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Perturbed or Not Perturbed?
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In this book structural and dynamic perturbations in DNA helices upon incorporation of fluorophores are characterized by NMR spectroscopy. Two fluorophores are examined in detail: 2-Aminopurine (2AP) and 2-Hydroxy-7-nitrofluorene(HNF) which function as base and base pair surrogates, respectively. Improved protocols for NMR data analysis and subsequent calculations are described. State-of-the-art NMR experiments such as measurement of Residual Dipolar Couplings, are demonstrated to enable detection of hitherto unknown, very slight structural perturbations of a DNA helix around the 2AP modification site. Advances at the experimental as well as data analysis levels are shown to allow for determination of base pair lifetimes 1 ms. Thus it has been possible to prove an extended, distributed effect of 2AP incorporation on the dynamics of at least three adjacent base pairs in either direction, in spite of the small structural influence. The HNF has been introduced as a base pair surrogate but proved to be of insufficient shape complementarity to a natural Watson-Crick-type base pair. Thus significant structural as well as dynamic perturbation around the incorporation site are observed.
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