Purification of synthetic oligomers
First Claim
1. In a method for preparing a polynucleotide containing a plurality of nucleotide monomers in a preselected order, in which (a) an individual terminal hydroxyl-blocked, O- and N-protected nucleotide monomer is added to available terminal hydroxyl groups on a fraction of a plurality of identical support-bound growing oligonucleotide chains, (b) terminal hydroxyl groups on the remaining fraction of growing oligonucleotide chains to which nucleotide monomer did not add and which chains thus constitute failure sequences, are capped with a capping group, (c) the terminal hydroxyl blocking group is selectively removed from the added terminal hydroxyl-blocked nucleotide monomer, steps (a), (b) and (c) are repeated serially until the last nucleotide in the preselected order has been added, and synthesis of the desired polynucleotide sequence is complete, and (d) thereafter the polynucleotide is cleaved from the support, the improvement which comprises:
- employing as the terminal hydroxyl blocking group, a group which (a) is retained when removing the capping group and (b) prevents enzymatic hydrolysis of the completed polynucleotide;
employing as the capping group for capping the failure sequences, a group which is selectively removable while retaining the terminal hydroxyl blocking group;
after the last repetition of step (a), wherein the last terminal hydroxyl-blocked nucleotide monomer is added, removing the capping group, thereby forming decapped failure sequence oligonucleotides and terminal hydroxyl-blocked polynucleotide bound to the support;
contacting the decapped failure sequence oligonucleotides and terminal hydroxyl-blocked polynucleotide bound to the support with an enzyme capable of hydrolyzing the decapped failure sequence oligonucleotides under conditions effective to degrade the decapped failure sequence oligonucleotides and give rise to a support having substantially pure terminally blocked polynucleotide bound thereto; and
removing the terminal blocking group and directly cleaving the polynucleotide in substantially pure form from the support.
1 Assignment
0 Petitions
Accused Products
Abstract
Oligomers and polymers are prepared substantially free of error sequences by sequentially adding monomers, which are terminally blocked and have active functionalities protected, to a growing chain bound to a support through a selectively cleavable linkage. After each addition, unblocked terminal groups are capped. At the completion of monomer addition, enzymatic hydrolysis interfering protecting groups are removed along with the capping group and failure sequences enzymatically degraded. The terminal blocking group may then be removed. The completed oligomer or polymer may be cleaved from the support prior or subsequent to enzymatic degradation but after completion of the sequence.
103 Citations
29 Claims
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1. In a method for preparing a polynucleotide containing a plurality of nucleotide monomers in a preselected order, in which (a) an individual terminal hydroxyl-blocked, O- and N-protected nucleotide monomer is added to available terminal hydroxyl groups on a fraction of a plurality of identical support-bound growing oligonucleotide chains, (b) terminal hydroxyl groups on the remaining fraction of growing oligonucleotide chains to which nucleotide monomer did not add and which chains thus constitute failure sequences, are capped with a capping group, (c) the terminal hydroxyl blocking group is selectively removed from the added terminal hydroxyl-blocked nucleotide monomer, steps (a), (b) and (c) are repeated serially until the last nucleotide in the preselected order has been added, and synthesis of the desired polynucleotide sequence is complete, and (d) thereafter the polynucleotide is cleaved from the support, the improvement which comprises:
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employing as the terminal hydroxyl blocking group, a group which (a) is retained when removing the capping group and (b) prevents enzymatic hydrolysis of the completed polynucleotide; employing as the capping group for capping the failure sequences, a group which is selectively removable while retaining the terminal hydroxyl blocking group; after the last repetition of step (a), wherein the last terminal hydroxyl-blocked nucleotide monomer is added, removing the capping group, thereby forming decapped failure sequence oligonucleotides and terminal hydroxyl-blocked polynucleotide bound to the support; contacting the decapped failure sequence oligonucleotides and terminal hydroxyl-blocked polynucleotide bound to the support with an enzyme capable of hydrolyzing the decapped failure sequence oligonucleotides under conditions effective to degrade the decapped failure sequence oligonucleotides and give rise to a support having substantially pure terminally blocked polynucleotide bound thereto; and removing the terminal blocking group and directly cleaving the polynucleotide in substantially pure form from the support. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. In a method for preparing and purifying polypeptides employing terminal blocked amino acids, where the polypeptide is prepared by the sequential addition of terminally blocked O-, S- and N-protected amino acids, where the growing polypeptide is bound to a support through a selectively cleavable linkage, each sequential addition followed by removal of the terminal blocking group and the addition of the next amino acid, where after the final amino acid addition, the terminal blocking group and any protective groups are removed and the polypeptide cleaved from the support;
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the improvement which comprises; prior to each sequential addition, capping terminal groups which are not blocked with a capping group which may be selectively removed while retaining the terminal blocking group and linkage to the support; after the last monomer addition, removing capping groups and any enzymatic hydrolysis interfering protective groups, while retaining the linkage to the support; and enzymatically hydrolyzing polypeptides lacking a terminal blocking group, prior to removal of the terminal blocking group and cleavage from the support. - View Dependent Claims (19, 20)
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21. In a method for preparing and purifying a polynucleotide, said method comprising the steps of:
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sequentially adding to a growing nucleotide chain (1) joined to a support through a carboxylic acid ester linkage and (2) having a free terminal hydroxyl group, and O-blocked nucleosidyl phosphoramidite, to form a phosphite triester, oxidizing the phosphite triester to a phosphate ester and capping failed sequences by reacting free hydroxyl groups with an activated carboxylic acid to form a carboxylate ester, in a predetermined sequence; removing O-blocking groups and repeating the above sequence, until addition of the terminal nucleosidyl phosphoramidite; removal of phosphate ester protecting groups; removal of amine protecting groups and capping groups; and removal of the polynucleotide chain from the support; the improvement which comprises; employing as nucleosidyl phosphoramidites, protected adenosine and guanosine, where the exocyclic amine is N,N-disubstituted aminomethylene substituted to form a formamidine, and protected cytosine, where the exocyclic amine is substituted with an aroyl group to form an amide; capping with an oxo-substituted aliphatic carboxylic acid capable of forming a ring of from five to six annular members with hydrazine; removal of the amine protecting groups and capping groups with hydrazine; and prior to removal of the terminal O-blocking group, digesting failure sequences with a phosphodiesterase. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29)
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Specification