Probing the Ionic Atmosphere of Single-Stranded DNA Using Continuous Flow Capillary Electrophoresis and Fluorescence Correlation Spectroscopy

Abstract

Two-beam fluorescence cross-correlation spectroscopy coupled with continuous flow capillary electrophoresis (2bFCCS-CFCE) was used to study the relationship between diffusion and effective charge of a fluorescently labeled 40-base polythymine single-stranded DNA (ssDNA) as a function of Mg2+ concentration. Cross-correlation analysis of the fluorescence monitored from two spatially offset microscopic detection volumes revealed the diffusion and electrophoretic migration of ssDNA at a range of Mg2+ concentrations and electric field strengths. The effective charge of the ssDNA could then be determined using simple calculations. It was found that as the Mg2+ concentration in the buffer solution increased, the diffusion of the ssDNA also increased, while the effective charge of the ssDNA decreased. This was believed to be caused by increased association of the Mg2+ counterions with the negatively charged backbone of the ssDNA, which partially neutralized the negatively charged functional groups and allowed the ssDNA to adopt a more compact structure. To our knowledge, this is the first demonstration of the measurement of effective charge of ssDNA in relation to Mg2+ concentration.