Measuring the folding transition time of single RNA molecules
Measuring the folding transition time of single RNA molecules
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DOI：
10.1529/biophysj.106.094623
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发表时间：
2007-05-01
影响因子：
3.4
通讯作者：
Chu, Steven
中科院分区：
文献类型：
--
作者：
Lee, Tae-Hee;Lapidus, Lisa J.;Chu, Steven
我们描述了一种新的时间孔径光子相关方法，用于解决RNA折叠中两种状态之间的跃迁时间，即，状态之间的转换时间，而不是在每个状态中花费的时间。单分子荧光共振能量转移和荧光相关光谱被用来获得这些测量。单个RNA分子用荧光团如Cy 3和Cy 5标记。然后将这些分子固定在表面上并观察许多秒，在此期间，分子自发地在具有不同水平的荧光共振能量转移效率的两种构象状态之间切换。从每个荧光团计数单个光子，并在跃迁周围的小窗口中交叉相关。超过1000个互相关的平均值可以拟合成多项式，这可以确定与平均光子发射间隔一样短的跃迁时间。我们将该方法应用于四膜虫I组自我剪接内含子的P4-P6结构域，得到240 μ s的折叠过渡时间。发现展开时间太短而不能用这种方法测量。
We describe a new, time-apertured photon correlation method for resolving the transition time between two states of RNA in folding-i.e., the time of the transition between states rather than the time spent in each state. Single molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy are used to obtain these measurements. Individual RNA molecules are labeled with fluorophores such as Cy3 and Cy5. Those molecules are then immobilized on a surface and observed for many seconds during which time the molecules spontaneously switch between two conformational states with different levels of flourescence resonance energy transfer efficiency. Single photons are counted from each fluorophore and cross correlated in a small window around a transition. The average of over 1000 cross correlations can be fit to a polynomial, which can determine transition times as short as the average photon emission interval. We applied the method to the P4-P6 domain of the Tetrahymena group I self-splicing intron to yield the folding transition time of 240 mu s. The unfolding time is found to be too short to measure with this method.