Method for enzymatic synthesis of oligonucleotides
First Claim
1. A method for synthesizing an oligonueleotide of a defined sequence, comprising the steps of:
- (a) combining (1) an oligonucleotide primer and (2) a blocked nucleotide or a blocked nucleotide precursor that forms a blocked nucleotide in situ, in a reaction mixture in the presence of a chain extending enzyme effective to couple the blocked nucleotide to the 3'"'"'-end of the oligonucleotide primer such that a primer-blocked nucleotide product is formed, wherein the blocked nucleotide comprises a nucleotide to be added to form part of the defined sequence and a blocking group attached to the 3'"'"'-end of the nucleotide effective to prevent the addition of more than one blocked nucleotide to the primer;
(b) removing the blocking group from the 3'"'"'-end of the primer-blocked nucleotide product to form a primer-nucleotide product, whereby the reaction mixture contains any unreacted starting materials that may remain, primer-nucleotide product and reaction by-products; and
(c) repeating at least one cycle of steps (a) and (b) using the primer-nucleotide product from step (b) as the oligonucleotide primer of step (a) in the subsequent cycle without separation of the primer-nucleotide product from the remainder of the reaction mixture.
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Abstract
Enzymatic synthesis of oligonucleotides is performed by the steps of: (a) combining a primer and a blocked nucleotide in the presence of a chain extending enzyme to form a primer-blocked nucleotide product containing the blocked nucleotide coupled to the primer at its 3'"'"'-end; (b) removing the blocking group from the 3'"'"' end of the primer-blocked nucleotide product; and (c) repeating the cycle of steps (a) and (b), using the primer-nucleotide product of step (b) as the primer for step (a) in the next cycle, for sufficient cycles to form the oligonucleotide product. Cycles may optionally include the step of converting any unreacted blocked nucleotide to an unreactive form which is substantially less active as a substrate for the chain extending enzyme. Cycles may also include the step of removing the blocking group from unreacted blocked nucleotide. This step is unnecessary, however, when the same nucleotide is added in two or more successive cycles. The synthetic cycles are preferably performed in a single vessel without intermediate purification of oligonucleotide product.
146 Citations
29 Claims
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1. A method for synthesizing an oligonueleotide of a defined sequence, comprising the steps of:
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(a) combining (1) an oligonucleotide primer and (2) a blocked nucleotide or a blocked nucleotide precursor that forms a blocked nucleotide in situ, in a reaction mixture in the presence of a chain extending enzyme effective to couple the blocked nucleotide to the 3'"'"'-end of the oligonucleotide primer such that a primer-blocked nucleotide product is formed, wherein the blocked nucleotide comprises a nucleotide to be added to form part of the defined sequence and a blocking group attached to the 3'"'"'-end of the nucleotide effective to prevent the addition of more than one blocked nucleotide to the primer; (b) removing the blocking group from the 3'"'"'-end of the primer-blocked nucleotide product to form a primer-nucleotide product, whereby the reaction mixture contains any unreacted starting materials that may remain, primer-nucleotide product and reaction by-products; and (c) repeating at least one cycle of steps (a) and (b) using the primer-nucleotide product from step (b) as the oligonucleotide primer of step (a) in the subsequent cycle without separation of the primer-nucleotide product from the remainder of the reaction mixture. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 19, 24, 26, 28, 29)
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13. A method for synthesizing a repeat region of an oligonucleotide having a defined sequence, said repeat region including a repeated nucleotide that appears more than once in succession, comprising the steps of:
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(a) enzymatically coupling an oligonucleotide primer with a 3'"'"'-phosphate-blocked repeated nucleotide to form a 3'"'"'-phosphate blocked primer-nucleotide; (b) removing the 3'"'"'-phosphate blocking group from the 3'"'"'-phosphate-blocked primer-nucleotide using 3'"'"'-phosphatase enzyme substantially without removing the 3'"'"'-phosphate blocking group from unreacted 3'"'"'-phosphate-blocked repeated nucleotide; and (c) repeating steps (a) and (b) using the unreacted 3'"'"'-phosphate-blocked repeated nucleotide from step (b) as the 3'"'"'-phosphate-blocked nucleotide of step (a) and using the deblocked primer-nucleotide product of step (b) as the oligonucleotide primer of step (a).
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- 14. A method for synthesizing an oligonucleotide, wherein the 3'"'"'-end of an oligonucleotide primer is coupled with a blocked nucleotide to form a primer-blocked nucleotide product in a reaction mixture, said blocked nucleotide comprising a nucleotide to be added to the oligonucleotide primer and a blocking group attached to the 3'"'"'-end of the nucleotide effective to prevent the addition of more than one blocked nucleotide to the oligonucleotide primer, comprising incubating the reaction mixture with an exonuclease, whereby any oligonucleotide primer which was not coupled is degraded, substantially without degrading the primer-blocked nucleotide product.
- 15. A method for synthesizing an oligonucleotide, wherein the 3'"'"'-end of an oligonucleotide primer is enzymatically coupled with a blocked nucleotide to form a primer-blocked nucleotide product in a reaction mixture, said blocked nucleotide comprising a nucleotide to be added to the oligonucleotide primer and a removable blocking group attached to the 3'"'"'-end of the nucleotide effective to prevent the addition of more than one blocked nucleotide to the oligonucleotide primer, comprising incubating the reaction mixture with a chain terminating nucleotide and an enzyme effective to couple the chain terminating nucleotide to the oligonucleotide primer, whereby oligonucleotide primer which was not coupled to a blocked nucleotide is end-capped to render it unreactive to further coupling, said chain terminating nucleotide being different from said blocked nucleotide and selected such that end-capped oligonucleotide primer remains end-capped and unreactive when the blocking group is removed from the primer-blocked nucleotide product.
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20. A method for coupling a blocked nucleotide AppNp to an oligonucleotide primer, characterized in that the blocked nucleotide is coupled to the primer using RNA Ligase in the absence of ATP, and in that pyrophosphate or unactivated nucleotide substrate, 3,5'"'"'-NDP, is used to regenerate free RNA Ligase from the inactivated adenylylated form, wherein N represents any nucleoside or nucleoside analog which RNA ligase can couple to an oligonucleotide primer.
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21. A method for coupling a blocked nucleotide to an oligonucleotide primer, characterized in that the coupling is performed using RNA Ligase in the presence of 5'"'"'-Nucleotidase, AMP Nucleotidase or AMP Deaminase, whereby AMP released in the coupling reaction is converted to a form which is less effective than AMP to inhibit the coupling reaction or participate as a substrate in a reverse coupling reaction.
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22. A method for synthesizing a selected oligonucleotide wherein an oligonucleotide primer is extended by enzymatically adding at least two nucleotides to the 3'"'"'-end of the oligonucleotide primer, characterized in that the primer is cleaved from the added nucleotides to form the selected oligonucleotide.
- 23. A method for converting a blocked nucleotide comprising a dinucleotide pyrophosphate moiety, a blocking group effective to prevent the enzymatic coupling of more than one blocked nucleotide to an oligonucleotide primer, and a nucleotide to be enzymatically coupled to the primer to a less reactive form, characterized in that the blocked nucleotide is treated with a dinucleotide pyrophosphate degrading enzyme.
Specification