Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
First Claim
1. A mononucleoside 5'"'"'-triphosphate having a removable blocking moiety protecting the 3'"'"' position which is an ester and which has the following formula:
- ##STR18## wherein R2 is a triphosphate and R is selected from the group consisting of;
formate, benzoylformate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, p-P-phenylacetate, 3-phenylpropionate, 3-benzoylpropionate, isobutyrate, 4-oxopentanoate, pivaloate, adamanioate, crotonate, 4-methoxycrotonate, (E)-2-methyl-2-butenoate, o-(dibromomethyl)benzoate, o-(methoxycarbonyl)benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate and α
-naphthanoate.
4 Assignments
0 Petitions
Reexamination
Accused Products
Abstract
A method for the stepwise creation of phosphodiester bonds between desired nucleosides resulting in the synthesis of polynucleotides having a predetermined nucleotide sequence by preparing an initiation substrate containing a free and unmodified 3'"'"'-hydroxyl group; attaching a mononucleotide selected according to the order of the predetermined nucleotide sequence to the 3'"'"'-hydroxyl of the initiating substrate in a solution containing a catalytic amount of an enzyme capable of catalyzing the 5'"'"' to 3'"'"' phosphodiester linkage of the 5'"'"'-phosphate of the mononucleotide to the 3'"'"'-hydroxyl of the initiating substrate, wherein the mononucleotide contains a protected 3'"'"'-hydroxyl group, whereby the protected mononucleotide is covalently linked to the initiating substrate and further additions are hindered by the 3'"'"'-hydroxyl protecting group. Methods in which a mononucleotide immobilized on a solid support is added to a free polynucleotide chain are also disclosed.
207 Citations
7 Claims
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1. A mononucleoside 5'"'"'-triphosphate having a removable blocking moiety protecting the 3'"'"' position which is an ester and which has the following formula:
- ##STR18## wherein R2 is a triphosphate and R is selected from the group consisting of;
formate, benzoylformate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, p-P-phenylacetate, 3-phenylpropionate, 3-benzoylpropionate, isobutyrate, 4-oxopentanoate, pivaloate, adamanioate, crotonate, 4-methoxycrotonate, (E)-2-methyl-2-butenoate, o-(dibromomethyl)benzoate, o-(methoxycarbonyl)benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate and α
-naphthanoate. - View Dependent Claims (3, 5, 6, 7)
- ##STR18## wherein R2 is a triphosphate and R is selected from the group consisting of;
-
2. A mononucleoside 5'"'"' triphosphate having a removable blocking moiety protecting the 3'"'"' position which is an ester and which has the following formula:
- ##STR19## wherein R2 is triphosphate; and
wherein R is selected from the group consisting of;
H, CH3, CH3 (CH2) where N is an integer from 1 to 12, (CH3)x+1 (CH)x where x is an integer from 1 to 12, (CH3)x+1 (CH)x (CH2)n where x and n are independent integers from 1 to 12, Cx (CH3)3x-(x)1) (CH2)n where x and n are independent integers from 1 to 12, and ##STR20## where R1, R3, R4, R5 and R6 are selected from the group consisting of CH3, H or NO2.
- ##STR19## wherein R2 is triphosphate; and
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4. A mononucleoside 5'"'"'-triphosphate having a removable blocking moiety protecting the 3'"'"' position which has the following formula:
- ##STR21## wherein R2 is triphosphate; and
wherein R1 is toluic, benzoic or acetic acid ester.
- ##STR21## wherein R2 is triphosphate; and
Specification