Edman L, Mets U, Rigler R
Department of Medical Biophysics, Karolinska Institute, Stockholm, Sweden.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6710-5. doi: 10.1073/pnas.93.13.6710.
Phenomena that can be observed for a large number of molecules may not be understood if it is not possible to observe the events on the single-molecule level. We measured the fluorescence lifetimes of individual tetramethylrhodamine molecules, linked to an 18-mer deoxyribonucleotide sequence specific for M13 DNA, by time-resolved, single-photon counting in a confocal fluorescence microscope during Brownian motion in solution. When many molecules were observed, a biexponential fluorescence decay was observed with equal amplitudes. However, on the single-molecule level, the fraction of one of the amplitudes spanned from 0 to unity for a collection of single-molecule detections. Further analysis by fluorescence correlation spectroscopy made on many molecules revealed a process that obeys a stretched exponential relaxation law. These facts, combined with previous evidence of the quenching effect of guanosine on rhodamines, indicate that the tetramethylrhodamine molecule senses conformational transitions as it associates and dissociates to a guanosine-rich area. Thus, our results reveal conformational transitions in a single molecule in solution under conditions that are relevant for biological processes.
如果无法在单分子水平上观察事件，那么对于大量分子可观察到的现象可能就无法理解。我们通过共聚焦荧光显微镜中的时间分辨单光子计数，在溶液中的布朗运动过程中，测量了与M13 DNA特异的18聚体脱氧核糖核苷酸序列相连的单个四甲基罗丹明分子的荧光寿命。当观察许多分子时，观察到具有相等幅度的双指数荧光衰减。然而，在单分子水平上，对于一系列单分子检测，其中一个幅度的分数范围从0到1。对许多分子进行的荧光相关光谱进一步分析揭示了一个遵循拉伸指数弛豫定律的过程。这些事实，结合先前关于鸟苷对罗丹明淬灭作用的证据，表明四甲基罗丹明分子在与富含鸟苷的区域缔合和解离时感知构象转变。因此，我们的结果揭示了在与生物过程相关的条件下溶液中单个分子的构象转变。
