JP6898236B2 - 核酸合成のための方法および装置 - Google Patents
核酸合成のための方法および装置 Download PDFInfo
- Publication number
- JP6898236B2 JP6898236B2 JP2017529593A JP2017529593A JP6898236B2 JP 6898236 B2 JP6898236 B2 JP 6898236B2 JP 2017529593 A JP2017529593 A JP 2017529593A JP 2017529593 A JP2017529593 A JP 2017529593A JP 6898236 B2 JP6898236 B2 JP 6898236B2
- Authority
- JP
- Japan
- Prior art keywords
- nucleotide
- inhibitor
- nucleotide analog
- analog
- seq
- 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.)
- Active
Links
- 238000000034 method Methods 0.000 title claims description 97
- 238000001668 nucleic acid synthesis Methods 0.000 title description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 128
- 239000003112 inhibitor Substances 0.000 claims description 88
- 239000002773 nucleotide Substances 0.000 claims description 61
- 108090000119 Nucleotidyltransferases Proteins 0.000 claims description 35
- 102000003832 Nucleotidyltransferases Human genes 0.000 claims description 35
- 239000007787 solid Substances 0.000 claims description 31
- 108091034117 Oligonucleotide Proteins 0.000 claims description 30
- 150000007523 nucleic acids Chemical class 0.000 claims description 27
- 102000039446 nucleic acids Human genes 0.000 claims description 23
- 108020004707 nucleic acids Proteins 0.000 claims description 23
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 17
- 238000003776 cleavage reaction Methods 0.000 claims description 16
- 230000007017 scission Effects 0.000 claims description 16
- 230000010354 integration Effects 0.000 claims description 15
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 14
- 125000004122 cyclic group Chemical group 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 230000002194 synthesizing effect Effects 0.000 claims description 10
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 10
- 229930024421 Adenine Natural products 0.000 claims description 8
- 229960000643 adenine Drugs 0.000 claims description 8
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 8
- 229940035893 uracil Drugs 0.000 claims description 8
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 229940113082 thymine Drugs 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 4
- 229940104302 cytosine Drugs 0.000 claims description 4
- 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 3
- 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 3
- 108060002716 Exonuclease Proteins 0.000 claims description 3
- 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 3
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 3
- 239000006227 byproduct Substances 0.000 claims description 3
- 102000013165 exonuclease Human genes 0.000 claims description 3
- 102000040430 polynucleotide Human genes 0.000 description 59
- 108091033319 polynucleotide Proteins 0.000 description 59
- 239000002157 polynucleotide Substances 0.000 description 59
- 125000005647 linker group Chemical group 0.000 description 46
- 239000003153 chemical reaction reagent Substances 0.000 description 39
- 238000006243 chemical reaction Methods 0.000 description 38
- 102000004190 Enzymes Human genes 0.000 description 35
- 108090000790 Enzymes Proteins 0.000 description 35
- 238000003786 synthesis reaction Methods 0.000 description 35
- 230000015572 biosynthetic process Effects 0.000 description 31
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 26
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 26
- 108020004414 DNA Proteins 0.000 description 19
- 102000053602 DNA Human genes 0.000 description 19
- 239000002585 base Substances 0.000 description 18
- 238000003860 storage Methods 0.000 description 18
- 238000010586 diagram Methods 0.000 description 17
- RGWHQCVHVJXOKC-SHYZEUOFSA-N dCTP Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO[P@](O)(=O)O[P@](O)(=O)OP(O)(O)=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-N 0.000 description 15
- 108010068698 spleen exonuclease Proteins 0.000 description 15
- 239000003999 initiator Substances 0.000 description 14
- 125000003275 alpha amino acid group Chemical class 0.000 description 13
- 125000002652 ribonucleotide group Chemical group 0.000 description 13
- 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 12
- 235000001014 amino acid Nutrition 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 230000002401 inhibitory effect Effects 0.000 description 11
- 229920000642 polymer Polymers 0.000 description 11
- 108090000765 processed proteins & peptides Proteins 0.000 description 11
- 102000004357 Transferases Human genes 0.000 description 10
- 108090000992 Transferases Proteins 0.000 description 10
- 230000000903 blocking effect Effects 0.000 description 10
- 238000007641 inkjet printing Methods 0.000 description 10
- 229920002477 rna polymer Polymers 0.000 description 10
- 108091028664 Ribonucleotide Proteins 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 230000008030 elimination Effects 0.000 description 9
- 238000003379 elimination reaction Methods 0.000 description 9
- 230000002441 reversible effect Effects 0.000 description 9
- 239000002336 ribonucleotide Substances 0.000 description 9
- 239000001226 triphosphate Substances 0.000 description 9
- 235000011178 triphosphate Nutrition 0.000 description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 8
- -1 nucleotide triphosphate analog Chemical class 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 8
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 7
- 229910019142 PO4 Inorganic materials 0.000 description 7
- 101710124239 Poly(A) polymerase Proteins 0.000 description 7
- 238000004140 cleaning Methods 0.000 description 7
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 7
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 7
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 6
- 239000002105 nanoparticle Substances 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 6
- 239000010452 phosphate Substances 0.000 description 6
- 150000008300 phosphoramidites Chemical class 0.000 description 6
- 239000000376 reactant Substances 0.000 description 6
- 108010043958 Peptoids Proteins 0.000 description 5
- 239000005547 deoxyribonucleotide Substances 0.000 description 5
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 5
- 230000001419 dependent effect Effects 0.000 description 5
- 238000010348 incorporation Methods 0.000 description 5
- 229920002521 macromolecule Polymers 0.000 description 5
- 230000005257 nucleotidylation Effects 0.000 description 5
- 238000002515 oligonucleotide synthesis Methods 0.000 description 5
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000003908 quality control method Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 239000002699 waste material Substances 0.000 description 5
- FRYULLIZUDQONW-IMJSIDKUSA-N Asp-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(O)=O FRYULLIZUDQONW-IMJSIDKUSA-N 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 150000001450 anions Chemical class 0.000 description 4
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 239000000377 silicon dioxide Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 3
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 3
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 244000309466 calf Species 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 238000002818 protein evolution Methods 0.000 description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 3
- XKMLYUALXHKNFT-UHFFFAOYSA-N rGTP Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O XKMLYUALXHKNFT-UHFFFAOYSA-N 0.000 description 3
- QUKPALAWEPMWOS-UHFFFAOYSA-N 1h-pyrazolo[3,4-d]pyrimidine Chemical class C1=NC=C2C=NNC2=N1 QUKPALAWEPMWOS-UHFFFAOYSA-N 0.000 description 2
- JTBBWRKSUYCPFY-UHFFFAOYSA-N 2,3-dihydro-1h-pyrimidin-4-one Chemical class O=C1NCNC=C1 JTBBWRKSUYCPFY-UHFFFAOYSA-N 0.000 description 2
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
- PHIYHIOQVWTXII-UHFFFAOYSA-N 3-amino-1-phenylpropan-1-ol Chemical compound NCCC(O)C1=CC=CC=C1 PHIYHIOQVWTXII-UHFFFAOYSA-N 0.000 description 2
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 2
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 description 2
- HCGHYQLFMPXSDU-UHFFFAOYSA-N 7-methyladenine Chemical compound C1=NC(N)=C2N(C)C=NC2=N1 HCGHYQLFMPXSDU-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- PCDQPRRSZKQHHS-CCXZUQQUSA-N Cytarabine Triphosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-CCXZUQQUSA-N 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 2
- 101150040913 DUT gene Proteins 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N Hypoxanthine Natural products O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- KYQCOXFCLRTKLS-UHFFFAOYSA-N Pyrazine Chemical compound C1=CN=CC=N1 KYQCOXFCLRTKLS-UHFFFAOYSA-N 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- RZCIEJXAILMSQK-JXOAFFINSA-N TTP Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 RZCIEJXAILMSQK-JXOAFFINSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- ISAOCJYIOMOJEB-UHFFFAOYSA-N benzoin Chemical compound C=1C=CC=CC=1C(O)C(=O)C1=CC=CC=C1 ISAOCJYIOMOJEB-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 239000004205 dimethyl polysiloxane Substances 0.000 description 2
- 150000002333 glycines Chemical class 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 150000002500 ions Chemical group 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000000269 nucleophilic effect Effects 0.000 description 2
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 2
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 2
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 2
- 229920001610 polycaprolactone Polymers 0.000 description 2
- 239000004632 polycaprolactone Substances 0.000 description 2
- 239000004926 polymethyl methacrylate Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 2
- PGAVKCOVUIYSFO-UHFFFAOYSA-N uridine-triphosphate Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-UHFFFAOYSA-N 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- 0 *OCC(C1)(C1(C1(C2)O)O)OC12N(C=CC(N1**C=CC*=*)=O)C1=O Chemical compound *OCC(C1)(C1(C1(C2)O)O)OC12N(C=CC(N1**C=CC*=*)=O)C1=O 0.000 description 1
- FYADHXFMURLYQI-UHFFFAOYSA-N 1,2,4-triazine Chemical compound C1=CN=NC=N1 FYADHXFMURLYQI-UHFFFAOYSA-N 0.000 description 1
- JIHQDMXYYFUGFV-UHFFFAOYSA-N 1,3,5-triazine Chemical compound C1=NC=NC=N1 JIHQDMXYYFUGFV-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- BNGVWAFGHGJATM-UHFFFAOYSA-N 1h-imidazo[1,5-a][1,3,5]triazin-2-one Chemical compound N1C(=O)N=CN2C=NC=C21 BNGVWAFGHGJATM-UHFFFAOYSA-N 0.000 description 1
- HUTNOYOBQPAKIA-UHFFFAOYSA-N 1h-pyrazin-2-one Chemical compound OC1=CN=CC=N1 HUTNOYOBQPAKIA-UHFFFAOYSA-N 0.000 description 1
- YQTCQNIPQMJNTI-UHFFFAOYSA-N 2,2-dimethylpropan-1-one Chemical group CC(C)(C)[C]=O YQTCQNIPQMJNTI-UHFFFAOYSA-N 0.000 description 1
- LOJNBPNACKZWAI-UHFFFAOYSA-N 3-nitro-1h-pyrrole Chemical compound [O-][N+](=O)C=1C=CNC=1 LOJNBPNACKZWAI-UHFFFAOYSA-N 0.000 description 1
- CKTSBUTUHBMZGZ-ULQXZJNLSA-N 4-amino-1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-tritiopyrimidin-2-one Chemical group O=C1N=C(N)C([3H])=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-ULQXZJNLSA-N 0.000 description 1
- ZLAQATDNGLKIEV-UHFFFAOYSA-N 5-methyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC1=CNC(=S)NC1=O ZLAQATDNGLKIEV-UHFFFAOYSA-N 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- OZFPSOBLQZPIAV-UHFFFAOYSA-N 5-nitro-1h-indole Chemical compound [O-][N+](=O)C1=CC=C2NC=CC2=C1 OZFPSOBLQZPIAV-UHFFFAOYSA-N 0.000 description 1
- UJBCLAXPPIDQEE-UHFFFAOYSA-N 5-prop-1-ynyl-1h-pyrimidine-2,4-dione Chemical compound CC#CC1=CNC(=O)NC1=O UJBCLAXPPIDQEE-UHFFFAOYSA-N 0.000 description 1
- HRYKDUPGBWLLHO-UHFFFAOYSA-N 8-azaadenine Chemical compound NC1=NC=NC2=NNN=C12 HRYKDUPGBWLLHO-UHFFFAOYSA-N 0.000 description 1
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
- 229960005508 8-azaguanine Drugs 0.000 description 1
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 1
- 101800000112 Acidic peptide Proteins 0.000 description 1
- 206010000599 Acromegaly Diseases 0.000 description 1
- OMLWNBVRVJYMBQ-YUMQZZPRSA-N Arg-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O OMLWNBVRVJYMBQ-YUMQZZPRSA-N 0.000 description 1
- WCFCYFDBMNFSPA-ACZMJKKPSA-N Asp-Asp-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(O)=O WCFCYFDBMNFSPA-ACZMJKKPSA-N 0.000 description 1
- CKAJHWFHHFSCDT-WHFBIAKZSA-N Asp-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O CKAJHWFHHFSCDT-WHFBIAKZSA-N 0.000 description 1
- XMWRBQBLMFGWIX-UHFFFAOYSA-N C60 fullerene Chemical class C12=C3C(C4=C56)=C7C8=C5C5=C9C%10=C6C6=C4C1=C1C4=C6C6=C%10C%10=C9C9=C%11C5=C8C5=C8C7=C3C3=C7C2=C1C1=C2C4=C6C4=C%10C6=C9C9=C%11C5=C5C8=C3C3=C7C1=C1C2=C4C6=C2C9=C5C3=C12 XMWRBQBLMFGWIX-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 108010049175 N-substituted Glycines Proteins 0.000 description 1
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 description 1
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 1
- 108091028733 RNTP Proteins 0.000 description 1
- 101500027983 Rattus norvegicus Octadecaneuropeptide Proteins 0.000 description 1
- 206010067171 Regurgitation Diseases 0.000 description 1
- 229910052581 Si3N4 Inorganic materials 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 244000028419 Styrax benzoin Species 0.000 description 1
- 235000000126 Styrax benzoin Nutrition 0.000 description 1
- 235000008411 Sumatra benzointree Nutrition 0.000 description 1
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 1
- PGAVKCOVUIYSFO-XVFCMESISA-N UTP Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-XVFCMESISA-N 0.000 description 1
- YWBULOYFCXZCGF-UHFFFAOYSA-N [1,3]thiazolo[4,5-d]pyrimidine Chemical compound C1=NC=C2SC=NC2=N1 YWBULOYFCXZCGF-UHFFFAOYSA-N 0.000 description 1
- XAKBSHICSHRJCL-UHFFFAOYSA-N [CH2]C(=O)C1=CC=CC=C1 Chemical group [CH2]C(=O)C1=CC=CC=C1 XAKBSHICSHRJCL-UHFFFAOYSA-N 0.000 description 1
- 125000004036 acetal group Chemical group 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229960002130 benzoin Drugs 0.000 description 1
- 230000008238 biochemical pathway Effects 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000001715 carbamic acids Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229910000420 cerium oxide Inorganic materials 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical group O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229910003472 fullerene Inorganic materials 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 235000019382 gum benzoic Nutrition 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 239000002920 hazardous waste Substances 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000002071 nanotube Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- BMMGVYCKOGBVEV-UHFFFAOYSA-N oxo(oxoceriooxy)cerium Chemical compound [Ce]=O.O=[Ce]=O BMMGVYCKOGBVEV-UHFFFAOYSA-N 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- IGFXRKMLLMBKSA-UHFFFAOYSA-N purine Chemical compound N1=C[N]C2=NC=NC2=C1 IGFXRKMLLMBKSA-UHFFFAOYSA-N 0.000 description 1
- FICMSTTYJICTDM-UHFFFAOYSA-N pyridazine;triazine Chemical compound C1=CC=NN=C1.C1=CN=NN=C1 FICMSTTYJICTDM-UHFFFAOYSA-N 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- HQVNEWCFYHHQES-UHFFFAOYSA-N silicon nitride Chemical compound N12[Si]34N5[Si]62N3[Si]51N64 HQVNEWCFYHHQES-UHFFFAOYSA-N 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- RAOIDOHSFRTOEL-UHFFFAOYSA-N tetrahydrothiophene Chemical compound C1CCSC1 RAOIDOHSFRTOEL-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007723 transport mechanism Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 229950010342 uridine triphosphate Drugs 0.000 description 1
Images
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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Saccharide Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Enzymes And Modification Thereof (AREA)
Description
本発明は、例えば、以下の項目を提供する。
(項目1)
オリゴヌクレオチドを合成する方法であって、前記方法は、
ヌクレオチジルトランスフェラーゼ酵素の存在下、核酸鋳型の非存在下で、固体支持体に付着しているオリゴヌクレオチドをヌクレオチド類似体に曝露し、その結果前記ヌクレオチド類似体が前記オリゴヌクレオチドに組み込まれるステップを含み、
前記ヌクレオチド類似体は、阻害剤が切断可能なリンカーの切断によって除去されるまで前記阻害剤へ前記切断可能なリンカーによって結合されるヌクレオチドを含み、前記阻害剤は前記ヌクレオチジルトランスフェラーゼが前記オリゴヌクレオチドへのヌクレオチドまたはさらなるヌクレオチド類似体の組み込みを触媒することを立体配置的に防止する、方法。
(項目2)
前記ヌクレオチド類似体が、以下の構造
(項目3)
前記ヌクレオチド類似体が、以下の構造
(項目4)
前記阻害剤が、以下の構造
を含む、項目1に記載の方法。
(項目5)
前記阻害剤が、ポリペプトイドを含む、項目1に記載の方法。
(項目6)
前記阻害剤が、ポリマーを含む、項目1に記載の方法。
(項目7)
前記ポリマーが、ポリエチレングリコールを含む、項目6に記載の方法。
(項目8)
前記阻害剤が、タンパク質である、項目1に記載の方法。
(項目9)
前記阻害剤が、ナノ粒子である、項目1に記載の方法。
(項目10)
前記阻害剤が、直径50Å超の巨大分子である、項目1に記載の方法。
(項目11)
前記固体支持体に付着している前記核酸が、pH約6.5〜8.5を有する水溶液の存在下で前記ヌクレオチド類似体に曝露される、項目1に記載の方法。
(項目12)
前記固体支持体に付着している前記核酸が、約35〜39℃の温度で水溶液の存在下で前記ヌクレオチド類似体に曝露される、項目1に記載の方法。
(項目13)
前記固体支持体が、ビーズ、ウェルまたはPEGである、項目1に記載の方法。
(項目14)
前記核酸が、一本鎖である、項目1に記載の方法。
(項目15)
前記切断可能なリンカーが、前記ヌクレオチド類似体から切断されるときに環状の副産物を形成する部分を含む、項目1に記載の方法。
(項目16)
天然のヌクレオチドを産生するために前記切断可能なリンカーを切断するステップと;
ヌクレオチジルトランスフェラーゼ酵素の存在下、核酸鋳型の非存在下で、前記天然のヌクレオチドを第2のヌクレオチド類似体に曝露するステップと
をさらに含む、項目1に記載の方法。
(項目17)
前記オリゴヌクレオチドをエキソヌクレアーゼ酵素に曝露するステップをさらに含む、項目1に記載の方法。
(項目18)
前記ヌクレオチド類似体および前記ヌクレオチジルトランスフェラーゼ酵素を含む水溶液を提供するステップをさらに含む、項目1に記載の方法。
(項目19)
前記阻害剤が、荷電部分を含む、項目1に記載の方法。
(項目20)
前記ヌクレオチド類似体が、リボース糖またはデオキシリボース糖を含む、項目1に記載の方法。
(項目21)
ヌクレオチド基質が、アデニン、グアニン、シトシン、チミンおよびウラシルからなる群から選択される塩基を含む、項目1に記載の方法。
(項目22)
前記ヌクレオチジルトランスフェラーゼが、配列番号1、配列番号3、または配列番号5と少なくとも約90%同一のタンパク質配列を含む、項目1に記載の方法。
(項目23)
前記ヌクレオチジルトランスフェラーゼが、配列番号2、配列番号4、または配列番号6と少なくとも約90%同一のヌクレオチド配列を有する生物に由来する、項目1に記載の方法。
(項目24)
前記ヌクレオチド類似体が、液滴で送達される、項目1に記載の方法。
NTP−リンカー−阻害剤
の形態のものであり、そこで、NTPはヌクレオチド三リン酸(すなわち、dNTPまたはrNTP)であり、リンカーは塩基のピリジンまたはピリミジン間の切断可能なリンカーであり、阻害剤は、酵素が次のヌクレオチドを組み込むことを防止する基である。各ステップにおいて、新しいヌクレオチド類似体が、成長中のポリヌクレオチド鎖に組み込まれ、阻害基(inhibitor group)によって、酵素がさらなるヌクレオチドを付加することが遮断される。酵素が止まったら、過剰なヌクレオチド類似体を成長中の鎖から除去することができ、阻害剤をNTPから切断することができ、鎖に次のヌクレオチドを付加するために新しいヌクレオチド類似体を導入することができる。このステップを連続して繰り返すことにより、所望の長さおよび配列のヌクレオチド配列をすばやく構築することができる。ポリヌクレオチド合成にヌクレオチジルトランスフェラーゼを使用する長所には:1)鋳型非依存的重合において一本鎖(ss)開始プライマーを使用する3’伸長活性、2)高効率の様式でプライマーを伸長させて数千のヌクレオチドを付加する能力、および3)効率的な基質としての多種多様な修飾および置換NTPの受容、が挙げられる。加えて、本発明は、ヌクレオチジルトランスフェラーゼの基質であるイニシエーター配列を使用することができる。イニシエーターは、固体支持体に付着され、酵素の結合部位として用いられる。イニシエーターは、好ましくは酵素に対する汎用のイニシエーター、例えばホモポリマー配列であり、固体支持体上で再利用可能であり、形成されたオリゴヌクレオチドがイニシエーターから切断可能である。
類似体
酵素
本発明に特に重要(import)なことは、ポリメラーゼ活性を有するトランスフェラーゼ、例えばターミナルデオキシヌクレオチジルトランスフェラーゼ(TdT)は、ヌクレオチド鎖の3’末端へのデオキシリボヌクレオチドの付加を触媒する能力があり、それによってDNAヌクレオチドの鎖長が増加する。TdTは、DNA鎖の成長末端への1〜2つのリボヌクレオチドの付加しか触媒しないことになり、これは部位特異的DNA−RNAキメラポリヌクレオチドの構築に有用になり得る。特に、操作した大腸菌から供給される仔ウシ胸腺TdTは、本発明での使用に適しており、市販品、例えばThermo Scientific(ピッツバーグ、PA)から入手可能である。仔ウシTdTに対応するアミノ酸配列を、配列番号1として表1に挙げる。
固体状態合成
合成機
参照による援用
等価物
Claims (17)
- オリゴヌクレオチドを合成する方法であって、前記方法は、
ヌクレオチジルトランスフェラーゼ酵素の存在下、核酸鋳型の非存在下で、固体支持体に付着しているオリゴヌクレオチドをヌクレオチド類似体に曝露し、その結果前記ヌクレオチド類似体が前記オリゴヌクレオチドに組み込まれるステップを含み、
前記ヌクレオチド類似体は、阻害剤が切断可能なリンカーの切断によって除去されるまで前記阻害剤へ前記切断可能なリンカーによって結合されるヌクレオチドを含み、前記阻害剤は前記ヌクレオチジルトランスフェラーゼによる前記オリゴヌクレオチドへの天然のヌクレオチドまたはヌクレオチド類似体のいずれの組み込みについても触媒することを立体配置的に防止し、
前記阻害剤が、直径50Å超のタンパク質である、方法。 - 前記固体支持体に付着している前記核酸が、pH6.5〜8.5を有する水溶液の存在下で前記ヌクレオチド類似体に曝露される、請求項1に記載の方法。
- 前記固体支持体に付着している前記核酸が、35〜39℃の温度で水溶液の存在下で前記ヌクレオチド類似体に曝露される、請求項1に記載の方法。
- 前記固体支持体が、ビーズ、ウェルまたはPEGである、請求項1に記載の方法。
- 前記核酸が、一本鎖である、請求項1に記載の方法。
- 前記切断可能なリンカーが、前記ヌクレオチド類似体から切断されるときに環状の副産物を形成する部分を含む、請求項1に記載の方法。
- 天然のヌクレオチドを産生するために前記切断可能なリンカーを切断するステップと;
ヌクレオチジルトランスフェラーゼ酵素の存在下、核酸鋳型の非存在下で、前記天然のヌクレオチドを第2のヌクレオチド類似体に曝露するステップと
をさらに含む、請求項1に記載の方法。 - 前記オリゴヌクレオチドをエキソヌクレアーゼ酵素に曝露するステップをさらに含む、請求項1に記載の方法。
- 前記ヌクレオチド類似体および前記ヌクレオチジルトランスフェラーゼ酵素を含む水溶液を提供するステップをさらに含む、請求項1に記載の方法。
- 前記阻害剤が、荷電部分を含む、請求項1に記載の方法。
- 前記ヌクレオチド類似体が、リボース糖またはデオキシリボース糖を含む、請求項1に記載の方法。
- ヌクレオチド基質が、アデニン、グアニン、シトシン、チミンおよびウラシルからなる群から選択される塩基を含む、請求項1に記載の方法。
- 前記ヌクレオチジルトランスフェラーゼが、配列番号1、配列番号3、または配列番号5と少なくとも90%同一のタンパク質配列を含む、請求項1に記載の方法。
- 前記ヌクレオチジルトランスフェラーゼが、配列番号2、配列番号4、または配列番号6と少なくとも90%同一のヌクレオチド配列を有する生物に由来する、請求項1に記載の方法。
- 前記ヌクレオチド類似体が、液滴で送達される、請求項1に記載の方法。
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201462038604P | 2014-08-18 | 2014-08-18 | |
US62/038,604 | 2014-08-18 | ||
US201462069067P | 2014-10-27 | 2014-10-27 | |
US62/069,067 | 2014-10-27 | ||
PCT/US2015/045730 WO2016028803A2 (en) | 2014-08-18 | 2015-08-18 | Methods and apparatus for synthesizing nucleic acids |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2021011173A Division JP2021061867A (ja) | 2014-08-18 | 2021-01-27 | 核酸合成のための方法および装置 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2017525391A JP2017525391A (ja) | 2017-09-07 |
JP6898236B2 true JP6898236B2 (ja) | 2021-07-07 |
Family
ID=55351200
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2017529593A Active JP6898236B2 (ja) | 2014-08-18 | 2015-08-18 | 核酸合成のための方法および装置 |
JP2021011173A Pending JP2021061867A (ja) | 2014-08-18 | 2021-01-27 | 核酸合成のための方法および装置 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2021011173A Pending JP2021061867A (ja) | 2014-08-18 | 2021-01-27 | 核酸合成のための方法および装置 |
Country Status (5)
Country | Link |
---|---|
EP (2) | EP3183365A4 (ja) |
JP (2) | JP6898236B2 (ja) |
CN (1) | CN107109452A (ja) |
CA (1) | CA2958581C (ja) |
WO (2) | WO2016028802A1 (ja) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10774316B2 (en) | 2014-10-20 | 2020-09-15 | Molecular Assemblies, Inc. | Modified template-independent enzymes for polydeoxynucleotide synthesis |
US11390858B2 (en) | 2014-10-20 | 2022-07-19 | Molecular Assemblies, Inc. | Modified template-independent enzymes for polydeoxynucleotide synthesis |
WO2016064880A1 (en) * | 2014-10-20 | 2016-04-28 | Molecular Assemblies, Inc. | Modified template-independent enzymes for polydeoxynucleotide systhesis |
WO2016182984A1 (en) | 2015-05-08 | 2016-11-17 | Centrillion Technology Holdings Corporation | Disulfide-linked reversible terminators |
CN109402202A (zh) * | 2018-11-30 | 2019-03-01 | 深圳华大生命科学研究院 | 核酸合成方法 |
KR20220114026A (ko) * | 2019-12-12 | 2022-08-17 | 디엔에이 스크립트 | 폴리뉴클레오타이드의 주형-부재 효소 합성을 위한 키메라 말단 데옥시뉴클레오타이드 전달효소 |
WO2022013094A1 (en) * | 2020-07-15 | 2022-01-20 | Dna Script | Massively parallel enzymatic synthesis of polynucleotides |
CN114410602B (zh) * | 2022-01-28 | 2024-01-19 | 赛纳生物科技(北京)有限公司 | 一种末端脱氧核苷酸转移酶的突变体及其应用 |
WO2024079270A1 (en) * | 2022-10-14 | 2024-04-18 | Dna Script | Method for synthesizing polynucleotides using a move-stop printer |
CN116286722B (zh) * | 2023-04-24 | 2024-05-24 | 中国科学院深圳先进技术研究院 | 一种全遗传编码的光控dna合成酶、核酸与应用 |
Family Cites Families (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6214987B1 (en) | 1994-09-02 | 2001-04-10 | Andrew C. Hiatt | Compositions for enzyme catalyzed template-independent formation of phosphodiester bonds using protected nucleotides |
AU7878301A (en) * | 2000-08-23 | 2002-03-04 | Takara Shuzo Co | Method of amplifying nucleic acid |
US7211654B2 (en) | 2001-03-14 | 2007-05-01 | Regents Of The University Of Michigan | Linkers and co-coupling agents for optimization of oligonucleotide synthesis and purification on solid supports |
AUPR975201A0 (en) * | 2001-12-24 | 2002-01-24 | Unisearch Limited | Enzymatic redox labelling of nucleic acids |
DE10215035A1 (de) * | 2002-04-05 | 2003-10-23 | Roche Diagnostics Gmbh | Rekombinante terminale Deoxynukleotidyltransferase mit verbesserter Funktionalität |
US7572581B2 (en) * | 2003-06-30 | 2009-08-11 | Roche Molecular Systems, Inc. | 2′-terminator nucleotide-related methods and systems |
US7947817B2 (en) * | 2003-06-30 | 2011-05-24 | Roche Molecular Systems, Inc. | Synthesis and compositions of 2'-terminator nucleotides |
US8637650B2 (en) * | 2003-11-05 | 2014-01-28 | Genovoxx Gmbh | Macromolecular nucleotide compounds and methods for using the same |
US8071755B2 (en) * | 2004-05-25 | 2011-12-06 | Helicos Biosciences Corporation | Nucleotide analogs |
US20070117104A1 (en) * | 2005-11-22 | 2007-05-24 | Buzby Philip R | Nucleotide analogs |
EP2074133A2 (de) * | 2006-09-20 | 2009-07-01 | Genovoxx GmbH | Komponenten und verfahren zur enzymatischen synthese von nukleinsäuren |
US9163053B2 (en) * | 2007-05-18 | 2015-10-20 | Fluidigm Corporation | Nucleotide analogs |
KR101107315B1 (ko) * | 2009-09-29 | 2012-01-20 | 한국과학기술연구원 | 3′―하이드록실기에 형광을 띄는 장애그룹이 부착된 뉴클레오시드 삼인산을 가역적 종결자로서 이용한 dna 염기서열 분석 방법 |
US9295965B2 (en) * | 2010-11-12 | 2016-03-29 | Gen9, Inc. | Methods and devices for nucleic acid synthesis |
EP2551354A1 (en) * | 2011-07-25 | 2013-01-30 | Universität Heidelberg | Functionalization of RNA oligonucleotides |
AU2012308518B2 (en) | 2011-09-13 | 2017-08-17 | Agilent Technologies, Inc. | 5-methoxy, 3'-oh unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing |
CN102604934B (zh) * | 2012-03-31 | 2015-04-08 | 盛司潼 | 一种基于固相载体进行扩增及进行核酸测序的方法 |
US8808989B1 (en) | 2013-04-02 | 2014-08-19 | Molecular Assemblies, Inc. | Methods and apparatus for synthesizing nucleic acids |
-
2015
- 2015-08-18 WO PCT/US2015/045729 patent/WO2016028802A1/en active Application Filing
- 2015-08-18 CA CA2958581A patent/CA2958581C/en active Active
- 2015-08-18 EP EP15833737.8A patent/EP3183365A4/en active Pending
- 2015-08-18 WO PCT/US2015/045730 patent/WO2016028803A2/en active Application Filing
- 2015-08-18 EP EP15833657.8A patent/EP3183356A4/en active Pending
- 2015-08-18 JP JP2017529593A patent/JP6898236B2/ja active Active
- 2015-08-18 CN CN201580054493.XA patent/CN107109452A/zh active Pending
-
2021
- 2021-01-27 JP JP2021011173A patent/JP2021061867A/ja active Pending
Also Published As
Publication number | Publication date |
---|---|
WO2016028802A8 (en) | 2017-04-06 |
WO2016028802A1 (en) | 2016-02-25 |
EP3183365A4 (en) | 2018-01-10 |
EP3183365A1 (en) | 2017-06-28 |
EP3183356A4 (en) | 2018-04-11 |
WO2016028803A3 (en) | 2016-04-14 |
WO2016028803A2 (en) | 2016-02-25 |
JP2021061867A (ja) | 2021-04-22 |
CA2958581C (en) | 2023-04-04 |
CA2958581A1 (en) | 2016-02-25 |
JP2017525391A (ja) | 2017-09-07 |
EP3183356A2 (en) | 2017-06-28 |
CN107109452A (zh) | 2017-08-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6898236B2 (ja) | 核酸合成のための方法および装置 | |
EP2796552B1 (en) | Methods and apparatus for synthesizing nucleic acids | |
US10683536B2 (en) | Reusable initiators for synthesizing nucleic acids | |
US9771613B2 (en) | Methods and apparatus for synthesizing nucleic acid | |
US9695470B2 (en) | Methods and apparatus for synthesizing nucleic acids | |
JP6448097B2 (ja) | 核酸を合成するための方法および装置 | |
US11331643B2 (en) | Reusable initiators for synthesizing nucleic acids | |
US20230227880A1 (en) | Reusable initiators for synthesizing nucleic acids | |
JP2022523711A (ja) | 核酸を合成するための再使用可能な開始剤 | |
CA3179701A1 (en) | Reusable initiators for synthesizing nucleic acids |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20170510 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20180815 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20190718 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20191017 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20191206 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200529 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200828 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20200929 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20210127 |
|
C60 | Trial request (containing other claim documents, opposition documents) |
Free format text: JAPANESE INTERMEDIATE CODE: C60 Effective date: 20210127 |
|
C11 | Written invitation by the commissioner to file amendments |
Free format text: JAPANESE INTERMEDIATE CODE: C11 Effective date: 20210212 |
|
A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20210323 |
|
C21 | Notice of transfer of a case for reconsideration by examiners before appeal proceedings |
Free format text: JAPANESE INTERMEDIATE CODE: C21 Effective date: 20210324 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20210517 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20210610 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6898236 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |