Jung Jaemyeong, Ihly Rachelle, Scott Eric, Yu Ming, Van Orden Alan
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, USA.
J Phys Chem B. 2008 Jan 10;112(1):127-33. doi: 10.1021/jp076248t. Epub 2007 Dec 13.
The conformational fluctuations of dye-quencher labeled DNA hairpin molecules in aqueous solution were investigated using dual probe beam fluorescence fluctuation spectroscopy. The measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset optical probe regions, the absolute and relative concentrations of each conformational substate of the DNA, and the kinetics of the DNA hairpin folding and unfolding reactions in the 1 micros to 10 ms time range. A DNA hairpin containing a 21-nucleotide polythymine loop and a 4-base pair stem exhibited double exponential relaxation kinetics, with time constants of 84 and 393 micros. This confirms that folding and melting of the DNA hairpin structure is not a two state process but proceeds by way of metastable intermediate states. The fast time constant corresponds to formation and unfolding of an intermediate, and the slow time constant is due to formation and disruption of the fully base-paired stem. This is consistent with a previous study of a similar DNA hairpin with a 5-base pair stem, in which the fast reaction was attributed to the fluctuations of an intermediate DNA conformation [J. Am. Chem. Soc. 2006, 128, 1240-1249]. In that case, reactions involving the native conformation could not be observed directly due to the limited observation time range of the fluorescence correlation spectroscopy experiment. The intermediate states of the DNA hairpins are suggested to be due to a collapsed ensemble of folded hairpins containing various partially folded or misfolded conformations.
使用双探针光束荧光涨落光谱法研究了染料猝灭剂标记的DNA发夹分子在水溶液中的构象涨落。测量揭示了DNA分子通过两个空间偏移的光学探针区域的流动和扩散时间、DNA每个构象亚态的绝对和相对浓度，以及在1微秒到10毫秒时间范围内DNA发夹折叠和展开反应的动力学。一个含有21个核苷酸的聚胸腺嘧啶环和一个4碱基对茎的DNA发夹呈现出双指数弛豫动力学，时间常数分别为84和393微秒。这证实了DNA发夹结构的折叠和熔解不是一个两态过程，而是通过亚稳中间态进行的。快速时间常数对应于一个中间体的形成和展开，而缓慢时间常数则归因于完全碱基配对茎的形成和破坏。这与之前对一个具有5碱基对茎的类似DNA发夹的研究一致，在该研究中，快速反应归因于中间DNA构象的涨落[《美国化学会志》2006年，128卷，1240 - 1249页]。在那种情况下，由于荧光相关光谱实验的观测时间范围有限，涉及天然构象的反应无法直接观测到。DNA发夹的中间态被认为是由于包含各种部分折叠或错误折叠构象的折叠发夹的塌缩集合。
