Investigating Biopolymer Function and Probe Dynamics Using Fluorescence Techniques
Dr. Brent Krueger, Hope College
The Hairpin Ribozyme is a small catalytic RNA, with both endonuclease and ligase activities, that undergoes a large structural change as part of its function. Fluorescence-detected resonance energy transfer (FRET) experiments have provided significant data about the function of the Hairpin Ribozyme, but are limited due to a number of assumptions. To better understand these assumptions and how they impact structural dynamics measured through FRET experiments, we examine model DNA and RNA motifs labeled with fluorescent probes. These motifs, based on the Hairpin Ribozyme, are examined using several spectroscopic fluorescence techniques: bulk steady-state, bulk time-resolved, and single-molecule, all of which provide FRET data. These methods are utilized to determine how the limiting FRET assumptions affect results and how the dynamics of the fluorescent labels contribute to the overall dynamics measured in FRET experiments. Thus far, our experiments have centered on the fluorescent probes Cy3 and Cy5. Preliminary analysis has demonstrated consistent FRET values among the three methods.
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