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Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. Here, we report the development of a new enzyme that cleaves the RNA strand in DNA-RNA hybrids 5 nt from a nonanucleotide recognition sequence. The enzyme was constructed by fusing two functionally independent domains, a RNase HI, that hydrolyzes RNA in DNA-RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA-RNA hybrids. The optimization of the fusion enzyme's specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.

Sequence-specific cleavage of the RNA strand in DNA–RNA hybrids by the fusion of ribonuclease H with a zinc finger