Department of Physics, University of Oxford, Oxford, OX1 3PU, UK.
Kavli Institute for Nanoscience Discovery, University of Oxford, Dorothy Crowfoot Hodgkin Building, Oxford, OX1 3QU, UK.
Chemphyschem. 2023 Jun 15;24(12):e202300175. doi: 10.1002/cphc.202300175. Epub 2023 Apr 12.
Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.
荧光探针的光漂白限制了典型的单分子荧光测量的观察时间范围，并阻碍了长时间尺度上的动力学观察。在这里，我们提出了一种通过将荧光 DNA 与附着在靶分子上的互补 DNA 链短暂结合来补充荧光探针的通用策略，以规避光漂白。我们的策略允许观察近连续的单分子荧光超过一个小时，这一时间尺度比我们条件下单个荧光团的典型光漂白时间长两个数量级。使用两个正交序列，我们表明我们的方法适用于Förster 共振能量转移 (FRET)，并且可以用于研究动态结构（如 DNA 霍利迪结）的构象动力学，持续时间较长。通过调整时间分辨率和观察范围，我们的方法能够在广泛的时间尺度内捕获蛋白质和核酸的构象动力学。
