Martin Kenward

The theory of DNA separation by capillary electrophoresis

The Human Genome has been sequenced in large part owing to the invention of capillary electrophoresis. Although this technology has matured enough to allow such amazing achievements, the physical mechanisms at play during separation have yet to be completely understood and optimized. Recently, new separation regimes and new physical mechanisms have been investigated. The use of free-flow electrophoresis and new modes of pulsed-field electrophoresis have been suggested, while we have observed a shift towards single nucleotide polymorphism analysis and microchip technologies. A strong theoretical basis remains essential for the efficient development of new methods.

Brownian dynamics simulations of single-stranded DNA hairpins

We present a Brownian dynamics model which we use to study the kinetics and thermodynamics of single-stranded DNA hairpins, gaining insights into the role of stem mismatches and the kinetics rates underlying the melting transition. The model is a base-backbone type in which the DNA bases and sugar-phosphate backbone are represented as single units (beads) in the context of the Brownian dynamics simulations. We employ a minimal number of bead-bead interactions, leading to a simple computational scheme. To demonstrate the veracity of our model for DNA hairpins, we show that the model correctly captures the effects of base stacking, hydrogen bonding, and temperature on both the thermodynamics and the kinetics of hairpin formation and melting. When cast in dimensionless form, the thermodynamic results obtained from the present model compare favorably with default predictions of the m-fold server, although the present model is not sufficiently robust to provide dimensional results. The kinetic data at low temperatures indicate frequent but short-lived opening events, consistent with the measured chain end-to-end probability distribution. The model is also used to study the effect of base mismatches in the stem of the hairpin. With the parameters used here, the model overpredicts the relative shift in the melting temperature due to mismatches. The melting transition can be primarily attributed to a rapid increase in the hairpin opening rate rather than an equivalent decrease in the closing rate, in agreement with single-molecule experimental data.

Coarse-Grained Brownian Dynamics Simulations of the 10-23 DNAzyme

Deoxyribozymes (DNAzymes) are single-stranded DNA that catalyze nucleic acid biochemistry. Although a number of DNAzymes have been discovered by in vitro selection, the relationship between their tertiary structure and function remains unknown. We focus here on the well-studied 10-23 DNAzyme, which cleaves mRNA with a catalytic efficiency approaching that of RNase A. Using coarse-grained Brownian dynamics simulations, we find that the DNAzyme bends its substrate away from the cleavage point, exposing the reactive site and buckling the DNAzyme catalytic core. This hypothesized transition state provides microscopic insights into experimental observations concerning the size of the DNAzyme/substrate complex, the impact of the recognition arm length, and the sensitivity of the enzymatic activity to point mutations of the catalytic core. Upon cleaving the pertinent backbone bond in the substrate, we find that the catalytic core of the DNAzyme unwinds and the overall complex rapidly extends, in agreement with experiments on the related 8-17 DNAzyme. The results presented here provide a starting point for interpreting experimental data on DNAzyme kinetics, as well as developing more detailed simulation models. The results also demonstrate the limitations of using a simple physical model to understand the role of point mutations.

Deformation, Stretching, and Relaxation of Single‐Polymer Chains: Fundamentals and Examples

#From the forthcoming book, Soft Materials: Structure, and Dynamics, Marangoni, A. G. and Dutcher, J., Eds., Marcel Dekker, Inc., in press.

A boundary element method/Brownian dynamics approach for simulating DNA electrophoresis in electrically insulating microfabricated devices

We present an approach for merging boundary element method (BEM) solutions of the electric field in electrically insulating complex geometries with Brownian dynamics (BD) simulations of DNA electrophoresis therein. Although a rote application of the standard BEM algorithm proves inaccurate and prohibitively expensive, we show that regularization of the near‐wall electric field and an updating scheme commensurate with the characteristic length scale of the BD simulation furnishes a robust, efficient simulation protocol. The accuracy of the BEM‐BD method is verified by simulating λ‐DNA collisions with an isolated, insulating cylindrical obstacle and comparing the results with equivalent BD simulations that employ the exact solution for the electric field. The computational overhead of our implementation of BEM‐BD is comparable to an existing finite element method/BD approach. The BEM‐BD algorithm is readily parallelized and well‐suited to time‐dependent and responsive electric fields, making it broadly applicable to simulating DNA electrophoresis in microfluidic devices.