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

Agata A Sulej,Krzysztof J Skowronek,Janusz M Bujnicki,Irina Tuszynska,Marcin Nowotny

doi:10.1093/nar/gks885

Agata A Sulej, Krzysztof J Skowronek + Show 3 more

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https://doi.org/10.1093/nar/gks885

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Abstract

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.

Highlights

Our understanding of the importance of RNA molecules in many life processes has changed dramatically in recent years
In order to further decrease the non-specific binding of the DNA–RNA hybrid by the RNase HI catalytic domain, we introduced substitutions of three Lys residues (K138, K146 and K180, numbering as in the original B. halodurans RNase HI sequence) that interact with phosphates of both strands of the substrate in the crystal structure of RNase HI in complex with a DNA– RNA hybrid [6]
The hybrid binding domain (HBD) was eliminated in order to lower the non-specific binding of substrates by the RNase HI moiety and to increase the overall sequence specificity of the fusion enzyme

Summary

Introduction

Our understanding of the importance of RNA molecules in many life processes has changed dramatically in recent years. The first step of analysis often involves the fragmentation of the RNA molecule(s) under study [1]. E.g., Type II restriction endonucleases (REases) that cleave DNA with high specificity toward particular sequences [2], most RNases are inherently non-specific. Those RNases that do exhibit specificity typically recognize either single bases or particular secondary structures in the RNA substrate such as helices, loops or bulges [3]. The procurement of new enzymes that are able to cleave RNA in a purely sequence-dependent manner, similar to REases, would greatly facilitate studies on the structure and function of RNA and manipulation of these molecules for various purposes

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