CN118127102A - Method for synthesizing nucleic acid with 3' -O-phosphate modification by enzyme method - Google Patents
Method for synthesizing nucleic acid with 3' -O-phosphate modification by enzyme method Download PDFInfo
- Publication number
- CN118127102A CN118127102A CN202410574630.3A CN202410574630A CN118127102A CN 118127102 A CN118127102 A CN 118127102A CN 202410574630 A CN202410574630 A CN 202410574630A CN 118127102 A CN118127102 A CN 118127102A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- natural
- seq
- modification
- oligonucleotide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 70
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 69
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 43
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 13
- 230000009144 enzymatic modification Effects 0.000 title description 3
- 238000012986 modification Methods 0.000 claims abstract description 71
- 230000004048 modification Effects 0.000 claims abstract description 70
- 239000002773 nucleotide Substances 0.000 claims abstract description 64
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 63
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 56
- 239000000178 monomer Substances 0.000 claims abstract description 49
- 102000004190 Enzymes Human genes 0.000 claims abstract description 8
- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
- 239000002777 nucleoside Substances 0.000 claims description 33
- -1 nucleoside monophosphates Chemical class 0.000 claims description 20
- 239000000758 substrate Substances 0.000 claims description 17
- 229910052799 carbon Inorganic materials 0.000 claims description 16
- 235000000346 sugar Nutrition 0.000 claims description 16
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 claims description 13
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 13
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 13
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 13
- 102100037458 Dephospho-CoA kinase Human genes 0.000 claims description 12
- 108010049285 dephospho-CoA kinase Proteins 0.000 claims description 12
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 12
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 9
- 229910019142 PO4 Inorganic materials 0.000 claims description 8
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 8
- 235000021317 phosphate Nutrition 0.000 claims description 8
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 229960000643 adenine Drugs 0.000 claims description 5
- 229940104302 cytosine Drugs 0.000 claims description 5
- 229930024421 Adenine Natural products 0.000 claims description 4
- 239000001177 diphosphate Substances 0.000 claims description 4
- 235000011180 diphosphates Nutrition 0.000 claims description 4
- 150000002772 monosaccharides Chemical class 0.000 claims description 4
- 150000002972 pentoses Chemical class 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 150000003013 phosphoric acid derivatives Chemical class 0.000 claims description 4
- 239000001226 triphosphate Substances 0.000 claims description 4
- 235000011178 triphosphate Nutrition 0.000 claims description 4
- 229940035893 uracil Drugs 0.000 claims description 4
- 108020004414 DNA Proteins 0.000 claims description 3
- 102000053602 DNA Human genes 0.000 claims description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical group N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 229920000388 Polyphosphate Polymers 0.000 claims description 2
- 239000001205 polyphosphate Substances 0.000 claims description 2
- 235000011176 polyphosphates Nutrition 0.000 claims description 2
- 150000003291 riboses Chemical class 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 18
- 238000003786 synthesis reaction Methods 0.000 abstract description 17
- 238000006555 catalytic reaction Methods 0.000 abstract description 8
- 238000006911 enzymatic reaction Methods 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 description 25
- 108091028664 Ribonucleotide Proteins 0.000 description 13
- 239000002336 ribonucleotide Substances 0.000 description 13
- 108010054404 Adenylyl-sulfate kinase Proteins 0.000 description 11
- 102100040152 Adenylyl-sulfate kinase Human genes 0.000 description 11
- 108010022348 Sulfate adenylyltransferase Proteins 0.000 description 11
- 125000002652 ribonucleotide group Chemical group 0.000 description 10
- 125000003835 nucleoside group Chemical group 0.000 description 9
- 230000002255 enzymatic effect Effects 0.000 description 8
- 238000001819 mass spectrum Methods 0.000 description 8
- 238000006366 phosphorylation reaction Methods 0.000 description 8
- 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 description 7
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 7
- 230000002441 reversible effect Effects 0.000 description 7
- 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 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- 238000004811 liquid chromatography Methods 0.000 description 6
- 230000026731 phosphorylation Effects 0.000 description 6
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 5
- 229960005305 adenosine Drugs 0.000 description 5
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 5
- 238000004949 mass spectrometry Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 239000007791 liquid phase Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 4
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 125000004429 atom Chemical group 0.000 description 3
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 3
- 239000005516 coenzyme A Substances 0.000 description 3
- 229940093530 coenzyme a Drugs 0.000 description 3
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 125000001153 fluoro group Chemical group F* 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 150000003290 ribose derivatives Chemical class 0.000 description 3
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- DJJCXFVJDGTHFX-XVFCMESISA-N uridine 5'-monophosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-XVFCMESISA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 2
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 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 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 150000004712 monophosphates Chemical group 0.000 description 2
- 230000000865 phosphorylative effect Effects 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 2
- 229940045145 uridine Drugs 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 description 1
- LNQVTSROQXJCDD-UHFFFAOYSA-N adenosine monophosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)C(OP(O)(O)=O)C1O LNQVTSROQXJCDD-UHFFFAOYSA-N 0.000 description 1
- IRLPACMLTUPBCL-FCIPNVEPSA-N adenosine-5'-phosphosulfate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@@H](CO[P@](O)(=O)OS(O)(=O)=O)[C@H](O)[C@H]1O IRLPACMLTUPBCL-FCIPNVEPSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical group OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 239000002920 hazardous waste Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 125000002264 triphosphate group Chemical group [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for synthesizing nucleic acid with 3 '-O-phosphate modification by an enzymatic method, which comprises the step of catalyzing a nucleic acid monomer or an oligonucleotide chain with a length of 1nt or more than or equal to 2nt by using phosphokinase to obtain the nucleic acid monomer or the oligonucleotide chain with the 3' -O-phosphate. Can fill the technical blank that the nucleic acid with 3' -O-phosphate modification is difficult to synthesize by enzyme catalysis in the prior art, and is suitable for the field of non-natural nucleotide synthesis.
Description
Technical Field
The invention relates to the field of non-natural nucleotide synthesis, in particular to a method for synthesizing nucleic acid with 3' -O-phosphate modification by an enzymatic method.
Background
In the prior art, the synthesis of the oligonucleotide mainly depends on the solid phase synthesis method of phosphoramidite at present, but the synthesis of a nucleic acid chain with the length of more than 200nt still has high difficulty, meanwhile, the accuracy of the synthesis cannot meet the demands of the current market on oligonucleotide products, and the theoretical success rate of the oligonucleotide is highest and is 37 percent. Meanwhile, a large amount of organic solvents are used in the process of synthesizing the oligonucleotides in the prior art, the use of toxic reagents is involved, the PMI (PMI: the ratio of the total mass of all substances in the whole production process of the product to the mass of a target product) is large, about 4000 kg of waste is generated per kg of the product in the process of synthesizing the oligonucleotides with 20nt sequences, the bottleneck such as limited production amplification and the like are faced, and along with the general application of oligonucleotide drugs, the demands for the oligonucleotides are growing, so that the development of a novel method for synthesizing the oligonucleotides is very necessary.
The enzymatic synthesis of oligonucleotide is a green synthesis process, has the advantages of mild condition, rapid coupling and no hazardous waste, and can synthesize oligonucleotide with longer chain, thus having better application prospect. In the synthesis of oligonucleotides using enzymatic methods, it is necessary to reversibly protect the 3 'end of a nucleotide or oligonucleotide to achieve specificity of the synthesized sequence, to avoid the synthesis of erroneous sequences, and to allow for traceless removal of the 3' protecting group after completion of the enzymatic process. In the synthesis of oligonucleotides by nucleic acid polymerases in a template-independent manner, it is necessary to reversibly cap the 3' end of the nucleotide, and after the coupling reaction is completed, it is necessary to remove the 3' end cap to enter the next round of synthesis, so that the reversible cap of the 3' end of the nucleic acid strand or nucleotide is important for enzymatic synthesis of the oligonucleotide. However, at present, no report on the 3 'end enzymatic reversible group modification of oligonucleotide or nucleotide exists, the main means of the prior art is that the chemical method is used for synthesizing the nucleotide with the 3' end reversible modification, and the synthesis process is complicated. Reversible modification of the 3' end of a nucleic acid strand remains in a state of technical blank.
Disclosure of Invention
The main purpose of the present invention is to provide a method for synthesizing a nucleic acid modified by 3 '-O-phosphate by an enzymatic method, which fills the technical gap that the prior art is difficult to synthesize the nucleic acid modified by 3' -O-phosphate by an enzymatic method.
In order to achieve the above object, according to a first aspect of the present invention, there is provided a method for enzymatically synthesizing a nucleic acid having a 3' -O-phosphate modification, the method comprising: the phosphokinase is used for catalyzing a nucleic acid monomer with the length of 1nt or an oligonucleotide chain with the length of more than or equal to 2nt to obtain the nucleic acid monomer or the oligonucleotide chain of 3' -O-phosphoric acid.
Further, the nucleic acid monomers include nucleosides or nucleotides; nucleosides consist of bases and five-carbon sugars; the nucleotide consists of a base, five-carbon sugar and phosphoric acid; five carbon sugars include ribose or deoxyribose; the base is adenine, guanine, cytosine or uracil; the number of phosphates in the nucleotides is 1, 2 or 3, and the corresponding nucleotides are nucleoside monophosphates, nucleoside diphosphate or nucleoside triphosphate, respectively.
Further, the oligonucleotide strand is an RNA strand or a DNA strand with a length of not less than 2 nt.
Further, the length of the oligonucleotide strand is 2-20nt.
Further, phosphokinase enzymes include APSK enzyme or DPCK enzyme.
Further, DPCK enzyme is SEQ ID NO: 1-SEQ ID NO:10, or a protein as set forth in any one of seq id nos; APSK enzyme is SEQ ID NO: 11-SEQ ID NO:20, and a protein as set forth in any one of seq id nos.
Further, the nucleic acid monomers include natural nucleic acid monomers or non-natural nucleic acid monomers, the five-carbon sugar of the natural nucleic acid monomers is ribose or deoxyribose, and the five-carbon sugar of the non-natural nucleic acid monomers is non-natural monosaccharides composed of 5C atoms having modified ribose or deoxyribose, or having a non-natural backbone.
Further, the oligonucleotide strand 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, the method comprises the steps of: the nucleic acid monomer or oligonucleotide chain of 3' -O-phosphoric acid is obtained by using ATP or polyphosphate and phosphokinase and catalyzing with the nucleic acid monomer or oligonucleotide chain as a substrate.
By applying the technical scheme of the invention, the phosphokinase can catalyze nucleic acid monomers or oligonucleotide chains to obtain 3 '-O-phosphoric acid, so that the enzymatic preparation of the nucleic acid modified by the 3' -O-phosphoric acid is realized, and the enzymatic preparation has various advantages of production scale, yield, cost, environmental protection, safety and the like compared with chemical synthesis in the prior art.
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 graph of the results of liquid chromatography using APSK-6 catalytic substrate for 3' -O-phosphate modification according to example 1 of the invention.
FIG. 2 shows a mass spectrum of the 3' -O-phosphate modification with APSK-6 catalytic substrate according to example 1 of the invention.
FIG. 3 shows a graph of liquid chromatography results of 3' -O-phosphate modification using APSK-3 catalytic AMP according to example 3 of the invention.
FIG. 4 is a graph showing the results of mass spectrometry of 3' -O-phosphate modification using APSK-3-catalyzed AMP according to example 3 of the invention.
FIG. 5 shows a graph of the results of liquid chromatography using DPCK-7 to catalyze the modification of 5' -Pi-GA-OH-3' with 3' -O-phosphoric acid according to example 2 of the present invention.
FIG. 6 shows a liquid chromatography result of modification of 3' -O-phosphate with DPCK-3 catalytic Pi-GmGfUmCf-OH according to example 5 of the present invention.
FIG. 7 shows a graph of liquid chromatography results of 3' -O-phosphate modification with APSK-6 catalyzed UMP according to example 6 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.
A nucleoside: a compound formed by condensing a base (purine or pyrimidine base) and a pentose (including, but not limited to, ribose or deoxyribose), wherein the nucleoside does not contain phosphoric acid, and the 5' -position of the pentose is connected with-OH instead of a phosphate group.
Nucleotide: bases (purine or pyrimidine bases), pentoses (including but not limited to ribose or deoxyribose), and phosphates, including nucleoside monophosphates, nucleoside diphosphate, and nucleoside triphosphates.
Taking uridine and 5' -uridylic acid as examples, the structures of nucleosides and nucleotides containing uracil are shown:
，/>。
Nucleic acid monomer: in the present application, a single nucleoside or mononucleotide having a length of 1nt is referred to.
As mentioned in the background art, the 3' -O-phosphate modification of oligonucleotides in the prior art can only be obtained by chemical reaction, and chemical synthesis is limited in various aspects such as chain length, productivity and yield in industrial scale-up production. Thus, in the present application, the inventors have attempted to develop a method capable of synthesizing a nucleotide having a 3' -O modification by enzyme catalysis, and have proposed a series of protection schemes of the present application.
In a first exemplary embodiment of the present application, there is provided a method for enzymatically synthesizing a nucleic acid having a 3' -O-phosphate modification, the method comprising: the phosphokinase is used for catalyzing a nucleic acid monomer with the length of 1nt or an oligonucleotide chain with the length of more than or equal to 2nt to obtain the nucleic acid monomer or the oligonucleotide chain of 3' -O-phosphoric acid.
In the above-described method of the present application, the inventors have unexpectedly found that a 3'-O of a nucleic acid monomer or an oligonucleotide chain having a length of 2nt or more can be modified by phosphorylating 3' -O of a nucleic acid monomer to obtain a3 '-O-phosphate, or a 3' -O of a nucleotide located at the 3 '-end of an oligonucleotide chain can be modified by phosphorylating 3' -O of a nucleotide located at the 3 '-end to obtain an oligonucleotide chain having a 3' -O-phosphate at the 3 '-end by catalyzing the 3' -end of a nucleic acid monomer with phosphokinase. The 3' -O-phosphate modification of the oligonucleotide strand or nucleic acid monomer can be achieved using the above-described method.
In a preferred embodiment, the nucleic acid monomers comprise nucleosides or nucleotides; nucleosides consist of bases and five-carbon sugars; the nucleotide consists of a base, five-carbon sugar and phosphoric acid; five carbon sugars include ribose or deoxyribose; the base is adenine, guanine, cytosine or uracil; the number of phosphates in the nucleotides is 1, 2 or 3, and the corresponding nucleotides are nucleoside monophosphates, nucleoside diphosphate or nucleoside triphosphate, respectively.
In a preferred embodiment, the oligonucleotide strand is an RNA strand or a DNA strand having a length of.gtoreq.2 nt.
In a preferred embodiment, the length of the oligonucleotide strand is from 2 to 20 nt.
The 3' -O phosphorylation modification can be performed on nucleic acid monomers such as adenine ribonucleotide, guanine ribonucleotide and cytosine ribonucleotide by the method. Single-stranded RNA of length including, but not limited to, 2-5nt (2 nt, 3nt, 4nt or 5 nt) can also be catalyzed, and the 3' -O position of the nucleotide at the 3' -end of the single-stranded RNA can be phosphorylated to obtain a nucleotide containing a 3' -O-phosphate modification. The 3' -O-phosphate modification can remove the phosphate radical in the subsequent catalysis methods such as alkaline phosphatase or polynucleotide kinase, etc., so as to realize the reversible modification of the original single nucleotide or oligonucleotide chain.
In a preferred embodiment, the phosphokinase comprises APSK enzyme or DPCK enzyme; preferably, DPCK enzyme is SEQ ID NO: 1-SEQ ID NO:10, or a protein as set forth in any one of seq id nos; preferably, the APSK enzyme is SEQ ID NO: 11-SEQ ID NO:20, and a protein as set forth in any one of seq id nos.
In the prior art, APSK (adenosine-5 '-phosphosulfate kinase) enzyme is one of key enzymes participating in sulfur metabolism in plants and prokaryotes, which is catalyzed by taking adenosine-5' -phosphosulfate as a substrate for phosphorylation; DPCK enzyme (dephosphorylated CoA kinase) is the final step in CoA (CoA) biosynthesis, catalyzed by phosphorylation of dephosphorylated CoA as a substrate. In the present application, the inventors have found that catalysis of a single nucleotide or oligonucleotide chain can be achieved using phosphokinase enzymes including, but not limited to, APSK enzymes or DPCK enzymes.
SEQ ID NO：1：MRYIVALTGGIGSGKSTVANAFADLGINVIDADIIARQVVEPGAPALHAIADHFGANMIAADGTLQRRALRERIFANPEEKNWLNALLHPLIQQETQHQIQQATSPYVLWVVPLLVENSLYKKANRVLVVDVSPETQLKRTMQRDDVTREHVEQILAAQATREARLAVADDVIDNNGAPDAIASDVARLHAHYLQLASQFVSQEKP.
SEQ ID NO：2：MLRIGLTGGIGAGKSLLSTTFSQCGGIVVDGDVLAREVVQPGTEGLASLVDAFGRDILLADGALDRQALAAKAFRDDESRGVLNGIVHPLVARRRSEIIAAVSGDAVVVEDIPLLVESGMAPLFPLVVVVHADVELRVRRLVEQRGMAEADARARIAAQASDQQRRAVADVWLDNSGSPEDLVRRARDVWNTRVQPFAHNLAQRQIARAPARLVPADPSWPDQARRIVNRLKIACGHKALRVDHIGSTAVSGFPDFLAKDVIDIQVTVESLDVADELAEPLLAAGYPRLEHITQDTEKTDARSTVGRYDHTDSAALWHKRVHASADPGRPTNVHLRVHGWPNQQFALLFVDWLAANPGAREDYLTVKCDADRRADGELARYVTAKEPWFLDAYQRAWEWADAVHWRP.
SEQ ID NO：3：MTYIVGLTGGIGSGKTTIANLFTDLGVPLVDADVVAREVVAKDSPLLSKIVEHFGAQILTEQGELNRAALRERVFNHDEDKLWLNNLLHPAIRERMKQKLAEQTAPYTLFVVPLLIENKLTALCDRILVVDVSPQTQLARSAQRDNNNFEQIQRIMNSQVSQQERLKWADDVINNDAELAQNLPHLQQKVLELHQFYLQQAENKNA.
SEQ ID NO：4：MGHEAKHPIIIGITGNIGSGKSTVAALLRSWGYPVLDLDALAARARENKEEELKRLFPEAVVGGRLDRRALARLVFSDPERLKALEAVVHPEVRRLLMEELSRLEAPLVFLEIPLLFEKGWEGRLHGTLLVAAPLEERVRRVMARSGLSREEVLARERAQMPEEEKRKRATWVLENTGSLEDLERALKAVLAELTGGAKGGRG.
SEQ ID NO：5：MRIVGLTGGIASGKSTVSNLFKASGIPVVDADVVARDVLKKGSGGWKRVVAAFGEEILLPSGEVDRPKLGQIVFSSDSKRQLLNKLMAPYISSGIFWEILKQWASGAKVIVVDIPLLFEVKMDKWTKPIVVVWVSQETQLKRLMERDGLSEEDARNRVMAQMPLDSKRSKADVVIDNNGSLDDLHQQFEKVLIEIRRPLTWIEFWRSRQGAFSVLGSVILGLSVCKQLKIGS.
SEQ ID NO：6：MKMFFRLTRELRDELKRPLGELVRGPIPEPYLKVRGELEKHPVVTVGDVVTENVLKIGVKPIIALYDLKTKRKEYSPEIEDTAVFLTVTNPPGTITKALLDTVRKAFGLAERGRNVHILVSGEEDLAAIPAVLYAPLGTLVLYGQPDEGVVLIKVTPECKRRCAKILASMEVVRDGD.
SEQ ID NO：7：MVYSVGLTGNIASGKSTVAEFFSELGINVIYADKIAKELTSKNTPCYQDIISHFGSSVVLNNGELDRKRIRDIIFSNSNERLWLESLLHPVIREKIEEQLIACTSPYCLIEIPLLFNKHHYPYLQKVLLVIAPLESQLDRIVKRDHCTKKQALAILATQPNLEQRLEAADDVLINESGLSELKAKVNKLHQKYLREAKIKQ.
SEQ ID NO：8：MKNAFFVTASIACGKSTFIEIANSLGFKSISADKIAHKILDENALELEKIFSPFNLKNLLTKEKKIDRKILGEIVFNNKEAKKILENFTHPKIRAKILEQMQILDKENKAFFVEIPLFFESGAYENLGKVIVIYTPKELSLKRIMQRDKLSLEAAKVRLDSQIDIEEKLKKADFIIKNTNSYVDFRQECVKVIQEISKGKM.
SEQ ID NO：9：MYAIGLTGGIGSGKTTVADLFAARGASLVDTDLIAHRITAPAGLAMPAIEQTFGPAFVAADGSLDRARMRALIFSDEDARRRLEAITHPLIRAETEREARDAQGPYVIFVVPLLVESRNWKARCDRVLVVDCPVDTQIARVMQRNGFTREQVEAIIARQATREARLAAADDVIVNDATTPDALAAQVDALHQRYLAFAAAKH.
SEQ ID NO：10：MLAIGITGSYASGKTFILDYLAEKGYKTFCADRCIKELYQDLSVQTQILKLLPELESFNIGKISNLIYNNDLAREKLQNFIYPLLIDKLILFKKENANSKFGFAEIPLLYEAKFDKYFDFVVTIYCSEEIRMQRAITRTSFDIEIYNKIKEIQLSQESKIAKADFAINSGVDMLDLEKQIEKLILVIARKL.
SEQ ID NO：11：MSTNITFHASALTRSERTELRNQRGLTIWLTGLSASGKSTLAVELEHQLVRDRRVHAYRLDGDNIRFGLNKDLGFSEADRNENIRRIAEVAKLFADSNSIAITSFISPYRKDRDTARQLHEVATPGEETGLPFVEVYVDVPVEVAEQRDPKGLYKKAREGVIKEFTGISAPYEAPANPEVHVKNYELPVQDAVKQIIDYLDTKGYLPAKKE.
SEQ ID NO：12：MALHDENVVWHSHPVTVQQRELHHGHRGVVLWFTGLSGSGKSTVAGALEEALHKLGVSTYLLDGDNVRHGLCSDLGFSDADRKENIRRVGEVANLMVEAGLVVLTAFISPHRAERQMVRERVGEGRFIEVFVDTPLAICEARDPKGLYKKARAGELRNFTGIDSVYEAPESAEIHLNGEQLVTNLVQQLLDLLRQNDIIRS.
SEQ ID NO：13：MATNITWHPNLTYDERKALRKQDGCTIWLTGLSASGKSTIACALEQLLLQKNLSAYRLDGDNIRFGLNKDLGFSEKDRNENIRRISEVSKLFADSCAISITSFISPYRVDRDRARELHKEAGLKFIEIFVDVPLEVAEQRDPKGLYKKAREGVIKEFTGISAPYEAPKAPELHLRTDQKTVEECATIIYEYLISEKIIRKHL.
SEQ ID NO：14：MSTVGNSTNIFWQESPIGKTERQKLLNQKGCVVWITGLSGSGKSTLACSLSRELNNRGKLSYILDGDNLRHGLNKDLGFKAEDRVENIRRVGEVAKLFADAGLICIASLISPYRKDRDACREMIQNSSFIEVFMNMSLQLCEARDPKGLYKLARAGKIKGFTGIDDPYESPLNCEIELKEKEGECPSPVAMAEEVISYLEDKGFLQNE.
SEQ ID NO：15：MATNITHHAGITRNERNQLRKQKGLTIWLTGLSASGKSTIAVELEHQLLQRGLHAYRLDGDNVRFGLNKDLGFSDADRNENIRRIAEVAKLFADSSSIAITSFISPFRADRDTARKLHEVPTPNDSTGLPFVEVFVDVPIEVAEKRDPKGLYKKAREGIIKEFTGISSPYEAPENPEVHVKNVDLPIQEAVKQIIDYLDSKKLLDA.
SEQ ID NO：16：MTHNPNIIWHPAAISKSDRQSLNGHKSCVLWFTGLSGSGKSVLANAVDEKLYRKGIQSYVLDGDNIRHGLNKDLGFQTGDRIENIRRIGEVAKLFVDSGQMILTAFISPFREDRDMVRALFPKGEFFEIYVKCPLHVCEQRDPKGLYKKARNGEIKHFTGIDSPYEAPLSPDFIIESDQTSISDGADLIINALQNRGII.
SEQ ID NO：17：MATSMSFVIWITGPSGAGKTTLANALYKKLESMGYRVELLDGDGVRRKLYPNLGFSEEERWMHNRVVVEMARRLSRNGIITIVSVVSPYRAWREYARKEIEKFVEVYPRCPLEVRMKRDPKGLYSKALRGEIKGLTGLDGEYEEPENPEVVVDTDKMTVEEEVEAVLKKLMELGYL.
SEQ ID NO：18：MSTNIVWHHTSVTKEDRRQRNGHHSAVLWFTGLSGSGKSTVANAVSRRLFELGIQNYVLDGDNIRHGLNKDLGFSAADRTENIRRIGEVAKLFVDSGQFVLTAFISPFAEDRALVRRLVEEDEFIEIYVNCPIEECEKRDPKELYQKARRGEIREFTGIDSPYEAPEAPELTIETHRYSVDECVEQVLAYLRERGFIPASETH.
SEQ ID NO：19：MQQRGVTIWLTGLSGAGKTTITHALEKKLRDSGYRLEVLDGDVVRTNLTKGLGFSKEDRDTNIRRIGFVSHLLTRNGVIVLVSAISPYAAIRQEVKHTIGDFLEVFVNAPLAVCEERDVKGLYAKARSGEIKGFTGIDDPYEPPTNPDVECRTDLEELDESVGKIWQKLVDLKYIEG.
SEQ ID NO：20：MTTYKCIEKGIVVWLTGLPGSGKTTIATRLADLLQKEGYRVEVLDGDWARTTVSEGAGFTREERLRHLKRIAWIARLLARNGVIVICSFVSPYKQARNMVRRIVEEEGIPFLEIYVKASLEEVIRRDPKGLYKKALKGELENFTGITDPYEPPENPQLVLDTESNTIEHNVSYLYSLVKAVIE.
In a preferred embodiment, the nucleic acid monomers include natural nucleic acid monomers or non-natural nucleic acid monomers, the five-carbon sugar of the natural nucleic acid monomers being ribose or deoxyribose, the five-carbon sugar of the non-natural nucleic acid monomers being a non-natural monosaccharide consisting of 5C atoms having a modified ribose or deoxyribose, or having a non-natural backbone.
The nucleic acid monomers include natural nucleosides or nucleotides, or non-natural nucleosides or nucleotides.
In the above method, the nucleic acid monomer or oligonucleotide chain to be catalyzed may be composed of natural nucleotides or may contain non-natural nucleotides. The method can carry out 3' -O-phosphorylation modification on the 3' -OH of the natural nucleotide and can also carry out the same modification on the 3' -OH of the non-natural nucleotide.
Non-natural nucleotides (XNA) are a class of nucleic acid molecules having a non-natural backbone or nucleobases, preferably the non-natural nucleotides comprise: ribonucleotides with one or more of the following modifications: ribose 2' -position modification, ribose skeleton modification, base modification or phosphate skeleton modification; preferably, the ribose 2 '-modification includes a 2' -methoxy modification, a2 '-fluoro modification, a 2' -hydrogen modification, a2 '-methoxyethyl modification, a 2' -FANA modification, a locked nucleic acid modification, or a hexitol nucleic acid modification; preferably, the ribose backbone modification comprises replacing ribose in a nucleotide with ribuloNA, TNA, tPhoNA or dXNA; preferably, the base modification comprises deazaadenine C7 modification, deazaguanosine C7 modification, cytosine C5 modification or uridine C5 modification; phosphate backbone modifications include PS modifications. The structure of the unnatural nucleotide differs from the unnatural nucleotide in the presence or absence of a phosphate group.
Wherein the locked nucleic acid is modified to; Modification of hexitol nucleic acids to;2' Methoxyethyl modification to/>;2' Methoxy modification to;2' Fluorine modification to/>;2' -FANA is/>; RibuloNA is/>; TNA is/>; TPhoNA is/>; DXNA is/>; PS modification to/>. In the above structures, base represents a Base. Modifications to non-natural nucleotides as described above are described in the literature ：Duffy K , Arangundy-Franklin S , Holliger P . Modified nucleic acids: replication, evolution, and next-generation therapeutics[J]. BMC Biology, 2020, 18(1):112.
In a preferred embodiment, the nucleic acid monomers include natural nucleic acid monomers or non-natural nucleic acid monomers, the five-carbon sugar of the natural nucleic acid monomers being ribose or deoxyribose, the five-carbon sugar of the non-natural nucleic acid monomers being a non-natural monosaccharide consisting of 5C atoms having a modified ribose or deoxyribose, or having a non-natural backbone.
The nucleic acid monomers include natural nucleosides or nucleotides, or non-natural nucleosides or nucleotides. The non-natural nucleoside or nucleotide contains ribose 2' -position modification, ribose skeleton modification, base modification or phosphate skeleton modification (the non-natural nucleoside has no phosphate group).
In the above method, the nucleic acid monomer to be catalyzed may be either a natural nucleotide or a natural nucleoside or an unnatural nucleotide or an unnatural nucleoside. The catalyzed oligonucleotide strand may be composed of either natural nucleotides or non-natural nucleotides.
The method can carry out 3' -O-phosphorylation modification on the 3' -OH of the natural nucleoside or nucleotide, and can also carry out the same modification on the 3' -OH of the non-natural nucleoside or nucleotide.
In a preferred embodiment, the non-natural oligonucleotide consists of non-natural nucleotides.
The advantageous effects of the present application will be explained in further detail below in connection with specific examples.
Example 1
Phosphokinase (APSK) using 5' -phosphosulfate-adenosine or phosphokinase (DPCK) dephosphorizing coa acts directly on the 3' end of a nucleic acid monomer or oligonucleotide chain, plus a removable protecting group 3' -O-phosphate.
Adding a reaction system into a clean container, enabling the concentration of a substrate (5 ' -UmCfUmCf-3 ') to be 100 mu M, enabling the final concentration of the phosphokinase of 5' -phosphosulfate-adenosine or the phosphokinase of dephosphorization coenzyme A to be 0.2mg/mL, adding 1mM ATP,10 mM MgCl 2, 100 mM Tris-HCl, adjusting the pH value to 8.0, reacting with 30 ℃ for 16 hours, and carrying out mass spectrum detection on the converted system.
M after A, C, G or U of the application represents a 2 'methoxy modification to the ribonucleotide and f represents a 2' fluoro modification to the ribonucleotide.
The APSK or DPCK used is shown in table 1.
TABLE 1
Wherein, -represents that the product is detected, the conversion rate is less than or equal to 0.1%, + represents that the product is detected, 0.1% < conversion rate is less than or equal to 1%, ++ represents that the product is detected, 1% < conversion rate is less than or equal to 10%.
Fig. 1 shows the use of SEQ ID NO:16, and FIG. 2 shows a mass spectrum of the results of catalysis using the kinase shown in SEQ ID NO. 16. Wherein the theoretical molecular weight of the substrate UmCfUmCf is 1192.20, the theoretical molecular weight of the product is 1271.17, the molecular weight (M-1:1271.16) of the product is detected, and the structure of the product is shown as a formula I.
。
Meanwhile, the product standard is used for comparison, and the same peak time is detected, which indicates that the reversible end capping of the 3' end is successfully carried out.
Example 2
Adding a reaction system into a clean container, enabling the substrate concentration to be 100 mu M, enabling the final concentration of the phosphokinase of 5' -phosphosulfate-adenosine or the phosphokinase of dephosphorization coenzyme A to be 0.2mg/mL, adding 1mM ATP,10 mM MgCl 2, 100 mM Tris-HCl, adjusting the pH value to 8.0, reacting with 30 ℃ for 16 hours, and carrying out mass spectrometry detection on the converted condition of the reacted system. The results show that the target products of 3' -O-phosphate are produced in both APSK-3 and DPCK-7 catalyzed phosphorylation of various oligonucleotide substrates. The phosphate can be removed after phosphorylation by alkaline phosphatase or polynucleotide kinase catalysis.
The substrate sequence for carrying out this reaction is shown in Table 2, for example.
TABLE 2
Wherein "+" indicates that the product is detected, and the conversion rate is less than or equal to 1%; "++" indicates that the product is detected, and the conversion rate is 1% < 10%; pi represents a monophosphate group; A. c, G or m after U represents the 2' methoxy modification to the ribonucleotide, f represents the 2' fluoro modification to the ribonucleotide, s represents the thio modification of the 5' phosphate to the ribonucleotide. A. C, G or d before T indicates that the nucleotide is deoxyribonucleotide, and the unlabeled d nucleotides are all ribonucleotides.
The LC-MS analysis of the reaction for 3' -O-phosphate modification using DPCK-7 to catalyze the 5' -Pi-GA-OH-3' is shown in FIG. 5. The theoretical product molecular weight was 772.08, the product molecular weight was detected (M-1:771.0685), and the product 5'-Pi-GA-OH-Pi-3', where Pi represents a phosphate group.
The above substrates can also be subjected to catalytic reactions using the above other APSK enzymes or DPCK enzymes.
Example 3
Adding a reaction system into a clean container, enabling the concentration of an AMP substrate to be 100 mu M, enabling the final concentration of phosphokinase APSK3 of 5' -phosphosulfuric acid-adenosine to be 0.2mg/mL, adding 1mM ATP,10 mM MgCl 2, 100 mM Tris-HCl, adjusting the pH value to 8.0, reacting with 30 ℃ for 16 hours, detecting conversion conditions by liquid phase and mass spectrum of the reacted system, and calibrating peak positions by using a standard substance. The results showed that a new peak was generated at 6.384min in the system, which was identical to the peak position of the standard 3' pi-AMP, while mass spectrometry identified molecular weight 428.1 and theoretical molecular weight 427.03, indicating the formation of the product.
LC-MS analysis spectra of 3 '-O-phosphate modification using APSK-3 catalytic AMP (adenosine monophosphate, 5' -adenine nucleotide) are shown in FIG. 3 and FIG. 4, wherein FIG. 3 is a liquid chromatography result diagram of the reaction system, and FIG. 4 is a mass spectrometry detection result diagram of the phosphorylated product (peak with peak time of 6.384).
Example 4
Adding a reaction system into a clean container, enabling the substrate concentration to be 100 mu M, enabling the final concentration of the phosphokinase of 5' -phosphosulfate-adenosine or the phosphokinase of dephosphorization coenzyme A to be 0.2mg/mL, adding 1mM ATP,10 mM MgCl 2, 100 mM Tris-HCl, adjusting the pH value to 8.0, reacting with 30 ℃ for 16 hours, and detecting the reaction condition of the reacted system by liquid phase and mass spectrum. The results showed that the formation of product molecules was examined in both APSK-3 and DPCK-7 catalyzed phosphorylation of various nucleotides or nucleoside substrates, indicating the formation of the target product of 3' -O-phosphate.
TABLE 3 Table 3
Wherein "+" indicates that the product is detected, and the conversion rate is less than or equal to 1%; "++" indicates that the product is detected, and the conversion rate is 1% < 10%; "+++". Representation of the presence of a product is detected and, the conversion rate is more than 10 percent and less than or equal to 20 percent; "+++". "means the presence of a product is detected and, the conversion rate is more than 20 percent and less than or equal to 30 percent; pi represents a monophosphate group; 2Pi represents a diphosphate group; 3Pi represents a triphosphate group; A. c, G or m after U represents the 2 'methoxy modification to the ribonucleotide and f represents the 2' fluoro modification to the ribonucleotide. A. C, G or d before T indicates that the nucleotide is deoxyribonucleotide, and the unlabeled d nucleotides are all ribonucleotides.
Example 5
Adding a reaction system into a clean container to enable the concentration of a Pi-GmGfUmCf-OH substrate to be 100 mu M, enabling the final concentration of DPCK-3 to be 0.2mg/mL, adding 1mM ATP,10 mM MgCl 2, 100 mM Tris-HCl, adjusting the pH value to 8.0, reacting with 30 ℃ for 16 hours, and detecting the reaction condition of the reacted system by liquid phase and mass spectrum. The results showed that the peaks of the products were generated in the reaction system, as shown in FIG. 6, and the molecular weights of the products were detected by mass spectrometry, indicating that the target products of 3' -O-phosphoric acid were generated.
Example 6
Adding a reaction system into a clean container, enabling the concentration of UMP substrate to be 100 mu M, enabling the final concentration of APSK-6 to be 0.2mg/mL, adding 1mM ATP,10 mM MgCl 2 and 100mM Tris-HCl, adjusting the pH value to 8.0, reacting with 30 ℃ for 16 hours, and detecting the reaction condition of the reacted system by liquid phase and mass spectrum. The result shows that a new peak is generated in the reaction system, and the molecular weight of the new peak is confirmed to be M-1 by mass spectrum: 403.0 the molecular weight of the theoretical product is 404.0, as shown in figure 7, and the peak time is the same as that of the product standard, which indicates that the target product of 3' -O-phosphoric acid is generated.
From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: by using the two enzyme catalysis methods, the phosphokinase such as APSK enzyme or DPCK enzyme can catalyze nucleic acid monomers or oligonucleotide chains to obtain the nucleic acid monomers or oligonucleotide chains of 3 '-O-phosphoric acid, so that the enzymatic preparation of the nucleotide containing the 3' -O-phosphoric acid modification is realized. The method can realize the enzymatic synthesis of the nucleotide modified by the 3' -O-phosphate, and can directly add the 3' -O-phosphate modification belonging to the reversible modification group at the 3' end of the nucleic acid chain relative to the chemical synthesis of the nucleic acid chain with the protecting group, and can easily remove the protection, thereby achieving the purposes of green environment protection and high efficiency.
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 (10)
1. A method of enzymatically synthesizing a nucleic acid having a 3' -O-phosphate modification, the method comprising:
The phosphokinase is used for catalyzing a nucleic acid monomer with the length of 1nt or an oligonucleotide chain with the length of more than or equal to 2nt to obtain the nucleic acid monomer or the oligonucleotide chain of 3' -O-phosphoric acid.
2. The method of claim 1, wherein the nucleic acid monomer comprises a nucleoside or nucleotide;
The nucleoside consists of a base and a five-carbon sugar;
the nucleotide consists of the base, the pentose and the phosphate;
the five-carbon sugar comprises ribose or deoxyribose;
the base is adenine, guanine, cytosine or uracil;
the number of the phosphates in the nucleotides is 1,2 or 3, and the corresponding nucleotides are nucleoside monophosphates, nucleoside diphosphate or nucleoside triphosphate respectively.
3. The method according to claim 1, wherein the oligonucleotide strand is an RNA strand or a DNA strand having a length of 2nt or more.
4. A method according to claim 3, wherein the oligonucleotide strand is 2-20nt in length.
5. The method of claim 1, wherein the phosphokinase comprises APSK enzyme or DPCK enzyme.
6. The method of claim 5, wherein the DPCK enzyme is SEQ ID NO: 1-SEQ ID NO:10, or a protein as set forth in any one of seq id nos;
The APSK enzyme is SEQ ID NO: 11-SEQ ID NO:20, and a protein as set forth in any one of seq id nos.
7. The method of claim 2, wherein the nucleic acid monomers comprise natural nucleic acid monomers or non-natural nucleic acid monomers, the five-carbon sugar of the natural nucleic acid monomers being ribose or deoxyribose, the five-carbon sugar of the non-natural nucleic acid monomers being non-natural monosaccharides consisting of 5C atoms containing modified ribose or deoxyribose, or having a non-natural backbone.
8. The method of claim 1, wherein the oligonucleotide strand comprises a natural oligonucleotide strand consisting of natural nucleotides, or a non-natural oligonucleotide strand comprising non-natural nucleotides.
9. The method of claim 8, wherein the non-natural oligonucleotide strand consists of the non-natural nucleotide.
10. The method according to any one of claims 1-9, characterized in that the method comprises: and catalyzing by using ATP or polyphosphate and the phosphokinase and taking the nucleic acid monomer or the oligonucleotide chain as a substrate to obtain the nucleic acid monomer or the oligonucleotide chain of the 3' -O-phosphate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410574630.3A CN118127102B (en) | 2024-05-10 | 2024-05-10 | Method for synthesizing nucleic acid with 3' -O-phosphate modification by enzyme method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410574630.3A CN118127102B (en) | 2024-05-10 | 2024-05-10 | Method for synthesizing nucleic acid with 3' -O-phosphate modification by enzyme method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118127102A true CN118127102A (en) | 2024-06-04 |
| CN118127102B CN118127102B (en) | 2024-08-06 |
Family
ID=91240995
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410574630.3A Active CN118127102B (en) | 2024-05-10 | 2024-05-10 | Method for synthesizing nucleic acid with 3' -O-phosphate modification by enzyme method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118127102B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119462803A (en) * | 2025-01-16 | 2025-02-18 | 天津凯莱英生物科技有限公司 | Synthesis method and application of nucleoside monomer analogs and oligonucleotides |
| CN119462803B (en) * | 2025-01-16 | 2025-04-18 | 天津凯莱英生物科技有限公司 | Synthesis method and application of nucleoside monomer analogs and oligonucleotides |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113272442A (en) * | 2019-01-03 | 2021-08-17 | Dna斯克瑞普特公司 | One-pot synthesis of pools of oligonucleotides |
| WO2022038386A1 (en) * | 2020-08-21 | 2022-02-24 | Nuclera Nucleics Ltd | Polyacrilamide type solid-supported nucleic acid synthesis |
| CN117070514A (en) * | 2023-10-16 | 2023-11-17 | 吉林凯莱英医药化学有限公司 | Preparation method of non-natural RNA and product |
-
2024
- 2024-05-10 CN CN202410574630.3A patent/CN118127102B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113272442A (en) * | 2019-01-03 | 2021-08-17 | Dna斯克瑞普特公司 | One-pot synthesis of pools of oligonucleotides |
| US20220356510A1 (en) * | 2019-01-03 | 2022-11-10 | Dna Script | One Pot Synthesis of Sets of Oligonucleotides |
| WO2022038386A1 (en) * | 2020-08-21 | 2022-02-24 | Nuclera Nucleics Ltd | Polyacrilamide type solid-supported nucleic acid synthesis |
| CN117070514A (en) * | 2023-10-16 | 2023-11-17 | 吉林凯莱英医药化学有限公司 | Preparation method of non-natural RNA and product |
Non-Patent Citations (2)
| Title |
|---|
| ACCESSION NO.：WP_010869082.1: "dephospho-CoA kinase [Haemophilus influenzae]", Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/protein/499171495?sat=5&satkey=758337272> * |
| 王启松: "多核苷酸的3’-端磷酰化", 生物化学与生物物理学报, vol. 11, no. 3, 30 September 1979 (1979-09-30), pages 207 - 214 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119462803A (en) * | 2025-01-16 | 2025-02-18 | 天津凯莱英生物科技有限公司 | Synthesis method and application of nucleoside monomer analogs and oligonucleotides |
| CN119462803B (en) * | 2025-01-16 | 2025-04-18 | 天津凯莱英生物科技有限公司 | Synthesis method and application of nucleoside monomer analogs and oligonucleotides |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118127102B (en) | 2024-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Burgess et al. | Syntheses of nucleoside triphosphates | |
| AU2006323745B2 (en) | Method for replicating nucleic acids and novel unnatural base pairs | |
| Barlow et al. | Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase | |
| R Kore et al. | Recent advances in the syntheses of nucleoside triphosphates | |
| US8153779B2 (en) | Nucleotide with an alpha-phosphate mimetic | |
| CN108366604A (en) | Compositions and methods for synthesizing 5' -capped RNA | |
| Samano et al. | Nucleic acid and related compounds. 75. Synthesis and radical-induced ring-opening reactions of 2'-deoxyadenosine-2'-spirocyclopropane and its uridine analogs. Mechanistic probes for ribonucleotide reductases | |
| US20220195424A1 (en) | Chemical Capping for Template Switching | |
| CN118127102B (en) | Method for synthesizing nucleic acid with 3' -O-phosphate modification by enzyme method | |
| Rammler et al. | Nucleoside phosphonic acids. II. The synthesis of 5'-deoxythymidine 5'-phosphonic acid and its pyrophosphate derivatives | |
| Taverna-Porro et al. | Chemoenzymatic preparation of nucleosides from furanoses | |
| Gillam et al. | Enzymatic synthesis of deoxyribo-oiigonucleotides of defined sequence. Deoxyribo-oligonucleotide synthesis | |
| Kore et al. | A new, facile, and protection-free one-pot chemical synthesis of 2′-deoxynucleoside-5′-tetraphosphates | |
| Jiang et al. | Synthesis and recognition of novel isonucleoside triphosphates by DNA polymerases | |
| Hollenstein | Enzymatic synthesis of base-modified nucleic acids | |
| US6329177B1 (en) | Enzymatic methods of preparing polymers from nucleotide and/or non-nucleotide monomers | |
| Caton-Williams et al. | Convenient synthesis of nucleoside 5′-(α-P-thio) triphosphates and phosphorothioate nucleic acids (DNA and RNA) | |
| CN119462803B (en) | Synthesis method and application of nucleoside monomer analogs and oligonucleotides | |
| CN113122593A (en) | Method for preparing nucleoside triphosphate and deoxynucleoside triphosphate by utilizing polyphosphate | |
| Lewkowicz et al. | Enzymatic synthesis of nucleic acid derivatives using whole cells | |
| R Kore et al. | Chemical and enzymatic synthesis of nucleoside tetraphosphates | |
| Kanavarioti | Preference for internucleotide linkages as a function of the number of constituents in a mixture | |
| WO2007144168A1 (en) | Enzymatic process for preparation of 5'-monophosphate-nucleotides | |
| Barpuzary et al. | Improved synthesis and polymerase recognition of 7-deaza-7-modified α-l-threofuranosyl guanosine analogs | |
| CN119462803A (en) | Synthesis method and application of nucleoside monomer analogs and oligonucleotides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |