Generation of artificial sequence-specific nucleases via a preassembled inert-template

Sequence specific nucleases are important tools for processing nucleic acids in a predictable way. Herein, we demonstrate a conceptually new approach for generating sequence-specific nucleases via a preassembled inert-template (PAIT). A fairly stable DNase I/inert-DNA complex was prepared with a customized sequence specificity for a target DNA which contains a sequence complementary to the inert-DNA template. The complex could efficiently cleave the targeted sequence within either a long double-stranded DNA or a single-stranded DNA without affecting other unrelated DNA strands. The discrimination factor against single-base mismatch strands within a 14 nt target region was as high as 25.3. The strategy was also successfully applied to RNase A. Our findings may hold great potential for the development of a number of new powerful enzymatic tools.