Abstract
Pepocin, isolated from Cucurbita pepo, is a ribosome-inactivating protein (RIP). RIPs site-specifically recognize and depurinate an adenosine at position 4324 in rat 28 S rRNA, rendering the ribosome incapable of interacting with essential elongation factors. Aptamers that target pepocin were isolated from a degenerate RNA pool by in vitro selection. A conserved hairpin motif, quite different from the sequence of the toxin-substrate domain in rat 28 S rRNA, was identified in the aptamer sequences. The aptamers selectively bind to pepocin with dissociation constants between 20 and 30 nM and inhibit the N-glycosidase activity of pepocin on rat liver 28 S rRNA. Competitive binding experiments using aptamer variants suggest that the conserved hairpin region in the anti-pepocin aptamer binds near the catalytic site on pepocin and prevents the interaction of pepocin and 28 S rRNA. Anti-RIP aptamers have potential use in diagnostic systems for the detection of pepocin or could be used as therapy to prevent the action of pepocin in mammalian cells.
Highlights
Ribosome-inactivating proteins (RIPs)1 are widely distributed throughout nature
Type II RIPs are heterodimeric proteins; an A-chain catalyzes the depurination of a specific adenosine in rRNA, and a disulfide linked B-chain binds cell surface galactosides
These results illustrate the importance of both sequence and structure for recognition in RNA-protein complexes; studies of the contacts between RNA hairpins and RIPs are informative as a model system for scrutinizing RNA-protein interactions
Summary
Ribosome-inactivating proteins (RIPs)1 are widely distributed throughout nature (for review, see Ref. 1). Aptamers that target pepocin were isolated from a degenerate RNA pool by in vitro selection. The aptamers selectively bind to pepocin with dissociation constants between 20 and 30 nM and inhibit the N-glycosidase activity of pepocin on rat liver 28 S rRNA.
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