Site-selective cleavage of structured RNA by a staphylococcal nuclease-DNA hybrid.

Abstract

A hybrid enzyme consisting of an oligodeoxyribonucleotide fused to a unique site on staphylococcal nuclease site-selectively cleaves a number of natural RNAs including Escherichia coli Ml RNA (377 bases), 16S rRNA (1542 bases), and yeast tRNAF'he. The oligonucleotide directs the nuclease activity of the enzyme to the nucleotides directly adjacent to the complementary target sequence on the substrate RNA. In the case of Ml RNA, hydrolysis occurs primarily at one phosphodiester bond, converting 50% of the starting material to product. Furthermore, the reaction products can be enzymatically manipulated: tRNArhe was cleaved in the anticodon region and was religated to form the full-length tRNA in high yield. Because the specificity of these hybrid enzymes can be easily altered, they should prove to be useful tools for probing RNA structure and function.The availability of a wide number of restriction enzymes capable of hydrolyzing duplex DNA at defined sequences has tremendously increased our ability to manipulate DNA structure (1). In contrast, studies of RNA structure and function have been hampered by the failure to isolate an analogous class of RNA restriction enzymes from nature. To overcome this obstacle, strategies are being developed for site-selectively hydrolyzing RNAs at specific sites (2-6). One such strategy involves introducing a high degree of specificity into naturally occurring relatively nonspecific enzymes, which efficiently hydrolyze RNA. Oligodeoxyribonucleotide binding sites of defined sequence (7) described (8,9). T4 polynucleotide kinase (11) and T7 RNA polymerase (12) were prepared as described; T4 RNA ligase was purchased from Takara Biomedicals, Japan. Melting temperature experiments (13) were performed with a Gilford 250 spectrophotometer in 50 mM sodium phosphate/50 mM NaCl/0.1 mM EGTA, pH 6.5; the temperature was increased from 100C to 90TC at a rate of 1.0LC/min. RNA Preparation. All RNAs were end-labeled on either the 5' termini with T4 polynucleotide kinase and ['y-32P]ATP (14) or the 3' termini with T4 RNA ligase and 5'-[32P]pCp (15).After purification from 7 M urea/5-8% polyacrylamide (acrylamide/bisacrylamide, 30:1) gels (16), the RNAs were recovered by elution overnight at 25TC in 0.5 M ammonium acetate/10 mM magnesium acetate/i mM EDTA/0.1% NaDodSO4, followed by extraction with an equal volume of chloroform/isoamyl alcohol (24:1) and two ethanol precipitations.E. coli Ml RNA was obtained by runoff transcription of SnaBI-linearized plasmid pDW27 with T7 RNA polymerase (17). The RNA product was separated from unincorporated NTPs by anion-exchange chromatography on a Pharmacia Mono Q HR 5/5 column (9) prior to end-labeling. E. coli 16S rRNA was purified on a 1.5% low melting point agarose gel (16)

Generation of a catalytic sequence-specific hybrid DNase

“…However with other substrates, the cleavage patterns were identical, or the enzyme with the direct linkage afforded slightly more specific cleavage. These results may indicate that the nuclease-DNA complex has considerable flexibility and that the structure of the substrate is the major factor in determining the exact nature of cleavage, an observation which has been made previously (Zuckermann et al, 1989).…”

“…We have used a combination of the latter techniques to generate sequence-specific single-stranded DNA and RNA phosphodiesterases. A surface cysteine (K116C) was intro-duced into staphylococcal nuclease by site-directed mutagenesis Zuckermann & Schultz, 1989). The K116C mutation did not significantly alter the catalytic properties of the enzyme but allowed the nuclease to be selectively cross-linked to oligonucleotides via disulfide exchange to generate hybrid nucleases with new specificities.…”

“…The hybrid nucleases selectively hydrolyzed single-stranded DNA and RNA at predefined target sites. This approach offers several potential advantages over oxidative cleavage strategies, most notably the high efficiency of the enzymatic cleavage reaction and the fact that hydrolysis generates termini that can be used in subsequent enzymatic reactions (Zuckermann & Schultz, 1989).…”

Targeted Drug Delivery

“…Their use as mere substitutes to oligodeoxyribonucleotides as antisense moieties could be envisaged as well, although no predictions can be made as far as relative efficiencies are concerned. Interesting results were obtained using hybrid molecules with staphylococcal nuclease (ZUCKERMANN and SCHULTZ 1989) and with the class II restriction endonuclease Fok I (KIM et al 1988). Another interesting prospect is the now well established catalytic activities associated with RNAs, recognition of which resulted from original studies of the sequence specific endoribonuclease activities of Tetrahymena ribozyme (MCSWIGGEN and CECH 1989) or RNase P associated Hi RNA (BARTKIEWICZ et al 1989).…”

Catalytic Knockdown of miR-21 by Artificial Ribonuclease: Biological Performance in Tumor Model

“…However, most of the developed metal-independent aRNases are less active and show low to negligible catalytic turnover (Mironova et al, 2006; Mironova et al, 2007). Hybrid enzymes engineered by the fusion of oligodeoxyribonucleotide to staphylococcal nuclease efficiently hydrolyzed complementary RNA targets but did not leave the substrate after cleavage (Zuckermann and Schultz, 1989). This was overcome by creating a hybrid of Escherichia coli RNase H and a 9-mer oligodeoxynucleotide, when the catalytic turnover of the reaction of sequence-specific cleavage was achieved (Kanaya et al, 1992), but never investigated in vivo .…”