Method for enzymatic synthesis of oligonucleotides using phosphate precipitation

Abstract

Enzymatic synthesis of a portion of an oligonucleotide is performed by a cycle of synthetic steps: (a) combining an oligonucleotide primer and a blocked nucleotide in a reaction mixture in the presence of a chain extending enzyme, such that a primer-blocked nucleotide product is formed, wherein the blocked nucleotide substrate comprises (i) a nucleotide to be added to form part of the defined sequence and (ii) a blocking group attached to the nucleotide effective to prevent the addition of more than one blocked nucleotide to the primer; and (b) removing the blocking group from the 3' end of the primer-blocked nucleotide product to form a primer-nucleotide product. Phosphate is generated in at least one synthetic step. A precipitate is formed in the cycle comprising phosphate and at least one precipitation cation. The precipitation of phosphate reduces its unfavorable effect on the method. Preferably, cycles of the method are repeated without intermediate purification of primer-nucleotide product or precursor. The precipitation cation may be a polyvalent elemental cation, spermine, or a cation which forms a poorly soluble salt with phosphate. Preferably, the chain extending enzyme is RNA Ligase, the blocked nucleotide is AppNp, and the blocking group is removed using a phosphatase. There is also provided a method for reducing the inhibitory effect of nucleoside 5'-monophosphate and of 3',5'-nucleoside diphosphate on phosphodiesterase I. This is accomplished by enzymatically converting these inhibitors to less inhibitory products.

Dependencies are inferred from the claim text.