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. 2009:(53):279-80.
doi: 10.1093/nass/nrp140.

Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease

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Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease

Yusuke Mineta et al. Nucleic Acids Symp Ser (Oxf). 2009.

Abstract

To refine zinc-finger nuclease (ZFN) technology, we constructed a sandwiched ZFN, in which a DNA cleavage enzyme was sandwiched with two artificial zinc-finger proteins (AZPs). Because the sandwiched ZFN is designed to cleave the DNA between the two AZP-binding sites, the sandwiched ZFN is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To prove the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two 3-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with control nucleases that possess a single AZP.

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