Analysis of birefringence decay profiles for nucleic acid helices possessing bends: the tau-ratio approach

Abstract

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.