The ability to rapidly identify small molecules that interact with RNA would have significant clinical and research applications. Low-molecular-weight molecules that bind to RNA have the potential to be used as drugs. Therefore, technologies facilitating the rapid and reliable identification of such activities become increasingly important. We have applied a fluorescence-based assay to screen for modulators of hammerhead ribozyme (HHR) catalysis from a small library of antibiotic compounds. Several unknown potent inhibitors of the hammerhead cleavage reaction were identified and further characterized. Tuberactinomycin A, for which positive cooperativity of inhibition in vitro was found, also reduced ribozyme cleavage in vivo. The assay is applicable to the screening of mixtures of compounds, as inhibitory activities were detected within a collection of 2,000 extracts from different actinomycete strains. This approach allows the rapid, reliable, and convenient identification and characterization of ribozyme modulators leading to insights difficult to obtain by classical methodology.
Low concentrations of the antibiotic doxycycline are sufficient to control the activity of an allosteric hammerhead ribozyme (shown schematically). This was obtained by allosteric selection of an RNA library, which was fused to helix II of the ribozyme. Such molecular switches may serve as a valuable tool for the development of tailored conditional gene expression systems that can be controlled by the presence or absence of any kind of small molecule.
The ribozyme we have isolated is an example of a catalytic RNA with ester transferase activity which uses a substrate that is not templated by Watson-Crick-pairing hydrogen bonds. The reaction catalyzed by the ribozyme expands the scope of RNA catalysis to include acyl transferase activity from an RNA 3' end to an internal 2' position and the reverse. Ribozymes with such activity have been postulated to be evolutionary precursors of ribosomal RNA.
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