Enzymatic synthesis of repeat regions of oligonucleotides
First Claim
1. 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:
- (a) enzymatically coupling an oligonucleotide primer with a 3'"'"'-phosphate-blocked repeated nucleotide to form a 3'"'"'-phosphate blocked primer;
(b) removing the 3'"'"'-phosphate blocking group from the 3'"'"'-phosphate-blocked primer using a 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 unreacted 3'"'"'-phosphate-blocked repeated nucleotide from step (b) as the 3'"'"'-phosphate-blocked repeated nucleotide of step (a) and the deblocked primer product of step (b) as the oligonucleotide primer of step (a) without prior separation of the unreacted 3'"'"'-phosphate-blocked repeated nucleotide from the deblocked primer product.
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Abstract
Enzymatic synthesis of a repeat region of an oligonucleotide may be performed by the steps of: (a) combining a primer and a blocked nucleotide in the presence of a chain extending enzyme whereby a primer-blocked nucleotide product is formed 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 using a 3'"'"'-phosphatase enzyme substantially without removing the 3'"'"'-phosphate blocking group from unreacted 3'"'"'-phosphate-blocked nucleotide; 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. These cycles are performed preferably in a single vessel without intermediate purification of oligonucleotide product.
Also disclosed is a process for synthesizing an oligonucleotide having a defined sequence including at least one repeat region and one non-repeating region, wherein at least one non-repeating region is synthesized by reaction cycles using the steps of extending a primer with a 3'"'"'-blocked nucleotide, inactivating unreacted 3'"'"'-blocked nucleotide, and removing the blocking group from the extended primer. The disclosed processes may be used to synthesize repeat regions of oligoribonucleotides.
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Citations
21 Claims
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1. 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; (b) removing the 3'"'"'-phosphate blocking group from the 3'"'"'-phosphate-blocked primer using a 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 unreacted 3'"'"'-phosphate-blocked repeated nucleotide from step (b) as the 3'"'"'-phosphate-blocked repeated nucleotide of step (a) and the deblocked primer product of step (b) as the oligonucleotide primer of step (a) without prior separation of the unreacted 3'"'"'-phosphate-blocked repeated nucleotide from the deblocked primer product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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8. A method according to claim 7, wherein the 3'"'"'-phosphate-blocked nucleoside moiety is nucleosidyl-3'"'"'-phosphate monoester.
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9. A method according to claim 7, further comprising the step of converting the adenosine monophosphate to a less reactive form, whereby any inhibitory effects of the adenosine monophosphate on the primer coupling reactions are reduced.
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10. A method according to claim 7, wherein the 3'"'"'-phosphatase is derived from bacteriophage T4.
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11. A method according to claim 7, wherein the 3'"'"'-phosphatase enzyme is derived from rye grass.
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12. A method according to claim 1, wherein a single reaction cycle of steps (a) and (b) comprises the steps of
incubating an oligonucleotide primer with RNA Ligase and AppNp or precursors thereof to form the extended primer; -
heat inactivating the RNA Ligase; incubating the heat-inactivated composition with 3'"'"'-Phosphatase to remove the phosphate blocking group from the 3'"'"'-end of the extended primer; and heat inactivating the 3'"'"'-Phosphatase, wherein App is an adenosine diphosphate moiety which is released as adenosine monophosphate upon extension of the primer, and Np is a 3'"'"'-phosphate-blocked nucleoside moiety.
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13. A method according to claim 1, further comprising the step of adding an exonuclease to the composition prior to incubation with 3'"'"'-phosphatase.
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14. A method according to claim 13, wherein the exonuclease is polynucleotide phosphorylase.
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15. A method for synthesizing an oligonucleotide having a defined sequence including at least one repeat region in which a repeated nucleotide appears more than once in succession and at least one non-repeating region, wherein at least one non-repeating region of the oligonucleotide is synthesized by reaction cycles comprising the steps of
extending a primer with a 3'"'"'-blocked nucleotide substrate to form an extended primer having a blocking group at the 3'"'"'-end; -
inactivating unreacted 3'"'"'-blocked nucleotide substrate to render it less reactive than blocked nucleotide as a substrate for the primer extension reaction of step (a); and removing the blocking group from the extended primer to render it available for subsequent reaction cycles; and
at least one repeat region is synthesized by reaction cycles comprising the steps ofenzymatically coupling an oligonucleotide primer with a 3'"'"'-phosphateblocked repeated nucleotide to form a 3'"'"'-phosphate blocked primer; and removing the 3'"'"'-blocking group from the 3'"'"'-phosphate-blocked primer using a 3'"'"'-phosphatase enzyme substantially without removing the 3'"'"'-phosphate blocking group from unreacted 3'"'"'-phosphate-blocked repeated nucleotide;
wherein unfeacted 3'"'"'-phosphate blocked repeated nucleotide from a first cycle is utilized as substrate in at least one successive cycle without prior separation from the primer from which the 3'"'"'-phosphate blocking group has been removed. - View Dependent Claims (16, 17, 18)
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19. A method for synthesizing a repeat region of an oligoribonucleotide having a defined sequence, said repeat region including a repeated ribonucleotide 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 ribonucleotide to form a 3'"'"'-phosphate blocked primer; (b) removing the 3'"'"'-phosphate blocking group from the 3'"'"'-phosphate-blocked primer using a 3'"'"'-phosphatase enzyme substantially without removing the 3'"'"'-phosphate blocking group from unreacted 3'"'"'-phosphate-blocked repeated ribonucleotide; and (c) repeating steps (a) and (b) using unreacted 3'"'"'-phosphate-blocked repeated ribonucleotide from step (b) as the 3'"'"'-phosphate-blocked repeated ribonucleotide of step (a) and the deblocked primer product of step (b) as the oligonucleotide primer of step (a) without prior separation of the unreacted 3'"'"'-phosphate-blocked repeated ribonucleotide from the deblocked primer product. - View Dependent Claims (20, 21)
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Specification