2-Aminopurine as a Real-Time Probe of Enzymatic Cleavage and Inhibition of Hammerhead Ribozymes
Sarah R. Kirk, Nathan W. Luedtke and Yitzhak Tor
The design, synthesis and study of internally fluorescent hammerhead (HH) ribozymes, where changes in fluorescence parameters directly reflect the progress of the ribozyme's cleavage chemistry, are described. The approach relies on a HH substrate modified at position 1.1, proximal to the cleavage site, with 2-aminopurine (2AP), an intensely fluorescent adenosine isoster. The incorporation of 2AP, an unnatural nucleoside, does not interfere with the ribozyme folding and catalysis. Since 2AP is highly sensitive to environmental changes, its fluorescence is dramatically altered upon ribozyme-mediated cleavage of the substrate. This generates a measurable signal that directly reflects the progress of the ribozyme's reaction in real time. Identical pseudo first order rate constants are obtained for HH constructs using both continuous fluorescence monitoring and radioactive labeling. This rapid and real-time monitoring facilitates the study of ribozyme activity under different conditions (e.g., ionic strength, pH, etc.), and provides a useful assay to rapidly screen potential inhibitors. Three hitherto unknown HH inhibitors are presented and compared to neomycin B and chlortetracycline, two previously studied HH inhibitors. All three new small molecules, neo-acridine, guanidino-neomycin B, and [D-(Eilatin)Ru(bpy)2]2+, prove to be better inhibitors than neomycin B or chlortetracycline. Investigating HH inhibition under different ionic strengths reveals that the binding of neo-acridine, [D-(Eilatin)Ru(bpy)2]2+, and chlortetracycline to the HH involves hydrophobic interactions as their RNA affinities are largely unaffected by increasing salt concentrations. In contrast, neomycin B loses more than 50-fold of its inhibitory ability as the NaCl concentration is increased from 50 to 500mM.