CA2144334A1 - Oligonucleotides having universal nucleoside spacers - Google Patents
Oligonucleotides having universal nucleoside spacersInfo
- Publication number
- CA2144334A1 CA2144334A1 CA002144334A CA2144334A CA2144334A1 CA 2144334 A1 CA2144334 A1 CA 2144334A1 CA 002144334 A CA002144334 A CA 002144334A CA 2144334 A CA2144334 A CA 2144334A CA 2144334 A1 CA2144334 A1 CA 2144334A1
- Authority
- CA
- Canada
- Prior art keywords
- nucleoside
- universal
- formula
- oligonucleotide according
- nucleosides
- 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.)
- Abandoned
Links
- 239000002777 nucleoside Substances 0.000 title claims abstract description 78
- 108091034117 Oligonucleotide Proteins 0.000 title claims abstract description 63
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 title abstract description 30
- 125000003835 nucleoside group Chemical group 0.000 claims abstract description 32
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 19
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 44
- 229910052760 oxygen Inorganic materials 0.000 claims description 16
- 229910052717 sulfur Inorganic materials 0.000 claims description 13
- 125000004122 cyclic group Chemical group 0.000 claims description 12
- 125000006575 electron-withdrawing group Chemical group 0.000 claims description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 229910052711 selenium Inorganic materials 0.000 claims description 7
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 6
- KAESVJOAVNADME-UHFFFAOYSA-N 1H-pyrrole Natural products C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 claims description 5
- 101100054666 Streptomyces halstedii sch3 gene Proteins 0.000 claims description 3
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims 2
- 150000002460 imidazoles Chemical class 0.000 claims 1
- 150000003233 pyrroles Chemical class 0.000 claims 1
- 150000003852 triazoles Chemical class 0.000 claims 1
- 108020004414 DNA Proteins 0.000 description 20
- 239000000203 mixture Substances 0.000 description 11
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 8
- 125000001424 substituent group Chemical group 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 7
- 102000039446 nucleic acids Human genes 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- LOJNBPNACKZWAI-UHFFFAOYSA-N 3-nitro-1h-pyrrole Chemical compound [O-][N+](=O)C=1C=CNC=1 LOJNBPNACKZWAI-UHFFFAOYSA-N 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 150000008300 phosphoramidites Chemical class 0.000 description 5
- VGONTNSXDCQUGY-RRKCRQDMSA-N 2'-deoxyinosine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 VGONTNSXDCQUGY-RRKCRQDMSA-N 0.000 description 4
- 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 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 239000005549 deoxyribonucleoside Substances 0.000 description 4
- VGONTNSXDCQUGY-UHFFFAOYSA-N desoxyinosine Natural products C1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 VGONTNSXDCQUGY-UHFFFAOYSA-N 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical group [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 125000000623 heterocyclic group Chemical group 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 239000000543 intermediate Substances 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 description 2
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical group N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-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
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 2
- CKTSBUTUHBMZGZ-UHFFFAOYSA-N Deoxycytidine Natural products O=C1N=C(N)C=CN1C1OC(CO)C(O)C1 CKTSBUTUHBMZGZ-UHFFFAOYSA-N 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 230000003466 anti-cipated effect Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000002515 oligonucleotide synthesis Methods 0.000 description 2
- -1 phosphite triester Chemical class 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000002342 ribonucleoside Substances 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- ZGYYPTJWJBEXBC-QYYRPYCUSA-N (2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-4-fluoro-2-(hydroxymethyl)oxolan-3-ol Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1F ZGYYPTJWJBEXBC-QYYRPYCUSA-N 0.000 description 1
- NSMOSDAEGJTOIQ-CRCLSJGQSA-N (2r,3s)-2-(hydroxymethyl)oxolan-3-ol Chemical compound OC[C@H]1OCC[C@@H]1O NSMOSDAEGJTOIQ-CRCLSJGQSA-N 0.000 description 1
- HYZJCKYKOHLVJF-UHFFFAOYSA-N 1H-benzimidazole Chemical compound C1=CC=C2NC=NC2=C1 HYZJCKYKOHLVJF-UHFFFAOYSA-N 0.000 description 1
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 description 1
- MZWDAEVXPZRJTQ-WUXMJOGZSA-N 4-[(e)-(4-fluorophenyl)methylideneamino]-3-methyl-1h-1,2,4-triazole-5-thione Chemical compound CC1=NNC(=S)N1\N=C\C1=CC=C(F)C=C1 MZWDAEVXPZRJTQ-WUXMJOGZSA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- WTKZEGDFNFYCGP-UHFFFAOYSA-N Pyrazole Chemical compound C=1C=NNC=1 WTKZEGDFNFYCGP-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- FJHSYOMVMMNQJQ-PAMZHZACSA-N [(2r,3s)-5-chloro-3-(4-methylbenzoyl)oxyoxolan-2-yl]methyl 4-methylbenzoate Chemical compound C1=CC(C)=CC=C1C(=O)OC[C@@H]1[C@@H](OC(=O)C=2C=CC(C)=CC=2)CC(Cl)O1 FJHSYOMVMMNQJQ-PAMZHZACSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 1
- 229960000961 floxuridine Drugs 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- AQSJGOWTSHOLKH-UHFFFAOYSA-N phosphite(3-) Chemical class [O-]P([O-])[O-] AQSJGOWTSHOLKH-UHFFFAOYSA-N 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/044—Pyrrole radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/052—Imidazole radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/056—Triazole or tetrazole radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/24—Heterocyclic radicals containing oxygen or sulfur as ring hetero atom
Abstract
Oligonucleotides are made having at least ten nucleosides, at least two of which are A, T, G or C and at least one is a uni-versal nucleoside.
Description
WO94/06810 ~ 3 3 ~ PCT/US93/08391 .
OLIGONUCL~O~ HAVIN~ UNlV~:~SAL NUCLEOSIDE SPACERS
Field of the Invention.
The present invention relates generally to oligonucleotides, and more particularly to oligonucleotides having universal nucleosides included therein.
Background to the Invention.
This invention was made with U.S. Government support under Grant #R01 GM45551 awarded by the National Institute of Health. The U.S. Government has certain rights in the invention. The invention was also developed with support from Purdue University, the University of ~ichigan and the Walther Cancer Institute.
The chemical synthesis of oligonucleotides has had tremendous biological application over the past seve~al decades. The simultaneous development of rapid and efficient methods of synthesis, together with advances in molecular biology techniques, has led to an increasing demand for synthetic oligonucleotides. Oligonucleotides can serve a multitude of purposes, including use as hybridization probes for DNA isolation, as primers in the enzymatic amplification of DNA, as mutagens for site-directed DNA alterations, and as sequencing primers.
A major use of synthetic oligonucleotides is the identification of naturally-occurring DNA sequences. The efficient isolation of specific DNA se~uences depends to a great extent on the ability to accurately identify the DNA or RNA sequence of interest. When amino acid sequence information is available, it is possible to approximately wog4/n68l0 2~44~ 2- PCT/US93/08391 deduce the nucleotide sequence and then synthesize an oligonucleotide that can be used to identify clones containing the desire sequence. This approach has been used very successful and is one of the most widely used methods for identifying specific DNA or RNA sequences.
Due to redundancy in the genetic code, it is almost always impossible to precisely predict a unique nucleotide sequence from an amino acid sequence. Mixtures of oligonucleotides that take this redundancy into account must be synthesized and used for screening potential DNA or RNA
candidates. However, the potential ambiguities or mismatches present in the sequences of a highly degenerate oligonucleotide mixture can result in the identification of colonies which contain sequences that are unrelated to the DNA or RNA sequence of interest. This may be partially overcome by modifying the stringency of the hybridization conditions. An additional problem occurs if the oligonucleotide mixture contains a large number of sequences. In that case, the correct sequence may be diluted to the point that the mixture becomes ineffective.
Unfortunately, there is often no alternative to the use of a complex mixture of oligonucleotides. The design of longer, unique oligonucleotides making use of species-specific codon frequencies to increase the 2s probabilities of correct base pairing is not always an option. Frequently the use of protein sequence information to screen DNA or RNA sequences is seriously limited due to either high degeneracy or incomplete or uncertain protein sequence information.
One solution to the problem has been to seek bases that would hybridize equally to more than one nucleic acid base and hence decrease the number of partially redundant probes required. This has led to the concept of a "universal base,"
a modified nucleic acid base that could base-pair with any of the common bases: deoxyadenosine (A), deoxythymidine (T), -2 1 ~
WO94/06810 PCT/US93/~8391 deoxycytidine (C) and deoxyguanosine (G). Reference herein to A, C, G and T is intended to additionally encompass the RNA analogs thereto, including uridine (U) as the analog to T. The use of a universal base should reduce the degeneracy ~ 5 to l and still preserve the uniqueness of the probe.
Successful development of a universal base could greatly reduce the element of risk and enhance success in screening DNA libraries.
A variety of compounds have been investigated as universal bases and examples of such compounds are shown in FIG. l. For example, Millican et al. proposed the use of either 1,2-dideoxyribofuranose or l,2-dideoxy-l(C)-pllenylribofuranose as a universal base in a paper published in 1984. Ikehara and Inaoka synthesized the deoxyriboside of benzimidazole, and suggested its use in oligonucleotides.
Hypoxan~hine, xanthine and guanine deoxyribonucleosi~es have been evaluated for their ability to hybridize to each of the four DNA bases in nonadecamers. The ninth base from the 5' end was modified in the sequence 5'-CGATGTTAYTACATGAGAC-3' and binding to the four sequences 5'-GTCTCATGTANTAACATCG-3' (N . A, C, G or T) was determined. Each of the substitutions destabilized the duplex relative to a control in which a G-C
base pair occurred at this position.
Although it has been widely promoted, deoxyinosine is not as discriminating in forming base pairs as is required for many applications and has not met widespread acceptance.
Since its introduction in 1985 as a "universal base" there have been some reports of its successful use in DNA probes, however many more studies have been published usiny oligonucleotide mixtures than using deoxyinosine - suggesting that the need for a truly universal base remains.
The feasibility of using 5-fluorodeoxyuridine (F) as a base analog has been examined in synthetic oligonucleotides.
The A-F base pair is actually more stable than an A-T base W O 94/06810 PC~r/US93/08391 3 ~ ~
pair and increases the Tm 1C above an A-T pair. A G-F
base pair is essentially neutral. Unfortunately, the application of 5-fluoracil as a universal base is limited to pairing with A and G.
The introduction of a universal base would have numerous advantages. As has been stated, the total number of seguences in a degenerate oligonucleotide mixture would be reduced.
This would increase the effective specific activity of the correct sequence by exactly the amount due to the reduction in degeneracy. One of the limiting factors in the use of highly degenerate oligonucleotide mixtures as probes for screening DNA or RNA sequences is the reduction in effective specific activity of the correct probe sequence in the large population of incorrect oligonucleotide sequences.
Second, a universal base would promote a uniform distribution of oligonucleotides. For example, when all four bases are incorporated into an oligonucleotide during chemical synthesis, all four bases are not equally represented due to different rates of degradation and to different degrees of phosphoramidite reactivity. This may cause under-representation of certain seguences in an oligonucleotide mixture.
A need therefore exists for oligonucleotides having universal bases at potentially degenerate positions so that the oligonucleotide will bond to ambiguous DNA or RNA
sequences. The present invention addresses that need.
W O 94/06810 2 I 4 4 ~ ~ ~ P~r/US93/08391 SU~L~RY OF TH~ INV~NTION
Briefly describing the.present invention, there are provided novel oligonucleotides comprising at least ten nucleosides, wherein at least two ~ifferent nucleosides are selected from t~le group consisting of A, T, C and C, and wherein at least one nucleoside is a universal nucleoside of the formula:
/\
\ Rl ~
~ /
wherein in the first cyclic structure illustrated above:
each Rn is H, OH, F or OCH3;
Z is a member of the group consisting of O, S and CH2;
and B is a second cyclic structure comprising a five-membered, cyclic base having at least two double bonds in one of its possible tautomeric forms, and further having an electron withdrawing group bonded thereto, said base with electron withdrawing group being epresented by the formula:
X~ X2 W
/
wherein:
said base with electron withdrawing group is bonded at X4 to the second cyclic structure of the _ W O 94/06810 PC~r/US93/08391 ~4~3~ -6-nucleoside;
Xl, X3 and X5 are each members of the group consisting of N, O, C, S and Se;
X2 and X4 are each members of the group consisting of N and C; and W is a member of the group consistiIlg of F, Cl, Br, I, O, S, OH, SII, NH2, NO2, C(O)H, C(O)NHOH, C(S)NHOEI, NO, C(NOCH3)NH2' CH3~ SCH3~ SeCH3' NH2' NEIOCH3, N3, CN, C(O)NE12, C(NOH)NH2, CSNEI2 and 10 C02H.
One object of the present invention is to provide oligonucleotides which include universal nucleosides at degenerate positions.
Further objects and advantages of the present invention will be apparent from the following description.
WO94/06810 ~ ~ 4 13 ~ ~ PCT/US93/08391 BRI~ DESCRIPTION OF THE DRAWINGS
FIG. l shows various compounds which have been proposed as universal nucleosides in the prior art.
E~IG. 2 shows nucleosides in which the cyclic oxygen of the sugar portion is replaced with S or CH2.
FIG. 3 shows nucleosides in which the base portion is a heterocycle including O, S or Se.
FIG. 4 shows nucleosides in which the base portion is a pyrrole, diazole or triazole derivative.
FIG. 5 shows potentially useful phosphonucleotide intermediates for use in constructing oligonucleotides of the present invention.
FIG. 6 shows a universal nucleoside according to the present invention.
W O 94/06810 PC~r/US93/08391 3~ ~ ~
DESCRIPTION OF T~IE PRE~ERRED EMBODIMENT
For the purposes of promoting an understanding of the principles of the invention, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated aspects of the invention, and such further applications of the principles of the invention as illustrated herein being contemplated as would normally occur to one skilled in the art to which the invention relates.
One aspect of the present invention relates to oligonucleotides comprising at least ten nucleosides, at least two of which are selected from the group consisting of A, T, C and G, and at least one nucleoside being a universal nucleoside. The incorporation of one or more universal nucleosides into the oligomer makes bonding to unknown bases possible and allows the oligonucleotide to match ambiguous or unknown nucleic acid sequences. In one preferred aspect, all f the common DNA nucleosides - deoxyadenosine (A), thymidine (T), deoxycytidine (C) and deoxyguanosine (G) - are combined with at least several of the universal nucleosides to make an oligonucleotide having at least l0 nucleosides therein.
Considering first the universal nucleoside portion of the present invention, the preferred nucleosides of the present invention include a first (sugar) portion and a second (base) portion. The first portion of the nucleoside can be represented by the formula:
W O 94/06810 2 1 4 4 ~ 3 ~ PC~r/US93/08391 _g _ ~z~J
~H R2 wherein each Rn is H, OH, F or OCH3, and Z is a member of the group consisting of O, S and CII2.
Most commonly, the first (suga~) portion will be D-deoxyribose or D-ribose as found in naturally occurring ribonucleosides and deoxyribonucleosides. The Z atom of the above formula will be O in these preferred cases.
Alternatively, the oxygen of this cyclic structure may be replaced by either S or CIl2 without unreasonably affecting the performance of the nucleoside in some applications.
Examples of nucleosides having a first "sugar" portion which is substituted with S or CH2 are shown in FIG. 2;
additional examples of such compounds may be developed by those skilled in the art without undue experimentation.
A variety of substituents may be included at Rl and/or R2. In particular, both Rl and R2 may be H as in the case of D-deoxyribose. If one of those substituents is H and the other is OH the first (sugar) portion may be D-ri~ose as in naturally-occurring ribonucleosides. Alternatively, either or both of Rl and R2 may be substituted with F or OC~3. The use of 1uorine substituted nucleosides has been suggested by the prior art as in, for example, 2'-fluoro-2'-deoxyadenosine, which was incorporated into oligonucleotides by M. Ikehara and coworkers (Ikehara, 21 He~erocycles 75, 1984).
The second (base) portion "B" of the universal W O 94/06810 ~ ~ ~ PC~r/US93/08391 nucleosides is preferably a five-membered, heterocyclic base having at least two double bonds in one of its possible tautomeric forms, and further having an electron withdrawing group bonded thereto. Preferred base portions are represented by the formula:
~Xl~
X5 X2_W
As was mentioned above, Xl, X3 and X5 are each members of the qroup consisting of C, N, O, S and Se. In the universal nucleosides preferred in testing to date, Xl, X3 and X5 are either C or N, with the most preferred compounds including at least one N. Alternative nucleosides including O, S or Se are shown in FIG. 3; additional alternatives may be prepared by one skilled in the art without undue experimentation.
As was stated above, X2 and X4 are each preferably members of the group consisting of N and C. In the nucleosides most preferred in testing to date at least one of these two atoms is N, although the exact location of the nitrogen may vary according to the particular application.
It is to be appreciated that there are some limitations as to which atoms can be located at Xl, X2, X3 and X5. In particular, when X4= N and X4 is the site of the glycosidic bond, then Xl, X3 and X5 can only be C
or N, and X2 must be C. O, S or Se can be tolorated at Xl, X3 and X5 only when X4= C, and even in that case there can be no more than one of these atoms (O, S and Se) in the five-membered heterocyclic ring.
When N is included in the nucleoside, the base may be, for example, a pyrrole, dia~ole or tria~ole. Examples o~
such nucleosides are shown in FIG. 4, and can be prepared by ~ 21443~
one skilled in the art without undue experimentation.
The electron withdrawing group W is a member of the group consisting of F, Cl, Br, I, O, S, OH, Sl~, N~I2, NO2, C(O)H, C(O)NHOH, C(S)NHOH, NO, C(NOCH3)NH2, OCH3, SCH3, SeCH3, ONH2, NHOCH3, N3, CN, C(O)NH2, C(NOH)NH2, CSNH2 and CO2H. NO2 has been especially effective in experiments to date, and is particularly preferred for Sanger sequencing. C(O)NH2 has also been particularly effective in certain applications.
A number of structural features of the preferred base portions should be mentioned. First, it is to be appreciated that the electron withdrawing group is bonded to the remainder of the base portion only at X2. In some unsatisfactory prior art nucleosides, such as deoxyinosine, the electron withdrawing group bonds to the remainder of the base portion at both X2 and X3 - which limits the ability of the base to assume a position necessary to optimize both hydrogen bonding and base stacking.
Adding substituents at Xl, X3 or X5 may be effective in some cases, although any substituents added at those positions must be small enough to avoid steric interference and must not prevent effective base stacking and bonding interactions. Small substituents which do not interfere with the coplanarity of the extended ring system are preferred. X1, X3 or X5 should be C if a substituent is added to that atom. Nucleosides in which such substituents are included at Xl, X3 or X5 are to be considered equivalent to the specifically disclosed nucleosides.
Preferably, the base-with-electron withdrawing group comprises an extended ~ system which favors base stacking interactions. Specifically, the preerred electron withdrawing groups enhance base stacking through interaction with adjacent pyrimidine and purine rin~ systems in a polynucleotide double helix.
-WO94/06810 ~ ~ PCT/US93/08391 The universal nucleosides of the present invention accordingly preferably possess the following chemical and structural properties:
l. One or more donor or acceptor sites for hydrogen bonding to A, C, G or T.
2. Planar aromatic ~ system capable of stacking interactions with A, C, G and T in duplex or triplex nucleic acids.
3. The molecule is sterically accommodated within duplex or triplex nucleic acid, while at the same time maintaining both base stacking and hydrogen bonding interactions.
OLIGONUCL~O~ HAVIN~ UNlV~:~SAL NUCLEOSIDE SPACERS
Field of the Invention.
The present invention relates generally to oligonucleotides, and more particularly to oligonucleotides having universal nucleosides included therein.
Background to the Invention.
This invention was made with U.S. Government support under Grant #R01 GM45551 awarded by the National Institute of Health. The U.S. Government has certain rights in the invention. The invention was also developed with support from Purdue University, the University of ~ichigan and the Walther Cancer Institute.
The chemical synthesis of oligonucleotides has had tremendous biological application over the past seve~al decades. The simultaneous development of rapid and efficient methods of synthesis, together with advances in molecular biology techniques, has led to an increasing demand for synthetic oligonucleotides. Oligonucleotides can serve a multitude of purposes, including use as hybridization probes for DNA isolation, as primers in the enzymatic amplification of DNA, as mutagens for site-directed DNA alterations, and as sequencing primers.
A major use of synthetic oligonucleotides is the identification of naturally-occurring DNA sequences. The efficient isolation of specific DNA se~uences depends to a great extent on the ability to accurately identify the DNA or RNA sequence of interest. When amino acid sequence information is available, it is possible to approximately wog4/n68l0 2~44~ 2- PCT/US93/08391 deduce the nucleotide sequence and then synthesize an oligonucleotide that can be used to identify clones containing the desire sequence. This approach has been used very successful and is one of the most widely used methods for identifying specific DNA or RNA sequences.
Due to redundancy in the genetic code, it is almost always impossible to precisely predict a unique nucleotide sequence from an amino acid sequence. Mixtures of oligonucleotides that take this redundancy into account must be synthesized and used for screening potential DNA or RNA
candidates. However, the potential ambiguities or mismatches present in the sequences of a highly degenerate oligonucleotide mixture can result in the identification of colonies which contain sequences that are unrelated to the DNA or RNA sequence of interest. This may be partially overcome by modifying the stringency of the hybridization conditions. An additional problem occurs if the oligonucleotide mixture contains a large number of sequences. In that case, the correct sequence may be diluted to the point that the mixture becomes ineffective.
Unfortunately, there is often no alternative to the use of a complex mixture of oligonucleotides. The design of longer, unique oligonucleotides making use of species-specific codon frequencies to increase the 2s probabilities of correct base pairing is not always an option. Frequently the use of protein sequence information to screen DNA or RNA sequences is seriously limited due to either high degeneracy or incomplete or uncertain protein sequence information.
One solution to the problem has been to seek bases that would hybridize equally to more than one nucleic acid base and hence decrease the number of partially redundant probes required. This has led to the concept of a "universal base,"
a modified nucleic acid base that could base-pair with any of the common bases: deoxyadenosine (A), deoxythymidine (T), -2 1 ~
WO94/06810 PCT/US93/~8391 deoxycytidine (C) and deoxyguanosine (G). Reference herein to A, C, G and T is intended to additionally encompass the RNA analogs thereto, including uridine (U) as the analog to T. The use of a universal base should reduce the degeneracy ~ 5 to l and still preserve the uniqueness of the probe.
Successful development of a universal base could greatly reduce the element of risk and enhance success in screening DNA libraries.
A variety of compounds have been investigated as universal bases and examples of such compounds are shown in FIG. l. For example, Millican et al. proposed the use of either 1,2-dideoxyribofuranose or l,2-dideoxy-l(C)-pllenylribofuranose as a universal base in a paper published in 1984. Ikehara and Inaoka synthesized the deoxyriboside of benzimidazole, and suggested its use in oligonucleotides.
Hypoxan~hine, xanthine and guanine deoxyribonucleosi~es have been evaluated for their ability to hybridize to each of the four DNA bases in nonadecamers. The ninth base from the 5' end was modified in the sequence 5'-CGATGTTAYTACATGAGAC-3' and binding to the four sequences 5'-GTCTCATGTANTAACATCG-3' (N . A, C, G or T) was determined. Each of the substitutions destabilized the duplex relative to a control in which a G-C
base pair occurred at this position.
Although it has been widely promoted, deoxyinosine is not as discriminating in forming base pairs as is required for many applications and has not met widespread acceptance.
Since its introduction in 1985 as a "universal base" there have been some reports of its successful use in DNA probes, however many more studies have been published usiny oligonucleotide mixtures than using deoxyinosine - suggesting that the need for a truly universal base remains.
The feasibility of using 5-fluorodeoxyuridine (F) as a base analog has been examined in synthetic oligonucleotides.
The A-F base pair is actually more stable than an A-T base W O 94/06810 PC~r/US93/08391 3 ~ ~
pair and increases the Tm 1C above an A-T pair. A G-F
base pair is essentially neutral. Unfortunately, the application of 5-fluoracil as a universal base is limited to pairing with A and G.
The introduction of a universal base would have numerous advantages. As has been stated, the total number of seguences in a degenerate oligonucleotide mixture would be reduced.
This would increase the effective specific activity of the correct sequence by exactly the amount due to the reduction in degeneracy. One of the limiting factors in the use of highly degenerate oligonucleotide mixtures as probes for screening DNA or RNA sequences is the reduction in effective specific activity of the correct probe sequence in the large population of incorrect oligonucleotide sequences.
Second, a universal base would promote a uniform distribution of oligonucleotides. For example, when all four bases are incorporated into an oligonucleotide during chemical synthesis, all four bases are not equally represented due to different rates of degradation and to different degrees of phosphoramidite reactivity. This may cause under-representation of certain seguences in an oligonucleotide mixture.
A need therefore exists for oligonucleotides having universal bases at potentially degenerate positions so that the oligonucleotide will bond to ambiguous DNA or RNA
sequences. The present invention addresses that need.
W O 94/06810 2 I 4 4 ~ ~ ~ P~r/US93/08391 SU~L~RY OF TH~ INV~NTION
Briefly describing the.present invention, there are provided novel oligonucleotides comprising at least ten nucleosides, wherein at least two ~ifferent nucleosides are selected from t~le group consisting of A, T, C and C, and wherein at least one nucleoside is a universal nucleoside of the formula:
/\
\ Rl ~
~ /
wherein in the first cyclic structure illustrated above:
each Rn is H, OH, F or OCH3;
Z is a member of the group consisting of O, S and CH2;
and B is a second cyclic structure comprising a five-membered, cyclic base having at least two double bonds in one of its possible tautomeric forms, and further having an electron withdrawing group bonded thereto, said base with electron withdrawing group being epresented by the formula:
X~ X2 W
/
wherein:
said base with electron withdrawing group is bonded at X4 to the second cyclic structure of the _ W O 94/06810 PC~r/US93/08391 ~4~3~ -6-nucleoside;
Xl, X3 and X5 are each members of the group consisting of N, O, C, S and Se;
X2 and X4 are each members of the group consisting of N and C; and W is a member of the group consistiIlg of F, Cl, Br, I, O, S, OH, SII, NH2, NO2, C(O)H, C(O)NHOH, C(S)NHOEI, NO, C(NOCH3)NH2' CH3~ SCH3~ SeCH3' NH2' NEIOCH3, N3, CN, C(O)NE12, C(NOH)NH2, CSNEI2 and 10 C02H.
One object of the present invention is to provide oligonucleotides which include universal nucleosides at degenerate positions.
Further objects and advantages of the present invention will be apparent from the following description.
WO94/06810 ~ ~ 4 13 ~ ~ PCT/US93/08391 BRI~ DESCRIPTION OF THE DRAWINGS
FIG. l shows various compounds which have been proposed as universal nucleosides in the prior art.
E~IG. 2 shows nucleosides in which the cyclic oxygen of the sugar portion is replaced with S or CH2.
FIG. 3 shows nucleosides in which the base portion is a heterocycle including O, S or Se.
FIG. 4 shows nucleosides in which the base portion is a pyrrole, diazole or triazole derivative.
FIG. 5 shows potentially useful phosphonucleotide intermediates for use in constructing oligonucleotides of the present invention.
FIG. 6 shows a universal nucleoside according to the present invention.
W O 94/06810 PC~r/US93/08391 3~ ~ ~
DESCRIPTION OF T~IE PRE~ERRED EMBODIMENT
For the purposes of promoting an understanding of the principles of the invention, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated aspects of the invention, and such further applications of the principles of the invention as illustrated herein being contemplated as would normally occur to one skilled in the art to which the invention relates.
One aspect of the present invention relates to oligonucleotides comprising at least ten nucleosides, at least two of which are selected from the group consisting of A, T, C and G, and at least one nucleoside being a universal nucleoside. The incorporation of one or more universal nucleosides into the oligomer makes bonding to unknown bases possible and allows the oligonucleotide to match ambiguous or unknown nucleic acid sequences. In one preferred aspect, all f the common DNA nucleosides - deoxyadenosine (A), thymidine (T), deoxycytidine (C) and deoxyguanosine (G) - are combined with at least several of the universal nucleosides to make an oligonucleotide having at least l0 nucleosides therein.
Considering first the universal nucleoside portion of the present invention, the preferred nucleosides of the present invention include a first (sugar) portion and a second (base) portion. The first portion of the nucleoside can be represented by the formula:
W O 94/06810 2 1 4 4 ~ 3 ~ PC~r/US93/08391 _g _ ~z~J
~H R2 wherein each Rn is H, OH, F or OCH3, and Z is a member of the group consisting of O, S and CII2.
Most commonly, the first (suga~) portion will be D-deoxyribose or D-ribose as found in naturally occurring ribonucleosides and deoxyribonucleosides. The Z atom of the above formula will be O in these preferred cases.
Alternatively, the oxygen of this cyclic structure may be replaced by either S or CIl2 without unreasonably affecting the performance of the nucleoside in some applications.
Examples of nucleosides having a first "sugar" portion which is substituted with S or CH2 are shown in FIG. 2;
additional examples of such compounds may be developed by those skilled in the art without undue experimentation.
A variety of substituents may be included at Rl and/or R2. In particular, both Rl and R2 may be H as in the case of D-deoxyribose. If one of those substituents is H and the other is OH the first (sugar) portion may be D-ri~ose as in naturally-occurring ribonucleosides. Alternatively, either or both of Rl and R2 may be substituted with F or OC~3. The use of 1uorine substituted nucleosides has been suggested by the prior art as in, for example, 2'-fluoro-2'-deoxyadenosine, which was incorporated into oligonucleotides by M. Ikehara and coworkers (Ikehara, 21 He~erocycles 75, 1984).
The second (base) portion "B" of the universal W O 94/06810 ~ ~ ~ PC~r/US93/08391 nucleosides is preferably a five-membered, heterocyclic base having at least two double bonds in one of its possible tautomeric forms, and further having an electron withdrawing group bonded thereto. Preferred base portions are represented by the formula:
~Xl~
X5 X2_W
As was mentioned above, Xl, X3 and X5 are each members of the qroup consisting of C, N, O, S and Se. In the universal nucleosides preferred in testing to date, Xl, X3 and X5 are either C or N, with the most preferred compounds including at least one N. Alternative nucleosides including O, S or Se are shown in FIG. 3; additional alternatives may be prepared by one skilled in the art without undue experimentation.
As was stated above, X2 and X4 are each preferably members of the group consisting of N and C. In the nucleosides most preferred in testing to date at least one of these two atoms is N, although the exact location of the nitrogen may vary according to the particular application.
It is to be appreciated that there are some limitations as to which atoms can be located at Xl, X2, X3 and X5. In particular, when X4= N and X4 is the site of the glycosidic bond, then Xl, X3 and X5 can only be C
or N, and X2 must be C. O, S or Se can be tolorated at Xl, X3 and X5 only when X4= C, and even in that case there can be no more than one of these atoms (O, S and Se) in the five-membered heterocyclic ring.
When N is included in the nucleoside, the base may be, for example, a pyrrole, dia~ole or tria~ole. Examples o~
such nucleosides are shown in FIG. 4, and can be prepared by ~ 21443~
one skilled in the art without undue experimentation.
The electron withdrawing group W is a member of the group consisting of F, Cl, Br, I, O, S, OH, Sl~, N~I2, NO2, C(O)H, C(O)NHOH, C(S)NHOH, NO, C(NOCH3)NH2, OCH3, SCH3, SeCH3, ONH2, NHOCH3, N3, CN, C(O)NH2, C(NOH)NH2, CSNH2 and CO2H. NO2 has been especially effective in experiments to date, and is particularly preferred for Sanger sequencing. C(O)NH2 has also been particularly effective in certain applications.
A number of structural features of the preferred base portions should be mentioned. First, it is to be appreciated that the electron withdrawing group is bonded to the remainder of the base portion only at X2. In some unsatisfactory prior art nucleosides, such as deoxyinosine, the electron withdrawing group bonds to the remainder of the base portion at both X2 and X3 - which limits the ability of the base to assume a position necessary to optimize both hydrogen bonding and base stacking.
Adding substituents at Xl, X3 or X5 may be effective in some cases, although any substituents added at those positions must be small enough to avoid steric interference and must not prevent effective base stacking and bonding interactions. Small substituents which do not interfere with the coplanarity of the extended ring system are preferred. X1, X3 or X5 should be C if a substituent is added to that atom. Nucleosides in which such substituents are included at Xl, X3 or X5 are to be considered equivalent to the specifically disclosed nucleosides.
Preferably, the base-with-electron withdrawing group comprises an extended ~ system which favors base stacking interactions. Specifically, the preerred electron withdrawing groups enhance base stacking through interaction with adjacent pyrimidine and purine rin~ systems in a polynucleotide double helix.
-WO94/06810 ~ ~ PCT/US93/08391 The universal nucleosides of the present invention accordingly preferably possess the following chemical and structural properties:
l. One or more donor or acceptor sites for hydrogen bonding to A, C, G or T.
2. Planar aromatic ~ system capable of stacking interactions with A, C, G and T in duplex or triplex nucleic acids.
3. The molecule is sterically accommodated within duplex or triplex nucleic acid, while at the same time maintaining both base stacking and hydrogen bonding interactions.
4. At least one of the following derivatives of the universal nucleoside is chemically accessible and stable: a phosphoramidite substituted by a protecting group such as cyanoethyl; H-phosphonate; a phosphodiester in which one of the phosphorus substituents is a protecting group such as O-chlorophenyl; or a phosphite triester in which two of the substituents can function as either transient protecting groups or leaving groups for phosphorus ester formation.
5. The universal nucleoside, once incorporated into oligonucleotide under construction, is completely stable to the reagents and conditions of oligonucleotide synthesis as well as stable to oxygen, light and water.
The synthesis of oligonucleotides from their nucleosidal components is accomplished in a straightforward manner using standard protocols on commercial DNA synthesizers. In general, the synthesis of the oligonucleotide proceeds through a phosphorus-based intermediate. Such syntheses can be accomplished by one skilled in the art without undue experimentation.
Examples of useful phosphorus-containing nucleotide intermediates are shown in FIG. 5 and include phosphotriesters according to Sproat and Gait (1984);
WO94/06810 -13- PCT/US93/~8391 phosphoramidites according to Beaucaye and Caruthers (1981);
H-phosphonates according to Froehler and Matteucci (1986);
and phosphites according to Hasaka et al. (1991). The selection of an appropriate pathway to oligonucleotide synthesis may be selected by one skilled in the art without undue experimentation.
The synthesis of 3-nitropyrrole deoxyribonucleoside (1) and its protected phosphoramidite (2) is outlined in Scheme 1 below. The two reactants, 3-nitropyrrole and 2-deoxy-3,5-di-O-p-toluoyl-D-erythro-pentofuranosyl chloride are prepared by literature methods.
WO 94/06810 -14- PCI~/USg3/08391 .
CH3~CO ~~ NaH Ho_ ~/N~ NO2 O ~2) NH3,MeOH OH
~C~H3 D~a C:l~ ~0~ py~ e ~ NO2 2 D~r~
DMT~ = ~C--~
WO~4/U681U ~1 4 4 3 ~ I PCT/US93/U8391 Modified nucleosides designed to function as universal nucleic acid bases were synthesized and their suitability as constituents of oligonucleotide probes were determined in physicochemical and molecular biological studies. As many as nine DNA bases of a 17-mer primer were replaced by 3-nitropyrrole deoxyribonucleoside without destroying the ability of the primer to initiate DNA synthesis.
The oligonucleotide sequences shown in Table 1 have been synthesized and tested as primers for DNA synthesis. T~le synthetic oligonucleotides were used as primers for sequencing single-stranded DNA by the Sanger method. Sanger dideoxy sequencing was performed using the United States Biochemical (USB) Sequenase version 2.0 sequencing kit. The DNA sequenced was a Hind III-Bluescript SK subclone of a Drosophila neural peptide gene described in Nichols et al. J.
Biol. Chem. 263: 12167 (1988). The template DNA was either ssDNA or dsDNA, and oligonucleotides were purified using 20%
acrylamide-8M urea eletrophoretic gels and size exclusion chromatography. Approximately 1 ~gDNA was sequenced with 0.1 ~Ig oligonucleotide according to conditions provided by the supplier using 35S-dATP (Amersham). Aliquots of the sequencing reactions were electrophoresed on 6% acrylamide-8M
urea sequencing gels after which the gel was dried and exposed to Kodak XAR X-ray film.
The results indicate that proper sequencing was achieved by oligonucleotides of the present invention. ln particular, it was shown that as many as nine bases in a 17-mer sequence can be substituted by 3-nitropyrrole and a readable sequencing ladder obtained.
As previously stated, there are a variety oE applicatiorls in addition to DNA sequencing for which the oligonucleotides of the present invention are particularly effective. For example, the use of such oligonucleotides in polymerase chain W O 94/06810 PC~r/US93/08391 ~ ~ -16-reaction (PCR) techniques would be extremely beneficial. PCR
is a powerful technique with many applications such as, for example, the screening of individuals for medically significant mutations.
Also, the oligonucleotides of the present invention may be effective for hybridization uses such as the screenillg of complex DNA or genomic libraries, the quantification of nucleic acids and the analysis of a Northern or Southern blot. The use of a universal nucleoside at the degenerate sites would allow a single oligonucleotide to be used as a probe instead of a complex mixture.
It is also anticipated that the oligonucleotides of the present invention will find widespread applicability in both clinical and therapeutic settings. For example, DNA
hybridization assays have become an important clinical tool for diagnosis of many disease states. Because of variations in the genetic sequence of virtually all pathogenic viruses, probes containing oligonucleotides having universal bases would be particularly effective. Therapeutic applications such as incorporation into triplex forming oligonucleotides and in antisense oligonucleotide therapeutics directed toward nucleic acid targets which have significant variability are also anticipated.
While the invention has been illustrated and described in detail in the foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.
WO94/06810 2 1 4 4 ~ ~ 4 PCT/US93/08391 .
Table 1. Modified Primers for Sanger Sequencing Primer No. Sequence 5'-CGT AAT CAG AAA ACA AT-3' 66 5'-CGT AAN CAN AAN ACN AT-3' (256 - degenerate primer mixture) 67 5'-CGT AAI CAI AAI ACI AT-3' 72 5'-CGT AAM CAM AAM ACM AT-3' 73 5'-CGT AAT CAG AAA ACA MT-3' 74 5'-CGT AAT CAG AAA ACA AT-3' (synthesized on a universal support) 5'-CGT AAT CAG AAA ACA AM-3' (synthesized on a universal support) 77 5'-CGT AAT CAG AAA MMM AT-3' 78 5'-CGT AAT CAG MMM MMM AT-3' 79 5'-CGT AAT CAG AAC ACA AT-3' 81 5'-CGT AAT CAG AAA ACG AT-3' 82 5'-CGT AAT CAG AAC AC~ AT-3' 83 5'-CGT AAT MMM MMM MMM AT-3' 84 5'-CGT A~T CAG AAA ACA AC-3' 5'-CGT AA~ CA_ AAC ACG AT-3' I = deoxyinosine M = 3-nitropyrrole deoxyribonucleoside All underlined bases are mismatches to target sequence.
The synthesis of oligonucleotides from their nucleosidal components is accomplished in a straightforward manner using standard protocols on commercial DNA synthesizers. In general, the synthesis of the oligonucleotide proceeds through a phosphorus-based intermediate. Such syntheses can be accomplished by one skilled in the art without undue experimentation.
Examples of useful phosphorus-containing nucleotide intermediates are shown in FIG. 5 and include phosphotriesters according to Sproat and Gait (1984);
WO94/06810 -13- PCT/US93/~8391 phosphoramidites according to Beaucaye and Caruthers (1981);
H-phosphonates according to Froehler and Matteucci (1986);
and phosphites according to Hasaka et al. (1991). The selection of an appropriate pathway to oligonucleotide synthesis may be selected by one skilled in the art without undue experimentation.
The synthesis of 3-nitropyrrole deoxyribonucleoside (1) and its protected phosphoramidite (2) is outlined in Scheme 1 below. The two reactants, 3-nitropyrrole and 2-deoxy-3,5-di-O-p-toluoyl-D-erythro-pentofuranosyl chloride are prepared by literature methods.
WO 94/06810 -14- PCI~/USg3/08391 .
CH3~CO ~~ NaH Ho_ ~/N~ NO2 O ~2) NH3,MeOH OH
~C~H3 D~a C:l~ ~0~ py~ e ~ NO2 2 D~r~
DMT~ = ~C--~
WO~4/U681U ~1 4 4 3 ~ I PCT/US93/U8391 Modified nucleosides designed to function as universal nucleic acid bases were synthesized and their suitability as constituents of oligonucleotide probes were determined in physicochemical and molecular biological studies. As many as nine DNA bases of a 17-mer primer were replaced by 3-nitropyrrole deoxyribonucleoside without destroying the ability of the primer to initiate DNA synthesis.
The oligonucleotide sequences shown in Table 1 have been synthesized and tested as primers for DNA synthesis. T~le synthetic oligonucleotides were used as primers for sequencing single-stranded DNA by the Sanger method. Sanger dideoxy sequencing was performed using the United States Biochemical (USB) Sequenase version 2.0 sequencing kit. The DNA sequenced was a Hind III-Bluescript SK subclone of a Drosophila neural peptide gene described in Nichols et al. J.
Biol. Chem. 263: 12167 (1988). The template DNA was either ssDNA or dsDNA, and oligonucleotides were purified using 20%
acrylamide-8M urea eletrophoretic gels and size exclusion chromatography. Approximately 1 ~gDNA was sequenced with 0.1 ~Ig oligonucleotide according to conditions provided by the supplier using 35S-dATP (Amersham). Aliquots of the sequencing reactions were electrophoresed on 6% acrylamide-8M
urea sequencing gels after which the gel was dried and exposed to Kodak XAR X-ray film.
The results indicate that proper sequencing was achieved by oligonucleotides of the present invention. ln particular, it was shown that as many as nine bases in a 17-mer sequence can be substituted by 3-nitropyrrole and a readable sequencing ladder obtained.
As previously stated, there are a variety oE applicatiorls in addition to DNA sequencing for which the oligonucleotides of the present invention are particularly effective. For example, the use of such oligonucleotides in polymerase chain W O 94/06810 PC~r/US93/08391 ~ ~ -16-reaction (PCR) techniques would be extremely beneficial. PCR
is a powerful technique with many applications such as, for example, the screening of individuals for medically significant mutations.
Also, the oligonucleotides of the present invention may be effective for hybridization uses such as the screenillg of complex DNA or genomic libraries, the quantification of nucleic acids and the analysis of a Northern or Southern blot. The use of a universal nucleoside at the degenerate sites would allow a single oligonucleotide to be used as a probe instead of a complex mixture.
It is also anticipated that the oligonucleotides of the present invention will find widespread applicability in both clinical and therapeutic settings. For example, DNA
hybridization assays have become an important clinical tool for diagnosis of many disease states. Because of variations in the genetic sequence of virtually all pathogenic viruses, probes containing oligonucleotides having universal bases would be particularly effective. Therapeutic applications such as incorporation into triplex forming oligonucleotides and in antisense oligonucleotide therapeutics directed toward nucleic acid targets which have significant variability are also anticipated.
While the invention has been illustrated and described in detail in the foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.
WO94/06810 2 1 4 4 ~ ~ 4 PCT/US93/08391 .
Table 1. Modified Primers for Sanger Sequencing Primer No. Sequence 5'-CGT AAT CAG AAA ACA AT-3' 66 5'-CGT AAN CAN AAN ACN AT-3' (256 - degenerate primer mixture) 67 5'-CGT AAI CAI AAI ACI AT-3' 72 5'-CGT AAM CAM AAM ACM AT-3' 73 5'-CGT AAT CAG AAA ACA MT-3' 74 5'-CGT AAT CAG AAA ACA AT-3' (synthesized on a universal support) 5'-CGT AAT CAG AAA ACA AM-3' (synthesized on a universal support) 77 5'-CGT AAT CAG AAA MMM AT-3' 78 5'-CGT AAT CAG MMM MMM AT-3' 79 5'-CGT AAT CAG AAC ACA AT-3' 81 5'-CGT AAT CAG AAA ACG AT-3' 82 5'-CGT AAT CAG AAC AC~ AT-3' 83 5'-CGT AAT MMM MMM MMM AT-3' 84 5'-CGT A~T CAG AAA ACA AC-3' 5'-CGT AA~ CA_ AAC ACG AT-3' I = deoxyinosine M = 3-nitropyrrole deoxyribonucleoside All underlined bases are mismatches to target sequence.
Claims (16)
1. An oligonucleotide comprising at least ten nucleosides, including at least two different nucleoside members of the group consisting of A, T, C and G, and wherein at least one nucleoside is a universal nucleoside of the formula:
wherein in the first cyclic structure illustrated above:
each Rn is H, OH, F or OCH3;
Z is a member of the group consisting of O, S and CH2;
and B is a second cyclic structure comprising a five-membered, cyclic base having at least two double bonds in one of the possible tautomeric forms of the cyclic structure, and further having an electron withdrawing group bonded thereto, said base with electron withdrawing group being represented by the formula:
wherein:
said base with electron withdrawing group is bonded at X4 to the second cyclic structure of the nucleoside;
X1, X3 and X5 are each members of the group consisting of N, O, C, S and Se;
X2 and X4 are each members of the group consisting of N and C; and W is a member of the group consisting of F, Cl, Br, I, O, S, OH, SH, NH2, NO2, C(O)H, C(O)NHOH, C(S)NHOH, NO, C(NOCH3)NH2, OCH3, SCH3, SeCH3, ONH2, NHOCH3, N3, CN, C(O)NH2, C(NOH)NH2, CSNH2 and CO2H.
wherein in the first cyclic structure illustrated above:
each Rn is H, OH, F or OCH3;
Z is a member of the group consisting of O, S and CH2;
and B is a second cyclic structure comprising a five-membered, cyclic base having at least two double bonds in one of the possible tautomeric forms of the cyclic structure, and further having an electron withdrawing group bonded thereto, said base with electron withdrawing group being represented by the formula:
wherein:
said base with electron withdrawing group is bonded at X4 to the second cyclic structure of the nucleoside;
X1, X3 and X5 are each members of the group consisting of N, O, C, S and Se;
X2 and X4 are each members of the group consisting of N and C; and W is a member of the group consisting of F, Cl, Br, I, O, S, OH, SH, NH2, NO2, C(O)H, C(O)NHOH, C(S)NHOH, NO, C(NOCH3)NH2, OCH3, SCH3, SeCH3, ONH2, NHOCH3, N3, CN, C(O)NH2, C(NOH)NH2, CSNH2 and CO2H.
2. An oligonucleotide according to claim 1 wherein at least four nucleosides are universal nucleosides of the formula of claim 1.
3. An oligonucleotide according to claim 2 wherein at least eight nucleosides are said universal nucleosides of the formula of claim 1.
4. An oligonucleotide according to claim 1 wherein at least one nucleoside is A, at least one nucleoside is T, at least one nucleoside is C, and at least one nucleoside is G.
5. An oligonucleotide according to claim 1 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein Z is O.
6. An oligonucleotide according to claim 1 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein each Rn is H.
7. An oligonucleotide according to claim 1 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein R1 is H and R2 is OH.
8. An oligonucleotide according to claim 1 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein R1 is H and R2 is F.
9. An oligonucleotide according to claim 1 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein R1 is H and R2 is OCH3.
10. An oligonucleotide according to claim 1 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein B is a substituted pyrrole.
11. An oligonucleotide according to claim 1 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein B is a substituted imidazole.
12. An oligonucleotide according to claim 1 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein B is a substituted triazole.
13. An oligonucleotide according to claim 1 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein X4 is N.
14. An oligonucleotide according to claim 13 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein W is NO2.
15. An oligonucleotide according to claim 1 wherein at least one universal nucleoside is a nucleoside of the formula of claim 1 wherein W is NO2.
16. A nucleoside of the formula:
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US07/946,971 US5438131A (en) | 1992-09-16 | 1992-09-16 | 3-nitropyrrole nucleoside |
US07/946,971 | 1992-09-16 |
Publications (1)
Publication Number | Publication Date |
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CA2144334A1 true CA2144334A1 (en) | 1994-03-31 |
Family
ID=25485273
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002144334A Abandoned CA2144334A1 (en) | 1992-09-16 | 1993-09-07 | Oligonucleotides having universal nucleoside spacers |
Country Status (5)
Country | Link |
---|---|
US (2) | US5438131A (en) |
EP (1) | EP0660842A4 (en) |
JP (1) | JPH08501308A (en) |
CA (1) | CA2144334A1 (en) |
WO (1) | WO1994006810A1 (en) |
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-
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- 1993-09-07 JP JP6508127A patent/JPH08501308A/en not_active Ceased
- 1993-09-07 WO PCT/US1993/008391 patent/WO1994006810A1/en not_active Application Discontinuation
- 1993-09-07 EP EP93922156A patent/EP0660842A4/en not_active Withdrawn
- 1993-09-07 CA CA002144334A patent/CA2144334A1/en not_active Abandoned
-
1994
- 1994-10-28 US US08/330,423 patent/US5681947A/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
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EP0660842A1 (en) | 1995-07-05 |
EP0660842A4 (en) | 1996-03-27 |
US5681947A (en) | 1997-10-28 |
WO1994006810A1 (en) | 1994-03-31 |
US5438131A (en) | 1995-08-01 |
JPH08501308A (en) | 1996-02-13 |
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