Scalettar B A, Selvin P R, Axelrod D, Hearst J E, Klein M P
Department of Biophysics, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Biophys J. 1988 Feb;53(2):215-26. doi: 10.1016/S0006-3495(88)83083-2.
We have conducted a polarized fluorescence photobleaching recovery (FPR) study of the rotational dynamics of ethidium azide labeled DNA. Polarized photobleaching experiments provide data on microsecond and millisecond molecular reorientation that complement the information available from nanosecond fluorescence depolarization studies. In polarized FPR experiments an anisotropic angular concentration of fluorophore is created by bleaching dye molecules in a preferred orientation with a short, intense pulse of polarized light. The sample is then weakly illuminated, and the temporal variation in the emitted fluorescence is monitored. The fluorescence signal will systematically change as molecules undergo post-bleach reorientation and the angular distribution of dye tends toward isotropy. We have observed that the time dependence of our microsecond FPR curves is also determined in part by nonrotational phenomena. To isolate the reorientational recovery we conduct our FPR experiments in two modes (called parallel and perpendicular) that differ only in the polarization of the bleaching light. A quotient function, R(t), is constructed from the data obtained in these two modes; the variation with time of this new quantity is governed solely by processes that are sensitive to the polarization of the incident light (e.g., molecular rotation). It is found experimentally that R(t) remains constant, as expected, for rotationally restricted DNA systems despite a temporal recovery in the parallel and perpendicular FPR curves. We also follow the dynamics of solutions of phage lambda DNA as revealed in the temporal dependence of R(t). This DNA system rotationally relaxes after approximately 100 microseconds and the dye/DNA complex reorients substantially during the 10-microseconds bleach period. Our FPR data are interpreted in terms of dynamic models of DNA motion.
我们进行了一项关于叠氮化乙锭标记DNA旋转动力学的偏振荧光光漂白恢复（FPR）研究。偏振光漂白实验提供了关于微秒和毫秒级分子重新定向的数据，这些数据补充了纳秒级荧光去极化研究中可得的信息。在偏振FPR实验中，通过用短而强的偏振光脉冲以优选取向漂白染料分子，产生荧光团的各向异性角浓度。然后对样品进行弱照明，并监测发射荧光的时间变化。随着分子经历漂白后重新定向且染料的角分布趋向各向同性，荧光信号将系统性地改变。我们观察到，我们的微秒级FPR曲线的时间依赖性部分也由非旋转现象决定。为了分离重新定向恢复，我们以两种模式（称为平行和垂直模式）进行FPR实验，这两种模式仅在漂白光的偏振方面有所不同。从这两种模式获得的数据构建一个商函数R(t)；这个新量随时间的变化仅由对入射光偏振敏感的过程（例如分子旋转）控制。实验发现，对于旋转受限的DNA系统，尽管平行和垂直FPR曲线有时间上的恢复，但R(t)如预期的那样保持恒定。我们还跟踪了噬菌体λ DNA溶液的动力学，这在R(t)的时间依赖性中有所体现。这个DNA系统在大约100微秒后旋转弛豫，并且染料/DNA复合物在10微秒的漂白期内大幅重新定向。我们的FPR数据根据DNA运动的动力学模型进行解释。
