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To assess the specificity of ribozyme binding, streptavidin-coated surfaces were incubated with ribozyme bound to biotinylated and non-biotinylated tethers. The former gave 100-fold greater surface density of fluorescently labeled molecules than the latter, demonstrating that the biotinylated ribozyme binds to the surface predominately through the biotin-streptavidin interaction.
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25
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In the total internal reflection microscope, fluorescent molecules were excited by an evanescent wave generated by total internal reflection of a 514-nm laser beam and detected by a CCD (charge-coupled device) camera (23). The microscope could detect several hundred individual molecules simultaneously with a time resolution of 100 ms. The cleavage kinetics and overall folding kinetics were measured using this apparatus. In the scanning confocal microscope, fluorescent molecules were excited by a focused 514-nm laser beam and detected by avalanche photodiodes. This apparatus can measure the fluorescence emission of one molecule with a time resolution of 1 ms. The docking kinetics were measured using this apparatus.
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27
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32P]-labeled S. After ≥1 min for binding of S, cleavage was initiated by addition of G. Reactions measuring the rate constant for S binding were carried out as previously described (26).
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33
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o is typically between 3 and 7 nm, depending on the spectral overlap between the donor emission and acceptor absorption, quantum yield of the donor, and the orientations of the donor and acceptor. Thus, the FRET efficiency strongly depends on the distance between donor and acceptor as well as their orientations. Although there is a strong expectation of a large decrease in distance between the donor and acceptor upon Pl docking or overall folding, the conclusions here are independent of whether the FRET efficiency responds to changes in distance, orientation, or both.
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0342896665
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Previous studies of the rate constants for docking (35, 45), using different substrates under different conditions, yielded values within the same order of magnitude as reported here.
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45
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0342896664
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-1, the same as the cleavage rate constant obtained from prefolded ribozyme.
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49
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0342462591
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note
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We are grateful to S. A. Scaringe and J. A. Orr for their advice on oligonucleotide preparation. This research was supported by an NSF grant (PHY-9970017) and an Air Force Office of Scientific Research grant (F49620-98-1-0219) to S.C. and an NIH grant (GM49423) to D.H. X.Z. was supported by the Chodorow Fellowship of the Stanford Applied Physics Department and an NIH postdoctoral fellowship. L.E.B. was supported in part by an NIH training grant. H.P.B. was supported in part by a Center on Polymer Interfaces and Macromolecular Assemblies grant. R.R. was supported by an NIH postdoctoral fellowship.
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