Generation of a hybrid sequence-specific single-stranded deoxyribonuclease

The relatively nonspecific single-stranded deoxyribonuclease, staphylococcal nuclease, was selectively fused to an oligonucleotide binding site of defined sequence to generate a hybrid enzyme. A cysteine was substituted for Lys¹¹⁶ in the enzyme by oligonucleotide-directed mutagenesis and coupled to an oligonucleotide that contained a 3′-thiol. The resulting hybrid enzyme cleaved single-stranded DNA at sites adjacent to the oligonucleotide binding site.