Vacano E, Hagerman P J
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver 80262, USA.
Biophys J. 1997 Jul;73(1):306-17. doi: 10.1016/S0006-3495(97)78071-8.
For nucleic acid helices in the 100-200-bp range, a central bend or point of flexibility increases the rate of rotational diffusion. In a transient electric birefringence (TEB) experiment, this increase is manifest as a reduction in the terminal (slowest) birefringence decay time. Previous experimental and theoretical work has demonstrated that the ratio of the decay times for a bent/flexible molecule and its fully duplex (linear) counterpart represents a sensitive, quantifiable measure of the apparent bend angle (tau-ratio approach). In the current work, we have examined the influence of helix parameters (e.g., persistence length, helix rise, diameter) on the tau-ratio for a given bend. The tau-ratio is found to be remarkably insensitive to variations and/or uncertainties in the helix parameters, provided that one employs bent and control molecules with the same sequence and length (apart from the bend itself). Although a single tau-ratio determination normally does not enable one to distinguish between fixed and flexible bends, such a distinction can be made from a set of tau-ratios for molecules possessing two variably phased bends. A number of additional uncertainties are examined, including errors in the estimation of the dimensions of nonhelix elements that are responsible for bends; such errors can, in principle, be estimated by performing a series of measurements for molecules of varying length.
对于长度在100 - 200个碱基对范围内的核酸螺旋，其中心弯曲或柔韧性点会增加旋转扩散速率。在瞬态电双折射（TEB）实验中，这种增加表现为末端（最慢）双折射衰减时间的缩短。先前的实验和理论工作表明，弯曲/柔性分子与其完全双链（线性）对应物的衰减时间之比代表了表观弯曲角度的一种灵敏、可量化的度量（τ-比率法）。在当前工作中，我们研究了螺旋参数（例如，持续长度、螺旋上升、直径）对给定弯曲的τ-比率的影响。结果发现，只要使用具有相同序列和长度（除弯曲本身外）的弯曲分子和对照分子，τ-比率对螺旋参数的变化和/或不确定性非常不敏感。尽管单次τ-比率测定通常无法区分固定弯曲和柔性弯曲，但对于具有两个可变相位弯曲的分子，可以通过一组τ-比率来进行这种区分。还研究了许多其他不确定性，包括对导致弯曲的非螺旋元件尺寸估计中的误差；原则上，可以通过对不同长度的分子进行一系列测量来估计此类误差。
