Kim Jiho, Doose Sören, Neuweiler Hannes, Sauer Markus
Applied Laser Physics and Laser Spectroscopy, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany.
Nucleic Acids Res. 2006 May 10;34(9):2516-27. doi: 10.1093/nar/gkl221. Print 2006.
Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.
通过监测鸟苷残基（dG）对恶嗪荧光团MR121的接触诱导荧光猝灭，研究了单链DNA（ssDNA）寡核苷酸在水溶液中的构象波动。我们应用荧光相关光谱以及稳态和时间分辨荧光光谱来分析DNA发夹折叠的动力学。我们首先通过研究MR121与鸟苷单磷酸在水溶液中的双分子猝灭相互作用来表征报告系统，估计猝灭复合物形成的速率常数、效率和稳定性。然后，我们研究了MR121与位点特异性掺入DNA发夹中的dG残基之间复合物形成的动力学。为了揭示DNA发夹折叠的初始步骤，我们研究了携带一个或两个互补碱基对（dC-dG对）的ssDNA中的复合物形成，这些碱基对可以杂交形成短茎。我们的数据表明，单个dC-dG对的掺入导致打开和关闭动力学的非指数衰减，并使速率常数降低一到两个数量级。我们发现正的活化焓与dC-dG对的数量无关。这些结果表明，DNA发夹折叠的速率限制步骤不是由环动力学或茎中的错配决定的，而是由茎和环核苷酸之间的相互作用决定的。
