Methods and compositions for synthesis of labelled oligonucleotides and analogs on solid-supports
First Claim
1. A method for 5′
- to 3′
synthesis of 5′
labelled oligonucleotides comprising the steps of;
a) providing a labelled solid-support having the structure where,S is a solid-support;
A is a cleavable linker;
X is a moiety with three or more attachment sites;
L is a label;
Y is selected from the group consisting of oxygen, NH, NR where R is methyl, lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl, and sulfur; and
P1 is an acid-cleavable protecting group;
b) reacting the labelled solid-support with acid to remove the acid-cleavable protecting group, P1; and
c) adding a 5′
-phosphoramidite, 3′
protected nucleoside and an activator, thereby forming a bond between Y 5′
terminus of the and the nucleoside.
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Accused Products
Abstract
Methods and compositions to label oligonucleotides and analogs directly on a solid-support having the structure
where S is a solid-support, A is a cleavable linker, X is a moiety with three or more attachment sites, L is a label, Y is a nucleophile, i.e. O, NH, NR or S, and P1 is an acid cleavable protecting group are provided. The labelled solid-support is reacted in a cyclical fashion to synthesize a labelled oligonucleotide on a solid-support in the 5′ to 3′ direction, having the structure:
Labelled oligonucleotides are also synthesized by reacting: (i) a label reagent bearing functionality consisting of carboxylic acid, sulfonic acid, phosphonic acid, or phosphoric acid, (ii) an oligonucleotide on solid support with nucleophilic functionality, and (iii) a coupling reagent, whereby an ester, amide, thioester, sulfonamide, sulfonate, phosphonate, phosphoramidate, phosphorothioate, or phosphate bond is formed. The labelling reaction may be conducted at label sites including the 5′ terminus, the 3′ terminus, a nucleobase, an internucleotide linkage, a sugar, amino, sulfide, hydroxyl, and carboxyl.
67 Citations
34 Claims
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1. A method for 5′
- to 3′
synthesis of 5′
labelled oligonucleotides comprising the steps of;a) providing a labelled solid-support having the structure where, S is a solid-support;
A is a cleavable linker;
X is a moiety with three or more attachment sites;
L is a label;
Y is selected from the group consisting of oxygen, NH, NR where R is methyl, lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl, and sulfur; and
P1 is an acid-cleavable protecting group;
b) reacting the labelled solid-support with acid to remove the acid-cleavable protecting group, P1; and
c) adding a 5′
-phosphoramidite, 3′
protected nucleoside and an activator, thereby forming a bond between Y 5′
terminus of the and the nucleoside.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
d) capping any unreacted sites on the solid-support; - and
e) adding an oxidizing reagent.
- to 3′
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3. The method of claim 1 further comprising the steps of
d) capping any unreacted sites on the solid-support; -
e) adding an oxidizing reagent; and
f) repeating steps (b) to (c) until the labelled oligonucleotide is completely synthesized.
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4. The method of claim 3 further comprising the step of deprotecting the labelled oligonucleotide.
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5. The method of claim 3 further comprising the steps of cleaving the labelled compound from the solid-support and deprotecting the labelled oligonucleotide.
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6. The method of claim 1 wherein the solid-support S is selected from the group consisting of polystyrene, controlled-pore-glass, silica gel, silica, polyacrylamide, magnetic beads, polyacrylate, hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, and copolymers and grafts of such.
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7. The method of claim 1 wherein the form of the solid support S is selected from the group consisting of small particles, beads, membranes, frits, slides, plates, micromachined chips, alkanethiol-gold layers, non-porous surfaces, addressable arrays, and polynucleotide-immobilizing media.
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8. The method of claim 1 wherein the cleavable linker A is selected from the group consisting of:
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9. The method of claim 1 wherein the linker X is selected from the group consisting of:
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where n is 1 to 12.
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10. The method of claim 1 wherein the label L is selected from the group consisting of hybridization-stabilizing moieties, fluorescent dyes, fluorescence quenchers, energy-transfer dye sets, chemiluminescent dyes, amino acids, proteins, peptides, enzymes, and affinity ligands.
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11. The method of claim 10 where the fluorescent dyes and fluorescence quenchers are selected from the group consisting of FAM, TET, HEX JOE, TAMRA, d-TAMRA, JODA, ROX, VIC, NED, dJON, dR139, DABCYL, DABSYL, malachite green, 4,7-dichloro-fluoresceins, 4,7-dichloro-rhodamines, NTB, and cyanines.
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12. The method of claim 10 where the hybridization-stabilizing moieties are selected from the group consisting of Hoechst 33258, CDPI1-3, MGB1, netropsin, and distamycin.
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13. The method of claim 1 wherein the acid-cleavable protecting group P1 is selected from the group consisting of DMT, MMT, trityl, substituted trityl, pixyl, and trialkylsilyl.
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14. The method of claim 1 wherein the 5′
- -phosphoramidite, 3′
protected nucleoside monomer has the structure;
where, R is selected from the group consisting of cyanoethyl, methyl, lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl;
R1 and R2 are individually selected from the group consisting of isopropyl, morpholino, methyl, ethyl, lower alkyl, cycloalkyl, and aryl;
P2 is an exocyclic nitrogen protecting group selected from the group consisting of benzoyl, isobutyryl, acetyl, phenoxyacetyl, aryloxyacetyl, dimethylformamidine, dialkylformamidine, and dialkylacetamidine; and
P3 is an acid-labile protecting group selected from the group consisting of DMT, MMT, pixyl, trityl, and trialkylsilyl.
- -phosphoramidite, 3′
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15. The method of claim 3 wherein the oligonucleotide comprises one or more DNA, RNA, and nucleic acid analog monomer units wherein the nucleic acid analog is selected from the group consisting of nucleobase analogs, sugar analogs, and internucleotide analogs.
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16. The method of claim 15 wherein nucleobase analogs are selected from the group consisting of C-5-alkyl pyrimidine, 2,6-diaminopurine, 2-thiopyrimidine, C-5-propyne pyrimidine, phenoxazine, 7-deazapurine, isocytidine, pseudo-isocytidine, isoguanosine, hypoxanthine, 8-oxopurine, and universal base.
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17. The method of claim 15 wherein sugar analogs are selected from the group consisting of 2′
- -O-alkyl-ribonucleotides, 2′
-O-methyl-ribonucleotides, 2′
-O-allyl-ribonucleotides, 2′
-allyl-ribonucleotides, 2′
-halo-ribonucleotides, 2′
-O-methoxyethyl-ribonucleotides, 2′
-branching group-ribonucleotides, 2′
-O-branching group-ribonucleotides, 4′
-α
-anomeric nucleotides, and 1′
-α
-anomeric nucleotides.
- -O-alkyl-ribonucleotides, 2′
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18. The method of claim 15 wherein internucleotide analogs are selected from the group consisting of 2-aminoethylglycine (PNA), 2′
- -5′
-linkage, inverted 3′
-3′
linkage, inverted 5′
-5′
linkage, phosphorothioate, methyl phosphonate, non-bridging N-substituted phosphoramidate, alkylated phosphotriester branched structure, and 3′
-N-phosphoramidate.
- -5′
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19. The method of claim 1 wherein the labelled solid-support comprises a compound of the formula
where S is polystyrene or controlled-pore-glass; - and
L is a label.
- and
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20. A solid-support comprising a compound of the formula
where, S is a solid-support; -
A is a cleavable linker;
X is a moiety with three or more attachment sites;
L is a label;
Y is selected from the group consisting of O, NH, NR, and S;
R is selected from the group consisting of cyanoethyl, methyl, lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl;
P2 is an exocyclic nitrogen protecting group selected from the group consisting of benzoyl, isobutyryl, acetyl, phenoxyacetyl, aryloxyacetyl, dimethylformamidine, dialkylformamidine, and dialkylacetamidine; and
P3 is acid-labile protecting group selected from the group consisting of DMT, MMT, trityl, substituted trityl, pixyl, and trialkylsilyl.
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21. A solid-support comprising a compound of the formula
where n is an interger from 0 to 100; -
S is a solid-support;
A is a cleavable linker;
X is a moiety with three or more attachment sites;
L is a label;
Y is selected from the group consisting of O, NH, NR, and S;
R is cyanoethyl, methyl, lower alkyl, substituted alkyl, phenyl, aryl, and substituted aryl;
P2 is an exocyclic nitrogen protecting group selected from the group consisting of benzoyl, isobutyryl, acetyl, phenoxyacetyl, aryloxyacetyl, dimethylformamidine, dialkylformamidine, and dialkylacetamidine.
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22. A method to synthesize a labelled oligonucleotide comprising:
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synthesizing an oligonucleotide on a solid support wherein the oligonucleotide includes nucleophilic functionality consisting of alcohol, amine, or thiol, and coupling a label reagent with functionality consisting of carboxylic acid, sulfonic acid, phosphonic acid, or phosphoric acid;
to the oligonucleotidebound to the solid support with a coupling reagent;
wherein the coupling reagent is HATU, HBTU, TBTU, BOP, PyBOP, EEDQ, DCC, DIPCDI, HOBt, MSNT, triisopropylbenzenesulfonyl chloride, or an aryl sulfonyl halide;
whereby an ester, amide, thioester, sulfonamide, sulfonate, phosphonate, phosphoramidate, phosphorothioate, or phosphate bond is formed. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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Specification