Disclosed is a method for synthesizing of stereospecific (Rp) phosphorothioate oligonucleotides. In this method, a primer comprising a plurality of deoxyribonucleotides and a ribonucleotide at the 5' terminal or 5' penultimate position, is annealed to a template. The structure is contacted with a mixture of deoxynucleoside alpha -triphosphate Sp diastereomers and a DNA polymerase to form a PS-Rp oligodeoxynucleotide extension which is liberated as a single-stranded PS-Rp oligonucleotide by cleavage after the ribonucleotide in the primer. Also disclosed are PS-Rp oligonucleotides and oligonucleotides prepared according to this method.
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