RESUMO
OBJECTIVE: Type C2H2 zinc fingers bind a variety of substrates, specific sequences in the double-stranded DNA counting among them. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA-RNA hybrids using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between the zinc finger modules. RESULTS: The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of the modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA-RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.
Assuntos
DNA/metabolismo , Evolução Molecular Direcionada , RNA/metabolismo , Dedos de Zinco/genética , Biblioteca Gênica , Ligação Proteica , Seleção GenéticaRESUMO
Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage Φ6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , Ribonuclease III/química , Ribonuclease III/genética , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
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.
