CN119462803A - Synthesis method and application of nucleoside monomer analogs and oligonucleotides - Google Patents
Synthesis method and application of nucleoside monomer analogs and oligonucleotides Download PDFInfo
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- CN119462803A CN119462803A CN202510069353.5A CN202510069353A CN119462803A CN 119462803 A CN119462803 A CN 119462803A CN 202510069353 A CN202510069353 A CN 202510069353A CN 119462803 A CN119462803 A CN 119462803A
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- 108091034117 Oligonucleotide Proteins 0.000 title claims abstract description 260
- 239000000178 monomer Substances 0.000 title claims abstract description 169
- 239000002777 nucleoside Substances 0.000 title claims abstract description 127
- 150000003833 nucleoside derivatives Chemical class 0.000 title claims abstract description 123
- 238000001308 synthesis method Methods 0.000 title claims abstract description 16
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 title abstract description 21
- 230000002441 reversible effect Effects 0.000 claims abstract description 46
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 20
- 239000001226 triphosphate Substances 0.000 claims abstract description 19
- 235000011178 triphosphate Nutrition 0.000 claims abstract description 19
- -1 methoxy, methoxyethyl Chemical group 0.000 claims abstract description 14
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 14
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000001177 diphosphate Substances 0.000 claims abstract description 10
- 235000011180 diphosphates Nutrition 0.000 claims abstract description 10
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims abstract description 9
- 150000004712 monophosphates Chemical class 0.000 claims abstract description 9
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 8
- 239000001257 hydrogen Substances 0.000 claims abstract description 8
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims abstract description 7
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims abstract description 7
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims abstract description 7
- 125000001153 fluoro group Chemical group F* 0.000 claims abstract description 5
- 239000000758 substrate Substances 0.000 claims description 144
- 125000003729 nucleotide group Chemical group 0.000 claims description 60
- 239000002773 nucleotide Substances 0.000 claims description 58
- 230000004048 modification Effects 0.000 claims description 56
- 238000012986 modification Methods 0.000 claims description 56
- 238000000034 method Methods 0.000 claims description 33
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 30
- 230000015572 biosynthetic process Effects 0.000 claims description 29
- 238000003786 synthesis reaction Methods 0.000 claims description 29
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 20
- 102000003960 Ligases Human genes 0.000 claims description 15
- 108090000364 Ligases Proteins 0.000 claims description 15
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 12
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 12
- 229940035893 uracil Drugs 0.000 claims description 12
- 230000002194 synthesizing effect Effects 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 102000001253 Protein Kinase Human genes 0.000 claims description 7
- 229940104302 cytosine Drugs 0.000 claims description 7
- PISWNSOQFZRVJK-XLPZGREQSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methyl-2-sulfanylidenepyrimidin-4-one Chemical compound S=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 PISWNSOQFZRVJK-XLPZGREQSA-N 0.000 claims description 6
- UHUHBFMZVCOEOV-UHFFFAOYSA-N 1h-imidazo[4,5-c]pyridin-4-amine Chemical compound NC1=NC=CC2=C1N=CN2 UHUHBFMZVCOEOV-UHFFFAOYSA-N 0.000 claims description 6
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 claims description 6
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 claims description 6
- ZTWYAIASAJSBMA-UHFFFAOYSA-N 8-azido-7h-purin-6-amine Chemical compound NC1=NC=NC2=C1NC(N=[N+]=[N-])=N2 ZTWYAIASAJSBMA-UHFFFAOYSA-N 0.000 claims description 6
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 claims description 6
- 229930024421 Adenine Natural products 0.000 claims description 6
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 229960000643 adenine Drugs 0.000 claims description 6
- JOZGNYDSEBIJDH-UHFFFAOYSA-N eniluracil Chemical compound O=C1NC=C(C#C)C(=O)N1 JOZGNYDSEBIJDH-UHFFFAOYSA-N 0.000 claims description 6
- 108020000161 polyphosphate kinase Proteins 0.000 claims description 6
- 238000010189 synthetic method Methods 0.000 claims description 6
- 229940113082 thymine Drugs 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 108010092060 Acetate kinase Proteins 0.000 claims description 3
- 102000002281 Adenylate kinase Human genes 0.000 claims description 3
- 108020000543 Adenylate kinase Proteins 0.000 claims description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
- 102000013901 Nucleoside diphosphate kinase Human genes 0.000 claims description 3
- 108010011356 Nucleoside phosphotransferase Proteins 0.000 claims description 3
- 229920000388 Polyphosphate Polymers 0.000 claims description 3
- 108020005115 Pyruvate Kinase Proteins 0.000 claims description 3
- 102000013009 Pyruvate Kinase Human genes 0.000 claims description 3
- 101710086015 RNA ligase Proteins 0.000 claims description 3
- 101710100179 UMP-CMP kinase Proteins 0.000 claims description 3
- 101710119674 UMP-CMP kinase 2, mitochondrial Proteins 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- 238000006555 catalytic reaction Methods 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 239000001205 polyphosphate Substances 0.000 claims description 3
- 235000011176 polyphosphates Nutrition 0.000 claims description 3
- 102000012410 DNA Ligases Human genes 0.000 claims 1
- 108010061982 DNA Ligases Proteins 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 125000003396 thiol group Chemical class [H]S* 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- 238000002515 oligonucleotide synthesis Methods 0.000 abstract description 3
- 125000004435 hydrogen atom Chemical class [H]* 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 77
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 54
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 28
- 238000001514 detection method Methods 0.000 description 27
- 239000000047 product Substances 0.000 description 25
- 102000004190 Enzymes Human genes 0.000 description 19
- 108090000790 Enzymes Proteins 0.000 description 19
- 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 description 18
- 239000006228 supernatant Substances 0.000 description 18
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 18
- 230000000415 inactivating effect Effects 0.000 description 17
- 238000005119 centrifugation Methods 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 239000011780 sodium chloride Substances 0.000 description 14
- 230000002255 enzymatic effect Effects 0.000 description 10
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 10
- 229910019142 PO4 Inorganic materials 0.000 description 9
- 235000021317 phosphate Nutrition 0.000 description 9
- 230000008878 coupling Effects 0.000 description 8
- 238000010168 coupling process Methods 0.000 description 8
- 238000005859 coupling reaction Methods 0.000 description 8
- 230000000903 blocking effect Effects 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 125000002264 triphosphate group Chemical group [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 7
- 238000006911 enzymatic reaction Methods 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 5
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 239000010452 phosphate Substances 0.000 description 5
- 235000011007 phosphoric acid Nutrition 0.000 description 5
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 4
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 4
- 108091028664 Ribonucleotide Proteins 0.000 description 4
- 229910052731 fluorine Inorganic materials 0.000 description 4
- 239000011737 fluorine Substances 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 239000002336 ribonucleotide Substances 0.000 description 4
- 125000002652 ribonucleotide group Chemical group 0.000 description 4
- 150000002431 hydrogen Chemical class 0.000 description 3
- 150000003016 phosphoric acids Chemical class 0.000 description 3
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
- 241000235346 Schizosaccharomyces Species 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
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- 239000003814 drug Substances 0.000 description 2
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- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 241000589496 Meiothermus ruber Species 0.000 description 1
- 241001460678 Napo <wasp> Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
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- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 108091008104 nucleic acid aptamers Proteins 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
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- 239000002904 solvent Substances 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
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- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical group [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
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- 230000002588 toxic effect Effects 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
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- Saccharide Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a synthesis method and application of nucleoside monomer analogues and oligonucleotides. R on the nucleoside monomer is a base, R 1 comprises one or more of methoxy, methoxyethyl, fluoro, hydroxy or hydrogen, R 2 comprises one or more of hydroxy, monophosphate, diphosphate, triphosphate or thio modified phosphate, and R 3 comprises one or more of methyl, ethyl, propyl or isopropyl. Can solve the problem of low reversible end-capping activity of the oligonucleotide in the prior art, and is suitable for the field of oligonucleotide synthesis.
Description
Technical Field
The invention relates to the field of oligonucleotide synthesis, in particular to a nucleoside monomer analogue, a synthesis method and application of an oligonucleotide.
Background
Oligonucleotides are basic tools for regulating gene expression in biomedical and life science research, and are used as gene targeting therapeutic drugs for treating various diseases such as genetic diseases, viruses, tumors and the like. The oligonucleotide medicine mainly comprises antisense oligonucleotide, small interfering ribonucleic acid, micro ribonucleic acid, nucleic acid aptamer and the like. Natural oligonucleotides are easily degraded in vivo and have low pharmaceutical properties, so that drug oligonucleotides usually have specific modification groups such as phosphorothioate linkages, fluoro groups, methyl groups, locked nucleic acids and the like, so as to enhance the stability of the oligonucleotides in vivo, improve the specificity and reduce the toxic and side effects thereof.
The synthesis method of the oligonucleotide mainly adopts a phosphoramidite method, but has the defects of low purity of an initial product, large chemical solvent consumption, difficult large-scale amplification and the like, so that the method for synthesizing the oligonucleotide by the novel enzyme method is focused on because of the advantages of green synthesis technology, high coupling efficiency, easy amplification and the like.
The nucleotide analogue with the reversible blocking group is a key raw material for synthesizing the oligonucleotide by an enzymatic method, the specific synthesis of the oligonucleotide is realized by utilizing the reversible blocking group, the introduction of an error sequence is prevented, and meanwhile, the blocking group can be removed without trace after the synthesis is finished so as to realize the synthesis of the target oligonucleotide.
However, the prior art has fewer reversible blocking group types and relatively low activity, which results in low coupling efficiency with the oligonucleotide chain, and thus the synthesis of the target oligonucleotide is hindered.
Disclosure of Invention
The invention mainly aims to provide a nucleoside monomer analogue, a synthesis method and application of an oligonucleotide, and aims to solve the problem of low reversible end capping activity of the oligonucleotide in the prior art.
In order to achieve the above object, according to a first aspect of the present invention, there is provided a nucleoside monomer analog having the structural formula shown in formula I;
A formula I;
wherein R is a base, R1 comprises one or more of methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen;
R2 comprises one or more of hydroxyl, monophosphate, diphosphate, triphosphate or thio-modified phosphate, and R3 comprises one or more of methyl, ethyl, propyl or isopropyl.
Further, the bases include natural bases or unnatural bases, the natural bases include adenine, guanine, cytosine, thymine, or uracil, the unnatural bases include one or more of 3-deazaadenine, 7-deazaguanine, 2, 6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyl uracil, C7 modified deazaadenine, C7 modified deazaguanine, C5 modified cytosine, or C5 modified uracil, wherein the modifications include one or more of methyl modifications, H modifications, cl modifications, or F modifications.
According to a second aspect of the present invention there is provided a nucleotide monomer analogue of formula II:
A formula II;
Wherein R is a base, R4 comprises one or more of methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen, R5 comprises one or more of methyl, ethyl, propyl or isopropyl, and R6 comprises hydroxyl or mercapto.
Further, the bases include natural bases or unnatural bases, the natural bases include adenine, guanine, cytosine, thymine, or uracil, the unnatural bases include one or more of 3-deazaadenine, 7-deazaguanine, 2, 6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyl uracil, C7 modified deazaadenine, C7 modified deazaguanine, C5 modified cytosine, or C5 modified uracil, wherein the modifications include one or more of methyl modifications, H modifications, cl modifications, or F modifications.
According to a third aspect of the present application there is provided a method of synthesizing an oligonucleotide having a 3' reversibly modified end, the method comprising, a) when the oligonucleotide chain substrate is a single piece, ligating the above nucleoside monomer analogue, or the above nucleotide monomer analogue, to the 3' end of the oligonucleotide chain substrate to obtain an oligonucleotide having a 3' reversibly modified end;
Or b) when the number of the oligonucleotide chain substrates is two, the oligonucleotide chain substrates comprise a first oligonucleotide chain substrate and a second oligonucleotide chain substrate, wherein the nucleotide monomer analogue or the nucleotide monomer analogue is connected to the 3 'end of the first oligonucleotide chain substrate, and the first oligonucleotide chain substrate and the second oligonucleotide chain substrate are connected to obtain the oligonucleotide with the 3' end reversible modified end.
Further, the oligonucleotide strand substrate includes a natural oligonucleotide strand composed of natural nucleotides, or a non-natural oligonucleotide strand containing non-natural nucleotides.
Further, the non-natural oligonucleotide strand consists of non-natural nucleotides.
Further, when R2 of the nucleoside monomer analogue is hydroxyl, monophosphate, diphosphate or thio-modified phosphate, the synthesis method comprises a-1) mixing the nucleoside monomer analogue with polyphosphate, catalyzing with a phosphokinase to form a nucleoside monomer analogue with R2 being triphosphate, when the oligonucleotide chain substrate is single, catalyzing with a polymerase a nucleoside monomer analogue with R2 being triphosphate to be linked to the 3 'end of the oligonucleotide chain substrate to obtain an oligonucleotide with a 3' end reversibly modified end, b-1) when the oligonucleotide chain substrate is two, wherein the nucleoside monomer analogue is linked to the 3 'end of the first oligonucleotide chain substrate, catalyzing with a ligase the first oligonucleotide chain substrate and the second oligonucleotide chain substrate to obtain an oligonucleotide with a 3' end reversibly modified end, wherein the phosphokinase comprises one or more of acetate kinase, pyruvate kinase, adenylate kinase, polyphosphate kinase, nucleoside kinase or nucleoside diphosphate kinase.
Further, when R2 of the nucleoside monomer analog is triphosphate, the synthesis method comprises a-2) when the oligonucleotide chain substrate is single, catalyzing the nucleoside monomer analog of which R2 is triphosphate to be linked to the 3 'end of the oligonucleotide chain substrate by using polymerase, thereby obtaining an oligonucleotide having a 3' end reversibly modified end, and b-2) when the oligonucleotide chain substrate is two, wherein the nucleoside monomer analog linked to the 3 'end of the first oligonucleotide chain substrate is linked to the second oligonucleotide chain substrate by using ligase, thereby obtaining an oligonucleotide having a 3' end reversibly modified end.
Further, the synthesis method comprises a-3) when the oligonucleotide chain substrate is a single strand, catalyzing the ligation of the nucleotide monomer analogue to the 3 'end of the oligonucleotide chain substrate using a polymerase, thereby obtaining an oligonucleotide having a reversibly modified end of the 3' end;
b-3) when there are two oligonucleotide chain substrates, wherein the above-mentioned nucleotide monomer analogue is attached to the 3 'end of the first oligonucleotide chain substrate, the first oligonucleotide chain substrate and the second oligonucleotide chain substrate are attached by using ligase to catalyze the attachment of the first oligonucleotide chain substrate and the second oligonucleotide chain substrate, thereby obtaining an oligonucleotide having a 3' end reversibly modified end.
Further, the polymerase includes a PUP polymerase and the ligase includes a T4 ligase.
According to a fourth aspect of the present application, there is provided a method for producing an oligonucleotide, comprising removing the reversibly modified end of the 3' -end of an oligonucleotide having a reversibly modified end synthesized by any one of the above synthetic methods, to obtain an oligonucleotide.
Further, the removing comprises mixing the oligonucleotide with the 3 '-end reversible modified end with the first solution to obtain a mixed system, and removing the 3' -end reversible modified end by using the mixed system.
Further, the pH of the mixed system is 4-6, and the mixed system is maintained at the temperature of 20-40 ℃ for 5-10 min, so that the removal of the 3' -end reversible modification end is realized.
Further, the first solution includes any one or more of hydrochloric acid, sulfuric acid, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, citric acid, or boric acid.
Further, the concentration of the solute in the first solution is 0.01 mM-10M.
According to a fifth aspect of the present application there is provided a method of synthesis of any one of the nucleoside monomer analogues described above, or any one of the nucleotide monomer analogues described above, or any one of the oligonucleotides having a 3' reversibly modified end described above, or a method of preparation of an oligonucleotide of any one of the above, for use in the preparation of an oligonucleotide.
By applying the technical scheme of the invention, the nucleoside monomer analogue with the structure shown in the formula I can be used for connecting a 3 '-reversible modified end to an oligonucleotide chain to successfully synthesize the oligonucleotide with the 3' -reversible modified end, and compared with the reversible end capping commonly used in the prior art, the oligonucleotide has higher activity and higher substrate conversion rate, and the target oligonucleotide is synthesized after the reversible modification is further removed.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 shows a schematic diagram of the results of UPLC detection of two products of phosphoric acid attached to reaction system 1 according to example 11 of the present invention.
FIG. 2 shows a schematic representation of the results of UPLC detection of the products of linked nucleoside monomers according to example 12 of the present invention.
FIG. 3 shows a schematic representation of the results of UPLC detection of a product with the 3' reversible closed end removed according to example 12 of the present invention.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to examples.
Term interpretation:
Nucleosides, which are compounds of a base (purine or pyrimidine base) condensed with a five-carbon sugar (including, but not limited to, ribose or deoxyribose), are free of phosphate.
Nucleotides are formed by the combination of bases (purine or pyrimidine bases), five-carbon sugars (including, but not limited to, ribose or deoxyribose) and phosphates, including nucleoside monophosphates, nucleoside diphosphate and nucleoside triphosphate.
Nucleoside monomer refers to a mononucleoside with a length of 1nt in the present application.
Nucleotide monomer refers to a single nucleotide with the length of 1nt in the application.
As mentioned in the background, the 3' -end reversible blocking group of the oligonucleotide is less active in the prior art, resulting in a low efficiency of ligation to the oligonucleotide strand, which prevents synthesis of the target oligonucleotide. Thus, the inventors tried to develop a new nucleoside monomer capable of connecting the 3' -end reversible blocking group with the oligonucleotide by an enzymatic method, improving the conversion rate of the substrate, having higher activity than the reversible blocking end capping of the prior art, and proposed a series of protection schemes of the present application.
In a first exemplary embodiment of the present application, there is provided a nucleoside monomer analog having the structural formula shown in formula I;
A formula I;
wherein R is a base, R1 comprises one or more of methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen, R2 comprises one or more of hydroxyl, monophosphate, diphosphate, triphosphate or thio-modified phosphate, and R3 comprises one or more of methyl, ethyl, propyl or isopropyl.
The nucleoside monomer analogue comprises a nucleoside monomer analogue, when the group at R2 is mono-, di-, tri-or thio-modified phosphoric acid, the nucleoside monomer is a nucleoside monomer analogue, and when the group at R2 is hydroxy, the nucleoside monomer is a nucleoside monomer analogue.
In the system of the oligonucleotide synthesized by the enzymatic method in the prior art, the early research of the application discovers that for certain specific reactions, the reaction efficiency and the reversible end capping of the 3' end are related, so that the commonly used end capping activity is poor, the substrate conversion rate is low, and the target oligonucleotide is difficult to synthesize in the follow-up process.
The application utilizes the nucleoside monomer analogue with the structural formula shown in the formula I to be coupled with the oligonucleotide chain substrate in a system for synthesizing the oligonucleotide by an enzymatic method, so that the 3' -end of the oligonucleotide chain substrate is successfully connected with the reversible closed end, and the substrate conversion rate is high. The method plays a role in synthesizing the oligonucleotide, prevents the connection of wrong sequences, can be removed by a simple acidolysis method, and the oligonucleotide product with the removed groups can be used as an oligonucleotide chain substrate of a next round of coupling nucleoside monomers, thereby realizing the specific synthesis of the target oligonucleotide of the specific sequence by an enzymatic method.
That is, the present application has developed a novel reversible end-capping which is different from the prior art, and when there is the above-mentioned phenomenon of low substrate conversion rate associated with the 3' -end reversible end-capping in the reaction, the present application provides a variety of reversible end-capping options, which can overcome the associated phenomenon, and facilitate the synthesis of various types of target oligonucleotides. When the R2 group of the nucleoside monomer analogue is a triphosphate group, the nucleoside monomer is a nucleotide monomer analogue, and the structural formula of the nucleoside monomer analogue is shown in a formula III. Under the catalysis of polymerase, a single oligonucleotide chain substrate is used as a template, and a nucleotide monomer shown in a formula III can be directly coupled with the oligonucleotide chain substrate to synthesize the oligonucleotide with the reversible closed end at the 3' end.
Formula III.
In a preferred embodiment, the base comprises a natural base or a non-natural base. The natural base comprises natural adenine, natural guanine, natural cytosine, natural thymine or natural uracil, and the unnatural base comprises one or more of 3-deazaadenine, 7-deazaguanine, 2, 6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyluracil, C7 modified deazaadenine, C7 modified deazaguanine, C5 modified cytosine or C5 modified uracil, wherein the modification comprises one or more of a methyl modification, an H modification, a Cl modification or an F modification.
In a second exemplary embodiment of the present application, there is provided a nucleotide monomer analog having the structural formula shown in formula II:
A formula II;
Wherein R is a base, R4 comprises one or more of methoxy, methoxyethyl, fluorine, hydroxyl or hydrogen, R5 comprises one or more of methyl, ethyl, propyl or isopropyl, and R6 comprises hydroxyl or mercapto.
By using the nucleotide monomer analogue shown as the formula II in the application, the oligonucleotide with the 3' -end reversibly modified end can be synthesized. Under the condition of a single oligonucleotide chain substrate, the nucleotide monomer analogue is mixed with polymerase, the oligonucleotide chain is taken as a synthetic chain, and the nucleotide monomer is catalyzed to be coupled to the oligonucleotide chain, so that the oligonucleotide with the reversible modified end at the 3' end can be obtained, and then the target oligonucleotide is further synthesized.
Or in the case of two oligonucleotide chain substrates, wherein the 3' end of one substrate is connected with the nucleotide monomer analogue, the 3' end of the other substrate does not contain the nucleotide monomer, and the ligase can be utilized to catalyze the two substrates to form phosphodiester bond connection, so that an oligonucleotide with a reversible modified end at the 3' end can be finally formed, and then the target oligonucleotide is further synthesized. The two methods are simple to operate, can be used for synthesizing the oligonucleotide with the reversible modification group at the 3' end in one step, are easy to remove, and are favorable for synthesizing the target oligonucleotide.
In a preferred embodiment, the base comprises a natural base or a non-natural base, the natural base comprising one or more of adenine, guanine, cytosine, thymine, or uracil, the non-natural base comprising 3-deazaadenine, 7-deazaguanine, 2, 6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyluracil, C7 modified deazaadenine, C7 modified deazaguanine, C5 modified cytosine, or C5 modified uracil, wherein the modification comprises a methyl modification, an H modification, a Cl modification, an F modification.
In a second exemplary embodiment of the present application, there is provided a method for synthesizing an oligonucleotide having a 3' -end reversibly modified end, the method comprising a) when the oligonucleotide chain substrate is a single piece, ligating the above nucleoside monomer analogue, or the above nucleotide monomer analogue, to the 3' -end of the oligonucleotide chain substrate to obtain an oligonucleotide having a 3' -end reversibly modified end;
Or b) when the number of the oligonucleotide chain substrates is two, the oligonucleotide chain substrates comprise a first oligonucleotide chain substrate and a second oligonucleotide chain substrate, wherein the nucleotide monomer analogue or the nucleotide monomer analogue is connected to the 3 'end of the first oligonucleotide chain substrate, and the first oligonucleotide chain substrate and the second oligonucleotide chain substrate are connected to obtain the oligonucleotide with the 3' end reversible modified end.
When there are two oligonucleotide chain substrates, the last nucleoside monomer at the 3 'end of one oligonucleotide chain substrate is connected with a reversible end cap, namely the first oligonucleotide chain substrate, the nucleoside monomer is connected with the rest of the oligonucleotide chain in front through a phosphate bond, the reversible end cap can be connected with the 3' end of the oligonucleotide chain substrate through the method or chemical synthesis method or other means known by the person skilled in the art, and the person can flexibly select the oligonucleotide chain substrate according to actual requirements.
Under the catalysis of ligase, the first oligonucleotide chain substrate and the second oligonucleotide chain substrate which are reversibly blocked are connected at the 3 'end, so that the oligonucleotide with the 3' end reversibly modified end is obtained.
In a preferred embodiment, the oligonucleotide strand substrate comprises a natural oligonucleotide strand consisting of natural nucleotides, or a non-natural oligonucleotide strand comprising non-natural nucleotides.
In a preferred embodiment, the non-natural oligonucleotide strand consists of non-natural nucleotides.
An unnatural nucleotide (XNA) is a class of nucleic acid molecules having an unnatural backbone or nucleobase, preferably the unnatural nucleotide comprises a ribonucleotide with one or more modifications at the ribose 2' position, ribose backbone, base or phosphate backbone, preferably the ribose 2' position comprises a 2' -methoxy, 2' -fluoro, 2' -hydrogen, 2' -methoxyethyl, 2' -FANA, locked or hexitol nucleic acid modification, preferably the ribose backbone comprises a substitution of ribose in the nucleotide for ribuloNA, TNA, tPhoNA or dXNA, preferably the base modification comprises a deadenosine C7, deazaguanosine C7, cytosine C5 or uridine C5 modification, and the phosphate backbone modification comprises a PS modification.
In a preferred embodiment, when R2 of the nucleoside monomer analog is hydroxy, monophosphate, diphosphate or thio-modified phosphate, the method of synthesis comprises mixing the nucleoside monomer analog with polyphosphate, catalyzed by a phosphokinase, to form a nucleoside monomer analog in which R2 is triphosphate, a-1) when the oligonucleotide chain substrate is single, catalyzed by a polymerase, the nucleoside monomer analog in which R2 is triphosphate is linked to the 3 'end of the oligonucleotide chain substrate to obtain an oligonucleotide having a 3' end reversibly modified end, b-1) when the oligonucleotide chain substrate is two, wherein the nucleoside monomer analog described above is linked to the 3 'end of the first oligonucleotide chain substrate, catalyzed by a ligase to the first oligonucleotide chain substrate and the second oligonucleotide chain substrate to obtain an oligonucleotide having a 3' end reversibly modified end, the phosphokinase including one or more of acetate kinase, pyruvate kinase, adenylate kinase, polyphosphate kinase, nucleoside kinase or nucleoside diphosphate kinase. When R2 of the nucleoside monomer analogue is one or more of hydroxyl, monophosphate, diphosphate or thio-modified phosphate, the R2 end of the nucleoside monomer analogue is linked to the phosphate using a polyphosphate kinase to form a nucleoside monomer analogue with R2 having three phosphates.
The synthesis method of the oligonucleotide chain substrate comprises the steps of mixing one or more nucleoside monomer analogues of hydroxyl, monophosphate, diphosphate or thio-modified phosphate with phosphate donors, tris-HCl and MgCl2, maintaining the pH value of a reaction system at 6-9, adding kinase, reacting at 0-80 ℃ for 0.1-100 h, adding acetonitrile inactivating enzyme with the volume being 1 times that of the reaction system after the reaction, and centrifuging to obtain supernatant containing nucleoside monomers with R2 containing three phosphates.
Mixing the system containing the nucleoside monomer with the R2 and the three phosphoric acids, an oligonucleotide chain substrate, tris-HCl, mnCl2 and NaCl, maintaining the pH value of the reaction system at 6-9, adding polymerase, reacting for 0.1-100 h at 0-100 ℃, adding acetonitrile inactivating enzyme with the volume being 1 time of that of the reaction system after the reaction is finished, and centrifuging to obtain a supernatant containing the oligonucleotide with the 3' -end reversible modified end.
In a preferred embodiment, when R2 of the nucleoside monomer analog is triphosphate, the synthesis method comprises a-2) when the oligonucleotide chain substrate is single, catalyzing the attachment of the nucleoside monomer analog of which R2 is triphosphate to the 3 'end of the oligonucleotide chain substrate by a polymerase to obtain an oligonucleotide having a 3' end reversibly modified end, and b-2) when the oligonucleotide chain substrate is two, wherein the above-mentioned nucleoside monomer analog is attached to the 3 'end of the first oligonucleotide chain substrate, catalyzing the attachment of the first oligonucleotide chain substrate and the second oligonucleotide chain substrate by a ligase to obtain an oligonucleotide having a 3' end reversibly modified end.
When R2 of the nucleoside monomer analogue is triphosphates, the nucleoside monomer can be directly mixed with polymerase and an oligonucleotide chain substrate to react, and coupled to the oligonucleotide chain substrate, so that the synthesis of the oligonucleotide with the 3' -end reversibly modified end is realized in one step.
In some preferred embodiments, the synthesis method comprises mixing a nucleoside monomer analogue with R2 having three phosphates, an oligonucleotide chain substrate, tris-HCl, mnCl2 and NaCl when the oligonucleotide chain substrate is single, maintaining the pH value of the reaction system at 6-9, adding polymerase, reacting at 1-100 ℃ for 0.1-100 h, adding acetonitrile inactivating enzyme with the volume of 1 time of the reaction system after the reaction is finished, and centrifuging to obtain supernatant containing the oligonucleotide with the 3' -end reversible modified end.
When the number of the oligonucleotide chain substrates is two, wherein the 3 '-end of the first oligonucleotide chain substrate is connected with the nucleoside monomer analogue with the R2 having three phosphoric acids, the first oligonucleotide chain substrate, the second oligonucleotide chain substrate, RNA ligase, mgCl2, ATP and Tris-HCl are mixed, the pH of a reaction system is maintained at 7-7.8, 1-50 ℃ is incubated for 1-50 h, acetonitrile inactivating enzyme with the volume being 1 times that of the reaction system is added after the reaction is finished, and the supernatant after centrifugation contains the oligonucleotide with the 3' -end reversible modification end.
In a preferred embodiment, the method of synthesis comprises:
a-3) when the oligonucleotide chain substrate is a single piece, catalyzing the nucleotide monomer analogue to be connected to the 3 'end of the oligonucleotide chain substrate by using polymerase, thereby obtaining the oligonucleotide with the 3' end reversibly modified end;
b-3) when there are two oligonucleotide chain substrates, wherein the above-mentioned nucleotide monomer analogue is attached to the 3 'end of the first oligonucleotide chain substrate, the first oligonucleotide chain substrate and the second oligonucleotide chain substrate are attached by using ligase to catalyze the attachment of the first oligonucleotide chain substrate and the second oligonucleotide chain substrate, thereby obtaining an oligonucleotide having a 3' end reversibly modified end.
The synthesis method of the oligonucleotide with the 3' -end reversibly modified end, which participates in the nucleotide monomer analogue, is the synthesis method when R2 of the nucleotide monomer analogue is triphosphates.
In a preferred embodiment, the polymerase comprises a PUP polymerase and the ligase comprises a T4 ligase.
The PUP polymerase is derived from Schizosaccharomyces NCBI accession No. NP-594901.1, and the T4 ligase is derived from ESCHERICHIA PHAGE T4, and NCBI accession No. NP-049790.1.
The polymerase adds a nucleoside monomer with a cap to the 3 'end of the nucleic acid strand, thereby providing the extended nucleic acid strand with a 3' reversible cap. Ligase is the addition of a short strand of oligonucleotides with end caps to the 3 'end of a nucleic acid strand, resulting in an extended oligonucleotide strand with 3' reversible end caps.
In a third exemplary embodiment of the present application, there is provided a method for preparing an oligonucleotide, comprising removing the reversibly modified end of the 3' -end of the oligonucleotide having the reversibly modified end synthesized by the above-described synthesis method to obtain the oligonucleotide.
By using the synthesis method, the synthesized oligonucleotide with the 3' -end reversible modified end can be removed by simple acidolysis in the subsequent process, thereby realizing reversible modification. Oligonucleotides with 3' end modified ends removed can also be used as substrates for the next round of coupling to nucleoside monomers. The nucleoside monomer analogue or the nucleotide monomer analogue can be used for realizing reversible modification of the 3' end of the oligonucleotide and realizing the specific synthesis of the target oligonucleotide with a specific sequence by an enzymatic method.
In a preferred embodiment, the removing comprises mixing the oligonucleotide having a 3 'reversibly modified end with the first solution to obtain a mixed system, and removing the 3' reversibly modified end using the mixed system.
In a preferred embodiment, the pH of the mixed system is 4-6, and the mixed system is maintained at 20-40 ℃ for 5-10 min, so that the removal of the 3' -end reversible modification end is realized. The conditions for removal include a pH of 4 to 6, including but not limited to 4,5, or 6, a temperature of 20 to 40 ℃, including but not limited to 20 ℃,25 ℃,30 ℃,35 ℃, or 40 ℃, for a period of 5 to 10 minutes, including but not limited to 5,6,7,8, 9, or 10 minutes.
In a preferred embodiment, the first solution comprises any one or more of hydrochloric acid, sulfuric acid, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, citric acid, or boric acid.
In a preferred embodiment, the concentration of the solute in the first solution is 0.01 mM-10M.
The application can realize the removal of the reversible modified end of the 3' -end of the coupled nucleoside monomer on the oligonucleotide chain by utilizing simple acidolysis, and can promote the effective removal of the reversible modified end by controlling the conditions of the acidolysis reaction of removal, thereby being convenient for the subsequent synthesis of target oligonucleotides.
In a fourth exemplary embodiment of the application, there is provided a method for synthesizing any one of the nucleoside monomers described above, or any one of the oligonucleotides having a reversibly modified 3' -end described above, or a method for preparing any one of the oligonucleotides described above, for use in preparing an oligonucleotide.
The advantageous effects of the present application will be explained in further detail below in connection with specific examples.
The reagents used in the examples of the present application are all conventional commercial products, except for the specific descriptions.
Example 1
The reaction system was placed in a clean vessel such that the oligonucleotide strand substrate was 50. Mu.M, PUP polymerase (derived from Schizosaccharomyces, NCBI accession No. NP-594901.1) was added to a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added, pH was adjusted to 8.0, and nucleoside monomer analogues were added to a concentration of 700. Mu.M, and the structural formula of the nucleoside monomer analogues of this example was as follows:
;
after 2h reaction at 37 ℃,1 volume of acetonitrile inactivating enzyme was added to the reaction system, and denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to UPLC detection.
The oligonucleotide chain substrates of this example are substrate 1, substrate 2 and substrate 3 (SEQ ID NO: 1), respectively, and the sequences thereof and the results of UPLC detection of the supernatant thereof by the synthetic linked nucleoside monomer analog are shown in Table 1.
The UPLC detection results of the embodiment show that 1 nucleoside monomer analogue is coupled to the 3' end of different oligonucleotide chain substrates, which shows that the nucleoside monomer analogue shown in the structural formula can be applied to the enzymatic synthesis of oligonucleotides.
TABLE 1
In the table 1, the contents of the components, "+++" "indicates that a product is detected, the conversion rate is 0.1% -30%;" +++ "" means the presence of a product was detected and, the conversion rate is 30% -90%.
"M" after A, C, G or U of the application means 2' methoxy modification to the ribonucleotide, "f" means 2' fluoro modification to the ribonucleotide, and "s" means thio modification of the 5' phosphate of the ribonucleotide.
Example 2
The reaction system was added to a clean vessel such that the 2 oligonucleotide substrate fragments were 50mM, and the 2 oligonucleotide substrate fragments of this example are shown in the following structural formula, wherein the 3' oh end of the first oligonucleotide substrate fragment was attached to the reversibly blocked modified end (nucleoside monomer analogue) of the present application, i.e., the framed portion of the following structural formula:
first oligonucleotide strand substrate fragment:
;
second oligonucleotide strand substrate fragment:
。
RNA ligase (from ESCHERICHIA PHAGE T, NCBI accession number NP-049790.1) was added to a concentration of 0.2mg/mL, 10mM MgCl2,250mM ATP,50mM Tris-HCl was added, the pH was adjusted to 7.5,16 ℃and incubated for 16h, 1 volume of acetonitrile inactivating enzyme was added to the reaction system, and denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to LCMS detection.
Since the 3' OH end of the first oligonucleotide fragment carries a reversible end-capping modification, only a molecular weight of 2618.83.+ -.2 could theoretically be detected, and not a molecular weight of 2 or more products in which one oligonucleotide fragment is linked. Only the molecular weight 2618.79 was detected by mass spectrometry, indicating a successful' ligation.
Example 3
The reaction system was added to a clean vessel so that the oligonucleotide strand substrate (5 'HO-Cms-Ams-Gm-Am-3' OH, substrate 1 in example 1) was 50. Mu.M, PUP polymerase was added to a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added, pH was adjusted to 8.0, and nucleotide monomer analogue was added to a concentration of 700. Mu.M, and the structural formula of the nucleotide monomer analogue of this example was as follows:
;
After 4h reaction at 37 ℃,1 volume of acetonitrile inactivating enzyme was added to the reaction system, and denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to UPLC detection. The detection of the 3' end coupled 1 nucleoside monomer oligonucleotide product, 20% conversion, shows the example of nucleoside monomer analogues can be applied to enzymatic synthesis of oligonucleotides.
Example 4
The reaction system was added to a clean vessel so that the oligonucleotide strand substrate (5 'HO-Cms-Ams-Gm-Am-3' OH, substrate 1 in example 1) was 50. Mu.M, PUP polymerase was added to a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added, pH was adjusted to 8.0, and nucleotide monomer analogue was added to a concentration of 700. Mu.M, and the structural formula of the nucleotide monomer analogue of this example was as follows:
;
After 4h reaction at 37 ℃,1 volume of acetonitrile inactivating enzyme was added to the reaction system, and denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to UPLC detection. The detection of the 3' -end coupled 1 nucleoside monomer analogue oligonucleotide product, the conversion rate of 9%, shows that the nucleoside monomer analogue can be applied to the enzymatic synthesis of oligonucleotides.
Example 5
The reaction system was added to a clean vessel so that the oligonucleotide strand substrate (5 'HO-Cms-Ams-Gm-Am-3' OH, substrate 1 in example 1) was 50. Mu.M, PUP polymerase was added to a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added, pH was adjusted to 8.0, and nucleotide monomer analogue was added to a concentration of 700. Mu.M, and the structural formula of the nucleotide monomer analogue of this example was as follows:
;
After 4h reaction at 37 ℃,1 volume of acetonitrile inactivating enzyme was added to the reaction system, and denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to UPLC detection. The detection of the 3' end coupled 1 nucleoside monomer analogue oligonucleotide product, conversion rate of 1%, shows that the embodiment of the nucleotide monomer analogue can be applied to enzymatic synthesis of oligonucleotides.
Example 6
The reaction system was charged in a clean vessel so that the oligonucleotide chain substrate (SEQ ID NO: 1) was 50. Mu.M, PUP polymerase was added so as to have a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added, pH was adjusted to 8.0, and nucleotide monomer analogue was added so as to have a concentration of 700. Mu.M, and the structural formula of the nucleotide monomer analogue of this example was as follows:
;
after 2h reaction at 37 ℃,1 volume of acetonitrile inactivating enzyme was added to the reaction system, and denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to UPLC detection. The product of the oligonucleotide with 1 nucleoside monomer analogue coupled to the 3' -end was detected, and the conversion rate was 43%, which indicates that the nucleoside monomer analogue of this example can be applied to the enzymatic synthesis of oligonucleotides.
Example 7
The reaction system was added to a clean vessel so that the oligonucleotide strand substrate (5 'HO-Cms-Ams-Gm-Am-3' OH, substrate 1 in example 1) was 50. Mu.M, PUP polymerase was added to a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added, pH was adjusted to 8.0, and nucleotide monomer analogue was added to a concentration of 700. Mu.M, and the structural formula of the nucleotide monomer analogue of this example was as follows:
;
After 16h reaction at 37 ℃,1 volume of acetonitrile inactivating enzyme was added to the reaction system, and denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to UPLC detection. The detection of the 3' end coupled 1 nucleoside monomer analogue oligonucleotide product, conversion rate of 0.6%, shows that the nucleoside monomer analogue can be applied to the enzymatic synthesis of oligonucleotides.
Example 8
The reaction system was charged in a clean vessel so that the oligonucleotide chain substrate (SEQ ID NO: 1) was 50. Mu.M, PUP polymerase was added so as to have a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added, pH was adjusted to 8.0, and nucleotide monomer analogue was added so as to have a concentration of 700. Mu.M, and the structural formula of the nucleotide monomer analogue of this example was as follows:
;
After 1h reaction at 37 ℃,1 volume of acetonitrile inactivating enzyme was added to the reaction system, and denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to UPLC detection. The detection of the 3' end coupled 1 nucleoside monomer analogue oligonucleotide product, the conversion rate of 83%, shows that the nucleoside monomer analogue can be applied to the enzymatic synthesis of oligonucleotides.
Example 9
The reaction system was added to a clean vessel so that the oligonucleotide strand substrate (5 'HO-Cms-Ams-Gm-Am-3' OH, substrate 1 in example 1) was 50. Mu.M, PUP polymerase was added to a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added, pH was adjusted to 8.0, and nucleoside monomer analogue was added to a concentration of 700. Mu.M, the structural formula of the nucleoside monomer analogue of this example was as follows:
;
After 2h reaction at 37℃1 volume of acetonitrile inactivating enzyme was added to the reaction system, and the denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to MS detection, resulting in detection of 1693.3 as a product molecular weight, which was an oligonucleotide chain substrate coupled to 1 molecule of nucleoside monomer analogue, and no product coupled to 2 or more nucleoside monomers. Nucleoside monomers of the formula are effectively capped.
Example 10
The nucleoside monomers of this example include three nucleotide monomer analogs as shown below:
First nucleoside monomer analog ;
Second nucleoside monomer analog;
Third nucleoside monomer analog;
The reaction system was placed in three clean containers, respectively, such that the oligonucleotide strand substrate (5 'HO-Cms-Ams-Gm-Am-3' OH, i.e., substrate 1in example 1) was 50. Mu.M, the polymerase was added to a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added, the pH was adjusted to 8.0, and the three nucleotide monomer analogues were added, respectively, to a concentration of 700. Mu.M.
After 16h reaction at 37 ℃,1 volume of acetonitrile inactivating enzyme was added to the reaction system, and the denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to mass spectrometry detection.
The molecular weight of theoretical products of the oligonucleotide synthesized by the three reaction systems is 1706.53,1720.37,1720.37 respectively, and the peaks of the molecular weight of 1705.17,1719.42,1719.40 are detected respectively through mass spectrum detection, which shows that the three nucleoside monomers shown in the structural formula can be applied to the enzymatic synthesis of the oligonucleotide.
Example 11
In a clean vessel, a nucleoside monomer analog was added to a concentration of 2000. Mu.M, i.e., reaction system 1, and the nucleoside monomer analog of this example is represented by the following formula:
;
15mM MnCl2,0.5mg/mL (NaPO 3) 6,100mM Tris-HCl was continuously added to the reaction system 1, the pH was adjusted to 8.0, polyphosphate kinase (the polyphosphate kinase was derived from Meiothermus ruber, PDB: 5LC 9) was added to a concentration of 0.5mg/mL, after 16 hours of reaction at 30 ℃, acetonitrile inactivating enzyme was added to the reaction sample by 1 time volume, and denatured proteins were removed by centrifugation at 12000rpm, and the conversion was examined using UPLC, the results of which are schematically shown in FIG. 1, and two products 1 and 2 produced by the system and nucleoside monomer analogues of the present application were detected.
The reaction system 2 was put into a clean vessel so that the oligonucleotide strand substrate (5 'HO-Cms-Ams-Gm-Am-3' OH, i.e., substrate 1 in example 1) was 50. Mu.M, PUP polymerase was added to a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added to adjust pH to 8.0, the above reaction system 1 was added so that the concentration of reaction system 2 was 700. Mu.M, after 2 hours of reaction at 37 ℃, acetonitrile of 1 volume of the reaction system was added to inactivate the enzyme, and denatured protein was removed by centrifugation at 12000rpm, and UPLC detection was performed on the supernatant to detect the formation of the target product of the oligonucleotide strand coupled with one product 2, indicating that the nucleoside monomer of this example was applicable to the enzymatic synthesis of oligonucleotides.
Example 12
The reaction system was added in a clean vessel such that the oligonucleotide strand substrate (5 ' HO-Cms-Ams-Gm-Am-3' OH, i.e., substrate 1 in example 1) was 50. Mu.M, PUP polymerase was added to a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added to adjust the pH to 8.0, and 35. Mu.M fourth nucleoside monomer analogue and 665. Mu.M fifth nucleoside monomer analogue (existing end-blocked, 3' end was-NH 2) of the formula:
fourth nucleoside monomer analog ;
Fifth nucleoside monomer analog;
After 16h reaction at 37 ℃,1 volume of acetonitrile inactivating enzyme was added to the reaction system, and denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to UPLC detection. Only 6% of the oligonucleotide strand substrate was detected, 28% of the oligonucleotide product coupled to one fifth nucleoside monomer analog was produced, and 36% of the oligonucleotide product coupled to one fourth nucleoside monomer analog was produced, and the UPLC detection results are shown in FIG. 2. The coupling efficiency of the polymerase PUP to the fourth nucleoside monomer analogue is much higher than to the existing blocked fifth nucleoside monomer analogue.
1M NaOAc and 0.7M NaNO2 are added into the reaction system, the pH is adjusted to 4-6, the temperature is increased to 30 ℃ and the temperature is increased for 5-10 min, so that the 3' -end modification group on the nucleoside monomer analogue is removed, and the reacted solution is sent to UPLC for detection, and the result is shown in figure 3. The fourth modified monomer has better polymerase catalytic activity, and can be effectively removed to realize reversible modification of the oligonucleotide. The oligonucleotide with the reversible modification groups removed can be used as an oligonucleotide chain substrate for coupling nucleoside monomers in the next round.
Example 13
The reaction system was placed in a clean vessel such that the oligonucleotide strand substrate (5 ' HO-Ams-Cfs-Am-Af-Af-3' OH, i.e., substrate 2 in example 1) was 50. Mu.M, the PUP polymerase was added to a concentration of 0.2mg/mL, 10mM MnCl2,50mM NaCl,50mM Tris-HCl was added, the pH was adjusted to 8.0, and a fifth nucleoside monomer analogue (now blocked, 3' at-NH 2) was added to a concentration of 300. Mu.M, and a fourth nucleoside monomer analogue was added to a concentration of 300. Mu.M, as shown in the following formula:
A fourth nucleoside monomer analog; ;
A fifth nucleoside monomer analog; ;
after 2h reaction at 37 ℃,1 volume of acetonitrile inactivating enzyme was added to the reaction system, and the denatured proteins were removed by centrifugation at 12000rpm, and the supernatant was subjected to UPLC detection, resulting in 67% of the product of the fourth nucleoside monomer analog 2 and 30% of the product of the conjugated fifth nucleoside monomer analog. The coupling efficiency of the polymerase PUP to the fourth nucleoside monomer analogue is much higher than to the existing blocked fifth nucleoside monomer analogue.
From the above description, it can be seen that the above embodiments of the present application achieve the technical effect that the 3' end of the oligonucleotide chain substrate can be connected to the reversible closed end under the system of enzymatic synthesis by using the nucleoside monomer of the present application, and the reversible closed end can be effectively removed by using simple acidolysis in the subsequent step, and the oligonucleotide from which the reversible modification group is removed can be used as the oligonucleotide chain substrate of the next round of coupling nucleoside monomer, which proves that the target oligonucleotide of specific sequence can be specifically synthesized by using the nucleoside monomer of the present application.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (16)
1. A nucleoside monomer analogue, which is characterized in that the structural formula of the nucleoside monomer analogue is shown in a formula I;
A formula I;
Wherein R is a base;
r 1 comprises one or more of methoxy, methoxyethyl, fluoro, hydroxy or hydrogen;
R 2 comprises one or more of hydroxyl, monophosphate, diphosphate, triphosphate, or thio modified phosphate;
r 3 includes one or more of methyl, ethyl, propyl, or isopropyl.
2. The nucleoside monomer analog according to claim 1, wherein the base comprises a natural base or a non-natural base;
the natural base includes adenine, guanine, cytosine, thymine or uracil;
the unnatural base comprises one or more of 3-deazaadenine, 7-deazaguanine, 2, 6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyl uracil, C7 modified deazaadenine, C7 modified deazaguanine, C5 modified cytosine, or C5 modified uracil;
Wherein the modification comprises one or more of a methyl modification, an H modification, a Cl modification, or an F modification.
3. A nucleotide monomer analogue, which is characterized in that the structural formula of the nucleotide analogue is shown in a formula II:
A formula II;
Wherein R is a base;
R 4 comprises one or more of methoxy, methoxyethyl, fluoro, hydroxy or hydrogen;
R 5 comprises one or more of methyl, ethyl, propyl or isopropyl;
R 6 includes hydroxy or mercapto.
4. The nucleotide monomer analogue according to claim 3, wherein the base comprises a natural base or a non-natural base;
the natural base includes adenine, guanine, cytosine, thymine or uracil;
the unnatural base comprises one or more of 3-deazaadenine, 7-deazaguanine, 2, 6-diaminopurine, 8-azidoadenine, 2-thiothymidine, 5-carboxamide uracil, 5-methylcytosine, 5-ethynyl uracil, C7 modified deazaadenine, C7 modified deazaguanine, C5 modified cytosine, or C5 modified uracil;
Wherein the modification comprises one or more of a methyl modification, an H modification, a Cl modification, or an F modification.
5. A method of synthesizing an oligonucleotide having a 3' reversibly modified end, the method comprising:
a) When the oligonucleotide chain substrate is a single piece, connecting the nucleoside monomer analogue of any one of claims 1-2 or the nucleotide monomer analogue of any one of claims 3-4 to the 3 'end of the oligonucleotide chain substrate to obtain the oligonucleotide with the 3' end reversibly modified end;
Or (b)
B) When the number of the oligonucleotide chain substrates is two, the oligonucleotide chain substrates comprise a first oligonucleotide chain substrate and a second oligonucleotide chain substrate, wherein the 3' end of the first oligonucleotide chain substrate is connected with the nucleoside monomer analogue of any one of claims 1-2 or the nucleotide monomer analogue of any one of claims 3-4;
Ligating the first oligonucleotide strand substrate and the second oligonucleotide strand substrate to obtain the oligonucleotide having a 3' reversibly modified end.
6. The method of synthesis according to claim 5, wherein the oligonucleotide strand substrate comprises a natural oligonucleotide strand composed of natural nucleotides or a non-natural oligonucleotide strand containing non-natural nucleotides.
7. The synthetic method according to claim 5, wherein when R 2 of the nucleoside monomer analog is hydroxy, monophosphate, diphosphate or thio modified phosphate, the synthetic method comprises:
a-1) mixing said nucleoside monomer analogue with a polyphosphate salt, using phosphokinase catalysis, to form said nucleoside monomer analogue wherein R 2 is triphosphate;
Catalyzing, with a polymerase, a nucleoside monomer analog in which the R 2 is triphosphate, when the oligonucleotide chain substrate is single, to attach to the 3 'end of the oligonucleotide chain substrate, thereby obtaining the oligonucleotide having a reversibly modified end at the 3' end;
Wherein the phosphokinase comprises one or more of acetate kinase, pyruvate kinase, adenylate kinase, polyphosphate kinase, nucleoside kinase or nucleoside diphosphate kinase.
8. The synthetic method of claim 5, wherein when R 2 of the nucleoside monomer analog is triphosphate, the synthetic method comprises:
a-2) when the oligonucleotide chain substrate is a single piece, catalyzing the ligation of the nucleoside monomer analogue, in which R 2 is triphosphate, to the 3 'end of the oligonucleotide chain substrate by using a polymerase, thereby obtaining the oligonucleotide with the 3' end reversibly modified end.
9. The method of synthesis according to claim 5, wherein the method of synthesis comprises:
a-3) when the oligonucleotide chain substrate is a single piece, catalyzing the nucleotide monomer analogue in any one of claims 3-4 to be connected to the 3 'end of the oligonucleotide chain substrate by using polymerase, thereby obtaining the oligonucleotide with the 3' end reversibly modified end.
10. The synthetic method according to any one of claims 7 to 9, wherein the polymerase comprises PUP polymerase;
the ligase includes RNA ligase or DNA ligase.
11. A method for preparing an oligonucleotide, comprising removing the reversibly modified end of the 3' -end of an oligonucleotide having a reversibly modified end synthesized by the synthesis method according to any one of claims 5 to 10, to obtain the oligonucleotide.
12. The method of preparing according to claim 11, wherein the removing comprises:
mixing the oligonucleotide with the 3' -end reversibly modified end with a first solution to obtain a mixed system,
And removing the reversible modification end of the 3' end by using the mixed system.
13. The preparation method of claim 12, wherein the pH of the mixed system is 4-6, and the mixed system is maintained at a temperature of 20-40 ℃ for 5-10 min, so as to remove the 3' -end reversible modification end.
14. The method of claim 13, wherein the first solution comprises any one or more of hydrochloric acid, sulfuric acid, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, citric acid, or boric acid.
15. The method of claim 14, wherein the concentration of the solute in the first solution is 0.01mm to 10m.
16. Use of the nucleoside monomer analogue of any one of claims 1 to 2, or the nucleotide monomer analogue of any one of claims 3 to 4, or the method of synthesis of an oligonucleotide having a 3' reversibly modified end of any one of claims 5 to 10, or the method of preparation of an oligonucleotide of any one of claims 11 to 15, in the preparation of an oligonucleotide.
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| US20080050780A1 (en) * | 2006-06-30 | 2008-02-28 | Applera Corporation | Reversible terminator nucleotides and methods of use |
| CN114040983A (en) * | 2019-06-26 | 2022-02-11 | 南京金斯瑞生物科技有限公司 | Oligonucleotide containing blocker |
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