Suitability of Double-Stranded DNA as a Molecular Standard for the Validation of Analytical Ultracentrifugation Instruments

Authors

Maduni Ranasinghe
Jonathan M Fogg
Daniel J Catanese
Lynn Zechiedrich
Borries Demeler

Publication Date

7-1-2023

Journal

European Biophysics Journal

DOI

10.1007/s00249-023-01671-y

PMID

37501021

PMCID

PMC10530205

PubMedCentral® Posted Date

7-27-2024

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes

Keywords

DNA, Superhelical, Reproducibility of Results, DNA, DNA, Circular, Ultracentrifugation, Analytical ultracentrifugation, Molecular standards, Double-stranded DNA, DNA minicircle, Hydrodynamics, Global analysis

Abstract

To 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 from bacteria is unsuitable as a molecular standard. Linear and nicked samples are well suited as a molecular standard for AUC and have exceptional colloidal stability in an AUC cell. Even after sixty experiments at different speeds and temperatures, measured over the course of 4 months, all topological states of DNA remained colloidal, and their concentrations remained essentially unchanged.

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