Suitability of double-stranded DNA as a molecular standard for the validation of analytical ultracentrifugation instruments

Eur Biophys J. 2023 Jul 27. doi: 10.1007/s00249-023-01671-y. Online ahead of print.ABSTRACTTo address the current lack of validated molecular standards for analytical ultracentrifugation (AUC), we investigated the suitability of double-stranded DNA molecules. We compared the hydrodynamic properties of linear and circular DNA as a function of temperature. Negatively supercoiled, nicked, and linearized 333 and 339 bp minicircles were studied. We quantified the hydrodynamic properties of these DNAs at five different temperatures, ranging from 4 to 37 °C. To enhance the precision of our measurements, each sample was globally fitted over triplicates and five rotor speeds. The exceptional stability of DNA allowed each sample to be sedimented repeatedly over the course of several months without aggregation or degradation, and with excellent reproducibility. The sedimentation and diffusion coefficients of linearized and nicked minicircle DNA demonstrated a highly homogeneous sample, and increased with temperature, indicating a decrease in friction. The sedimentation of linearized DNA was the slowest; supercoiled DNA sedimented the fastest. With increasing temperature, the supercoiled samples shifted to slower sedimentation, but sedimented faster than nicked minicircles. These results suggest that negatively supercoiled DNA becomes less compact at higher temperatures. The supercoiled minicircles, as purified from bacteria, displayed heterogeneity. Therefore, supercoiled DNA isolated ...

Source: European Biophysics Journal : EBJ - July 27, 2023 Category: Physics Authors: Maduni Ranasinghe Jonathan M Fogg Daniel J Catanese Lynn Zechiedrich Borries Demeler Source Type: research