Enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
First Claim
1. A method for synthesizing a polynucleotide of a predetermined sequence, comprising:
- (a) providing an initiating substrate comprising a nucleoside having an unprotected 3'"'"'-hydroxyl group; and
(b) reacting said initiating substrate with a nucleoside 5'"'"'-triphosphate having its 3'"'"' position protected with a removable blocking moiety, wherein said nucleoside 5'"'"'-triphosphate is selected according to the order of said predetermined sequence, in the presence of an enzyme that catalyzes the formation of a 5'"'"' to 3'"'"' phosphodiester linkage between said unprotected 3'"'"'-hydroxyl group of said initiating substrate and a 5'"'"'-phosphate of said nucleoside 5'"'"'-triphosphate, so as to add said nucleoside to said initiating substrate.
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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.
71 Citations
29 Claims
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1. A method for synthesizing a polynucleotide of a predetermined sequence, comprising:
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(a) providing an initiating substrate comprising a nucleoside having an unprotected 3'"'"'-hydroxyl group; and (b) reacting said initiating substrate with a nucleoside 5'"'"'-triphosphate having its 3'"'"' position protected with a removable blocking moiety, wherein said nucleoside 5'"'"'-triphosphate is selected according to the order of said predetermined sequence, in the presence of an enzyme that catalyzes the formation of a 5'"'"' to 3'"'"' phosphodiester linkage between said unprotected 3'"'"'-hydroxyl group of said initiating substrate and a 5'"'"'-phosphate of said nucleoside 5'"'"'-triphosphate, so as to add said nucleoside to said initiating substrate. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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Specification