim][dca]、[Cho][Cl]、[Cho][BF 4 ]Or [ C 4 mim][Cl]Any one or a combination of at least two of them.
7. A ribonucleic acid purification partner according to any one of claims 1 to 6, characterised in that when the ribonucleic acid is mRNA, the ionic liquid comprises [ Cho][Cl]、[Cho][Br]、[C 4 mim][Cl]、[C 2 mim][Br]、[C 6 mim][Tf 2 N]Or [ C 4 mim][dca]Any one or a combination of at least two of them;
preferably, the ionic liquid comprises [ Cho][BF 4 ]And/or [ C 4 mim][Cl]；
Preferably, said [ Cho][BF 4 ]And [ C 4 mim][Cl]The concentration ratio of (1) to (4) is (3-6) to (7).
8. Use of a ribonucleic acid purification partner according to any one of claims 1 to 7 for the preparation of a chromatography eluent for the purification of ribonucleic acid.
9. A method for purifying ribonucleic acid, comprising feeding ribonucleic acid to be purified to a chromatography column containing a chromatography medium, eluting with an eluent comprising a ribonucleic acid purification partner of any of claims 1 to 7, and collecting the eluted fractions to complete the purification.
10. The method for purifying ribonucleic acid according to claim 9, wherein the elution solution is obtained by mixing water or a buffer solution comprising any one or a combination of at least two of acetate buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer or carbonate buffer with the ribonucleic acid purification partner;
preferably, the concentration of the buffer solution is 5-200 mM;
preferably, the pH value of the buffer solution is 6.0-8.5;
preferably, the concentration of the ribonucleic acid purification partner in the buffer solution is 0.05M-2M;
preferably, the eluent also contains inorganic salt, and the inorganic salt comprises any one or the combination of at least two of sodium chloride, ammonium sulfate, potassium chloride or ammonium sulfate;
preferably, the concentration of the inorganic salt in the eluent is 0.5-2M;
preferably, the manner of elution comprises isocratic elution or gradient elution, the gradient elution comprising linear gradient elution or step gradient elution;
preferably, the elution further comprises rinsing the chromatography medium with an equilibration buffer comprising any one or a combination of at least two of an acetate buffer, a phosphate buffer, a Tris-HCl buffer, a HEPES buffer, or a carbonate buffer.
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