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Key to catalytic function of RNA is its folding into a precise three-dimensional (native) structure, which we study using the following techniques.
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One of the techniques we use to study the folding of ribozymes is fluorescence resonance energy transfer (FRET). Here, two fluorophores, called donor and acceptor, are chemically coupled to defined sites on the RNA (Fig. 1). If these sites get closer upon folding of the RNA, then the resonance interaction between the fluorophores increases, and more energy is being transferred from donor to acceptor. This results in a change in the steady-state fluorescence signal of the labeled RNA that can be monitored over time to calculate the folding rate constant kfold. |
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Another technique we use to measure distances in catalytic RNA is called time-resolved FRET. Here, we illuminate the donor-acceptor labeled RNA with laser pulses of picosecond duration (Fig. 2) and observe the nanosecond fluorescence decay of the donor. The associated lifetime of the fluorophore in the excited state is dependent on the distance to the acceptor and allows us to precisely measure this distance, or distances, when multiple RNA conformations are present. |
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Fluorescence techniques are very powerful. In a collaboration with Steven Chu at Stanford (Physics department), we have been able to push the detection limit to single RNA molecules. Using the donor-acceptor labeled catalytic RNA depicted in Fig. 1, we have been able to observe time trajectories reflecting the folding of individual ribozyme molecules between two conformations, with low FRET (high donor fluorescence, green) and with high FRET (high acceptor fluorescence, red), respectively (Fig. 3). |
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Yet another single-molecule technique we employ is atomic force microscopy (AFM). AFM obtains data by gently tapping a sharply pointed probe attached to a flexible cantilever in a raster scan over a surface, recording the height deflections of the probe. The collected data can be used to compose a contour plot of the scanned area that may, for example, consist of RNA molecules bound to an atomically flat surface. In this fashion, we have been able to observe individual ribozyme molecules in aqueous buffer (Fig. 4). |
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In summary, we are employing a broad range of techniques from biophysics, chemistry, and molecular biology for the exploration of structure and function of catalytic RNAs. Pursuing this in the top-rated Chemistry department at the University of Michigan in Ann Arbor gives us access to excellent research facilities in a very enjoyable work environment. Any questions or comments? Please
email Nils G. Walter. |
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