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Abstract-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, guanidinoneomycin B, and [Á-(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, [Á-(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 500 mM. #

Research experiences are widely understood to be valuable for the intellectual and professional development of undergraduate science students. Course-based undergraduate research experiences (CUREs) have become popular as a means of engaging large numbers of students in research by leveraging institutional supports for laboratory courses. At Willamette University, we have adapted a guided-inquiry experiment that gave unexpected results into a CURE in the lower-division organic chemistry curriculum. This experience has engaged several cohorts of students in a 7 week investigation focused on identifying the mechanistic origin of a nonstereospecific hydroxybromination of Z-stilbene, an observation that contradicts how this addition reaction is presented in standard organic chemistry curricula. Each year, the cohort experimentally “discovers” the anomalous stereochemical outcome of the addition reaction. Students then participate in a workshop focused on developing mechanistic hypotheses that can account for the result and on designing experiments that will address their hypotheses. Over the next three lab sessions, students execute their experiments, reconsidering and revising their hypotheses as their data dictate. The experience culminates in formal oral and written presentations of results. Student perceptions of the pedagogical value of the CURE, the effect of the lab on their self-efficacy, and the novelty of research being conducted were assessed in multiple class cohorts, while faculty perceptions of changes in student behavior and skill were also documented.

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