| Abstract: |
A deeper understanding of how different topoisomers of DNA minicircles behave in solution is immensely beneficial in the fields of gene expression, DNA replication, gene therapy, and essentially every aspect of DNA structure and function. In this study, we investigated the hydrodynamic properties of DNA minicircle topoisomers. A 336 bp minicircle (mc336) was measured as a linear, relaxed, nicked, supercoiled and hyper-negatively supercoiled topoisomer. A 672 bp minicircle (mc672) was measured in a nicked and supercoiled conformation. All experiments were performed at 20°C at five different rotor speeds of 10, 14, 25, 35, and 45 krpm to maximize sedimentation and diffusion signal. A global analysis was performed over all speeds to simultaneously capture the improved signal from all experiments.
We show how prior knowledge of the sequence-derived molar masses of each minicircle can be combined with the measured hydrodynamic parameters (sedimentation and diffusion coefficients), to derive both the anisotropy and the partial specific volume (PSV) of different DNA topoisomers. Our measurements indicate that the PSV is largely constant within the error of measurement for different topoisomers of DNA, and can be reliably determined with good reproducibility. Furthermore, the PSV appears to be independent of size, molar mass and anisotropy under buffer conditions used in this experiment. The anisotropy can be used to reliably predict the supercoiling state of minicircles, and a prediction for the change in anisotropy as a function of mass is provided. |
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