CN107151262A - Functional polypeptide and its pharmaceutical applications - Google Patents
Functional polypeptide and its pharmaceutical applications Download PDFInfo
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- CN107151262A CN107151262A CN201710182954.2A CN201710182954A CN107151262A CN 107151262 A CN107151262 A CN 107151262A CN 201710182954 A CN201710182954 A CN 201710182954A CN 107151262 A CN107151262 A CN 107151262A
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- Prior art keywords
- functional polypeptide
- pyr
- polypeptide according
- quencher
- mirna
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 39
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 34
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 32
- 230000009368 gene silencing by RNA Effects 0.000 claims abstract description 21
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 claims abstract description 12
- 238000003745 diagnosis Methods 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 9
- 238000012216 screening Methods 0.000 claims abstract description 6
- 108091005804 Peptidases Proteins 0.000 claims abstract description 5
- 239000004365 Protease Substances 0.000 claims abstract description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 5
- 229920000724 poly(L-arginine) polymer Polymers 0.000 claims abstract description 4
- 239000004475 Arginine Substances 0.000 claims abstract description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract description 3
- 235000009697 arginine Nutrition 0.000 claims abstract description 3
- 210000004899 c-terminal region Anatomy 0.000 claims abstract description 3
- 125000005647 linker group Chemical group 0.000 claims abstract description 3
- 125000002755 pyrazolinyl group Chemical group 0.000 claims abstract description 3
- 238000010791 quenching Methods 0.000 claims abstract description 3
- 230000000171 quenching effect Effects 0.000 claims abstract description 3
- 238000010008 shearing Methods 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 239000000126 substance Substances 0.000 claims description 8
- OVBPIULPVIDEAO-LBPRGKRZSA-N Folic acid Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims description 5
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 claims description 4
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 claims description 4
- 235000019152 folic acid Nutrition 0.000 claims description 4
- 239000011724 folic acid Substances 0.000 claims description 4
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 125000003368 amide group Chemical group 0.000 claims description 3
- 229960000304 folic acid Drugs 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 2
- AOUOVFRSCMDPFA-QSDJMHMYSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-carboxypropanoyl]amino]-4-carboxybutanoyl]amino]-3-methylbutanoyl]amino]butanedioic acid Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(O)=O AOUOVFRSCMDPFA-QSDJMHMYSA-N 0.000 claims description 2
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 2
- 108010047702 MPG peptide Proteins 0.000 claims description 2
- 102100039560 Microtubule-associated protein RP/EB family member 1 Human genes 0.000 claims description 2
- 101710099411 Microtubule-associated protein RP/EB family member 1 Proteins 0.000 claims description 2
- 108010033276 Peptide Fragments Proteins 0.000 claims description 2
- 102000007079 Peptide Fragments Human genes 0.000 claims description 2
- 101710149951 Protein Tat Proteins 0.000 claims description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 2
- 101710107943 Trans-activator protein BZLF1 Proteins 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 2
- 125000003929 folic acid group Chemical group 0.000 claims description 2
- 229920002674 hyaluronan Chemical group 0.000 claims description 2
- 229960003160 hyaluronic acid Drugs 0.000 claims description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 claims description 2
- 150000002790 naphthalenes Chemical class 0.000 claims description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
- 108010059128 rabies virus glycoprotein peptide Proteins 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 150000003852 triazoles Chemical class 0.000 claims description 2
- HDBMWBJHOWINSB-UHFFFAOYSA-N O1C=NCC1.N1C=CC=C1 Chemical compound O1C=NCC1.N1C=CC=C1 HDBMWBJHOWINSB-UHFFFAOYSA-N 0.000 claims 1
- 239000002679 microRNA Substances 0.000 abstract description 25
- 230000003834 intracellular effect Effects 0.000 abstract description 24
- 108091070501 miRNA Proteins 0.000 abstract description 24
- 108020004459 Small interfering RNA Proteins 0.000 abstract description 19
- 230000008685 targeting Effects 0.000 abstract description 10
- 230000001225 therapeutic effect Effects 0.000 abstract description 8
- 229920002477 rna polymer Polymers 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 26
- 108091029119 miR-34a stem-loop Proteins 0.000 description 25
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 18
- 230000032258 transport Effects 0.000 description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 10
- 108091030071 RNAI Proteins 0.000 description 9
- 239000013642 negative control Substances 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 108060001084 Luciferase Proteins 0.000 description 7
- 239000005089 Luciferase Substances 0.000 description 7
- 230000006870 function Effects 0.000 description 7
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- 108090000623 proteins and genes Proteins 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 0 CC*(C)CCC(C)C(*(C)C)=*=CC1C=CC(*(CC2C=*C(*=C(C)C3IC3)=C(CC)*2)=C)=CC1 Chemical compound CC*(C)CCC(C)C(*(C)C)=*=CC1C=CC(*(CC2C=*C(*=C(C)C3IC3)=C(CC)*2)=C)=CC1 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 238000000034 method Methods 0.000 description 5
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 4
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 4
- 102000003952 Caspase 3 Human genes 0.000 description 4
- 108090000397 Caspase 3 Proteins 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- 239000006143 cell culture medium Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
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- 239000000243 solution Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
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- 239000002299 complementary DNA Substances 0.000 description 3
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- 230000010189 intracellular transport Effects 0.000 description 3
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
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- 238000012360 testing method Methods 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical class C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 102100031455 NAD-dependent protein deacetylase sirtuin-1 Human genes 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 108010041191 Sirtuin 1 Proteins 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 102000005936 beta-Galactosidase Human genes 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
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- -1 hexafluorophosphate Chemical compound 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000005292 vacuum distillation Methods 0.000 description 2
- VAKXPQHQQNOUEZ-UHFFFAOYSA-N 3-[4-[[bis[[1-(3-hydroxypropyl)triazol-4-yl]methyl]amino]methyl]triazol-1-yl]propan-1-ol Chemical compound N1=NN(CCCO)C=C1CN(CC=1N=NN(CCCO)C=1)CC1=CN(CCCO)N=N1 VAKXPQHQQNOUEZ-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- QUNWWUMHZUYPJM-UHFFFAOYSA-N C(C1=CC=CC=C1)(C1=CC=CC=C1)(C1=CC=CC=C1)Cl.[Cl] Chemical compound C(C1=CC=CC=C1)(C1=CC=CC=C1)(C1=CC=CC=C1)Cl.[Cl] QUNWWUMHZUYPJM-UHFFFAOYSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 1
- 108091007780 MiR-122 Proteins 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical class CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 1
- TZRXHJWUDPFEEY-UHFFFAOYSA-N Pentaerythritol Tetranitrate Chemical compound [O-][N+](=O)OCC(CO[N+]([O-])=O)(CO[N+]([O-])=O)CO[N+]([O-])=O TZRXHJWUDPFEEY-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229920003180 amino resin Polymers 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- LUEHNHVFDCZTGL-UHFFFAOYSA-N but-2-ynoic acid Chemical compound CC#CC(O)=O LUEHNHVFDCZTGL-UHFFFAOYSA-N 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- IBOVDNBDQHYNJI-UHFFFAOYSA-N dabcyl SE dye Chemical class C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(=O)ON2C(CCC2=O)=O)C=C1 IBOVDNBDQHYNJI-UHFFFAOYSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- FHIVAFMUCKRCQO-UHFFFAOYSA-N diazinon Chemical compound CCOP(=S)(OCC)OC1=CC(C)=NC(C(C)C)=N1 FHIVAFMUCKRCQO-UHFFFAOYSA-N 0.000 description 1
- 239000012954 diazonium Substances 0.000 description 1
- 150000001989 diazonium salts Chemical class 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- VDQVEACBQKUUSU-UHFFFAOYSA-M disodium;sulfanide Chemical compound [Na+].[Na+].[SH-] VDQVEACBQKUUSU-UHFFFAOYSA-M 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 229940064302 folacin Drugs 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
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- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- UORYAOZFMGBGAC-UHFFFAOYSA-N n-methylbenzenesulfonohydrazide Chemical compound CN(N)S(=O)(=O)C1=CC=CC=C1 UORYAOZFMGBGAC-UHFFFAOYSA-N 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000019624 protein content Nutrition 0.000 description 1
- DNXIASIHZYFFRO-UHFFFAOYSA-N pyrazoline Chemical compound C1CN=NC1 DNXIASIHZYFFRO-UHFFFAOYSA-N 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 229910052979 sodium sulfide Inorganic materials 0.000 description 1
- 235000019187 sodium-L-ascorbate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
Abstract
Functional polypeptide and its pharmaceutical applications, structure are as follows：Wherein R represents the arginine of L configurations, and X is linking group, and Y is targeted molecular, and Pyr is pyrazoline fluorogen, and Z is can be recognized the small peptide of shearing by different protease, and Quencher is the fluorescent quenching group corresponding to Pyr；Y or Pyr Z Quencher can be connected to the N-terminal or C-terminal of nine poly arginines.Functional polypeptide and its pharmaceutical applications that the present invention is provided, available for the intracellular targeting transport of siRNA and miRNA, treatment and diagnosis.The real time imagery of intracellular targeting transport, treatment and the therapeutic effect of small interference ribonucleic acid and miRNA can be realized.Suitable for preparing the RNA interference diagnosis and treatment medicine, and screening the RNA interference diagnosis and treatment medicine.
Description
Technical field
The invention belongs to chemical biology field, and in particular to a kind of functional polypeptide and its pharmaceutical applications, more particularly to：
(1) parcel, the targeting intracellular transport using functional polypeptide to above-mentioned two classes ribonucleic acid；(2) transported using functional polypeptide
The RNA interference treatment that defeated above-mentioned two classes ribonucleic acid is realized；(3) the RNA interference realized using functional polypeptide is controlled
The real-time assessment of therapeutic effect.
Background technology
The RNA interference (RNA interference, RNAi) is entering for internal specific target gene for discovered in recent years
The bioprocess of row regulation.During RNAi, small molecule non-coding RNA, such as endogenic miRNA
(microRNA, miRNA) and exogenous small interference ribonucleic acid (small interfering RNA, siRNA) can pass through alkali
The principle of base complementary pairing makes their target messenger RNA (mRNA) be degraded or suppress their translation.RNAi not only with
Body physiological process is closely related, and also plays the role of in numerous diseases develop very important.Thus,
SiRNA and miRNA have been considered as that the lead drug that a class of therapeutic purposes is new can be realized by RNAi in recent years.However,
Due to the elecrtonegativity of siRNA and miRNA in itself and the unstability in the complex physiologic environment such as serum, siRNA and miRNA's
Transport is there is still a need for extra carrier is realized, this requires carrier used to need with excellent biocompatibility, degradable
The simplicity of property and transport operation.In addition, still there is two key issues for needing to solve in RNAi therapeutic processes：Transport
During how to realize siRNA and miRNA targeting transport with improve RNAi treatment efficiency；How to be realized in transportation
The efficiency that the real time imagery of RNAi therapeutic effects is treated for assessment RNAi.
Polypeptide is the chemical molecular that a class has excellent biocompatibility, its can easily be modified by chemical means it is all kinds of not
Same functional group, with good biologic applications potentiality.In the present invention, inventor has invented can gather smart ammonia in cation nine
The two ends of acid introduce the molecule of target cell membrane surface receptor by specified chemical method, and RNAi therapeutic effects can be assessed
Image probe, the intracellular targeting that can realize small interference ribonucleic acid and miRNA using obtained functional polypeptide transports
Defeated, treatment and the real time imagery of therapeutic effect.
The content of the invention
The technical problem of solution：The present invention provides a kind of functional polypeptide and its pharmaceutical applications, and small interference ribose core can be achieved
Acid and miRNA intracellular targeting transport, treatment and therapeutic effect real time imagery.
Technical scheme：Functional polypeptide, structure is as follows：
Wherein R represents the arginine of L- configurations, and X is linking group, and YY is targeted molecular, and Pyr is pyrazoline fluorogen, Z
For that can be recognized the small peptide of shearing by different protease, Quencher is the fluorescent quenching group corresponding to Pyr；Y or Pyr-Z-
Quencher can be connected to the N-terminal or C-terminal of nine poly arginines.
Above-mentioned X be the alkyl containing one to ten carbon, oligomeric or many polyethylene glycol, disulfide bond, amido link, ester bond, ehter bond or
Triazole.
Above-mentioned Y is folic acid, hyaluronic acid, chain RGD peptide, ring-type RGD peptide, RVG peptides, tat peptide, MPG peptides, EB1 peptides, EGFR
Antibody or HER2 antibody.
Above-mentioned Z is protease substrate peptide fragment DEVD, PLGVR, GPGPNQ or KK.
Above-mentioned Quencher is Dabcyl, QSY21 or BHQ.
Above-mentioned Pyr is that luminous point hits chemical reaction fluorescence-causing substance pyrazoline, and chemical structure characteristic is as follows：
Wherein X or Z can be connected at an arbitrary position, Ar1And Ar2Respectively phenyl ring or naphthalene nucleus.
The preferred structure of functional polypeptide such as FA-R9-FPcas3It is shown：
Application of the above-mentioned functions polypeptide in the RNA interference diagnosis and treatment medicine is prepared.
Application of the above-mentioned functions polypeptide in screening the RNA interference diagnosis and treatment medicine.
Beneficial effect：Functional polypeptide and its pharmaceutical applications that the present invention is provided, available for the intracellular targets of siRNA and miRNA
To transport, treatment and diagnosis.Can realize the intracellular targeting transport of small interference ribonucleic acid and miRNA, treat with
And the real time imagery of therapeutic effect.Suitable for preparing the RNA interference diagnosis and treatment medicine, and screening the RNA interference diagnosis and treatment medicine
Thing.
Brief description of the drawings
Fig. 1 is FA-R9-FPcas3Mass spectrogram；
Fig. 2 is gel electrophoresis analysis figure；
Fig. 3 is nanoparticle size and current potential tendency chart；
Fig. 4 is the intracellular miR-34a transports quantitative analysis figures of HeLa；
Fig. 5 is the intracellular miR-34a transports quantitative analysis figure of HepG2, MCF-7, A549；Every group of columnar data is from left to right
It is followed successively by Blank, MIRNC-nc, MIRNC-34a, Lipo-34a.
Fig. 6 is HeLa intracellular luciferase experimental result pictures；
Fig. 7 is intracellular SIRT1, Caspase-3 protein expression the test result figures of HeLa；
Fig. 8 is HeLa Apoptosis result figures；
Fig. 9 is the intracellular miR-34a of HeLa function real time imagery result figure；
Figure 10 is that the intracellular Caspase-3 protein contents of HeLa change over time result figure.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.It is only exemplary for however, it should be understood that the embodiment, does not constitute any limitation to protection scope of the present invention.
, can be to the details and form of technical scheme it will be understood by those skilled in the art that without departing from the spirit of the invention
Modify or replace, but these modifications or substitutions each fall within protection scope of the present invention.
(1) preparation of functional polypeptide and siRNA or miRNA nano-complex
Functional polypeptide and siRNA or miRNA are diluted in high glucose medium or phosphate buffer with certain proportion, mixed
Room temperature is placed in after even to place 30 minutes.
(2) siRNA and miRNA targeting transport
The cell line that cell membrane surface is rich in above-mentioned targeting radical acceptor is chosen, by RNA nano-complexes obtained above
It is added to intracellular, after certain time, extracts intracellular rna and the expression with quantitative RT-PCR to intracellular siRNA or miRNA
Amount is measured.
(3) luciferase assay
The siRNA and miRNA transported by functional polypeptide stimulates intracellular luciferase signal change to judge that it is biological
Activity, idiographic flow is as follows：
(1) luciferase plasmids containing siRNA or miRNA binding sites are built；
(2) by after in plasmid transfection to test cell, corresponding siRNA or miRNA is transported with functional polypeptide；
Intracellular luciferase signal is determined after (3) 72 hours.
(4) protein blot experiment
Corresponding siRNA or miRNA is transported to intracellular using functional polypeptide, after culture different time, extracts cell
Interior total protein, is separated using SDS-PAGE, and by albumen be transferred on pvdf membrane after detected using corresponding antibodies.
(5) fluidic cell apoptosis is tested
Corresponding siRNA or miRNA is transported to intracellular using functional polypeptide.After culture different time, cell is collected,
Apoptotic cell is dyed using cell apoptosis detection kit, and quantitative analysis is carried out with flow cytometer.
(6) confocal fluorescent is imaged
Corresponding siRNA or miRNA is transported to intracellular using functional polypeptide.After culture different time, utilize Pyr's
Fluorescence carries out direct imaging to the function of the siRNA or miRNA of transport in the cell.
Embodiment 1, FA-R9-FPcas3Synthesis
1st, PA-R (Pbf) 9 synthesis
By standard solid-phase synthetic method, nine poly arginines are synthesized by solid phase carrier of chlorine trityl chloride resin, finally
Tetrolic acid is connected by O- BTAs-tetramethylurea hexafluorophosphate (HBTU) in end, 1% trifluoroacetic acid is subsequently added
(TFA)/dichloromethane (volume ratio) is cut off from resin, then with the isolated PA-R of HPLC (Pbf) 9.
2nd, 1B synthesis
By standard solid-phase synthetic method, DEVDK sequences are synthesized by solid phase carrier of amino resins, are finally passed through in end
HBTU connection 3- maleimidoproprionic acids, are subsequently added TFA excision blocking groups, then with the isolated intermediate products of HPLC.
By 37mg intermediate products (0.049mmol) and 18mg Dabcyl NHS esters (0.049mmol, lark prestige) (be dissolved in DMSO,
87 μ L DIPEAs (DIPEA, 0.5mmol) are added, room temperature reaction is stayed overnight, then with the isolated 1B of HPLC
(31mg, 62.9%).
3、Tet-NH2Synthesis
175mg compounds 1a (1mmol) is dissolved in dichloromethane, 205mg chloromethanes are then added under condition of ice bath
Sour isobutyl ester (1.5mmol) and 152mg 4- methyl morpholines (1.5mmol), are stirred at room temperature addition 237mg compounds after 4 hours
1b(1mmol).Continue at room temperature after reacting 4 hours, column chromatography for separation obtains 283mg 1c (71%).1c is dissolved in dichloromethane
In alkane, 50% (volume ratio) TFA is added, after reacting at room temperature 1 hour, 196mgTet-NH is obtained after solvent evaporated2(92.8%).
Wherein 1b synthetic method:
(1) compound 1b-1 (1.5g, 10mmol) and methanol is dissolved in Methyl benzenesulfonyl hydrazine (1.86g, 10mmol)
In (50mL) and water (50mL), it is stirred at room temperature 5 minutes, separates out a large amount of white solids, filter to obtain 1b-2 3g (yield 93%).
(2) diazonium salt of aniline is by the way that sodium nitrite solution (5mmol) cold 2mL is poured into containing 1.3mL below 5 DEG C
Prepared in the 50wt.% ethanol waters of the aniline (5mmol) of concentrated hydrochloric acid.Diazol is added drop-wise to 30mL dissolved with 1b
In the pyridine solution of (1.6g, 5mmol), controlling reaction temperature is at -10 DEG C to -15 DEG C.Follow-up continuous reaction completely 3 hours is added dropwise
Afterwards, with dichloromethane and water extractive reaction, organic layer is washed with watery hydrochloric acid after merging and uses anhydrous sodium sulfate drying.Vacuum distillation
Remove after solvent, propose to obtain yellow powder 1b-3 0.7g (yield 53%) by column chromatography.
(3) 1b-3 (0.6g, 2.5mmol) is dissolved in Dioxane (10mL), is heated to reflux (80 DEG C) Na2S (3g,
The aqueous solution (5mL) 7.5mmol) is added drop-wise in Dioxane.Continue to be heated to reflux 2 hours, dichloromethane and water extractive reaction,
Organic layer uses anhydrous sodium sulfate drying after merging.Vacuum distillation is removed after solvent, proposes to obtain yellow powder 1b by column chromatography
0.5g (yield 80%).
4、FA-N3Synthesis
44mg folic acid (2a, 0.1mmol) is dissolved in DMSO, addition 51mg dicyclohexylcarbodiimides (DCC,
0.2mmol) and 22mg n-hydroxysuccinimides (NHS, 0.2mmol).After room temperature reaction overnight, precipitation is filtered off, is added
The polyethylene glycol nitrine (2b, 0.2mmol, TCI) of 44mg amino three and 61mg DMAPs (DMAP, 0.5mmol),
After room temperature reaction 4 hours, with the isolated FA-N of HPLC3(15mg, 23.4%).
5th, 1A synthesis
By 175mg PA-R (Pbf) 9 (0.046mmol) and 14mg amido modified tetrazole molecule (Tet-NH2)
(0.046mmol) is dissolved in dichloromethane, is subsequently added 30mg HBTU (0.08mmol) and 28 μ L N, N- diisopropyls
Base ethamine (DIPEA, lark prestige) (0.016mmol).Mixed liquor is stirred at room temperature overnight, and is boiled off after solvent, adds TFA excisions
Blocking group, then with the isolated 1A of HPLC (41mg, 50.1%).
5、R9-FPcas3Synthesis
18mg 1A (0.01mmol) and 20mg 1B (0.02mmol) are dissolved in acetonitrile/PBS mixed solution
(CH3CN/PBS, volume ratio 1/1), the portable UV illumination for being then 302nm with launch wavelength 30 minutes uses HPLC immediately
Isolated R9-FPcas3(18mg, 65%).
6、FA-R9-FPcas3Synthesis
By 10mg R9-FPcas3(0.0036mmol) and 5mg FA-N3(0.0072mmol) is dissolved in DMSO/H2O's is mixed
Close (DMSO/H in solution2O, volume ratio 2/1), it is subsequently added 200mg sodium ascorbates, 3.2mg copper sulphate, 50mg's
THPTA, reacts 4 hours at 30 DEG C.Then with the isolated FA-R9-FP of HPLCcas3(3.6mg, 29.4%).Molecular weight is theoretical
It is worth for 3398.7425, actual measured value is 3399.4939 (Fig. 1).
FA-R9-FPcas3To miR-34a parcel
100pmole miR-34a are dissolved in 100 μ L DMEM, after by 0.2 μ L, 0.4 μ L, 0.6 μ L, 0.8 μ L 5mM
FA-R9-FPcas3It is added separately in DMEM, obtains the mixed liquor of several groups of different mol ratios, mixed liquor is placed in room temperature and placed
30 minutes.
Gel electrophoresis analysis
2% Ago-Gel is prepared, takes above-mentioned mixed liquor to carry out electrophoretic analysis respectively, and with ethidium bromide to miR-34a
Developed the color.As shown in Fig. 2 in FA-R9-FPcas3Ratio when gradually increasing, free miR-34a fades away, and shows two
The nano-complex of person is increasingly generated, and the size and current potential of compound also change (Fig. 3) therewith.
Target intracellular transport
HeLa cell kinds are entered in 12 orifice plates, after 24 hours, by miR-34a or negative control nc and FA-R9-FPcas3's
Mixed liquor is added in cell culture medium, miR-34a or negative control nc final concentration of 100nM, after cultivating 24 hours, is used
TRIZOL extracts intracellular rna.As shown in figure 4, working as FA-R9-FPcas3Molar ratio when gradually increasing, into intracellular
MiR-34a amount also gradually increases, and optimal transhipment effect is reached when molar ratio is 30/1, and the compound of the ratio is defined as
MIRNC-34a, negative control is MIRNC-nc, and transport efficacy is suitable with commercialized reagent Lipofectamine 2000.
Take negative HepG2, MCF-7, A549 cell of folacin receptor as control, carry out above-mentioned identical experiment.Such as Fig. 5 institutes
Show, MIRNC-34a transports the less efficient of miR-34a into negative cells, demonstrate the ability of targeting transport.
The measure of miRNA expression quantity
The RNA progress reverse transcription reactions for taking out 1 μ g prepare corresponding cDNA samples.The specific μ L systems of reverse transcription reaction 10
For：
5×AMV buffer(Takara)2μL
AMV(Takara)0.5μL
RT primer(ABI)0.5μL
dNTP mixture(Takara)1μL
DEPC H2O 5μL
RNA 1μL
Response procedures are：16 DEG C of 30min, 42 DEG C of 30min, 85 DEG C of 5min, 4 DEG C of preservations.The cDNA prepared uses probe again
Method (Taqman) PCR carries out real-time quantitative to miR-122.
20 μ L Taqman PCR reaction systems are：
10×buffer(Takara)2μL
MgCl2(25mM)(Takara)1.2μL
Taq(Takara)0.3μL
dNTP mixture(10mM)0.4μL
Probe(ABI)0.33μL
cDNA 1μL
Distilled H2O 14.77μL
PCR response procedures are 60 DEG C of 1min, 40 cyclic amplifications after 95 DEG C of 5min, 95 DEG C of 15s, and real-time fluorescent signals exist
The 60 DEG C of collections each circulated.
Luciferase assay
(1) structure of reporter plasmid
The 3 ' of Luciferase reporter plasmids (Promega) is sheared using HindIII and Spel (I Takara) restriction endonuclease
UTR ends, electrophoretic separation purifying is carried out by obtained segment.MiR-34a complementary series respectively by DNA ligase,
T4ligase (Takara), is inserted into the luciferase reporter plasmids that purifying is obtained, and be transformed into competence Escherichia coli
It is interior, then possible positive plasmid is obtained by the screening flat board screening containing ammonia benzyl.Most this report plasmid be sequenced really at last
Recognize its correctness (Invitrogen).
(2) luciferase assay
Reporter plasmid after identification is entered in HeLa cells by transfecting.Cell kind is worked as into cell into 24 orifice plates first
When density reaches 70-80%, 0.25 μ g miR-34a reporter plasmids and 0.15 μ g β-galactosidase internal references plasmid are used
The reagents of Lipofectamine 2000 are transfected into cell, and transfection procedure follows the Invitrogen descriptions of product.In transfection 4 hours
Afterwards, by miR-34a or negative control nc FA-R9-FPcas3Transport in cell (mol ratio 1/30), miR-34a or negative is right
According to nc final concentration of 100nM, continue after cultivating 72 hours, determine luciferase and β-galactosidase internal reference signals.
As shown in fig. 6, MIRNC-34a still has the function of suppressing expression of target gene to the miR-34a of intracellular transport.
Protein blot experiment
HeLa cell kinds are entered in 6 cm dishes, after 24 hours, by miR-34a or negative control nc and FA-R9-
FPcas3 mixed liquor (mol ratio 1/30) is added in cell culture medium, and miR-34a or negative control nc's is final concentration of
300nM, after cultivating 24 hours to 72 hours, extracts protein lysate.By protein lysate and 1 × SDS loading buffer
After mixing, selecting various concentrations according to the size of albumen, (SDS-PAGE of general 10%) carries out Protein Separation to sample.Albumen
After being successfully separated, go to albumen is wet on pvdf membrane, then entered with the TBST/Tween-20 buffer solutions containing 5wt.% skimmed milk powers
1 hour sealer of row, then with 4 DEG C of night incubations of primary antibody, TBST/Tween-20 10min are washed four times, and secondary antibody incubation at room temperature 1 is small
When, then washed four times with TBST/Tween-20 10min, finally with ECL reagents development protein band.As shown in fig. 7, compared to
Blanc cell or the cell of MIRNC-nc processing, the intracellular miR-34a of MIRNC-34a processing direct target SIRT1 table
It is remarkably decreased up to amount, and the Caspase-3 expression quantity in downstream significantly rises, and shows that miR-34a has adjusting function.
Streaming apoptosis test experience
HeLa cell kinds are entered in 6 orifice plates, after 24 hours, by miR-34a or negative control nc and FA-R9-FPcas3
Mixed liquor (mol ratio 1/30) is added in cell culture medium, miR-34a or negative control nc final concentration of 300nM, culture
After 24 hours to 72 hours, collect cell and dyed with streaming apoptosis detection kit, then entered with fluidic cell ceremony
The quantitative detection of row.As shown in figure 8, the cell handled compared to blanc cell or MIRNC-nc, the cell of MIRNC-34a processing withers
Die notable rising.
Intracellular miR-34a functions real time imagery experiment
HeLa cell kinds are entered in the burnt culture dish of 35 millimeters of copolymerization, after 24 hours, by miR-34a's and FA-R9-FPcas3
Mixed liquor (mol ratio 1/30) is added in cell culture medium, miR-34a final concentration of 300nM, is cultivated 24 hours small to 72
Shi Hou, directly observes Pyr fluorescence under confocal fluorescent microscope, and nucleus is dyed from DRAQ5.As shown in figure 9,
After MIRNC-34a processing, also gradually strengthen with incubation time corresponding to the Pyr fluorescence of activation Caspase-3 expression quantity, and cell
Phenotype also points out cell gradually apoptosis.And western blot result also demonstrates imaging results (Figure 10).
The above-mentioned embodiment technical scheme that the invention is not limited in any way, every use equivalent substitution or is waited
The technical scheme that the mode of effect conversion is obtained all falls within protection scope of the present invention.
Claims (9)
1. functional polypeptide, it is characterised in that structure is as follows：
Wherein R represents the arginine of L- configurations, and X is linking group, and Y is targeted molecular, and Pyr is pyrazoline fluorogen, and Z is can quilt
The small peptide of different protease identification shearings, Quencher is the fluorescent quenching group corresponding to Pyr；Y or Pyr-Z-Quencher
The N-terminal or C-terminal of nine poly arginines can be connected to.
2. functional polypeptide according to claim 1, it is characterised in that the X is the alkyl containing one to ten carbon, it is oligomeric or
Many polyethylene glycol, disulfide bond, amido link, ester bond, ehter bond or triazole.
3. functional polypeptide according to claim 1, it is characterised in that the Y is folic acid, hyaluronic acid, chain RGD peptide, ring
Shape RGD peptide, RVG peptides, tat peptide, MPG peptides, EB1 peptides, EGFR antibody or HER2 antibody.
4. functional polypeptide according to claim 1, it is characterised in that the Z be protease substrate peptide fragment DEVD, PLGVR,
GPGPNQ or KK.
5. functional polypeptide according to claim 1, it is characterised in that the Quencher is Dabcyl, QSY21 or BHQ.
6. functional polypeptide according to claim 1, it is characterised in that the Pyr is that luminous point hits chemical reaction fluorescence-causing substance pyrrole
Oxazoline, chemical structure characteristic is as follows：
Wherein X or Z can be connected at an arbitrary position, Ar1And Ar2Respectively phenyl ring or naphthalene nucleus.
7. functional polypeptide according to claim 1, it is characterised in that structure such as FA-R9-FPcas3It is shown：
8. application of any functional polypeptide of claim 1~7 in the RNA interference diagnosis and treatment medicine is prepared.
9. application of any functional polypeptide of claim 1~7 in screening the RNA interference diagnosis and treatment medicine.
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| WO2011133948A3 (en) * | 2010-04-22 | 2012-01-05 | Longevity Biotech, Inc. | Highly active polypeptides and methods of making and using the same |
| CN105315345A (en) * | 2015-10-29 | 2016-02-10 | 南京大学 | Targeting arginine nonamer and application thereof to RNA interference |
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2017
- 2017-03-24 CN CN201710182954.2A patent/CN107151262A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011133948A3 (en) * | 2010-04-22 | 2012-01-05 | Longevity Biotech, Inc. | Highly active polypeptides and methods of making and using the same |
| CN105315345A (en) * | 2015-10-29 | 2016-02-10 | 南京大学 | Targeting arginine nonamer and application thereof to RNA interference |
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