US20120302453A1 - Oxocarbonamide peptide nucleic acids and methods of using same - Google Patents
Oxocarbonamide peptide nucleic acids and methods of using same Download PDFInfo
- Publication number
- US20120302453A1 US20120302453A1 US11/947,705 US94770507A US2012302453A1 US 20120302453 A1 US20120302453 A1 US 20120302453A1 US 94770507 A US94770507 A US 94770507A US 2012302453 A1 US2012302453 A1 US 2012302453A1
- Authority
- US
- United States
- Prior art keywords
- nucleic acid
- formula
- oxocarbonamide
- peptide nucleic
- heteroatom
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108091093037 Peptide nucleic acid Proteins 0.000 title claims abstract description 85
- 238000000034 method Methods 0.000 title claims description 58
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 79
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 70
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 70
- 238000009396 hybridization Methods 0.000 claims abstract description 37
- 239000002679 microRNA Substances 0.000 claims abstract description 26
- 108091070501 miRNA Proteins 0.000 claims abstract 3
- -1 azaadenine Chemical compound 0.000 claims description 43
- MWUXSHHQAYIFBG-UHFFFAOYSA-N nitrogen oxide Inorganic materials O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 claims description 33
- 239000003446 ligand Substances 0.000 claims description 29
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 25
- 108020004414 DNA Proteins 0.000 claims description 23
- 102000053602 DNA Human genes 0.000 claims description 23
- 230000000295 complement effect Effects 0.000 claims description 22
- 229910052739 hydrogen Inorganic materials 0.000 claims description 21
- 125000000217 alkyl group Chemical group 0.000 claims description 18
- 150000001412 amines Chemical class 0.000 claims description 17
- 239000001257 hydrogen Substances 0.000 claims description 17
- 239000004005 microsphere Substances 0.000 claims description 16
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 15
- 239000007787 solid Substances 0.000 claims description 13
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 12
- 150000007942 carboxylates Chemical class 0.000 claims description 12
- 150000001298 alcohols Chemical class 0.000 claims description 11
- 150000001408 amides Chemical class 0.000 claims description 11
- 235000013877 carbamide Nutrition 0.000 claims description 11
- 150000002148 esters Chemical class 0.000 claims description 11
- 150000004820 halides Chemical class 0.000 claims description 11
- 229910052760 oxygen Inorganic materials 0.000 claims description 11
- XTQHKBHJIVJGKJ-UHFFFAOYSA-N sulfur monoxide Chemical class S=O XTQHKBHJIVJGKJ-UHFFFAOYSA-N 0.000 claims description 11
- 229910052815 sulfur oxide Inorganic materials 0.000 claims description 11
- 150000003672 ureas Chemical class 0.000 claims description 11
- 239000012625 DNA intercalator Substances 0.000 claims description 9
- 238000003556 assay Methods 0.000 claims description 9
- 229910052717 sulfur Inorganic materials 0.000 claims description 9
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 6
- CCGKOQOJPYTBIH-UHFFFAOYSA-N ethenone Chemical compound C=C=O CCGKOQOJPYTBIH-UHFFFAOYSA-N 0.000 claims description 6
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 6
- 125000005647 linker group Chemical group 0.000 claims description 6
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 6
- PTDZLXBJOJLWKG-UHFFFAOYSA-N 5-(bromomethyl)-1h-pyrimidine-2,4-dione Chemical compound BrCC1=CNC(=O)NC1=O PTDZLXBJOJLWKG-UHFFFAOYSA-N 0.000 claims description 3
- 229930024421 Adenine Natural products 0.000 claims description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 3
- 229960000643 adenine Drugs 0.000 claims description 3
- 150000001555 benzenes Chemical class 0.000 claims description 3
- 229940104302 cytosine Drugs 0.000 claims description 3
- 229940113082 thymine Drugs 0.000 claims description 3
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 claims description 2
- 125000006357 methylene carbonyl group Chemical group [H]C([H])([*:1])C([*:2])=O 0.000 claims description 2
- 150000001299 aldehydes Chemical class 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 34
- 108091032973 (ribonucleotides)n+m Proteins 0.000 abstract description 30
- 239000000523 sample Substances 0.000 abstract description 30
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 108091027963 non-coding RNA Proteins 0.000 abstract description 6
- 102000042567 non-coding RNA Human genes 0.000 abstract description 6
- 238000010230 functional analysis Methods 0.000 abstract description 3
- 108700011259 MicroRNAs Proteins 0.000 description 55
- 210000004027 cell Anatomy 0.000 description 46
- 108090000623 proteins and genes Proteins 0.000 description 35
- 125000005842 heteroatom Chemical group 0.000 description 27
- 125000002252 acyl group Chemical group 0.000 description 21
- 125000004432 carbon atom Chemical group C* 0.000 description 21
- 108091034117 Oligonucleotide Proteins 0.000 description 20
- 108090000765 processed proteins & peptides Proteins 0.000 description 20
- 108020004459 Small interfering RNA Proteins 0.000 description 17
- 102000004169 proteins and genes Human genes 0.000 description 17
- PWEBUXCTKOWPCW-UHFFFAOYSA-N squaric acid Chemical compound OC1=C(O)C(=O)C1=O PWEBUXCTKOWPCW-UHFFFAOYSA-N 0.000 description 17
- 125000003729 nucleotide group Chemical group 0.000 description 16
- 125000003545 alkoxy group Chemical group 0.000 description 15
- 230000014509 gene expression Effects 0.000 description 15
- 239000002773 nucleotide Substances 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 14
- 125000003118 aryl group Chemical group 0.000 description 14
- 230000027455 binding Effects 0.000 description 14
- 239000000203 mixture Substances 0.000 description 14
- 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 13
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 13
- 102000004196 processed proteins & peptides Human genes 0.000 description 13
- 150000003254 radicals Chemical class 0.000 description 12
- 239000000758 substrate Substances 0.000 description 12
- 230000000692 anti-sense effect Effects 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 229920000642 polymer Polymers 0.000 description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 description 10
- 229910052799 carbon Inorganic materials 0.000 description 10
- 201000010099 disease Diseases 0.000 description 10
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 10
- 125000002768 hydroxyalkyl group Chemical group 0.000 description 10
- 230000000670 limiting effect Effects 0.000 description 10
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 10
- 0 *CC(=O)N(CCNC(C)=O)[Y]CC Chemical compound *CC(=O)N(CCNC(C)=O)[Y]CC 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 9
- 150000001720 carbohydrates Chemical class 0.000 description 9
- 235000014633 carbohydrates Nutrition 0.000 description 9
- 239000002738 chelating agent Substances 0.000 description 9
- 238000000684 flow cytometry Methods 0.000 description 9
- 150000002632 lipids Chemical class 0.000 description 9
- 239000000178 monomer Substances 0.000 description 9
- 125000006239 protecting group Chemical group 0.000 description 9
- 239000004055 small Interfering RNA Substances 0.000 description 9
- 150000003431 steroids Chemical class 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- 108091032955 Bacterial small RNA Proteins 0.000 description 8
- 150000001721 carbon Chemical group 0.000 description 8
- 125000004423 acyloxy group Chemical group 0.000 description 7
- 229920006317 cationic polymer Polymers 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 125000004356 hydroxy functional group Chemical group O* 0.000 description 7
- 239000002853 nucleic acid probe Substances 0.000 description 7
- VGIRNWJSIRVFRT-UHFFFAOYSA-N 2',7'-difluorofluorescein Chemical compound OC(=O)C1=CC=CC=C1C1=C2C=C(F)C(=O)C=C2OC2=CC(O)=C(F)C=C21 VGIRNWJSIRVFRT-UHFFFAOYSA-N 0.000 description 6
- 241000282414 Homo sapiens Species 0.000 description 6
- 229910006069 SO3H Inorganic materials 0.000 description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 239000007801 affinity label Substances 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 238000002493 microarray Methods 0.000 description 6
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 6
- 125000002861 (C1-C4) alkanoyl group Chemical group 0.000 description 5
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 5
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical class [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 5
- 241000124008 Mammalia Species 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 238000000636 Northern blotting Methods 0.000 description 5
- 239000004793 Polystyrene Substances 0.000 description 5
- 125000003302 alkenyloxy group Chemical group 0.000 description 5
- 125000005133 alkynyloxy group Chemical group 0.000 description 5
- 125000004104 aryloxy group Chemical group 0.000 description 5
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 5
- 125000004122 cyclic group Chemical group 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 210000001161 mammalian embryo Anatomy 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 229920002223 polystyrene Polymers 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 229920002873 Polyethylenimine Polymers 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 4
- 125000002015 acyclic group Chemical group 0.000 description 4
- 125000003710 aryl alkyl group Chemical group 0.000 description 4
- 125000002102 aryl alkyloxo group Chemical group 0.000 description 4
- 229910052794 bromium Inorganic materials 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 229910052801 chlorine Inorganic materials 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- RBSLJAJQOVYTRQ-UHFFFAOYSA-N croconic acid Chemical compound OC1=C(O)C(=O)C(=O)C1=O RBSLJAJQOVYTRQ-UHFFFAOYSA-N 0.000 description 4
- SPXGBDTUWODGLI-UHFFFAOYSA-N deltic acid Chemical compound OC1=C(O)C1=O SPXGBDTUWODGLI-UHFFFAOYSA-N 0.000 description 4
- 229910052731 fluorine Inorganic materials 0.000 description 4
- 229910052740 iodine Inorganic materials 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000002777 nucleoside Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 229920000962 poly(amidoamine) Polymers 0.000 description 4
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 4
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 4
- WCJLIWFWHPOTAC-UHFFFAOYSA-N rhodizonic acid Chemical compound OC1=C(O)C(=O)C(=O)C(=O)C1=O WCJLIWFWHPOTAC-UHFFFAOYSA-N 0.000 description 4
- 229910052710 silicon Inorganic materials 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 239000003298 DNA probe Substances 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 3
- 102000039471 Small Nuclear RNA Human genes 0.000 description 3
- 108020004566 Transfer RNA Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 3
- 238000012875 competitive assay Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 239000000412 dendrimer Substances 0.000 description 3
- 229920000736 dendritic polymer Polymers 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- 238000010195 expression analysis Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000009368 gene silencing by RNA Effects 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 3
- 125000000623 heterocyclic group Chemical group 0.000 description 3
- 108091005601 modified peptides Proteins 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 239000002096 quantum dot Substances 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 108020004418 ribosomal RNA Proteins 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 229940035893 uracil Drugs 0.000 description 3
- ZDGOIRWJWVZKAQ-UHFFFAOYSA-N 5-[2-(aminomethyl)-4-(9h-fluoren-9-ylmethoxycarbonyl)-3,5-dimethoxyphenoxy]pentanoic acid Chemical compound COC1=CC(OCCCCC(O)=O)=C(CN)C(OC)=C1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZDGOIRWJWVZKAQ-UHFFFAOYSA-N 0.000 description 2
- KLAPVGNSPQNDTM-UHFFFAOYSA-N 5-[[1-amino-9-(9h-fluoren-9-ylmethoxycarbonyl)-9h-xanthen-2-yl]oxy]pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)C1C2=CC=CC=C2OC2=C1C(N)=C(OCCCCC(O)=O)C=C2 KLAPVGNSPQNDTM-UHFFFAOYSA-N 0.000 description 2
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 2
- IDLISIVVYLGCKO-UHFFFAOYSA-N 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein Chemical compound O1C(=O)C2=CC=C(C(O)=O)C=C2C21C1=CC(OC)=C(O)C(Cl)=C1OC1=C2C=C(OC)C(O)=C1Cl IDLISIVVYLGCKO-UHFFFAOYSA-N 0.000 description 2
- 208000035657 Abasia Diseases 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- 239000012099 Alexa Fluor family Substances 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- MWYQBMFDQCSFBJ-UHFFFAOYSA-N CC(C)C1=C(C(C)C)C(=[W])C(=[W])C(=[W])C1=[W].CC(C)C1=C(C(C)C)C(=[W])C(=[W])C1=[W].CC(C)C1=C(C(C)C)C(=[W])C1=[W].CC(C)C1=C(C(C)C)C1=[W].CC(C)C1=C(CC2=C(C(C)C)C(=[W])C(=[W])C(=[W])C2=[W])C(=[W])C(=[W])C(=[W])C1=[W].CC(C)C1=C(CC2=C(C(C)C)C(=[W])C(=[W])C2=[W])C(=[W])C(=[W])C1=[W].CC(C)C1=C(CC2=C(C(C)C)C(=[W])C2=[W])C(=[W])C1=[W].CC(C)C1=C(CC2=C(C(C)C)C2=[W])C1=[W] Chemical compound CC(C)C1=C(C(C)C)C(=[W])C(=[W])C(=[W])C1=[W].CC(C)C1=C(C(C)C)C(=[W])C(=[W])C1=[W].CC(C)C1=C(C(C)C)C(=[W])C1=[W].CC(C)C1=C(C(C)C)C1=[W].CC(C)C1=C(CC2=C(C(C)C)C(=[W])C(=[W])C(=[W])C2=[W])C(=[W])C(=[W])C(=[W])C1=[W].CC(C)C1=C(CC2=C(C(C)C)C(=[W])C(=[W])C2=[W])C(=[W])C(=[W])C1=[W].CC(C)C1=C(CC2=C(C(C)C)C(=[W])C2=[W])C(=[W])C1=[W].CC(C)C1=C(CC2=C(C(C)C)C2=[W])C1=[W] MWYQBMFDQCSFBJ-UHFFFAOYSA-N 0.000 description 2
- JFOJKICGKQTMKO-UHFFFAOYSA-N CC(C)N1CCN(C(C)C)CC1.CC(C)N1CN(C(C)C)C1 Chemical compound CC(C)N1CCN(C(C)C)CC1.CC(C)N1CN(C(C)C)C1 JFOJKICGKQTMKO-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108020003215 DNA Probes Proteins 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 102000007338 Fragile X Mental Retardation Protein Human genes 0.000 description 2
- 108010032606 Fragile X Mental Retardation Protein Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 2
- 229930010555 Inosine Natural products 0.000 description 2
- 108010008212 Integrin alpha4beta1 Proteins 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 108010004729 Phycoerythrin Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 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 2
- GYDJEQRTZSCIOI-UHFFFAOYSA-N Tranexamic acid Chemical compound NCC1CCC(C(O)=O)CC1 GYDJEQRTZSCIOI-UHFFFAOYSA-N 0.000 description 2
- 108010046334 Urease Proteins 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 108010004469 allophycocyanin Proteins 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000003491 array Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 2
- CREMABGTGYGIQB-UHFFFAOYSA-N carbon carbon Chemical compound C.C CREMABGTGYGIQB-UHFFFAOYSA-N 0.000 description 2
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 2
- 239000003283 colorimetric indicator Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 125000004093 cyano group Chemical group *C#N 0.000 description 2
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical compound C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 2
- 238000005401 electroluminescence Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 210000003754 fetus Anatomy 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- 238000009830 intercalation Methods 0.000 description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000006193 liquid solution Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000002082 metal nanoparticle Substances 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 210000004498 neuroglial cell Anatomy 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 2
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 2
- 125000003367 polycyclic group Chemical group 0.000 description 2
- 239000004926 polymethyl methacrylate Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 229920000915 polyvinyl chloride Polymers 0.000 description 2
- 239000004800 polyvinyl chloride Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 108091029842 small nuclear ribonucleic acid Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 208000002320 spinal muscular atrophy Diseases 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- WGTODYJZXSJIAG-UHFFFAOYSA-N tetramethylrhodamine chloride Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C(O)=O WGTODYJZXSJIAG-UHFFFAOYSA-N 0.000 description 2
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 2
- ZEMGGZBWXRYJHK-UHFFFAOYSA-N thiouracil Chemical compound O=C1C=CNC(=S)N1 ZEMGGZBWXRYJHK-UHFFFAOYSA-N 0.000 description 2
- 229950000329 thiouracil Drugs 0.000 description 2
- XXJGBENTLXFVFI-UHFFFAOYSA-N 1-amino-methylene Chemical compound N[CH2] XXJGBENTLXFVFI-UHFFFAOYSA-N 0.000 description 1
- 125000006345 2,2,2-trifluoroethoxymethyl group Chemical group [H]C([H])(*)OC([H])([H])C(F)(F)F 0.000 description 1
- 125000004206 2,2,2-trifluoroethyl group Chemical group [H]C([H])(*)C(F)(F)F 0.000 description 1
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-UHFFFAOYSA-N 0.000 description 1
- PIINGYXNCHTJTF-UHFFFAOYSA-N 2-(2-azaniumylethylamino)acetate Chemical compound NCCNCC(O)=O PIINGYXNCHTJTF-UHFFFAOYSA-N 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- 241000239223 Arachnida Species 0.000 description 1
- 102000008682 Argonaute Proteins Human genes 0.000 description 1
- 108010088141 Argonaute Proteins Proteins 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108050001427 Avidin/streptavidin Proteins 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
- QNGDNRZIUPRAKA-PLZCWAABSA-N C.C.CCC(=O)N(CCNC(=O)CN(CCNC)C(=O)CC)CC(C)=O.COC[C@@]12CO[C@@H](C(C)O1)[C@H]2OP(=O)(O)OC[C@@]12CO[C@@H](C(C)O1)[C@H]2OP(C)(=O)O.COC[C@H]1OC(C)C[C@H]1OP(=O)(O)OC[C@H]1OC(C)C[C@H]1OP(C)(=O)O Chemical compound C.C.CCC(=O)N(CCNC(=O)CN(CCNC)C(=O)CC)CC(C)=O.COC[C@@]12CO[C@@H](C(C)O1)[C@H]2OP(=O)(O)OC[C@@]12CO[C@@H](C(C)O1)[C@H]2OP(C)(=O)O.COC[C@H]1OC(C)C[C@H]1OP(=O)(O)OC[C@H]1OC(C)C[C@H]1OP(C)(=O)O QNGDNRZIUPRAKA-PLZCWAABSA-N 0.000 description 1
- HJYPZCGBPVGNCV-XJIVYGFWSA-N CC1=CN([C@H]2CC(NC3=C(NC[C@H]4O[C@@H](N5C=C(C)C(=O)NC5=O)CC4O)C(=O)C3=O)[C@@H](CO)O2)C(=O)NC1=O Chemical compound CC1=CN([C@H]2CC(NC3=C(NC[C@H]4O[C@@H](N5C=C(C)C(=O)NC5=O)CC4O)C(=O)C3=O)[C@@H](CO)O2)C(=O)NC1=O HJYPZCGBPVGNCV-XJIVYGFWSA-N 0.000 description 1
- XHNIFDXYGLPJLP-UHFFFAOYSA-N CCC(C)CC(C)CC(C)C Chemical compound CCC(C)CC(C)CC(C)C XHNIFDXYGLPJLP-UHFFFAOYSA-N 0.000 description 1
- 125000006519 CCH3 Chemical group 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108020004394 Complementary RNA Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241001559589 Cullen Species 0.000 description 1
- WEAHRLBPCANXCN-UHFFFAOYSA-N Daunomycin Natural products CCC1(O)CC(OC2CC(N)C(O)C(C)O2)c3cc4C(=O)c5c(OC)cccc5C(=O)c4c(O)c3C1 WEAHRLBPCANXCN-UHFFFAOYSA-N 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 208000003098 Ganglion Cysts Diseases 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000980898 Homo sapiens Cell division cycle-associated protein 4 Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- DZLNHFMRPBPULJ-VKHMYHEASA-N L-thioproline Chemical compound OC(=O)[C@@H]1CSCN1 DZLNHFMRPBPULJ-VKHMYHEASA-N 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 208000036626 Mental retardation Diseases 0.000 description 1
- 108091093189 Mir-375 Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- OKIZCWYLBDKLSU-UHFFFAOYSA-M N,N,N-Trimethylmethanaminium chloride Chemical compound [Cl-].C[N+](C)(C)C OKIZCWYLBDKLSU-UHFFFAOYSA-M 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- WDVSHHCDHLJJJR-UHFFFAOYSA-N Proflavine Chemical compound C1=CC(N)=CC2=NC3=CC(N)=CC=C3C=C21 WDVSHHCDHLJJJR-UHFFFAOYSA-N 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 229910007161 Si(CH3)3 Inorganic materials 0.000 description 1
- 229910003828 SiH3 Inorganic materials 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 108020004688 Small Nuclear RNA Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 208000005400 Synovial Cyst Diseases 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 229910009447 Y1-Yn Inorganic materials 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 125000002723 alicyclic group Chemical group 0.000 description 1
- 125000004453 alkoxycarbonyl group Chemical group 0.000 description 1
- 125000005194 alkoxycarbonyloxy group Chemical group 0.000 description 1
- 125000004448 alkyl carbonyl group Chemical group 0.000 description 1
- 125000005196 alkyl carbonyloxy group Chemical group 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 125000005199 aryl carbonyloxy group Chemical group 0.000 description 1
- 125000005200 aryloxy carbonyloxy group Chemical group 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 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
- 125000005841 biaryl group Chemical group 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000001951 carbamoylamino group Chemical group C(N)(=O)N* 0.000 description 1
- 125000002837 carbocyclic group Chemical group 0.000 description 1
- 239000002041 carbon nanotube Substances 0.000 description 1
- 229910021393 carbon nanotube Inorganic materials 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 229920006037 cross link polymer Polymers 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 125000004465 cycloalkenyloxy group Chemical group 0.000 description 1
- 125000000000 cycloalkoxy group Chemical group 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000001511 cyclopentyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- 125000001559 cyclopropyl group Chemical group [H]C1([H])C([H])([H])C1([H])* 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 150000001470 diamides Chemical class 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000003372 endocrine gland Anatomy 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 125000000219 ethylidene group Chemical group [H]C(=[*])C([H])([H])[H] 0.000 description 1
- 241001233957 eudicotyledons Species 0.000 description 1
- 210000003499 exocrine gland Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 150000002243 furanoses Chemical class 0.000 description 1
- 125000002541 furyl group Chemical group 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000003197 gene knockdown Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000007274 generation of a signal involved in cell-cell signaling Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 125000004475 heteroaralkyl group Chemical group 0.000 description 1
- 125000001072 heteroaryl group Chemical group 0.000 description 1
- 125000005553 heteroaryloxy group Chemical group 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 102000044493 human CDCA4 Human genes 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000000138 intercalating agent Substances 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 108091023663 let-7 stem-loop Proteins 0.000 description 1
- 108091063478 let-7-1 stem-loop Proteins 0.000 description 1
- 108091049777 let-7-2 stem-loop Proteins 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 108091053735 lin-4 stem-loop Proteins 0.000 description 1
- 108091032363 lin-4-1 stem-loop Proteins 0.000 description 1
- 108091028008 lin-4-2 stem-loop Proteins 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004777 loss-of-function mutation Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 210000003593 megakaryocyte Anatomy 0.000 description 1
- 108091057645 miR-15 stem-loop Proteins 0.000 description 1
- 108091027943 miR-16 stem-loop Proteins 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000002991 molded plastic Substances 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 210000002161 motor neuron Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- 239000002070 nanowire Substances 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000007472 neurodevelopment Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 125000006574 non-aromatic ring group Chemical group 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000003499 nucleic acid array Methods 0.000 description 1
- 108700020942 nucleic acid binding protein Proteins 0.000 description 1
- 102000044158 nucleic acid binding protein Human genes 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 238000002966 oligonucleotide array Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- WSHJJCPTKWSMRR-RXMQYKEDSA-N penam Chemical compound S1CCN2C(=O)C[C@H]21 WSHJJCPTKWSMRR-RXMQYKEDSA-N 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 125000000286 phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])* 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 229960000286 proflavine Drugs 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 125000003373 pyrazinyl group Chemical group 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 125000005493 quinolyl group Chemical group 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 125000000547 substituted alkyl group Chemical group 0.000 description 1
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical compound [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 229960003433 thalidomide Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 125000003944 tolyl group Chemical group 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 125000002348 vinylic group Chemical group 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
- C07K14/003—Peptide-nucleic acids (PNAs)
Definitions
- the present invention relates generally to nucleic acid probes useful in the detection and analysis of target nucleic acid sequences. More particularly, the present invention concerns nucleic acid probes wherein naturally occurring nucleobases or other nucleobase-binding moieties are covalently bound to an oxocarbonamide containing peptide backbone. In certain aspects, the present invention concerns methods employing nucleic acid probes in the detection and analysis of target nucleic acid sequences including, for example, mRNAs, miRNAs, and siRNAs.
- miRNAs A large number of small, non-coding RNAs have been identified and designated as microRNAs (miRNAs) (Ke et al., 2003). miRNAs have been shown to regulate gene expression at many levels, representing a novel gene regulatory mechanism. Understanding this RNA-based regulation will be useful to understand the complexity of the genome in higher eukaryotes as well as understand the complex gene regulatory networks.
- miRNAs are 18-25 nucleotide (nt) RNAs that are processed from longer endogenous hairpin transcripts by the enzymes Dicer and Argonaute (Ambros et al., 2003, Grishok et al., 2001). To date more than 4160 microRNAs have been identified in mammals, birds, fish, worms, flies, plants, and viruses according to the miRNA registry database release 9.0 in October 2006, hosted by Sanger Institute, UK. Some miRNAs have multiple loci in the genome (Reinhart et al., 2002) and may be arranged in tandem clusters (Lagos-Quintana et al., 2001).
- the first miRNAs to be discovered base-pair incompletely to repeated elements in the 3′ untranslated regions (UTRs) of other heterochrony genes, and regulate the translation directly and negatively by antisense RNA-RNA interaction (Lee et al., 1993; Reinhart et al., 2000).
- Some miRNAs are thought to interact with target mRNAs by limited complementary and suppressed translation as well (Lagos-Quintana et al., 2001; Lee and Ambros, 2001). Perfect complementarity between miRNAs and their target RNA may lead to target RNA degradation rather than inhibit translation (Hutvagner and Zamore, 2002), which suggests that the degree of complementarity determines function.
- one study determined that two different miRNA (miR15 and miR16) genes are clustered and located within the deleted minimal region of the B-cell chronic lymphocytic leukemia (B-CLL) tumor suppressor locus, and both genes are deleted or down-regulated in the majority of CLL cases (Calin et al., 2002).
- B-CLL B-cell chronic lymphocytic leukemia
- RNA interference in which double-stranded RNA leads to the degradation of any RNA that is homologous (Fire et al., 1998), relies on a mechanism that probably evolved for protection against viral attack and mobile genetic elements.
- One step in the RNAi mechanism is the generation of short interfering RNAs (siRNAs), double-stranded RNAs that are about 22 nt long.
- siRNAs short interfering RNAs
- the siRNAs lead to the degradation of homologous target RNA and the production of more siRNAs against the same target RNA (Lipardi et al., 2001; Zhang et al., 2002; Nykanen et al., 2001).
- RNAs The involvement of short RNAs in gene regulation has resulted in high interest among researchers in the discovery of siRNAs, miRNAs, their targets and mechanism of action.
- detection and analysis of these small RNAs is not trivial.
- the size and often low level of expression of miRNAs require the use of sensitive analysis tools.
- the use of conventional quantitative real-time PCR for monitoring expression of mature miRNAs is excluded due to their small size.
- Most miRNA researchers use Northern blot analysis combined with polyacrylamide gels to examine expression of both the mature and pre-miRNAs (Reinhart et al., 2000; Lagos-Quintana et al., 2001; Lee and Ambros, 2001). Primer extension has also been used to detect the mature miRNA (Zeng and Cullen, 2003).
- Microarrays are an alternative to Northern blot analysis for analyzing miRNA expression.
- Krichevsky et al. (2003) used cDNA microarrays to monitor the expression of miRNAs during neuronal development; however, the mature miRNAs had to be separated from the miRNA precursors using micro concentrators prior to microarray hybridization.
- Liu et al (2004) developed a microarray for expression profiling of 245 human and mouse miRNAs using 40-mer DNA oligonucleotide capture probes.
- Thomson et al. described the development of a oligonucleotide microarray platform for expression profiling of 124 mammalian miRNAs using oligonucleotide capture probes complementary to the mature microRNAs.
- DNA-based oligonucleotide arrays may include: the requirement of high concentrations of labeled input target RNA for efficient hybridization and signal generation, low sensitivity for rare and low-abundant miRNAs, and the necessity for post-array validation using more sensitive assays.
- a PCR-based approach has also been used to determine the expression levels of mature miRNAs (Grad et al., 2003).
- this method is cumbersome for routine miRNA expression profiling, since it involves gel isolation of small RNAs and ligation to linker oligonucleotides.
- Schmittgen et al. (2004) described an alternative method to Northern blot analysis, in which real-time PCR assays were used to quantify the expression of miRNA precursors.
- the disadvantage of this method is that it only allows quantification of the precursor miRNAs, which does not necessarily reflect the expression levels of mature miRNAs.
- LNA locked nucleic acid
- PNA peptide nucleic acid
- the present invention addresses these needs by providing novel oligonucleotide compositions for the accurate, sensitive, and specific detection and functional analysis of miRNAs and other non-coding RNAs.
- the compositions of the present invention will also be useful as biomarkers for disease diagnostics as well as for antisense-based intervention targeted against disease-associated miRNAs and other non-coding RNAs.
- the present invention provides a novel class of compounds that bind complementary DNA and RNA strands.
- the compounds of the invention generally comprise ligands linked to a oxocarbon acid amide modified peptide backbone.
- ligands include thymine, cytosine, adenine, guanine, uracil, inosine, 5-methylcytosine, thiouracil, bromothymine, azaadenine, or azaguanine.
- oxocarbonamides include deltic acid amide, thio-deltic acid amide, squaric acid amide, thio-squaric acid amide, croconic acid amide, thio-croconic acid amide, rhodizonic acid amide, and thio-rhodizonic acid amide.
- the present invention provides oxocarbonamide peptide nucleic acids having the formula (I):
- n is at least 1;
- each of L 1 -L n is independently selected from the group consisting of heteroatom substituted aryls, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, aromatic moieties, and reporter ligands, wherein at least one of L 1 -L n is a naturally occurring nucleobase, non-naturally occurring nucleobase, nucleobase binding group, or DNA intercalator;
- each of X 1 -X n is independently selected from the group consisting of R 1 NH and NHR 2 , wherein R 1 and R 2 are independently selected from the group consisting of H, CH 2 NH 2 , (CH 2 ) (1-10) —NH 2 , (CH 2 ) 2 (OCH 2 CH 2 ) (1-10) NH 2 , (CH 2 ) 2 (OCH 2 CH 2 ) (1-10) CO 2 H, (CH 2 ) (1-10) ;
- each of Y 1 -Y n is independently selected from the group consisting of CH 2 CO, formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh), and where at least one Y is formula (IVa), formula (Nb), formula (IVc), formula (Nd), formula (We), formula (IVf), formula (IVg), and formula (IVh),
- each W is independently selected from the group consisting of O and S, and M is selected from the group consisting of no linker, benzene, substituted benzene, formula (IVi), and formula (IVj);
- Q is selected from the group consisting of CO 2 H, CONR′R′′, SO 3 H, NH 2 , SH, or SO 2 NR′R′′ or an activated derivative of CO 2 H or SO 2 H;
- Z is selected from the group consisting of CO 2 H, CONR′R′′, SO 3 H, NH 2 , SH, SO 2 NR′R′′, NHR′′R′′′, or NR′′′COR′′′′, where R′, R′′, R′′′, and R′′′′ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyls, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteratom substituted alkyls, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers.
- the substituted benzene may be substituted with, for example, alkyl, amine, substituted amine, amide, branched amine, PEG, etc.
- the present invention provides oxocarbonamide peptide nucleic acids having the formula (II):
- each L is independently selected from the group consisting of heteroatom substituted aryls, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, aromatic moieties, and reporter ligands, wherein at least one of L 1 -L n is a heteroatom substituted acyl, naturally occurring nucleobase, non-naturally occurring nucleobase, nucleobase binding group, or DNA intercalator;
- each Y is independently selected from the group consisting of CH 2 CO, formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh), and where at least one Y is formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh);
- R′ is selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, heteroatom substituted acyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers;
- Z is selected from the group consisting of CO 2 H, CONR′R′′, SO 3 H, NH 2 , SH, SO 2 NR′R′′, NHR′′R′′′, or NR′′′COR′′′′, where R′, R′′, R′′′, and R′′′′ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, heteroatom substituted acyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers; and
- n is an integer from 1 to 100.
- the present invention provides an oxocarbonamide peptide nucleic acid having the formula (II):
- n is an integer from 1 to 100;
- each L is independently selected from the group consisting of heteroatom substituted aryl, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, and DNA intercalators;
- each Y is independently selected from the group consisting of CH 2 CO, formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh), and where at least one Y is formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh);
- R′ is selected from the group consisting of hydrogen, alkyl, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, and halides; and
- Z is selected from the group consisting of CO 2 H, NH 2 , and SH.
- the present invention provides an oxocarbonamide peptide nucleic acid having the formula (IIa):
- W is O or S
- each L is independently selected from the group consisting of heteroatom substituted aryl, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, aromatic moieties, and reporter ligands, wherein at least one of L 1 -L n is a heteroatom substituted aryl, naturally occurring nucleobase, non-naturally occurring nucleobase, nucleobase binding group, or DNA intercalator;
- R′ is selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, heteroatom substituted amide, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers;
- Z is selected from the group consisting of CO 2 H, CONR′R′′, SO 3 H, NH 2 , SH, SO 2 NR′R′′, NHR′′R′′′, or NR′′′COR′′′′, where R′, R′′, R′′′, and R′′′′ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers; and
- n is an integer from 1 to 100.
- the present invention provides an oxocarbonamide peptide nucleic acid having the formula (III):
- each L is independently selected from the group consisting of heteroatom substituted aryl, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, aromatic moieties, and reporter ligands, wherein at least one of L 1 -L n is a heteroatom substituted aryl, naturally occurring nucleobase, non-naturally occurring nucleobase, nucleobase binding group, or DNA intercalator;
- each Y is independently selected from the group consisting of CH 2 CO, formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh), and where at least one Y is formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh);
- R′ is selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers;
- R 3 is selected from the group consisting of H, CH 3 , and cationic polymers
- Z is selected from the group consisting of CO 2 H, CONR′R′′, SO 3 H, NH 2 , SH, SO 2 NR′R′′, NHR′′R′′′, or NR′′′COR′′′′, where R′, R′′, R′′′, and R′′′′ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers; and
- n is an integer from 1 to 100.
- the cationic polymer is a branched amine such as, for example, a polyamidoamine (PMAM) dendrimer or a polyethyleneimine (PEI).
- the cationic polymer is a polyammonium group (e.g., (CH 2 ) n NR 3 ). Due to their structure and charge, cationic polymers are useful nucleic acid transfection agents and drug carriers.
- the present invention provides an oxocarbonamide peptide nucleic acid having the formula (III):
- n is an integer from 1 to 100;
- each L is independently selected from the group consisting of naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, and DNA intercalators;
- each Y is independently selected from the group consisting of CH 2 CO, formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (We), formula (IVf), formula (IVg), and formula (IVh), and where at least one Y is formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh);
- R′ is selected from the group consisting of hydrogen, alkyl, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, heteroatom substituted acyls, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, and halides;
- R 3 is selected from the group consisting of H, CH 3 , and cationic polymers
- Z is selected from the group consisting of CO 2 H, NH 2 , and SH.
- the present invention provides an oxocarbonamide peptide nucleic acid having the formula (IIIa):
- W is O or S
- each L is independently selected from the group consisting of heteroatom substituted aryl, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, aromatic moieties, and reporter ligands, wherein at least one of L 1 -L n is a naturally occurring nucleobase, non-naturally occurring nucleobase, nucleobase binding group, or DNA intercalator;
- R′ is selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers;
- R 3 is selected from the group consisting of H, CH 3 , and cationic polymers
- Z is selected from the group consisting of CO 2 H, CONR′R′′, SO 3 H, NH 2 , SH, SO 2 NR′R′′, NHR′′R′′′, or NR′′′COR′′′′, where R′, R′′, R′′′, and R′′′′ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers; and
- n is an integer from 1 to 100.
- n may be an integer from 8 to 60, 10 to 50, 15 to 30, or 18 to 25. In some embodiments, n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or any range derivable therein.
- no Y is CH 2 CO. In some aspects of the invention all Ys are formula (IVa), formula (IVe), or a combination of formula (IVa) and formula (IVe).
- the present invention provides a method for detecting a target nucleic acid molecule, comprising: (a) providing an oxocarbonamide peptide nucleic acid comprising a sequence complementary to a sequence of a target nucleic acid molecule; (b) contacting the oxocarbonamide peptide nucleic acid with the target nucleic molecule under conditions that allow the oxocarbonamide peptide nucleic acid to hybridize with the target molecule; and (c) detecting the hybridization.
- the target nucleic acid molecule may be, for example, a DNA or an RNA molecule.
- the RNA molecule may be, for example, an mRNA, rRNA, tRNA, miRNA, or siRNA.
- one or both of the oxocarbonamide peptide nucleic acid probe or the target molecule may be labeled.
- a number of different labels may be used in the present invention such as fluorophores, chromophores, radiophores, enzymatic tags, antibodies, chemiluminescence, electroluminescence, metal nanoparticles, quantum dots, magnetic particles, and affinity labels.
- affinity labels include, but are not limited to the following: an antibody, an antibody fragment, a receptor protein, a hormone, biotin, DNP, a molecular imprint, or any polypeptide/protein molecule that binds to an affinity label.
- enzyme tags include enzymes such as urease, alkaline phosphatase or peroxidase to mention a few.
- Colorimetric indicator substrates can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples. All of these examples are generally known in the art and the skilled artisan will recognize that the invention is not limited to the examples described above.
- fluorophores include, but are not limited to the following: all of the Alexa Fluor® dyes, AMCA, BODIPY® 630/650, BODIPY® 650/665, BODIPY®-FL, BODIPY®-R6G, BODIPY®-TMR, BODIPY®-TRX, Cascade Blue®, CyDyesTM, including but not limited to Cy2TM, Cy3TM, and Cy5TM, DNA intercalating dyes, 6-FAMTM, Fluorescein, HEXTM, 6-JOE, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific BlueTM, REG, phycobilliproteins including, but not limited to, phycoerythrin and allophycocyanin, Rhodamine GreenTM, Rhodamine RedTM, ROXTM, TAMRATM, TETTM, Tetramethylrhodamine, and Texas Red®.
- the oxocarbonamide peptide nucleic acid probe or the target molecule is immobilized on a solid support.
- solid supports include: nitrocellulose, nylon membrane, glass, activated quartz, activated glass, polyvinylidene difluoride (PVDF) membrane, polystyrene substrates, polyacrylamide-based substrate, other polymers, copolymers, or crosslinked polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules).
- PVDF polyvinylidene difluoride
- a solid support may be in the form of, for example, a bead, a column, or a chip.
- the present invention provides an array comprising a plurality of oxocarbonamide peptide nucleic acid probes immobilized on a solid support.
- the solid support is a chip or a bead.
- the array comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000, or any range derivable therein, different oxocarbonamide peptide oligonucleotide probes.
- the present invention provides a method for detecting one or more target nucleic acid molecules in a multiplexed assay, comprising: (a) providing a plurality of different oxocarbonamide peptide nucleic acids, wherein each different oxocarbonamide peptide nucleic acid is covalently attached to a defined location on an array; (b) contacting a sample comprising the one or more target nucleic acid molecules with the array under conditions that allow the one or more target nucleic acid molecules to hybridize to complementary oxocarbonamide peptide nucleic acids on the array; and (c) detecting the hybridization.
- the plurality of nucleic acid molecules is a plurality of miRNA molecules.
- the array comprises a plurality of fluorescently encoded microspheres (“beads”).
- the invention provides a method for amplifying a target nucleic acid molecule.
- the method involves (a) incubating a first oxocarbonamide peptide nucleic acid of the invention with a target molecule under conditions that allow the first oxocarbonamide peptide nucleic acid to bind the target molecule; and (b) extending the first nucleic acid with the target molecule as a template.
- the method may further comprise contacting the target molecule with a second oxocarbonamide peptide nucleic acid that binds to a different region of the target molecule than the first oxocarbonamide peptide nucleic acid.
- the sequence of the target molecule is known or unknown.
- the association constant (K a ) of the oxocarbonamide peptide nucleic acid toward a complementary target molecule is higher than the association constant of the complementary strands of the double stranded target molecule.
- the melting temperature of a duplex between the oxocarbonamide peptide nucleic acid and a complementary target molecule is higher than the melting temperature of the complementary strands of the double stranded target molecule.
- the present invention provides pharmaceutical composition comprising an oxocarbonamide peptide nucleic acid for treatment of a disease curable by an antisense technology.
- the invention provides a method for inhibiting the expression of a target nucleic acid in a cell. The method comprises introducing into the cell a oxocarbonamide peptide nucleic acid of the invention in an amount sufficient to specifically attenuate expression of the target nucleic acid.
- the introduced oxocarbonamide peptide nucleic acid has a nucleotide sequence that is complementary to a region of the target nucleic acid sequence.
- the invention provides a method for preventing, stabilizing, or treating a disease, disorder, or condition associated with a target nucleic acid in a mammal.
- This method comprises introducing into the mammal oxocarbonamide peptide nucleic acid of the invention in an amount sufficient to specifically attenuate expression of the target nucleic acid, wherein the introduced oxocarbonamide peptide nucleic acid has a nucleotide sequence that is complementary to a region of the target nucleic acid.
- the oxocarbonamide peptide nucleic acid has a nucleotide sequence that is complementary to a region of between about 10 to about 100, about 10 to about 50, about 10 to about 30, about 15 to about 30, or about 17 to about 25, nucleotides of the target nucleic acid sequence.
- the introduced oxocarbonamide peptide nucleic acid is single stranded or double stranded.
- both strands may be oxocarbonamide peptide nucleic acids or one strand may be an oxocarbonamide peptide nucleic acid and the other strand may be a DNA, RNA, or an oligonucleotide analog such as a PNA or LNA.
- Exemplary mammals that can be treated using the methods of the invention include humans, primates, animals of veterinary interest (e.g., cows, sheep, goats, buffalos, and horses), and domestic pets (e.g., dogs and cats).
- Exemplary cells in which one or more target genes can be silenced using the methods of the invention include invertebrate, plant, bacteria, yeast, and vertebrate (e.g., mammalian) cells.
- FIG. 1 shows the oxocarbon acids: deltic acid, thio-deltic acid, squaric acid, thio-squaric acid, croconic acid, thio-croconic acid, rhodizonic acid, and thio-rhodizonic acid.
- FIG. 2 shows abasic monomers of squaric acid amides and reactive derivatives of squaric acid.
- FIG. 3 shows squaric acid amide peptide nucleic acid (SquarPNA) monomers.
- FIG. 4 shows one scheme for the solid phase synthesis of OxoPNAs.
- FIG. 5 shows one scheme for the synthesis of morpholino nucleosides for incorporation in OxoPNAs. Morpholino nucleotide synthesis may be performed according to the method of Girault et al. (1996) (incorporated by reference).
- FIGS. 6A , 6 B, and 6 C show one scheme for the synthesis of peptide nucleic acid monomers according to the method of Howarth et al. (1997).
- FIG. 6B shows examples of additional peptide nucleic acid monomers that may be prepared according to the scheme shown in FIG. 6A .
- FIG. 6C shows two examples of peptide nucleic acid monomers modified with squaric acid.
- FIGS. 7A and 7B show a scheme for the synthesis of a ⁇ -aminoalainine monomer modified with a nucleobase according to the protocol of Fujii et al. (1998).
- FIG. 7B shows the ⁇ -aminoalainine nucleoside monomer modified with squaric acid.
- FIG. 8 shows one scheme for the solid phase synthesis of OxoPNAs using reactive derivatives of squaric acid.
- difluoro squaric acid is used in this example, other reactive halogen derivatives, such as dichloro squaric acid, could be used.
- Dilkyl ester derivatives of squaric acid could also be used.
- FIG. 9 shows abasic dimers of squaric acid amides.
- the present invention provides novel oxocarbon acid incorporated peptide nucleic acids (OxoPNAs) that provide increased stability, sensitivity, and specificity as compared to their natural DNA and RNA counterparts.
- the OxoPNAs of the invention generally comprise nucleobases linked to an oxocarbon acid amide incorporated peptide backbone.
- the nucleobases may include any of the four main naturally occurring DNA bases (i.e., thymine, cytosine, adenine, or guanine) or other naturally occurring nucleobases (e.g., inosine, uracil, 5-methylcytosine, or thiouracil) or artificial bases (e.g., bromothymine, azaadenines, or azaguanines, etc.) attached to a peptide backbone through a suitable linker.
- the oxocarbon acid may be squaric acid, deltic acid, croconic acid, rhodizonic acid, or their corresponding thioxocarbon acids.
- the present invention also provides a variety of methods employing the OxoPNAs of the present invention.
- the oligonucleotide analogs of the present invention may be employed in a wide range of applications, particularly in applications involving hybridization.
- the present invention provides methods for the detection and functional analysis of nucleic acid molecules, including miRNAs and other non-coding RNAs.
- the present invention also provides methods for antisense-based intervention targeted against disease-associated nucleic acid molecules.
- the present invention provides novel oxocarbon acid amide (i.e., “oxocarbonamide”) incorporated peptide oligonucleotides.
- Oxocarbon acids are a class of organic compounds that are vinylogs of carboxylic acids, that is the OH and CO groups are joined through a vinylic unsaturation forming a cyclic non-aromatic ring. Furthermore, the carbon atoms not involved in the acidic moiety are substituted by oxygen and are present as carbonyl or hydroxy functions.
- Cyclic oxocarbon compounds have the general formula C x O x , wherein x ⁇ 3. Examples of oxocarbon acids are deltic acid, squaric acid, croconic acid, and rhodizonic acid ( FIG. 1 ).
- Squaric acid derivatives have been shown to function as amino acid-like analogs.
- Porter et al. demonstrated that a squaric acid derivative of the thioproline CT5219, a small molecule VLA-4 antagonist, was also a potent VLA-4 antagonist and had an improved pharmokinetic profile compared to CT5219 (Porter et al., 2002).
- the squaryl group has been evaluated as a mimic of the phosphate group in modified oligodeoxynucleotides (Sato et al., 2002).
- Squaric acid is a dibasic acid with two acidic hydroxyl groups and two carbonyl groups. Nucleophilic substitution of the squaric acid esters with amines gives the corresponding diamides.
- Sato et al. reported the synthesis of oligonucleotide analogues containing a single squaryldiamide internucleotide linkage between two thymidines (TsqT). The structure of the TsqT as reported by Sato et al. is as follows:
- a squaryldiamide modified peptide nucleic acid of the present invention has the structure:
- a PNA typically comprises one or more nucleotides or nucleosides that comprise a nucleobase moiety, a nucleobase linker moiety that is not a 5-carbon sugar, and/or a backbone moiety that is not a phosphate backbone moiety.
- nucleobase linker moieties described for PNAs include aza nitrogen atoms, amido and/or ureido tethers (see for example, U.S. Pat. No. 5,539,082).
- backbone moieties described for PNAs include an aminoethylglycine, polyamide, polyethyl, polythioamide, polysulfinamide or polysulfonamide backbone moiety.
- Various PNAs have been described in U.S. Pat. Nos. 5,786,461, 5,891,625, 5,773,571, 5,766,855, 5,736,336, 5,719,262, 5,714,331, 5,539,082, and WO 92/20702, each of which is incorporated herein by reference.
- nucleotide analogs include, for example, a locked nucleic acid or “LNA.”
- LNA monomer is a bi-cyclic compound that is structurally similar to RNA nucleosides. LNAs have a furanose conformation that is restricted by a methylene linker that connects the 2′-O position to the 4′-C position, as described in Koshkin et al., 1998a and 1998b and Wahlestedt et al., 2000.
- Typical structures of a deoxyribonucleic acid (DNA), locked nucleic acid (LNA), and a peptide nucleic acid (PNA) are:
- PNA oligomers may be based on standard solid phase peptide synthesis protocols such as those disclosed in WO 92/20702; U.S. Pat. No. 5,539,082; and Mc Cairn et al. (2006).
- Solid phase synthesis is also a convenient strategy for making the OxoPNAs of the present invention.
- Examples of reactive derivatives of squaric acid and OxoPNA monomers, which may be used in the synthesis of OxoPNAs are illustrated in FIGS. 1 and 2 , respectively.
- Examples of synthesis schemes for OxoPNAs are provided in FIGS. 4 to 8 .
- the exemplary synthesis schemes in FIGS. 4 to 8 show squaric acid amides, it will be understood by those in the art that other oxocarbon acid amides could also be used to synthesize OxoPNAs.
- the synthesis of OxoPNAs may be performed on commercially available synthesizers using commercially available reagents.
- resins such as 5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL) or 5-(9-Fmoc-aminoxanthen-2-oxy)valeric acid (XAL) may be employed.
- the resins may be immobilized with functional groups such as NH 2 and COOH.
- the amino or carboxy end of the OxoPNA may be attached to the resin by establishing an amide bond.
- commercially available microsphere e.g., Luminex beads
- oxocarbonamide peptide nucleic acids of the present invention have improved stability, sensitivity, and specificity for their target sequences, which make them well suited to a variety of applications including, for example, the detection, analysis, and capture of miRNAs, and other small nucleic acids; and as antisense molecules for the selective gene knock-down.
- amino means —NH 2 ; the term “nitro” means —NO 2 ; the term “halo” designates —F, —Cl, —Br or —I; the term “mercapto” means —SH; the term “cyano” means —CN; the term “azido” means —N 3 ; the term “silyl” means —SiH 3 , and the term “hydroxy” means —OH.
- alkyl includes straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), cyclic alkyl, heteroatom-unsubstituted alkyl, heteroatom-substituted alkyl, heteroatom-unsubstituted alkyl (Cn) , and heteroatom-substituted alkyl (Cn) .
- heteroatom-unsubstituted alkyl (Cn) refers to a radical, having a linear or branched, cyclic or acyclic structure, further having no carbon-carbon double or triple bonds, further having a total of n carbon atoms, all of which are nonaromatic, 3 or more hydrogen atoms, and no heteroatoms.
- a heteroatom-unsubstituted alkyl (C1-C10) has 1 to 10 carbon atoms.
- heteroatom-substituted alkyl (Cn) refers to a radical, having a single saturated carbon atom as the point of attachment, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1, or more than one hydrogen atom, at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
- a heteroatom-substituted alkyl (C1-C10) has 1 to 10 carbon atoms.
- heteroatom-substituted alkyl groups trifluoromethyl, —CH 2 F, —CH 2 Cl, —CH 2 Br, —CH 2 OH, —CH 2 OCH 3 , —CH 2 OCH 2 CF 3 , —CH 2 OC(O)CH 3 , —CH 2 NH 2 , —CH 2 NHCH 3 , —CH 2 N(CH 3 ) 2 , —CH 2 CH 2 Cl, —CH 2 CH 2 OH, CH 2 CH 2 OC(O)CH 3 , —CH 2 CH 2 NHCO 2 C(CH 3 ) 3 , and —CH 2 Si(CH 3 ) 3 .
- aryl includes heteroatom-unsubstituted aryl, heteroatom-substituted aryl, heteroatom-unsubstituted aryl (Cn) , heteroatom-substituted aryl (Cn) , heteroaryl, heterocyclic aryl groups, carbocyclic aryl groups, biaryl groups, and single-valent radicals derived from polycyclic fused hydrocarbons (PAHs).
- PAHs polycyclic fused hydrocarbons
- heteroatom-unsubstituted aryl (Cn) refers to a radical, having a single carbon atom as a point of attachment, wherein the carbon atom is part of an aromatic ring structure containing only carbon atoms, further having a total of n carbon atoms, 5 or more hydrogen atoms, and no heteroatoms.
- a heteroatom-unsubstituted aryl (C6-C10) has 6 to 10 carbon atoms.
- Non-limiting examples of heteroatom-unsubstituted aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, —C 6 H 4 CH 2 CH 3 , —C 6 H 4 CH 2 CH 2 CH 3 , —C 6 H 4 CH(CH 3 ) 2 , —C 6 H 4 CH(CH 2 ) 2 , —C 6 H 3 (CH 3 )CH 2 CH 3 , —C 6 H 4 CH—CH 2 , —C 6 H 4 CH ⁇ CHCH 3 , —C 6 H 4 C ⁇ CH, —C 6 H 4 C ⁇ CCH 3 , naphthyl, and the radical derived from biphenyl.
- heteroatom-substituted aryl refers to a radical, having either a single aromatic carbon atom or a single aromatic heteroatom as the point of attachment, further having a total of n carbon atoms, at least one hydrogen atom, and at least one heteroatom, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
- a heteroatom-unsubstituted heteroaryl (C1-C10) has 1 to 10 carbon atoms.
- Non-limiting examples of heteroatom-substituted aryl groups include the groups: —C 6 H 4 F, —C 6 H 4 Cl, —C 6 H 4 Br, —C 6 H 4 OH, —C 6 H 4 OCH 3 , —C 6 H 4 OCH 2 CH 3 , —C 6 H 4 OC(O)CH 3 , —C 6 H 4 NH 2 , —C 6 H 4 NHCH 3 , —C 6 H 4 N(CH 3 ) 2 , —C 6 H 4 CH 2 OH, —C 6 H 4 CH 2 OC(O)CH 3 , —C 6 H 4 CH 2 NH 2 , —C 6 H 4 CF 3 , —C 6 H 4 CN, —C 6 H 4 CHO, —C 6 H 4 CHO, —C 6 H 4 C(O)CH 3 , —C 6 H 4 C(O)C 6 H 5 , —C 6 H 4 CO 2 H, —C 6
- aralkyl includes heteroatom-unsubstituted aralkyl, heteroatom-substituted aralkyl, heteroatom-unsubstituted aralkyl (Cn) , heteroatom-substituted aralkyl (Cn) , heteroaralkyl, and heterocyclic aralkyl groups.
- heteroatom-unsubstituted aralkyl (Cn) ” refers to a radical, having a single saturated carbon atom as the point of attachment, further having a total of n carbon atoms, wherein at least 6 of the carbon atoms form an aromatic ring structure containing only carbon atoms, 7 or more hydrogen atoms, and no heteroatoms.
- a heteroatom-unsubstituted aralkyl (C7-C10) has 7 to 10 carbon atoms.
- Non-limiting examples of heteroatom-unsubstituted aralkyls are: phenylmethyl (benzyl, Bn) and phenylethyl.
- heteroatom-substituted aralkyl refers to a radical, having a single saturated carbon atom as the point of attachment, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one heteroatom, wherein at least one of the carbon atoms is incorporated an aromatic ring structures, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
- a heteroatom-substituted heteroaralkyl C2-C10 has 2 to 10 carbon atoms.
- acyl includes straight-chain acyl, branched-chain acyl, cycloacyl, cyclic acyl, heteroatom-unsubstituted acyl, heteroatom-substituted acyl, heteroatom-unsubstituted acyl (Cn) , heteroatom-substituted acyl (Cn) , alkylcarbonyl, alkoxycarbonyl and aminocarbonyl groups.
- heteroatom-unsubstituted acyl (Cn) refers to a radical, having a single carbon atom of a carbonyl group as the point of attachment, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 1 or more hydrogen atoms, a total of one oxygen atom, and no additional heteroatoms.
- a heteroatom-unsubstituted acyl (C1-C10) has 1 to 10 carbon atoms.
- the groups, —CHO, —C(O)CH 3 , —C(O)CH 2 CH 3 , —C(O)CH 2 CH 2 CH 3 , C(O)CH(CH 3 ) 2 , —C(O)CH(CH 2 ) 2 , —C(O)C 6 H 5 , —C(O)C 6 H 4 CH 3 , —C(O)C 6 H 4 CH 2 CH 3 , and —COC 6 H 3 (CH 3 ) 2 are non-limiting examples of heteroatom-unsubstituted acyl groups.
- heteroatom-substituted acyl (Cn) refers to a radical, having a single carbon atom as the point of attachment, the carbon atom being part of a carbonyl group, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, at least one additional heteroatom, in addition to the oxygen of the carbonyl group, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
- a heteroatom-substituted acyl (C1-C10) has 1 to 10 carbon atoms.
- the groups, —C(O)CH 2 CF 3 , —CO 2 H, —CO 2 CH 3 , —CO 2 CH 2 CH 3 , —CO 2 CH 2 CH 2 CH 3 , —CO 2 CH(CH 3 ) 2 , —CO 2 CH(CH 2 ) 2 , —C(O)NH 2 (carbamoyl), —C(O)NHCH 3 , —C(O)NHCH 2 CH 3 , —CONHCH(CH 3 ) 2 , —CONHCH(CH 2 ) 2 , —CON(CH 3 ) 2 , and —CONHCH 2 CF 3 , are non-limiting examples of heteroatom-substituted acyl groups.
- alkoxy includes straight-chain alkoxy, branched-chain alkoxy, cycloalkoxy, cyclic alkoxy, heteroatom-unsubstituted alkoxy, heteroatom-substituted alkoxy, heteroatom-unsubstituted alkoxy (Cn) , and heteroatom-substituted alkoxy (Cn) .
- heteroatom-unsubstituted alkoxy (Cn) refers to a group, having the structure —OR, in which R is a heteroatom-unsubstituted alkyl (Cn) , as that term is defined above.
- Heteroatom-unsubstituted alkoxy groups include: —OCH 3 , —OCH 2 CH 3 , —OCH 2 CH 2 CH 3 , —OCH(CH 3 ) 2 , and —OCH(CH 2 ) 2 .
- the term “heteroatom-substituted alkoxy (Cn) ” refers to a group, having the structure —OR, in which R is a heteroatom-substituted alkyl (Cn) , as that term is defined above.
- —OCH 2 CF 3 is a heteroatom-substituted alkoxy group.
- alkenyloxy includes straight-chain alkenyloxy, branched-chain alkenyloxy, cycloalkenyloxy, cyclic alkenyloxy, heteroatom-unsubstituted alkenyloxy, heteroatom-substituted alkenyloxy, heteroatom-unsubstituted alkenyloxy (Cn) , and heteroatom-substituted alkenyloxy (Cn) .
- heteroatom-unsubstituted alkenyloxy (Cn) refers to a group, having the structure —OR, in which R is a heteroatom-unsubstituted alkenyl (Cn) , as that term is defined above.
- heteroatom-substituted alkenyloxy refers to a group, having the structure —OR, in which R is a heteroatom-substituted alkenyl (Cn) , as that term is defined above.
- alkynyloxy includes straight-chain alkynyloxy, branched-chain alkynyloxy, cycloalkynyloxy, cyclic alkynyloxy, heteroatom-unsubstituted alkynyloxy, heteroatom-substituted alkynyloxy, heteroatom-unsubstituted alkynyloxy (Cn) , and heteroatom-substituted alkynyloxy (Cn) .
- heteroatom-unsubstituted alkynyloxy (Cn) refers to a group, having the structure —OR, in which R is a heteroatom-unsubstituted alkynyl (Cn) , as that term is defined above.
- heteroatom-substituted alkynyloxy (Cn) refers to a group, having the structure —OR, in which R is a heteroatom-substituted alkynyl (Cn) , as that term is defined above.
- aryloxy includes heteroatom-unsubstituted aryloxy, heteroatom-substituted aryloxy, heteroatom-unsubstituted aryloxy (Cn) , heteroatom-substituted aryloxy (Cn) , heteroaryloxy, and heterocyclic aryloxy groups.
- heteroatom-unsubstituted aryloxy (Cn) refers to a group, having the structure —OAr, in which Ar is a heteroatom-unsubstituted aryl (Cn) , as that term is defined above.
- a non-limiting example of a heteroatom-unsubstituted aryloxy group is —OC 6 H 5 .
- heteroatom-substituted aryloxy refers to a group, having the structure —OAr, in which Ar is a heteroatom-substituted aryl (Cn) , as that term is defined above.
- aralkyloxy includes heteroatom-unsubstituted aralkyloxy, heteroatom-substituted aralkyloxy, heteroatom-unsubstituted aralkyloxy (Cn) , heteroatom-substituted aralkyloxy (Cn) , heteroaralkyloxy, and heterocyclic aralkyloxy groups.
- heteroatom-unsubstituted aralkyloxy (Cn) refers to a group, having the structure —OAr, in which Ar is a heteroatom-unsubstituted aralkyl (Cn) , as that term is defined above.
- heteroatom-substituted aralkyloxy refers to a group, having the structure —OAr, in which Ar is a heteroatom-substituted aralkyl (Cn) , as that term is defined above.
- acyloxy includes straight-chain acyloxy, branched-chain acyloxy, cycloacyloxy, cyclic acyloxy, heteroatom-unsubstituted acyloxy, heteroatom-substituted acyloxy, heteroatom-unsubstituted acyloxy (Cn) , heteroatom-substituted acyloxy (Cn) , alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups.
- heteroatom-unsubstituted acyloxy (Cn) refers to a group, having the structure —OAc, in which Ac is a heteroatom-unsubstituted acyl (Cn) , as that term is defined above.
- —OC(O)CH 3 is a non-limiting example of a heteroatom-unsubstituted acyloxy group.
- heteroatom-substituted acyloxy (Cn) refers to a group, having the structure —OAc, in which Ac is a heteroatom-substituted acyl (Cn) , as that term is defined above.
- —OC(O)OCH 3 and —OC(O)NHCH 3 are non-limiting examples of heteroatom-unsubstituted acyloxy groups.
- a DNA intercalating agent is a ligand of an appropriate size and chemical nature to fit in between base pairs of DNA. These ligands are mostly polycyclic, aromatic, and planar.
- Non-limiting examples of DNA intercalators include ethidium bromide, proflavine, daunomycin, doxorubicin, and thalidomide.
- the stability, sensitivity, and specificity of the oxocarbonamide peptide nucleic acids of the present invention make them a useful tool in diagnostics and molecular biology. While the compounds of the present invention are useful in the detection and analysis of any nucleic acids, it is contemplated that they will be particularly useful in the detection of small RNA molecules such as miRNA molecules, which often require the use of sensitive analysis tools due to their size and low level of expression.
- the oxocarbonamide peptide nucleic acids of the present invention may be used as hybridization probes for the detection of complementary nucleic acid sequences.
- Sequence-specific nucleic acid hybridization assays e.g., Northern blotting, Southern blotting, and microarray analysis
- they are used for the detection of various biological agents and infectious pathogens.
- hybridization As used herein, “hybridization,” “hybridizes” or “capable of hybridizing” is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature.
- anneal as used herein is synonymous with “hybridize.”
- hybridization “hybridizes” or “capable of hybridizing” encompasses the terms “stringent conditions” or “high stringency” and the terms “low stringency” or “low stringency conditions.”
- stringent conditions or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strands containing complementary sequences, but preclude hybridization of random sequences.
- Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA, miRNA, or siRNA transcript or a nucleic acid segment thereof, and the like.
- Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acids, the length and nucleobase content of the target sequences, the charge composition of the nucleic acids, the presence of nucleic acid analogues in the nucleic acid molecules, and to the presence or concentration of formamide, tetramethylammonium chloride or other solvents in a hybridization mixture.
- low stringency or “low stringency conditions,” and non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20° C. to about 50° C.
- hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20° C. to about 50° C.
- the present invention may be employed in solution hybridization as well as in solid phase hybridization.
- the hybridization conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Optimization of hybridization conditions for the particular application of interest is well known to those of skill in the art.
- Representative solid phase hybridization methods are disclosed in U.S. Pat. Nos. 5,843,663, 5,900,481 and 5,919,626.
- Other methods of hybridization that may be used in the practice of the present invention are disclosed in U.S. Pat. Nos. 5,849,486 and 5,851,772.
- Recognition moieties incorporated into primers, incorporated into the amplified product during amplification, or attached to probes are useful in the identification of nucleic acid molecules.
- a number of different labels may be used for this purpose such as fluorophores, chromophores, radiophores, enzymatic tags, antibodies, chemiluminescence, electroluminescence, affinity labels, noble metal nanoparticles, quantum dots, magnetic particles, etc.
- affinity labels include, but are not limited to the following: an antibody, an antibody fragment, a receptor protein, a hormone, biotin, DNP, or any polypeptide/protein molecule that binds to an affinity label.
- enzyme tags include enzymes such as urease, alkaline phosphatase, or peroxidase to mention a few.
- Colorimetric indicator substrates can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples. All of these examples are generally known in the art and the skilled artisan will recognize that the invention is not limited to the examples described above.
- fluorophores include, but are not limited to the following: all of the Alexa Fluor® dyes, AMCA, BODIPY® 630/650, BODIPY® 650/665, BODIPY®-FL, BODIPY®-R6G, BODIPY®-TMR, BODIPY®-TRX, Cascade Blue®, CyDyesTM, including but not limited to Cy2TM, Cy3TM, and Cy5TM, DNA intercalating dyes, 6-FAMTM, Fluorescein, HEXTM, 6-JOE, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific BlueTM, REG, phycobilliproteins including, but not limited to, phycoerythrin and allophycocyanin, Rhodamine GreenTM, Rhodamine RedTM, ROXTM, TAMRATM, TETTM, Tetramethylrhodamine, and Texas Red®.
- Detection can result in qualitative identification, semi-quantitative identification, or quantitative identification of the target molecule.
- Qualitative detection includes detection of the presence of the molecule, without any correlation to an amount of the molecule in the sample that was tested.
- Semi-quantitative detection permits not only detection of the target molecule, but correlation of the signal to a basal level of target molecule in the sample that was tested. For example, it may indicate a minimum threshold amount of the target molecule was present in the sample.
- Quantitative detection permits the practitioner to determine the amount of target molecule present in the original sample over a wide range of amounts.
- an array or gene chip consists of a solid substrate upon which an array of single stranded oxocarbonamide peptide nucleic acid molecules have been attached. For screening, the chip or array is contacted with a single stranded DNA or RNA sample, which is allowed to hybridize under stringent conditions. The chip or array is then scanned to determine which probes have hybridized.
- Exemplary methods include: the immobilization of biotinylated nucleic acid molecules to avidin/streptavidin coated supports (Holmstrom, 1993), the direct covalent attachment of short, 5′-phosphorylated primers to chemically modified polystyrene plates (Rasmussen et al., 1991), or the precoating of the polystyrene or glass solid phases with poly-L-Lys or poly L-Lys, Phe, followed by the covalent attachment of either amino- or sulfhydryl-modified oligonucleotides using bi-functional crosslinking reagents (Running et al., 1990; Newton et al., 1993).
- the probes When immobilized onto a substrate, the probes are stabilized and therefore may be used repeatedly.
- hybridization is performed on an immobilized nucleic acid target or a probe molecule that is attached to a solid surface such as nitrocellulose, nylon membrane, or glass.
- nitrocellulose membrane reinforced nitrocellulose membrane, activated quartz, activated glass, polyvinylidene difluoride (PVDF) membrane, polystyrene substrates, polyacrylamide-based substrate, other polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules.
- PVDF polyvinylidene difluoride
- PVDF polystyrene substrates
- polyacrylamide-based substrate other polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules.
- the present invention is used in conjunction with Luminex® xMAP® technology.
- the Luminex technology allows the quantitation of nucleic acid products immobilized on fluorescently encoded microspheres. By dyeing microspheres with 10 different intensities each of two spectrally distinct fluorochromes, 100 fluorescently distinct populations of microspheres are produced. By using three or more spectrally distinct fluorochromes at different intensity levels, even greater numbers of fluorescently distinct populations can be created. These individual populations (sets) can represent individual detection sequences and the magnitude of hybridization on each set can be detected individually. The magnitude of the hybridization reaction is measured using a third spectrally distinct fluorochrome called a reporter.
- the reporter molecule signals the extent of the reaction by attaching to the molecules on the microspheres. As both the microspheres and the reporter molecules are labeled, digital signal processing allows the translation of signals into real-time, quantitative data for each reaction.
- the Luminex technology is described, for example, in U.S. Pat. Nos. 5,736,330, 5,981,180, and 6,057,107, all of which are specifically incorporated by reference.
- the present invention may also be used in conjunction with a competitive binding assay format.
- this format involves a detection sequence coupled to a solid surface, and a labeled sequence complementary to the detection sequence in solution.
- the target sequence in the sample being assayed does not need to be labeled. Rather, the target sequence's presence in the sample is detected because it competes with the labeled complement for hybridization with the immobilized detection sequence. Thus, if the target sequence is present in the sample, the signal decreases as compared to a sample lacking the target sequence.
- Luminex xMAP technology can be used in a competitive binding assay format.
- this format would comprise an oxocarbonamide peptide nucleic acid detection molecule immobilized on a labeled bead, a labeled sequence complementary to the detection sequence, exposing the immobilized detection sequence and the labeled complement to a nucleic acid sample under hybridizing conditions, and detecting the presence or absence of the target sequence in the sample.
- the use of the Luminex technology in a competitive binding assay format is described in U.S. Pat. Nos. 5,736,330 and 6,057,107, incorporated herein by reference.
- Flow cytometry is a useful tool in the analysis of biomolecules.
- flow cytometry is particularly useful in the analysis of microsphere based assays, such as the Luminex xMAP system.
- Flow cytometry involves the separation of cells or other particles, such as microspheres, in a liquid sample.
- the purpose of flow cytometry is to analyze the separated particles for one or more characteristics.
- the basic steps of flow cytometry involve the direction of a fluid sample through an apparatus such that a liquid stream passes through a sensing region. The particles should pass one at a time by the sensor and are categorized based on size, refraction, light scattering, opacity, roughness, shape, fluorescence, etc.
- flow cytometry can be used for simultaneous sequence identification and hybridization quantification.
- Internal dyes in the microspheres are detected by flow cytometry and used to identify the specific nucleic acid sequence to which a microsphere is coupled.
- the label on the target nucleic acid molecule is also detected by flow cytometry and used to quantify target hybridization to the microsphere.
- the oxocarbonamide peptide nucleic acids of the present invention are particularly useful in the detection of small RNA molecules, such as miRNA, in a sample.
- miRNAs are 18-25 nucleotide (nt) RNAs that are processed from longer endogenous hairpin transcripts.
- nt nucleotide
- OxoPNAs of the present invention overcome the limitations of their DNA-probe counterparts for the detection of short nucleotide targets.
- Small RNA molecules may be isolated from a sample, such as a cell sample, by a variety of methods known in the art.
- the most commonly used method is to co-purify the miRNA with total RNA using a combination of acidified phenol and guanidine isothiocyanate using care not to remove the highly-soluble short RNA (see, e.g., Pfeffer et al., 2003).
- This method isolates total RNA, which comprises transfer RNA (tRNA), ribosomal RNA (rRNA), polyA messenger RNA (mRNA), short interfering RNA (siRNA), small nuclear RNA (snRNA), and microRNA (miRNA).
- the miRNA can be enriched from the total RNA by size selection using gel purification (Pfeffer, Id.).
- RNA preparation (less than about 200 nucleotides) is enriched for miRNAs, siRNAs, and/or snRNAs.
- the Absolutely RNA® Miniprep Kit (Stratagene) may be used to isolate total RNA comprising miRNA. Removal of genomic DNA is desirable as its presence in the total RNA could lead to false or misleading results.
- the oxocarbonamide peptide nucleic acids of the present invention may be used as double-stranded siRNA molecules, single-stranded antisense molecules, or as decoy molecules for nucleic acid binding proteins. These uses may be for therapeutic or research purposes. Naturally occurring DNA molecules are generally not well suited to these applications due to the instability of unmodified DNA in vivo. However, chemically modified oligonucleotides have been shown to be effective inhibitors of coding and non-coding RNAs (see Weiler et al., 2006).
- Oxocarbonamide peptide siRNA, antisense, or decoy molecules may be prepared by solid phase synthesis as described above.
- hydrophilic groups such as polyethylene glycol (PEG)
- PEG polyethylene glycol
- Cationic polymers such as polyamidoamine (PMAM) dendrimers or a polyethyleneimine (PEI) may be combined with OxoPNAs for DNA delivery.
- PMAM polyamidoamine
- PEI polyethyleneimine
- the nucleotide sequence of the siRNA or antisense molecule is defined by the nucleotide sequence of its target gene.
- the siRNA or antisense molecule contains a nucleotide sequence that is essentially identical to at least a portion of the target gene.
- the siRNA or antisense contains a nucleotide sequence that is completely identical to at least a portion of the target gene.
- an “identical” RNA sequence will contain ribonucleotides where the DNA sequence contains deoxyribonucleotides, and further that the RNA sequence will typically contain a uracil at positions where the DNA sequence contains thymidine.
- the nucleotide sequence of the decoy molecule is defined by the recognition sequence of the target protein.
- the cell containing the target gene or protein may be derived from or contained in any organism (e.g., plant, animal, protozoan, virus, bacterium, or fungus).
- the plant may be a monocot, dicot or gynmosperm; the animal may be a vertebrate or invertebrate.
- Preferred microbes are those used in agriculture or by industry, and those that a pathogenic for plants or animals.
- Fungi include organisms in both the mold and yeast morphologies. Examples of vertebrates include fish and mammals, including cattle, goat, pig, sheep, hamster, mouse, rat, and human; invertebrate animals include nematodes, insects, arachnids, and other arthropods.
- the cell is a vertebrate cell. More preferably, the cell is a mammalian cell.
- the cell having the target gene or protein may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like.
- the cell can be a gamete or an embryo; if an embryo, it can be a single cell embryo or a constituent cell or cells from a multicellular embryo.
- embryo thus encompasses fetal tissue.
- the cell having the target gene or protein may be an undifferentiated cell, such as a stem cell, or a differentiated cell, such as from a cell of an organ or tissue, including fetal tissue, or any other cell present in an organism.
- Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells, of the endocrine or exocrine glands.
- the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations formed by cell division. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
- a host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
- a transformed cell includes the primary subject cell and its progeny.
- the terms “engineered” and “recombinant” cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence has been introduced. Therefore, recombinant cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced nucleic acid.
- a tissue may comprise a host cell or cells to be transformed or contacted with a nucleic acid delivery composition and/or an additional agent.
- the tissue may be part or separated from an organism.
- a tissue and its constituent cells may comprise, but is not limited to, blood (e.g., hematopoietic cells (such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34 + cells CD4 + cells), lymphocytes and other blood lineage cells), bone marrow, brain, stem cells, blood vessel, liver, lung, bone, breast, cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial, epithelial, esophagus, facia, fibroblast, follicular, ganglion cells, glial cells, goblet cells, kidney, lymph node, muscle, neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, small intestine, spleen, stomach, or test
- the oxocarbonamide peptide nucleic acids of the present invention may be used as anchor molecules for the attachment of different functional molecules to DNA via specific Watson-Crick base pairing.
- the coupling of molecules with different biological function to plasmids or other nucleic acid molecules of interest can improve the targeting of genetic material in non-viral gene delivery systems. Coupling methods based on chemical linkage of peptides to plasmid DNA can interfere with gene expression. Thus, coupling via specific Watson-Crick base pairing provides an attractive alternative to chemical linkage.
- nucleic acid analogs such as those described for PNA, bisPNA, and LNA (see e.g., Lundin et al., 2005; Branden et al., 1999; Rebuff et al., 2002; Hertoghs et al., 2003; Branden et al., 2002).
- the oxocarbonamide peptide nucleic acids of the present invention may also be used in the programmed assembly of nanoscale devices. Nucleic acid guided assembly has been used to organize gold nanoparticles (Mirkin et al., 1996; Alivisatos et al., 1996; Mucic et al., 1998), nanowires (Mbindyo et al., 2003), quantum dots (Parak et al., 2002); Mitchell et al., 1999), carbon nanotubes (Dwyer et al., 2004), dendrimers (DeMattei et al., 2004), micron-size polystyrene beads (Milam et al., 2003), virus particles (Strable et al., 2004), and to attach nano- and microparticles to substrates (Kannan et al., 2004; Hartmann et al., 2002); Niemeyer et al., 2001).
- kits comprising one or more oxocarbonamide peptide nucleic acid molecules.
- the kit comprises a plurality of oxocarbonamide peptide nucleic acid molecules having at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 different nucleic acid sequences.
- the kit may include components for making a nucleic acid array, and thus, may include, for example, a solid support.
- kits will comprise pre-fabricated arrays, such as, for example, microspheres coupled to oxocarbonamide peptide nucleic acid probes. It may also include one or more buffers, such as hybridization buffer or a wash buffer.
- kits may comprise suitably aliquoted nucleic acid compositions of the present invention, whether labeled or unlabeled, as may be used to isolate, separate, detect, or amplify a targeted nucleic acid.
- the components of the kits may be packaged either in aqueous media or in lyophilized form.
- the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional containers into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
- kits of the present invention also will typically include a means for containing the oxocarbonamide peptide nucleic acids, and any other reagent containers in close confinement for commercial sale.
- Such containers may include cardboard or injection or blow-molded plastic containers into which the desired vials, bottles, etc. are retained.
- the liquid solution may be an aqueous solution, with a sterile aqueous solution being particularly preferred.
- the components of the kit may be provided as dried powder(s).
- the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
- a kit may also include instructions for employing the kit components. Instructions may include variations that can be implemented.
- compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of certain embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
- This application claims priority to U.S. Application No. 60/868,514, filed on Dec. 4, 2006, the entire disclosure of which is incorporated by reference.
- 1. Field of the Invention
- The present invention relates generally to nucleic acid probes useful in the detection and analysis of target nucleic acid sequences. More particularly, the present invention concerns nucleic acid probes wherein naturally occurring nucleobases or other nucleobase-binding moieties are covalently bound to an oxocarbonamide containing peptide backbone. In certain aspects, the present invention concerns methods employing nucleic acid probes in the detection and analysis of target nucleic acid sequences including, for example, mRNAs, miRNAs, and siRNAs.
- 2. Description of Related Art
- A large number of small, non-coding RNAs have been identified and designated as microRNAs (miRNAs) (Ke et al., 2003). miRNAs have been shown to regulate gene expression at many levels, representing a novel gene regulatory mechanism. Understanding this RNA-based regulation will be useful to understand the complexity of the genome in higher eukaryotes as well as understand the complex gene regulatory networks.
- miRNAs are 18-25 nucleotide (nt) RNAs that are processed from longer endogenous hairpin transcripts by the enzymes Dicer and Argonaute (Ambros et al., 2003, Grishok et al., 2001). To date more than 4160 microRNAs have been identified in mammals, birds, fish, worms, flies, plants, and viruses according to the miRNA registry database release 9.0 in October 2006, hosted by Sanger Institute, UK. Some miRNAs have multiple loci in the genome (Reinhart et al., 2002) and may be arranged in tandem clusters (Lagos-Quintana et al., 2001).
- The first miRNAs to be discovered, lin-4 and let-7, base-pair incompletely to repeated elements in the 3′ untranslated regions (UTRs) of other heterochrony genes, and regulate the translation directly and negatively by antisense RNA-RNA interaction (Lee et al., 1993; Reinhart et al., 2000). Some miRNAs are thought to interact with target mRNAs by limited complementary and suppressed translation as well (Lagos-Quintana et al., 2001; Lee and Ambros, 2001). Perfect complementarity between miRNAs and their target RNA may lead to target RNA degradation rather than inhibit translation (Hutvagner and Zamore, 2002), which suggests that the degree of complementarity determines function.
- Several human diseases have been identified in which miRNAs or their processing machinery might be implicated. One such disease is spinal muscular atrophy (SMA), a pediatric neurodegenerative disease caused by reduced protein levels or loss-of-function mutations of the survival of motor neurons (SMN) gene (Paushkin et al., 2002). Another disease linked to mi/siRNAs is fragile X mental retardation (FXMR) caused by absence or mutations of the fragile X mental retardation protein (FMRP) (Nelson et al., 2003). Poy et al. (2004) concluded that miR-375 is a regulator of insulin secretion and could constitute a novel pharmacological target for the treatment of diabetes. Links between cancer and miRNAs have also been described. For example, one study determined that two different miRNA (miR15 and miR16) genes are clustered and located within the deleted minimal region of the B-cell chronic lymphocytic leukemia (B-CLL) tumor suppressor locus, and both genes are deleted or down-regulated in the majority of CLL cases (Calin et al., 2002).
- RNA interference (RNAi), in which double-stranded RNA leads to the degradation of any RNA that is homologous (Fire et al., 1998), relies on a mechanism that probably evolved for protection against viral attack and mobile genetic elements. One step in the RNAi mechanism is the generation of short interfering RNAs (siRNAs), double-stranded RNAs that are about 22 nt long. The siRNAs lead to the degradation of homologous target RNA and the production of more siRNAs against the same target RNA (Lipardi et al., 2001; Zhang et al., 2002; Nykanen et al., 2001).
- The involvement of short RNAs in gene regulation has resulted in high interest among researchers in the discovery of siRNAs, miRNAs, their targets and mechanism of action. However, the detection and analysis of these small RNAs is not trivial. The size and often low level of expression of miRNAs require the use of sensitive analysis tools. The use of conventional quantitative real-time PCR for monitoring expression of mature miRNAs is excluded due to their small size. Most miRNA researchers use Northern blot analysis combined with polyacrylamide gels to examine expression of both the mature and pre-miRNAs (Reinhart et al., 2000; Lagos-Quintana et al., 2001; Lee and Ambros, 2001). Primer extension has also been used to detect the mature miRNA (Zeng and Cullen, 2003). Disadvantages of all the gel-based assays (Northern blotting, primer extension, RNase protection assays etc.) for monitoring miRNA expression include low throughput and poor sensitivity. Consequently, a large amount of total RNA per sample is required for gel-based methods, which is not feasible when the cell or tissue source is limited.
- Microarrays are an alternative to Northern blot analysis for analyzing miRNA expression. Krichevsky et al. (2003) used cDNA microarrays to monitor the expression of miRNAs during neuronal development; however, the mature miRNAs had to be separated from the miRNA precursors using micro concentrators prior to microarray hybridization. Liu et al (2004) developed a microarray for expression profiling of 245 human and mouse miRNAs using 40-mer DNA oligonucleotide capture probes. Thomson et al. (2004) described the development of a oligonucleotide microarray platform for expression profiling of 124 mammalian miRNAs using oligonucleotide capture probes complementary to the mature microRNAs.
- Although microarrays can provide high throughput, the disadvantages of DNA-based oligonucleotide arrays may include: the requirement of high concentrations of labeled input target RNA for efficient hybridization and signal generation, low sensitivity for rare and low-abundant miRNAs, and the necessity for post-array validation using more sensitive assays.
- A PCR-based approach has also been used to determine the expression levels of mature miRNAs (Grad et al., 2003). However, this method is cumbersome for routine miRNA expression profiling, since it involves gel isolation of small RNAs and ligation to linker oligonucleotides. Schmittgen et al. (2004) described an alternative method to Northern blot analysis, in which real-time PCR assays were used to quantify the expression of miRNA precursors. The disadvantage of this method, however, is that it only allows quantification of the precursor miRNAs, which does not necessarily reflect the expression levels of mature miRNAs.
- Many limitations of DNA probes for the detection of short nucleotide targets have been overcome by using locked nucleic acid (LNA) based probes or peptide nucleic acid (PNA) based probes. The use of LNAs and PNAs in oligonucleotide probes has been shown to increase sensitivity and selectivity for small RNA targets compared to their DNA-probe counterparts (see e.g., Valoczi et al., 2004). Nevertheless, additional compositions and methods are needed to increase the sensitivity and specificity of oligonucleotide sequences for the detection and analysis of miRNAs and other small RNAs, as well as for use in disease diagnostics and for antisense-based therapies.
- The present invention addresses these needs by providing novel oligonucleotide compositions for the accurate, sensitive, and specific detection and functional analysis of miRNAs and other non-coding RNAs. The compositions of the present invention will also be useful as biomarkers for disease diagnostics as well as for antisense-based intervention targeted against disease-associated miRNAs and other non-coding RNAs.
- The present invention provides a novel class of compounds that bind complementary DNA and RNA strands. The compounds of the invention generally comprise ligands linked to a oxocarbon acid amide modified peptide backbone. Non-limiting examples of ligands include thymine, cytosine, adenine, guanine, uracil, inosine, 5-methylcytosine, thiouracil, bromothymine, azaadenine, or azaguanine. Representative oxocarbon acid amides (“oxocarbonamides”) include deltic acid amide, thio-deltic acid amide, squaric acid amide, thio-squaric acid amide, croconic acid amide, thio-croconic acid amide, rhodizonic acid amide, and thio-rhodizonic acid amide.
- In one embodiment, the present invention provides oxocarbonamide peptide nucleic acids having the formula (I):
- wherein:
- n is at least 1;
- each of L1-Ln is independently selected from the group consisting of heteroatom substituted aryls, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, hydrogen, hydroxy, (C1-C4)alkanoyl, aromatic moieties, and reporter ligands, wherein at least one of L1-Ln is a naturally occurring nucleobase, non-naturally occurring nucleobase, nucleobase binding group, or DNA intercalator;
- each of X1-Xn is independently selected from the group consisting of R1NH and NHR2, wherein R1 and R2 are independently selected from the group consisting of H, CH2NH2, (CH2)(1-10)—NH2, (CH2)2(OCH2CH2)(1-10)NH2, (CH2)2(OCH2CH2)(1-10)CO2H, (CH2)(1-10);
- each of Y1-Yn is independently selected from the group consisting of CH2CO, formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh), and where at least one Y is formula (IVa), formula (Nb), formula (IVc), formula (Nd), formula (We), formula (IVf), formula (IVg), and formula (IVh),
- wherein each W is independently selected from the group consisting of O and S, and M is selected from the group consisting of no linker, benzene, substituted benzene, formula (IVi), and formula (IVj);
- Q is selected from the group consisting of CO2H, CONR′R″, SO3H, NH2, SH, or SO2NR′R″ or an activated derivative of CO2H or SO2H; and
- Z is selected from the group consisting of CO2H, CONR′R″, SO3H, NH2, SH, SO2NR′R″, NHR″R′″, or NR′″COR″″, where R′, R″, R′″, and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyls, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteratom substituted alkyls, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers.
- The substituted benzene may be substituted with, for example, alkyl, amine, substituted amine, amide, branched amine, PEG, etc.
- In certain embodiments, the present invention provides oxocarbonamide peptide nucleic acids having the formula (II):
- wherein:
- each L is independently selected from the group consisting of heteroatom substituted aryls, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, hydrogen, hydroxy, (C1-C4)alkanoyl, aromatic moieties, and reporter ligands, wherein at least one of L1-Ln is a heteroatom substituted acyl, naturally occurring nucleobase, non-naturally occurring nucleobase, nucleobase binding group, or DNA intercalator;
- each Y is independently selected from the group consisting of CH2CO, formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh), and where at least one Y is formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh);
- R′ is selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, heteroatom substituted acyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers;
- Z is selected from the group consisting of CO2H, CONR′R″, SO3H, NH2, SH, SO2NR′R″, NHR″R′″, or NR′″COR″″, where R′, R″, R′″, and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, heteroatom substituted acyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers; and
- n is an integer from 1 to 100.
- In certain embodiments, the present invention provides an oxocarbonamide peptide nucleic acid having the formula (II):
- wherein:
- n is an integer from 1 to 100;
- each L is independently selected from the group consisting of heteroatom substituted aryl, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, and DNA intercalators;
- each Y is independently selected from the group consisting of CH2CO, formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh), and where at least one Y is formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh);
- R′ is selected from the group consisting of hydrogen, alkyl, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, and halides; and
- Z is selected from the group consisting of CO2H, NH2, and SH.
- In particular embodiments, the present invention provides an oxocarbonamide peptide nucleic acid having the formula (IIa):
- wherein W is O or S;
- each L is independently selected from the group consisting of heteroatom substituted aryl, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, hydrogen, hydroxy, (C1-C4)alkanoyl, aromatic moieties, and reporter ligands, wherein at least one of L1-Ln is a heteroatom substituted aryl, naturally occurring nucleobase, non-naturally occurring nucleobase, nucleobase binding group, or DNA intercalator;
- R′ is selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, heteroatom substituted amide, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers;
- Z is selected from the group consisting of CO2H, CONR′R″, SO3H, NH2, SH, SO2NR′R″, NHR″R′″, or NR′″COR″″, where R′, R″, R′″, and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers; and
- n is an integer from 1 to 100.
- In certain embodiments, the present invention provides an oxocarbonamide peptide nucleic acid having the formula (III):
- wherein:
- each L is independently selected from the group consisting of heteroatom substituted aryl, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, hydrogen, hydroxy, (C1-C4)alkanoyl, aromatic moieties, and reporter ligands, wherein at least one of L1-Ln is a heteroatom substituted aryl, naturally occurring nucleobase, non-naturally occurring nucleobase, nucleobase binding group, or DNA intercalator;
- each Y is independently selected from the group consisting of CH2CO, formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh), and where at least one Y is formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh);
- R′ is selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers;
- R3 is selected from the group consisting of H, CH3, and cationic polymers;
- Z is selected from the group consisting of CO2H, CONR′R″, SO3H, NH2, SH, SO2NR′R″, NHR″R′″, or NR′″COR″″, where R′, R″, R′″, and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers; and
- n is an integer from 1 to 100. In certain aspects of the invention, the cationic polymer is a branched amine such as, for example, a polyamidoamine (PMAM) dendrimer or a polyethyleneimine (PEI). In some aspects of the invention, the cationic polymer is a polyammonium group (e.g., (CH2)nNR3). Due to their structure and charge, cationic polymers are useful nucleic acid transfection agents and drug carriers.
- In certain embodiments, the present invention provides an oxocarbonamide peptide nucleic acid having the formula (III):
- wherein:
- n is an integer from 1 to 100;
- each L is independently selected from the group consisting of naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, and DNA intercalators;
- each Y is independently selected from the group consisting of CH2CO, formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (We), formula (IVf), formula (IVg), and formula (IVh), and where at least one Y is formula (IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg), and formula (IVh);
- R′ is selected from the group consisting of hydrogen, alkyl, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, heteroatom substituted acyls, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, and halides;
- R3 is selected from the group consisting of H, CH3, and cationic polymers; and
- Z is selected from the group consisting of CO2H, NH2, and SH.
- In particular embodiments, the present invention provides an oxocarbonamide peptide nucleic acid having the formula (IIIa):
- wherein W is O or S;
- each L is independently selected from the group consisting of heteroatom substituted aryl, naturally occurring nucleobases, non-naturally occurring nucleobases, nucleobase binding groups, hydrogen, hydroxy, (C1-C4)alkanoyl, aromatic moieties, and reporter ligands, wherein at least one of L1-Ln is a naturally occurring nucleobase, non-naturally occurring nucleobase, nucleobase binding group, or DNA intercalator;
- R′ is selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers;
- R3 is selected from the group consisting of H, CH3, and cationic polymers;
- Z is selected from the group consisting of CO2H, CONR′R″, SO3H, NH2, SH, SO2NR′R″, NHR″R′″, or NR′″COR″″, where R′, R″, R′″, and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, heteroatom substituted acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted alkyl, carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides, and soluble and non-soluble polymers; and
- n is an integer from 1 to 100.
- In certain aspects of the invention, n may be an integer from 8 to 60, 10 to 50, 15 to 30, or 18 to 25. In some embodiments, n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or any range derivable therein.
- In certain aspects of the invention, no Y is CH2CO. In some aspects of the invention all Ys are formula (IVa), formula (IVe), or a combination of formula (IVa) and formula (IVe).
- The oligonucleotide analogs of the present invention may be employed in a wide range of applications, particularly in applications involving hybridization. In one embodiment, the present invention provides a method for detecting a target nucleic acid molecule, comprising: (a) providing an oxocarbonamide peptide nucleic acid comprising a sequence complementary to a sequence of a target nucleic acid molecule; (b) contacting the oxocarbonamide peptide nucleic acid with the target nucleic molecule under conditions that allow the oxocarbonamide peptide nucleic acid to hybridize with the target molecule; and (c) detecting the hybridization. The target nucleic acid molecule may be, for example, a DNA or an RNA molecule. The RNA molecule may be, for example, an mRNA, rRNA, tRNA, miRNA, or siRNA.
- To facilitate the detection of a target molecule, one or both of the oxocarbonamide peptide nucleic acid probe or the target molecule may be labeled. A number of different labels may be used in the present invention such as fluorophores, chromophores, radiophores, enzymatic tags, antibodies, chemiluminescence, electroluminescence, metal nanoparticles, quantum dots, magnetic particles, and affinity labels. One of skill in the art will recognize that these and other labels not mentioned herein can be used with success in this invention.
- Examples of affinity labels include, but are not limited to the following: an antibody, an antibody fragment, a receptor protein, a hormone, biotin, DNP, a molecular imprint, or any polypeptide/protein molecule that binds to an affinity label.
- Examples of enzyme tags include enzymes such as urease, alkaline phosphatase or peroxidase to mention a few. Colorimetric indicator substrates can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples. All of these examples are generally known in the art and the skilled artisan will recognize that the invention is not limited to the examples described above.
- Examples of fluorophores include, but are not limited to the following: all of the Alexa Fluor® dyes, AMCA, BODIPY® 630/650, BODIPY® 650/665, BODIPY®-FL, BODIPY®-R6G, BODIPY®-TMR, BODIPY®-TRX, Cascade Blue®, CyDyes™, including but not limited to Cy2™, Cy3™, and Cy5™, DNA intercalating dyes, 6-FAM™, Fluorescein, HEX™, 6-JOE, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific Blue™, REG, phycobilliproteins including, but not limited to, phycoerythrin and allophycocyanin, Rhodamine Green™, Rhodamine Red™, ROX™, TAMRA™, TET™, Tetramethylrhodamine, and Texas Red®.
- In certain aspects of the invention, the oxocarbonamide peptide nucleic acid probe or the target molecule is immobilized on a solid support. Non-limiting examples of solid supports include: nitrocellulose, nylon membrane, glass, activated quartz, activated glass, polyvinylidene difluoride (PVDF) membrane, polystyrene substrates, polyacrylamide-based substrate, other polymers, copolymers, or crosslinked polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules). A solid support may be in the form of, for example, a bead, a column, or a chip.
- In one embodiment, the present invention provides an array comprising a plurality of oxocarbonamide peptide nucleic acid probes immobilized on a solid support. In particular embodiments, the solid support is a chip or a bead. In certain aspects of the invention, the array comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000, or any range derivable therein, different oxocarbonamide peptide oligonucleotide probes.
- In certain embodiments, the present invention provides a method for detecting one or more target nucleic acid molecules in a multiplexed assay, comprising: (a) providing a plurality of different oxocarbonamide peptide nucleic acids, wherein each different oxocarbonamide peptide nucleic acid is covalently attached to a defined location on an array; (b) contacting a sample comprising the one or more target nucleic acid molecules with the array under conditions that allow the one or more target nucleic acid molecules to hybridize to complementary oxocarbonamide peptide nucleic acids on the array; and (c) detecting the hybridization. In particular embodiments, the plurality of nucleic acid molecules is a plurality of miRNA molecules. In certain embodiments, the array comprises a plurality of fluorescently encoded microspheres (“beads”).
- In another aspect, the invention provides a method for amplifying a target nucleic acid molecule. The method involves (a) incubating a first oxocarbonamide peptide nucleic acid of the invention with a target molecule under conditions that allow the first oxocarbonamide peptide nucleic acid to bind the target molecule; and (b) extending the first nucleic acid with the target molecule as a template. The method may further comprise contacting the target molecule with a second oxocarbonamide peptide nucleic acid that binds to a different region of the target molecule than the first oxocarbonamide peptide nucleic acid. In various embodiments, the sequence of the target molecule is known or unknown.
- In certain aspects, the association constant (Ka) of the oxocarbonamide peptide nucleic acid toward a complementary target molecule is higher than the association constant of the complementary strands of the double stranded target molecule. In some embodiments, the melting temperature of a duplex between the oxocarbonamide peptide nucleic acid and a complementary target molecule is higher than the melting temperature of the complementary strands of the double stranded target molecule.
- In one aspect, the present invention provides pharmaceutical composition comprising an oxocarbonamide peptide nucleic acid for treatment of a disease curable by an antisense technology. In one embodiment, the invention provides a method for inhibiting the expression of a target nucleic acid in a cell. The method comprises introducing into the cell a oxocarbonamide peptide nucleic acid of the invention in an amount sufficient to specifically attenuate expression of the target nucleic acid. The introduced oxocarbonamide peptide nucleic acid has a nucleotide sequence that is complementary to a region of the target nucleic acid sequence. In another aspect, the invention provides a method for preventing, stabilizing, or treating a disease, disorder, or condition associated with a target nucleic acid in a mammal. This method comprises introducing into the mammal oxocarbonamide peptide nucleic acid of the invention in an amount sufficient to specifically attenuate expression of the target nucleic acid, wherein the introduced oxocarbonamide peptide nucleic acid has a nucleotide sequence that is complementary to a region of the target nucleic acid. In particular embodiments, the oxocarbonamide peptide nucleic acid has a nucleotide sequence that is complementary to a region of between about 10 to about 100, about 10 to about 50, about 10 to about 30, about 15 to about 30, or about 17 to about 25, nucleotides of the target nucleic acid sequence. In some embodiments, the introduced oxocarbonamide peptide nucleic acid is single stranded or double stranded. Where the oxocarbonamide peptide nucleic acid is double stranded, both strands may be oxocarbonamide peptide nucleic acids or one strand may be an oxocarbonamide peptide nucleic acid and the other strand may be a DNA, RNA, or an oligonucleotide analog such as a PNA or LNA.
- Exemplary mammals that can be treated using the methods of the invention include humans, primates, animals of veterinary interest (e.g., cows, sheep, goats, buffalos, and horses), and domestic pets (e.g., dogs and cats). Exemplary cells in which one or more target genes can be silenced using the methods of the invention include invertebrate, plant, bacteria, yeast, and vertebrate (e.g., mammalian) cells.
- It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
- The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
- Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
- Following long-standing patent law, the words “a” and “an,” when used in conjunction with the word “comprising” in the claims or specification, denotes one or more, unless specifically noted.
- Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
- The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
-
FIG. 1 shows the oxocarbon acids: deltic acid, thio-deltic acid, squaric acid, thio-squaric acid, croconic acid, thio-croconic acid, rhodizonic acid, and thio-rhodizonic acid. -
FIG. 2 shows abasic monomers of squaric acid amides and reactive derivatives of squaric acid. -
FIG. 3 shows squaric acid amide peptide nucleic acid (SquarPNA) monomers. -
FIG. 4 shows one scheme for the solid phase synthesis of OxoPNAs. -
FIG. 5 shows one scheme for the synthesis of morpholino nucleosides for incorporation in OxoPNAs. Morpholino nucleotide synthesis may be performed according to the method of Girault et al. (1996) (incorporated by reference). -
FIGS. 6A , 6B, and 6C.FIG. 6A shows one scheme for the synthesis of peptide nucleic acid monomers according to the method of Howarth et al. (1997).FIG. 6B shows examples of additional peptide nucleic acid monomers that may be prepared according to the scheme shown inFIG. 6A .FIG. 6C shows two examples of peptide nucleic acid monomers modified with squaric acid. -
FIGS. 7A and 7B .FIG. 7A shows a scheme for the synthesis of a β-aminoalainine monomer modified with a nucleobase according to the protocol of Fujii et al. (1998).FIG. 7B shows the β-aminoalainine nucleoside monomer modified with squaric acid. -
FIG. 8 shows one scheme for the solid phase synthesis of OxoPNAs using reactive derivatives of squaric acid. Although difluoro squaric acid is used in this example, other reactive halogen derivatives, such as dichloro squaric acid, could be used. Dilkyl ester derivatives of squaric acid could also be used. -
FIG. 9 shows abasic dimers of squaric acid amides. - The present invention provides novel oxocarbon acid incorporated peptide nucleic acids (OxoPNAs) that provide increased stability, sensitivity, and specificity as compared to their natural DNA and RNA counterparts. The OxoPNAs of the invention generally comprise nucleobases linked to an oxocarbon acid amide incorporated peptide backbone. The nucleobases may include any of the four main naturally occurring DNA bases (i.e., thymine, cytosine, adenine, or guanine) or other naturally occurring nucleobases (e.g., inosine, uracil, 5-methylcytosine, or thiouracil) or artificial bases (e.g., bromothymine, azaadenines, or azaguanines, etc.) attached to a peptide backbone through a suitable linker. The oxocarbon acid may be squaric acid, deltic acid, croconic acid, rhodizonic acid, or their corresponding thioxocarbon acids.
- The present invention also provides a variety of methods employing the OxoPNAs of the present invention. As described herein, the oligonucleotide analogs of the present invention may be employed in a wide range of applications, particularly in applications involving hybridization. For example, the present invention provides methods for the detection and functional analysis of nucleic acid molecules, including miRNAs and other non-coding RNAs. In addition, the present invention also provides methods for antisense-based intervention targeted against disease-associated nucleic acid molecules.
- The present invention provides novel oxocarbon acid amide (i.e., “oxocarbonamide”) incorporated peptide oligonucleotides. Oxocarbon acids are a class of organic compounds that are vinylogs of carboxylic acids, that is the OH and CO groups are joined through a vinylic unsaturation forming a cyclic non-aromatic ring. Furthermore, the carbon atoms not involved in the acidic moiety are substituted by oxygen and are present as carbonyl or hydroxy functions. Cyclic oxocarbon compounds have the general formula CxOx, wherein x≧3. Examples of oxocarbon acids are deltic acid, squaric acid, croconic acid, and rhodizonic acid (
FIG. 1 ). - Squaric acid derivatives have been shown to function as amino acid-like analogs. For example, Porter et al. demonstrated that a squaric acid derivative of the thioproline CT5219, a small molecule VLA-4 antagonist, was also a potent VLA-4 antagonist and had an improved pharmokinetic profile compared to CT5219 (Porter et al., 2002).
- The squaryl group has been evaluated as a mimic of the phosphate group in modified oligodeoxynucleotides (Sato et al., 2002). Squaric acid is a dibasic acid with two acidic hydroxyl groups and two carbonyl groups. Nucleophilic substitution of the squaric acid esters with amines gives the corresponding diamides. Sato et al. reported the synthesis of oligonucleotide analogues containing a single squaryldiamide internucleotide linkage between two thymidines (TsqT). The structure of the TsqT as reported by Sato et al. is as follows:
- The present invention employs squaryldiamides, and other oxocarbonamides, in the design of modified peptide nucleic acids. In one embodiment, a squaryldiamide modified peptide nucleic acid of the present invention has the structure:
- A “peptide nucleic acid,” also known as a “PNA,” “peptide-based nucleic acid analog,” or “PENAM,” generally has enhanced sequence specificity, binding properties, and resistance to enzymatic degradation in comparison to molecules such as DNA and RNA (Egholm et al., 1993; PCT/EP/01219). A PNA typically comprises one or more nucleotides or nucleosides that comprise a nucleobase moiety, a nucleobase linker moiety that is not a 5-carbon sugar, and/or a backbone moiety that is not a phosphate backbone moiety. Examples of nucleobase linker moieties described for PNAs include aza nitrogen atoms, amido and/or ureido tethers (see for example, U.S. Pat. No. 5,539,082). Examples of backbone moieties described for PNAs include an aminoethylglycine, polyamide, polyethyl, polythioamide, polysulfinamide or polysulfonamide backbone moiety. Various PNAs have been described in U.S. Pat. Nos. 5,786,461, 5,891,625, 5,773,571, 5,766,855, 5,736,336, 5,719,262, 5,714,331, 5,539,082, and WO 92/20702, each of which is incorporated herein by reference.
- Other nucleotide analogs include, for example, a locked nucleic acid or “LNA.” An LNA monomer is a bi-cyclic compound that is structurally similar to RNA nucleosides. LNAs have a furanose conformation that is restricted by a methylene linker that connects the 2′-O position to the 4′-C position, as described in Koshkin et al., 1998a and 1998b and Wahlestedt et al., 2000.
- Typical structures of a deoxyribonucleic acid (DNA), locked nucleic acid (LNA), and a peptide nucleic acid (PNA) are:
- The preparation of PNA oligomers may be based on standard solid phase peptide synthesis protocols such as those disclosed in WO 92/20702; U.S. Pat. No. 5,539,082; and Mc Cairn et al. (2006).
- Solid phase synthesis is also a convenient strategy for making the OxoPNAs of the present invention. Examples of reactive derivatives of squaric acid and OxoPNA monomers, which may be used in the synthesis of OxoPNAs are illustrated in
FIGS. 1 and 2 , respectively. Examples of synthesis schemes for OxoPNAs are provided inFIGS. 4 to 8 . Although the exemplary synthesis schemes inFIGS. 4 to 8 show squaric acid amides, it will be understood by those in the art that other oxocarbon acid amides could also be used to synthesize OxoPNAs. The synthesis of OxoPNAs may be performed on commercially available synthesizers using commercially available reagents. In certain embodiments, resins such as 5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL) or 5-(9-Fmoc-aminoxanthen-2-oxy)valeric acid (XAL) may be employed. The resins may be immobilized with functional groups such as NH2 and COOH. The amino or carboxy end of the OxoPNA may be attached to the resin by establishing an amide bond. In addition, commercially available microsphere (e.g., Luminex beads) are functionalized with, for example, COOH and can be modified to make an amide bond for attachment of OxoPNAs. - The oxocarbonamide peptide nucleic acids of the present invention have improved stability, sensitivity, and specificity for their target sequences, which make them well suited to a variety of applications including, for example, the detection, analysis, and capture of miRNAs, and other small nucleic acids; and as antisense molecules for the selective gene knock-down.
- As used herein, the term “amino” means —NH2; the term “nitro” means —NO2; the term “halo” designates —F, —Cl, —Br or —I; the term “mercapto” means —SH; the term “cyano” means —CN; the term “azido” means —N3; the term “silyl” means —SiH3, and the term “hydroxy” means —OH.
- The term “alkyl” includes straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), cyclic alkyl, heteroatom-unsubstituted alkyl, heteroatom-substituted alkyl, heteroatom-unsubstituted alkyl(Cn), and heteroatom-substituted alkyl(Cn). The term “heteroatom-unsubstituted alkyl(Cn)” refers to a radical, having a linear or branched, cyclic or acyclic structure, further having no carbon-carbon double or triple bonds, further having a total of n carbon atoms, all of which are nonaromatic, 3 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted alkyl(C1-C10) has 1 to 10 carbon atoms. The groups, —CH3 (Me), —CH2CH3 (Et), —CH2CH2CH3 (n-Pr), —CH(CH3)2 (iso-Pr), —CH(CH2)2 (cyclopropyl), —CH2CH2CH2CH3 (n-Bu), —CH(CH3)CH2CH3 (sec-butyl), —CH2CH(CH3)2 (iso-butyl), —C(CH3)3 (tert-butyl), —CH2C(CH3)3 (neo-pentyl), cyclobutyl, cyclopentyl, and cyclohexyl, are all non-limiting examples of heteroatom-unsubstituted alkyl groups. The term “heteroatom-substituted alkyl(Cn)” refers to a radical, having a single saturated carbon atom as the point of attachment, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1, or more than one hydrogen atom, at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted alkyl(C1-C10) has 1 to 10 carbon atoms. The following groups are all non-limiting examples of heteroatom-substituted alkyl groups: trifluoromethyl, —CH2F, —CH2Cl, —CH2Br, —CH2OH, —CH2OCH3, —CH2OCH2CF3, —CH2OC(O)CH3, —CH2NH2, —CH2NHCH3, —CH2N(CH3)2, —CH2CH2Cl, —CH2CH2OH, CH2CH2OC(O)CH3, —CH2CH2NHCO2C(CH3)3, and —CH2Si(CH3)3.
- The term “aryl” includes heteroatom-unsubstituted aryl, heteroatom-substituted aryl, heteroatom-unsubstituted aryl(Cn), heteroatom-substituted aryl(Cn), heteroaryl, heterocyclic aryl groups, carbocyclic aryl groups, biaryl groups, and single-valent radicals derived from polycyclic fused hydrocarbons (PAHs). The term “heteroatom-unsubstituted aryl(Cn)” refers to a radical, having a single carbon atom as a point of attachment, wherein the carbon atom is part of an aromatic ring structure containing only carbon atoms, further having a total of n carbon atoms, 5 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted aryl(C6-C10) has 6 to 10 carbon atoms. Non-limiting examples of heteroatom-unsubstituted aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, —C6H4CH2CH3, —C6H4CH2CH2CH3, —C6H4CH(CH3)2, —C6H4CH(CH2)2, —C6H3(CH3)CH2CH3, —C6H4CH—CH2, —C6H4CH═CHCH3, —C6H4C≡CH, —C6H4C≡CCH3, naphthyl, and the radical derived from biphenyl. The term “heteroatom-substituted aryl(Cn)” refers to a radical, having either a single aromatic carbon atom or a single aromatic heteroatom as the point of attachment, further having a total of n carbon atoms, at least one hydrogen atom, and at least one heteroatom, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-unsubstituted heteroaryl(C1-C10) has 1 to 10 carbon atoms. Non-limiting examples of heteroatom-substituted aryl groups include the groups: —C6H4F, —C6H4Cl, —C6H4Br, —C6H4OH, —C6H4OCH3, —C6H4OCH2CH3, —C6H4OC(O)CH3, —C6H4NH2, —C6H4NHCH3, —C6H4N(CH3)2, —C6H4CH2OH, —C6H4CH2OC(O)CH3, —C6H4CH2NH2, —C6H4CF3, —C6H4CN, —C6H4CHO, —C6H4CHO, —C6H4C(O)CH3, —C6H4C(O)C6H5, —C6H4CO2H, —C6H4CO2CH3, —C6H4CONH2, —C6H4CONHCH3, —C6H4CON(CH3)2, furanyl, thienyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, indolyl, and imidazoyl.
- The term “aralkyl” includes heteroatom-unsubstituted aralkyl, heteroatom-substituted aralkyl, heteroatom-unsubstituted aralkyl(Cn), heteroatom-substituted aralkyl(Cn), heteroaralkyl, and heterocyclic aralkyl groups. The term “heteroatom-unsubstituted aralkyl(Cn)” refers to a radical, having a single saturated carbon atom as the point of attachment, further having a total of n carbon atoms, wherein at least 6 of the carbon atoms form an aromatic ring structure containing only carbon atoms, 7 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted aralkyl(C7-C10) has 7 to 10 carbon atoms. Non-limiting examples of heteroatom-unsubstituted aralkyls are: phenylmethyl (benzyl, Bn) and phenylethyl. The term “heteroatom-substituted aralkyl(Cn)” refers to a radical, having a single saturated carbon atom as the point of attachment, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one heteroatom, wherein at least one of the carbon atoms is incorporated an aromatic ring structures, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted heteroaralkyl(C2-C10) has 2 to 10 carbon atoms.
- The term “acyl” includes straight-chain acyl, branched-chain acyl, cycloacyl, cyclic acyl, heteroatom-unsubstituted acyl, heteroatom-substituted acyl, heteroatom-unsubstituted acyl(Cn), heteroatom-substituted acyl(Cn), alkylcarbonyl, alkoxycarbonyl and aminocarbonyl groups. The term “heteroatom-unsubstituted acyl(Cn)” refers to a radical, having a single carbon atom of a carbonyl group as the point of attachment, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 1 or more hydrogen atoms, a total of one oxygen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted acyl(C1-C10) has 1 to 10 carbon atoms. The groups, —CHO, —C(O)CH3, —C(O)CH2CH3, —C(O)CH2CH2CH3, C(O)CH(CH3)2, —C(O)CH(CH2)2, —C(O)C6H5, —C(O)C6H4CH3, —C(O)C6H4CH2CH3, and —COC6H3(CH3)2, are non-limiting examples of heteroatom-unsubstituted acyl groups. The term “heteroatom-substituted acyl(Cn)” refers to a radical, having a single carbon atom as the point of attachment, the carbon atom being part of a carbonyl group, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, at least one additional heteroatom, in addition to the oxygen of the carbonyl group, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted acyl(C1-C10) has 1 to 10 carbon atoms. The groups, —C(O)CH2CF3, —CO2H, —CO2CH3, —CO2CH2CH3, —CO2CH2CH2CH3, —CO2CH(CH3)2, —CO2CH(CH2)2, —C(O)NH2 (carbamoyl), —C(O)NHCH3, —C(O)NHCH2CH3, —CONHCH(CH3)2, —CONHCH(CH2)2, —CON(CH3)2, and —CONHCH2CF3, are non-limiting examples of heteroatom-substituted acyl groups.
- The term “alkoxy” includes straight-chain alkoxy, branched-chain alkoxy, cycloalkoxy, cyclic alkoxy, heteroatom-unsubstituted alkoxy, heteroatom-substituted alkoxy, heteroatom-unsubstituted alkoxy(Cn), and heteroatom-substituted alkoxy(Cn). The term “heteroatom-unsubstituted alkoxy(Cn)” refers to a group, having the structure —OR, in which R is a heteroatom-unsubstituted alkyl(Cn), as that term is defined above. Heteroatom-unsubstituted alkoxy groups include: —OCH3, —OCH2CH3, —OCH2CH2CH3, —OCH(CH3)2, and —OCH(CH2)2. The term “heteroatom-substituted alkoxy(Cn)” refers to a group, having the structure —OR, in which R is a heteroatom-substituted alkyl(Cn), as that term is defined above. For example, —OCH2CF3 is a heteroatom-substituted alkoxy group.
- The term “alkenyloxy” includes straight-chain alkenyloxy, branched-chain alkenyloxy, cycloalkenyloxy, cyclic alkenyloxy, heteroatom-unsubstituted alkenyloxy, heteroatom-substituted alkenyloxy, heteroatom-unsubstituted alkenyloxy(Cn), and heteroatom-substituted alkenyloxy(Cn). The term “heteroatom-unsubstituted alkenyloxy(Cn)” refers to a group, having the structure —OR, in which R is a heteroatom-unsubstituted alkenyl(Cn), as that term is defined above. The term “heteroatom-substituted alkenyloxy(Cn)” refers to a group, having the structure —OR, in which R is a heteroatom-substituted alkenyl(Cn), as that term is defined above.
- The term “alkynyloxy” includes straight-chain alkynyloxy, branched-chain alkynyloxy, cycloalkynyloxy, cyclic alkynyloxy, heteroatom-unsubstituted alkynyloxy, heteroatom-substituted alkynyloxy, heteroatom-unsubstituted alkynyloxy(Cn), and heteroatom-substituted alkynyloxy(Cn). The term “heteroatom-unsubstituted alkynyloxy(Cn)” refers to a group, having the structure —OR, in which R is a heteroatom-unsubstituted alkynyl(Cn), as that term is defined above. The term “heteroatom-substituted alkynyloxy(Cn)” refers to a group, having the structure —OR, in which R is a heteroatom-substituted alkynyl(Cn), as that term is defined above.
- The term “aryloxy” includes heteroatom-unsubstituted aryloxy, heteroatom-substituted aryloxy, heteroatom-unsubstituted aryloxy(Cn), heteroatom-substituted aryloxy(Cn), heteroaryloxy, and heterocyclic aryloxy groups. The term “heteroatom-unsubstituted aryloxy(Cn)” refers to a group, having the structure —OAr, in which Ar is a heteroatom-unsubstituted aryl(Cn), as that term is defined above. A non-limiting example of a heteroatom-unsubstituted aryloxy group is —OC6H5. The term “heteroatom-substituted aryloxy(Cn)” refers to a group, having the structure —OAr, in which Ar is a heteroatom-substituted aryl(Cn), as that term is defined above.
- The term “aralkyloxy” includes heteroatom-unsubstituted aralkyloxy, heteroatom-substituted aralkyloxy, heteroatom-unsubstituted aralkyloxy(Cn), heteroatom-substituted aralkyloxy(Cn), heteroaralkyloxy, and heterocyclic aralkyloxy groups. The term “heteroatom-unsubstituted aralkyloxy(Cn)” refers to a group, having the structure —OAr, in which Ar is a heteroatom-unsubstituted aralkyl(Cn), as that term is defined above. The term “heteroatom-substituted aralkyloxy(Cn)” refers to a group, having the structure —OAr, in which Ar is a heteroatom-substituted aralkyl(Cn), as that term is defined above.
- The term “acyloxy” includes straight-chain acyloxy, branched-chain acyloxy, cycloacyloxy, cyclic acyloxy, heteroatom-unsubstituted acyloxy, heteroatom-substituted acyloxy, heteroatom-unsubstituted acyloxy(Cn), heteroatom-substituted acyloxy(Cn), alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups. The term “heteroatom-unsubstituted acyloxy(Cn)” refers to a group, having the structure —OAc, in which Ac is a heteroatom-unsubstituted acyl(Cn), as that term is defined above. For example, —OC(O)CH3 is a non-limiting example of a heteroatom-unsubstituted acyloxy group. The term “heteroatom-substituted acyloxy(Cn)” refers to a group, having the structure —OAc, in which Ac is a heteroatom-substituted acyl(Cn), as that term is defined above. For example, —OC(O)OCH3 and —OC(O)NHCH3 are non-limiting examples of heteroatom-unsubstituted acyloxy groups.
- A DNA intercalating agent is a ligand of an appropriate size and chemical nature to fit in between base pairs of DNA. These ligands are mostly polycyclic, aromatic, and planar. Non-limiting examples of DNA intercalators include ethidium bromide, proflavine, daunomycin, doxorubicin, and thalidomide.
- The stability, sensitivity, and specificity of the oxocarbonamide peptide nucleic acids of the present invention, make them a useful tool in diagnostics and molecular biology. While the compounds of the present invention are useful in the detection and analysis of any nucleic acids, it is contemplated that they will be particularly useful in the detection of small RNA molecules such as miRNA molecules, which often require the use of sensitive analysis tools due to their size and low level of expression.
- In certain embodiments, the oxocarbonamide peptide nucleic acids of the present invention may be used as hybridization probes for the detection of complementary nucleic acid sequences. Sequence-specific nucleic acid hybridization assays (e.g., Northern blotting, Southern blotting, and microarray analysis) are commonly used for the detection of specific genetic sequences as indicators of genetic anomalies, mutations, and disease propensity. In addition, they are used for the detection of various biological agents and infectious pathogens.
- As used herein, “hybridization,” “hybridizes” or “capable of hybridizing” is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature. The term “anneal” as used herein is synonymous with “hybridize.” The term “hybridization,” “hybridizes” or “capable of hybridizing” encompasses the terms “stringent conditions” or “high stringency” and the terms “low stringency” or “low stringency conditions.” As used herein “stringent conditions” or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strands containing complementary sequences, but preclude hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA, miRNA, or siRNA transcript or a nucleic acid segment thereof, and the like.
- Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acids, the length and nucleobase content of the target sequences, the charge composition of the nucleic acids, the presence of nucleic acid analogues in the nucleic acid molecules, and to the presence or concentration of formamide, tetramethylammonium chloride or other solvents in a hybridization mixture.
- It is also understood that these ranges, compositions and conditions for hybridization are mentioned by way of non-limiting examples only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to one or more positive or negative controls. Depending on the application envisioned it is preferred to employ varying conditions of hybridization to achieve varying degrees of selectivity of a nucleic acid towards a target sequence. In a non-limiting example, identification or isolation of a related target nucleic acid that does not hybridize to a nucleic acid under stringent conditions may be achieved by hybridization at low temperature and/or high ionic strength. Such conditions are termed “low stringency” or “low stringency conditions,” and non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20° C. to about 50° C. Of course, it is within the skill of one in the art to further modify the low or high stringency conditions to suite a particular application.
- The present invention may be employed in solution hybridization as well as in solid phase hybridization. The hybridization conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Optimization of hybridization conditions for the particular application of interest is well known to those of skill in the art. Representative solid phase hybridization methods are disclosed in U.S. Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods of hybridization that may be used in the practice of the present invention are disclosed in U.S. Pat. Nos. 5,849,486 and 5,851,772.
- To detect hybridization, it will be advantageous to employ an appropriate detection moiety. Recognition moieties incorporated into primers, incorporated into the amplified product during amplification, or attached to probes are useful in the identification of nucleic acid molecules. A number of different labels may be used for this purpose such as fluorophores, chromophores, radiophores, enzymatic tags, antibodies, chemiluminescence, electroluminescence, affinity labels, noble metal nanoparticles, quantum dots, magnetic particles, etc. One of skill in the art will recognize that these and other labels not mentioned herein can be used with success in this invention.
- Examples of affinity labels include, but are not limited to the following: an antibody, an antibody fragment, a receptor protein, a hormone, biotin, DNP, or any polypeptide/protein molecule that binds to an affinity label.
- Examples of enzyme tags include enzymes such as urease, alkaline phosphatase, or peroxidase to mention a few. Colorimetric indicator substrates can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples. All of these examples are generally known in the art and the skilled artisan will recognize that the invention is not limited to the examples described above.
- Examples of fluorophores include, but are not limited to the following: all of the Alexa Fluor® dyes, AMCA, BODIPY® 630/650, BODIPY® 650/665, BODIPY®-FL, BODIPY®-R6G, BODIPY®-TMR, BODIPY®-TRX, Cascade Blue®, CyDyes™, including but not limited to Cy2™, Cy3™, and Cy5™, DNA intercalating dyes, 6-FAM™, Fluorescein, HEX™, 6-JOE, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific Blue™, REG, phycobilliproteins including, but not limited to, phycoerythrin and allophycocyanin, Rhodamine Green™, Rhodamine Red™, ROX™, TAMRA™, TET™, Tetramethylrhodamine, and Texas Red®.
- Detection can result in qualitative identification, semi-quantitative identification, or quantitative identification of the target molecule. Qualitative detection includes detection of the presence of the molecule, without any correlation to an amount of the molecule in the sample that was tested. Semi-quantitative detection permits not only detection of the target molecule, but correlation of the signal to a basal level of target molecule in the sample that was tested. For example, it may indicate a minimum threshold amount of the target molecule was present in the sample. Quantitative detection permits the practitioner to determine the amount of target molecule present in the original sample over a wide range of amounts. In general, quantitative detection compares the amount detected to a reference or standard that is either previously generated (e.g., a standard curve) or generated at the time of the assay for the target molecule using internal controls. Numerous techniques for performing quantitative and semi-quantitative analyses are known to those of skill in the art.
- Arrays and gene chip technology provide a means of rapidly screening a large number of nucleic acid samples for their ability to hybridize to oxocarbonamide peptide nucleic acid molecules immobilized on a solid substrate. These techniques involve quantitative methods for analyzing large numbers of miRNA molecules, or other nucleic acid sequences, rapidly and accurately. Basically, an array or gene chip consists of a solid substrate upon which an array of single stranded oxocarbonamide peptide nucleic acid molecules have been attached. For screening, the chip or array is contacted with a single stranded DNA or RNA sample, which is allowed to hybridize under stringent conditions. The chip or array is then scanned to determine which probes have hybridized.
- The ability to directly synthesize on or attach polynucleotide probes to solid substrates is well known in the art. See U.S. Pat. Nos. 5,837,832 and 5,837,860, both of which are expressly incorporated by reference. A variety of methods have been utilized to either permanently or removably attach the probes to the substrate. Exemplary methods include: the immobilization of biotinylated nucleic acid molecules to avidin/streptavidin coated supports (Holmstrom, 1993), the direct covalent attachment of short, 5′-phosphorylated primers to chemically modified polystyrene plates (Rasmussen et al., 1991), or the precoating of the polystyrene or glass solid phases with poly-L-Lys or poly L-Lys, Phe, followed by the covalent attachment of either amino- or sulfhydryl-modified oligonucleotides using bi-functional crosslinking reagents (Running et al., 1990; Newton et al., 1993). When immobilized onto a substrate, the probes are stabilized and therefore may be used repeatedly. In general terms, hybridization is performed on an immobilized nucleic acid target or a probe molecule that is attached to a solid surface such as nitrocellulose, nylon membrane, or glass. Numerous other matrix materials may be used, including reinforced nitrocellulose membrane, activated quartz, activated glass, polyvinylidene difluoride (PVDF) membrane, polystyrene substrates, polyacrylamide-based substrate, other polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules.
- In certain embodiments, the present invention is used in conjunction with Luminex® xMAP® technology. The Luminex technology allows the quantitation of nucleic acid products immobilized on fluorescently encoded microspheres. By dyeing microspheres with 10 different intensities each of two spectrally distinct fluorochromes, 100 fluorescently distinct populations of microspheres are produced. By using three or more spectrally distinct fluorochromes at different intensity levels, even greater numbers of fluorescently distinct populations can be created. These individual populations (sets) can represent individual detection sequences and the magnitude of hybridization on each set can be detected individually. The magnitude of the hybridization reaction is measured using a third spectrally distinct fluorochrome called a reporter. The reporter molecule signals the extent of the reaction by attaching to the molecules on the microspheres. As both the microspheres and the reporter molecules are labeled, digital signal processing allows the translation of signals into real-time, quantitative data for each reaction. The Luminex technology is described, for example, in U.S. Pat. Nos. 5,736,330, 5,981,180, and 6,057,107, all of which are specifically incorporated by reference.
- The present invention may also be used in conjunction with a competitive binding assay format. In general, this format involves a detection sequence coupled to a solid surface, and a labeled sequence complementary to the detection sequence in solution. With this format, the target sequence in the sample being assayed does not need to be labeled. Rather, the target sequence's presence in the sample is detected because it competes with the labeled complement for hybridization with the immobilized detection sequence. Thus, if the target sequence is present in the sample, the signal decreases as compared to a sample lacking the target sequence.
- The Luminex xMAP technology described above can be used in a competitive binding assay format. In general, this format would comprise an oxocarbonamide peptide nucleic acid detection molecule immobilized on a labeled bead, a labeled sequence complementary to the detection sequence, exposing the immobilized detection sequence and the labeled complement to a nucleic acid sample under hybridizing conditions, and detecting the presence or absence of the target sequence in the sample. The use of the Luminex technology in a competitive binding assay format is described in U.S. Pat. Nos. 5,736,330 and 6,057,107, incorporated herein by reference.
- Flow cytometry is a useful tool in the analysis of biomolecules. In the context of the present invention, flow cytometry is particularly useful in the analysis of microsphere based assays, such as the Luminex xMAP system. Flow cytometry involves the separation of cells or other particles, such as microspheres, in a liquid sample. Generally, the purpose of flow cytometry is to analyze the separated particles for one or more characteristics. The basic steps of flow cytometry involve the direction of a fluid sample through an apparatus such that a liquid stream passes through a sensing region. The particles should pass one at a time by the sensor and are categorized based on size, refraction, light scattering, opacity, roughness, shape, fluorescence, etc.
- In the context of the Luminex xMAP system, flow cytometry can be used for simultaneous sequence identification and hybridization quantification. Internal dyes in the microspheres are detected by flow cytometry and used to identify the specific nucleic acid sequence to which a microsphere is coupled. The label on the target nucleic acid molecule is also detected by flow cytometry and used to quantify target hybridization to the microsphere.
- Methods of flow cytometry are well know in the art and are described, for example, in U.S. Pat. Nos. 5,981,180, 4,284,412; 4,989,977; 4,498,766; 5,478,722; 4,857,451; 4,774,189; 4,767,206; 4,714,682; 5,160,974; and 4,661,913, all of which are specifically incorporated by reference.
- E. miRNA Isolation
- As discussed above, the oxocarbonamide peptide nucleic acids of the present invention are particularly useful in the detection of small RNA molecules, such as miRNA, in a sample. miRNAs are 18-25 nucleotide (nt) RNAs that are processed from longer endogenous hairpin transcripts. The increased sensitivity and selectivity of OxoPNAs of the present invention overcome the limitations of their DNA-probe counterparts for the detection of short nucleotide targets.
- Small RNA molecules may be isolated from a sample, such as a cell sample, by a variety of methods known in the art. The most commonly used method is to co-purify the miRNA with total RNA using a combination of acidified phenol and guanidine isothiocyanate using care not to remove the highly-soluble short RNA (see, e.g., Pfeffer et al., 2003). This method isolates total RNA, which comprises transfer RNA (tRNA), ribosomal RNA (rRNA), polyA messenger RNA (mRNA), short interfering RNA (siRNA), small nuclear RNA (snRNA), and microRNA (miRNA). If desired, the miRNA can be enriched from the total RNA by size selection using gel purification (Pfeffer, Id.).
- Other methods for isolating miRNA include the mirVana™ miRNA Isolation Kit (Ambion). The resulting RNA preparation (less than about 200 nucleotides) is enriched for miRNAs, siRNAs, and/or snRNAs. In addition, the Absolutely RNA® Miniprep Kit (Stratagene) may be used to isolate total RNA comprising miRNA. Removal of genomic DNA is desirable as its presence in the total RNA could lead to false or misleading results.
- The oxocarbonamide peptide nucleic acids of the present invention may be used as double-stranded siRNA molecules, single-stranded antisense molecules, or as decoy molecules for nucleic acid binding proteins. These uses may be for therapeutic or research purposes. Naturally occurring DNA molecules are generally not well suited to these applications due to the instability of unmodified DNA in vivo. However, chemically modified oligonucleotides have been shown to be effective inhibitors of coding and non-coding RNAs (see Weiler et al., 2006).
- Oxocarbonamide peptide siRNA, antisense, or decoy molecules may be prepared by solid phase synthesis as described above. For therapeutic application it may be desirable to incorporate hydrophilic groups, such as polyethylene glycol (PEG), in to the OxoPNA in order to increase its hydrophilicity. Cationic polymers such as polyamidoamine (PMAM) dendrimers or a polyethyleneimine (PEI) may be combined with OxoPNAs for DNA delivery.
- The nucleotide sequence of the siRNA or antisense molecule is defined by the nucleotide sequence of its target gene. The siRNA or antisense molecule contains a nucleotide sequence that is essentially identical to at least a portion of the target gene. Preferably, the siRNA or antisense contains a nucleotide sequence that is completely identical to at least a portion of the target gene. Of course, when comparing an RNA sequence to a DNA sequence, an “identical” RNA sequence will contain ribonucleotides where the DNA sequence contains deoxyribonucleotides, and further that the RNA sequence will typically contain a uracil at positions where the DNA sequence contains thymidine. The nucleotide sequence of the decoy molecule is defined by the recognition sequence of the target protein.
- The cell containing the target gene or protein may be derived from or contained in any organism (e.g., plant, animal, protozoan, virus, bacterium, or fungus). The plant may be a monocot, dicot or gynmosperm; the animal may be a vertebrate or invertebrate. Preferred microbes are those used in agriculture or by industry, and those that a pathogenic for plants or animals. Fungi include organisms in both the mold and yeast morphologies. Examples of vertebrates include fish and mammals, including cattle, goat, pig, sheep, hamster, mouse, rat, and human; invertebrate animals include nematodes, insects, arachnids, and other arthropods. Preferably, the cell is a vertebrate cell. More preferably, the cell is a mammalian cell.
- The cell having the target gene or protein may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell can be a gamete or an embryo; if an embryo, it can be a single cell embryo or a constituent cell or cells from a multicellular embryo. The term “embryo” thus encompasses fetal tissue. The cell having the target gene or protein may be an undifferentiated cell, such as a stem cell, or a differentiated cell, such as from a cell of an organ or tissue, including fetal tissue, or any other cell present in an organism. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells, of the endocrine or exocrine glands.
- As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations formed by cell division. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms “engineered” and “recombinant” cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence has been introduced. Therefore, recombinant cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced nucleic acid.
- A tissue may comprise a host cell or cells to be transformed or contacted with a nucleic acid delivery composition and/or an additional agent. The tissue may be part or separated from an organism. In certain embodiments, a tissue and its constituent cells may comprise, but is not limited to, blood (e.g., hematopoietic cells (such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34+ cells CD4+ cells), lymphocytes and other blood lineage cells), bone marrow, brain, stem cells, blood vessel, liver, lung, bone, breast, cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial, epithelial, esophagus, facia, fibroblast, follicular, ganglion cells, glial cells, goblet cells, kidney, lymph node, muscle, neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, small intestine, spleen, stomach, or testes.
- The oxocarbonamide peptide nucleic acids of the present invention may be used as anchor molecules for the attachment of different functional molecules to DNA via specific Watson-Crick base pairing. The coupling of molecules with different biological function to plasmids or other nucleic acid molecules of interest can improve the targeting of genetic material in non-viral gene delivery systems. Coupling methods based on chemical linkage of peptides to plasmid DNA can interfere with gene expression. Thus, coupling via specific Watson-Crick base pairing provides an attractive alternative to chemical linkage. Those in the art are familiar with methods of coupling peptides and other molecules to plasmid DNA using nucleic acid analogs, such as those described for PNA, bisPNA, and LNA (see e.g., Lundin et al., 2005; Branden et al., 1999; Rebuff et al., 2002; Hertoghs et al., 2003; Branden et al., 2002).
- The oxocarbonamide peptide nucleic acids of the present invention may also be used in the programmed assembly of nanoscale devices. Nucleic acid guided assembly has been used to organize gold nanoparticles (Mirkin et al., 1996; Alivisatos et al., 1996; Mucic et al., 1998), nanowires (Mbindyo et al., 2003), quantum dots (Parak et al., 2002); Mitchell et al., 1999), carbon nanotubes (Dwyer et al., 2004), dendrimers (DeMattei et al., 2004), micron-size polystyrene beads (Milam et al., 2003), virus particles (Strable et al., 2004), and to attach nano- and microparticles to substrates (Kannan et al., 2004; Hartmann et al., 2002); Niemeyer et al., 2001). The oxocarbonamide peptide nucleic acids of the present invention provide advantages in programmed assembly over natural DNA due to increased stability and greater affinity between complementary oligomers.
- Any of the compositions described herein may be comprised in a kit. In one embodiment, the present invention provides a kit comprising one or more oxocarbonamide peptide nucleic acid molecules. In certain aspects of the invention, the kit comprises a plurality of oxocarbonamide peptide nucleic acid molecules having at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 different nucleic acid sequences. In another embodiment of the invention, the kit may include components for making a nucleic acid array, and thus, may include, for example, a solid support. In some aspects of the invention, the kits will comprise pre-fabricated arrays, such as, for example, microspheres coupled to oxocarbonamide peptide nucleic acid probes. It may also include one or more buffers, such as hybridization buffer or a wash buffer.
- The kits may comprise suitably aliquoted nucleic acid compositions of the present invention, whether labeled or unlabeled, as may be used to isolate, separate, detect, or amplify a targeted nucleic acid. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional containers into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the oxocarbonamide peptide nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include cardboard or injection or blow-molded plastic containers into which the desired vials, bottles, etc. are retained.
- When the components of the kit are provided in one or more liquid solutions, the liquid solution may be an aqueous solution, with a sterile aqueous solution being particularly preferred.
- However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
- A kit may also include instructions for employing the kit components. Instructions may include variations that can be implemented.
- All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of certain embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
- The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
- U.S. Pat. No. 4,284,412
- U.S. Pat. No. 4,498,766
- U.S. Pat. No. 4,661,913
- U.S. Pat. No. 4,714,682
- U.S. Pat. No. 4,767,206
- U.S. Pat. No. 4,774,189
- U.S. Pat. No. 4,857,451
- U.S. Pat. No. 4,989,977
- U.S. Pat. No. 5,160,974
- U.S. Pat. No. 5,478,722
- U.S. Pat. No. 5,539,082
- U.S. Pat. No. 5,714,331
- U.S. Pat. No. 5,719,262
- U.S. Pat. No. 5,736,330
- U.S. Pat. No. 5,736,336
- U.S. Pat. No. 5,766,855
- U.S. Pat. No. 5,773,571
- U.S. Pat. No. 5,786,461
- U.S. Pat. No. 5,837,832
- U.S. Pat. No. 5,837,860
- U.S. Pat. No. 5,843,663
- U.S. Pat. No. 5,849,486
- U.S. Pat. No. 5,851,772
- U.S. Pat. No. 5,891,625
- U.S. Pat. No. 5,900,481
- U.S. Pat. No. 5,919,626
- U.S. Pat. No. 5,981,180
- U.S. Pat. No. 6,057,107
- Alivisatos et al., Nature, 382:609-611, 1996.
- Ambros et al. RNA, 9:277-279, 2003.
- Branden et al., Methods Enzymol., 346:106-124, 2002.
- Branden et al., Nat. Biotechnol., 17:784-787, 1999.
- Calin et al., Proc. Natl. Acad. Sci. USA, 99:15524-15529, 2002.
- DeMattei et al., Nano Lett., 4:771-777, 2004.
- Dwyer et al., Nanotechnology, 15:1240-1245, 2004.
- Egholm et al., Nature, 365(6446):566-568, 1993.
- Fire et al., Nature, 391:806-811, 1998.
- Fujii et al., Chem. Commun., 717-718, 1998.
- Girault et al., Bioconj. Chem., 7:445-450, 1996.
- Grad et al., Mol. Cell, 11:1253-1263, 2003.
- Grishok et al., Cell, 106:23-24, 2001.
- Hartmann et al., Mater. Res., 17:473-478, 2002.
- Hertoghs et al., Nucleic Acids Res., 31:5817-5830, 2003.
- Holmstrom et al., Anal. Biochem. 209:278-283, 1993.
- Howarth et al., J. Org. Chem., 62:5441-5450, 1997.
- Hutvagner and Zamore, Science, 297:2056-2060, 2002.
- Kannan et al., Nano Lett., 4:1521-1524, 2004.
- Ke et al., Curr. Opin. Chem. Biol., 7:516-523, 2003.
- Koshkin and Dunford, J. Biol. Chem., 273(11):6046-6049, 1998a.
- Koshkin and Wengel, J. Org. Chem., 63(8):2778-2781, 1998b.
- Krichevsky et al., RNA 9:1274-1281, 2003.
- Lagos-Quintana et al., Science, 294:853-858, 2001.
- Lee and Ambros, Science, 294:858-862, 2001a.
- Lee and Ambros, Science, 294:862-864, 2001b.
- Lee et al., Cell, 75:843-854, 1993.
- Lipardi et al., Cell, 107:297-230, 2001.
- Liu et al., Proc. Natl. Acad. Sci, USA, 101:9740-9744, 2004.
- Lundin et al., Biomolecular Engineering, 22:185-192, 2005.
- Mbindyo et al., Adv. Mater., 13:249-254, 2003.
- Mc Cairn et al., J. Combinatorial Chem., 8(1):1-3, 2006.
- Milam et al., Langmuir, 19:10317-10323, 2003.
- Mirkin et al., Nature, 382:607-609, 1996.
- Mitchell et al., J. Am. Chem. Soc., 121:8122-8123, 1999.
- Mucic et al., J. Am. Chem. Soc., 120:12674-12675, 1998.
- Nelson et al., TIBS, 28:534-540, 2003.
- Newton et al., Nucl. Acids Res. 21:1155-1162, 1993.
- Niemeyer et al., Colloid. Polym., 279:68-72, 2001.
- Nykanen et al., Cell, 107:309-321, 2001.
- Parak et al., Chem. Mater., 14:2113-2119, 2002.
- Paushkin et al., Curr. Opin. Cell Biol., 14:305-312, 2002.
- PCT Appln. PCT/EP/01219
- PCT Appln. WO 92/20702
- Pfeffer et al., In: Cloning of Small RNA Molecules in Current Protocols in Molecular Biology, Ausubel et al. (Eds), Ch. 26.4.1-26.4.18, Wiley Interscience, NY, 2003.
- Porter et al., Bioorganic Med. Chem. Ltrs., 12:1051-1054, 2002.
- Poy et al., Nature, 432:226-230, 2004.
- Rasmussen et al., Anal. Biochem, 198:138-142, 1991.
- Rebuff et al., FASEB J, 16:1426-1428, 2002.
- Reinhart et al. Genes Dev., 16:1616-1626, 2002.
- Reinhart et al., Nature, 403:901-906, 2000.
- Running et al., BioTechniques 8:276-277, 1990.
- Sato et al., J. Am. Chem. Soc., 124:12715-12724, 2002.
- Schmittgen et al., Nucleic Acids Res., 32:e43, 2004.
- Strable et al., Nano Lett., 4:1385-1389, 2004.
- Thomson et al., Nature Methods, 1:1-6, 2004.
- Valoczi et al., Nuc. Acids Res., 32(22):e175, 2004.
- Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97(10):5633-5638, 2000.
- Weiler et al., Gene Therapy, 13:496-502, 2006.
- Zeng and Cullen, RNA, 9:112-123, 2003.
- Zhang et al., EMBO J., 21:5875-5885, 2002.
Claims (27)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/947,705 US8324359B1 (en) | 2006-12-04 | 2007-11-29 | Oxocarbonamide peptide nucleic acids and methods of using same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US86851406P | 2006-12-04 | 2006-12-04 | |
US11/947,705 US8324359B1 (en) | 2006-12-04 | 2007-11-29 | Oxocarbonamide peptide nucleic acids and methods of using same |
Publications (2)
Publication Number | Publication Date |
---|---|
US20120302453A1 true US20120302453A1 (en) | 2012-11-29 |
US8324359B1 US8324359B1 (en) | 2012-12-04 |
Family
ID=39272510
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/947,705 Active 2031-10-04 US8324359B1 (en) | 2006-12-04 | 2007-11-29 | Oxocarbonamide peptide nucleic acids and methods of using same |
Country Status (3)
Country | Link |
---|---|
US (1) | US8324359B1 (en) |
EP (1) | EP2099816A1 (en) |
WO (1) | WO2008070525A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016138035A1 (en) * | 2015-02-24 | 2016-09-01 | Agilent Technologies, Inc. | Preparation of long synthetic oligonucleotides by squarate conjugation chemistry |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5539082A (en) | 1993-04-26 | 1996-07-23 | Nielsen; Peter E. | Peptide nucleic acids |
US5714331A (en) | 1991-05-24 | 1998-02-03 | Buchardt, Deceased; Ole | Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility |
DK51092D0 (en) | 1991-05-24 | 1992-04-15 | Ole Buchardt | OLIGONUCLEOTIDE ANALOGUE DESCRIBED BY PEN, MONOMERIC SYNTHONES AND PROCEDURES FOR PREPARING THEREOF, AND APPLICATIONS THEREOF |
US5766855A (en) | 1991-05-24 | 1998-06-16 | Buchardt, Deceased; Ole | Peptide nucleic acids having enhanced binding affinity and sequence specificity |
US5719262A (en) | 1993-11-22 | 1998-02-17 | Buchardt, Deceased; Ole | Peptide nucleic acids having amino acid side chains |
GB9211979D0 (en) | 1992-06-05 | 1992-07-15 | Buchard Ole | Uses of nucleic acid analogues |
DE4331011A1 (en) | 1993-09-13 | 1995-03-16 | Bayer Ag | C-branched oligomers that bind nucleic acids for therapy and diagnostics |
US5981180A (en) | 1995-10-11 | 1999-11-09 | Luminex Corporation | Multiplexed analysis of clinical specimens apparatus and methods |
US5736330A (en) | 1995-10-11 | 1998-04-07 | Luminex Corporation | Method and compositions for flow cytometric determination of DNA sequences |
GB9621367D0 (en) * | 1996-10-14 | 1996-12-04 | Isis Innovation | Chiral peptide nucleic acids |
-
2007
- 2007-11-29 WO PCT/US2007/085927 patent/WO2008070525A1/en active Application Filing
- 2007-11-29 US US11/947,705 patent/US8324359B1/en active Active
- 2007-11-29 EP EP07864909A patent/EP2099816A1/en not_active Withdrawn
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016138035A1 (en) * | 2015-02-24 | 2016-09-01 | Agilent Technologies, Inc. | Preparation of long synthetic oligonucleotides by squarate conjugation chemistry |
US9890190B2 (en) | 2015-02-24 | 2018-02-13 | Agilent Technologies, Inc. | Preparation of long synthetic oligonucleotides by squarate conjugation chemistry |
Also Published As
Publication number | Publication date |
---|---|
WO2008070525A1 (en) | 2008-06-12 |
US8324359B1 (en) | 2012-12-04 |
EP2099816A1 (en) | 2009-09-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9714446B2 (en) | Compositions and methods for the detection of small RNAs | |
Kam et al. | Detection of endogenous K-ras mRNA in living cells at a single base resolution by a PNA molecular beacon | |
US9121065B2 (en) | Nanoparticle-oligonucleotide hybrid structures and methods of use thereof | |
US20130225432A1 (en) | Solution-based methods for rna expression profiling | |
US20150376695A1 (en) | Detecting nucleic acids | |
Shi et al. | A review: Fabrications, detections and applications of peptide nucleic acids (PNAs) microarray | |
US20070065840A1 (en) | Novel oligonucleotide compositions and probe sequences useful for detection and analysis of microRNAS and their target mRNAS | |
SK4292001A3 (en) | Methods of nucleic acid amplification and sequencing | |
GB2456669A (en) | Assay ligates random primers to microarray probes to increase efficiency | |
Siddiquee et al. | A review of peptide nucleic acid | |
US20090326049A1 (en) | Blocking oligos for inhibition of microrna and sirna activity and uses thereof | |
US20140296092A1 (en) | Methods, Reagents and Kits for Detection of Nucleic Acid Molecules | |
US11591646B2 (en) | Small RNA detection method based on small RNA primed xenosensor module amplification | |
WO2008079303A2 (en) | Detection of organ rejection | |
US20080293580A1 (en) | Methods for Detecting and Measuring Specific Nucleic Acid Sequences | |
WO2001066804A2 (en) | Methods for optimizing hybridization performance of polynucleotide probes and localizing and detecting sequence variations | |
US20090221435A1 (en) | Microarray for detecting and quantifying microrna | |
US8324359B1 (en) | Oxocarbonamide peptide nucleic acids and methods of using same | |
JP2011511776A (en) | Cationic siRNA for RNA interference, synthesis and use | |
WO2010087409A1 (en) | Method for detecting nucleic acid | |
EP2824180B1 (en) | Method for detecting target nucleic acid | |
Pellestor et al. | The peptide nucleic acids (PNAs): introduction to a new class of probes for chromosomal investigation | |
EP4257684A1 (en) | Reagents for subcellular delivery of cargo to target cells | |
Kotorić | Peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) application in bacterial gene editing and biosensor production | |
Saba et al. | MicroRNA Profiling in CNS Tissue Using Microarrays |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: LUMINEX CORPORATION, TEXAS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LUGADE, ANANDA G.;JACOBSON, JAMES W.;SIGNING DATES FROM 20080107 TO 20080108;REEL/FRAME:020751/0533 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 8TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1552); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY Year of fee payment: 8 |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 12TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1553); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY Year of fee payment: 12 |