A real-time QCM-D approach to monitoring mammalian DNA damage using DNA adsorbed to a polyelectrolyte surface

Abstract

We have successfully demonstrated that the quartz crystal microbalance with dissipation monitoring (QCM-D) can be used to monitor real-time damage to genomic mammalian DNA adsorbed to a polyelectrolyte surface. To reveal the capabilities of this technique, we exposed DNA surfaces to quercetin, an agent that has been implicated in causing DNA strand breaks in a Cu(II)-dependent fashion in vitro. We show that the QCM-D frequency and dissipation patterns that result from exposure of the DNA surfaces to quercetin-Cu(II) are consistent with the induction of DNA strand scission. We use QCM-D to furthermore demonstrate that this process is dependent on Cu(II) and that the DNA damage induced by quercetin can still be detected if Cu(II) is in situ with the DNA surface and not in solution phase.

Figure 1

Gel electrophoresis data demonstrating the copper-dependent DNA damage induced by quercetin. pRS414 plasmid DNA (0.015μg/μL) was incubated at 23°C for 1hr in the presence of the indicated additives: Lane 1, no additive; Lane 2, 0.1mM CuSO₄; Lane 3, 0.1mM quercetin; Lanes 4 and 5, 0.1mM quercetin and 0.1mM CuSO₄. Aliquots of each sample were run on a 0.8% agarose gel and visualized with ethidium bromide. Super-coiled, open circular, and linear forms of the plasmid DNA are represented schematically and their corresponding bands are indicated. This identification of the band patterns is consistent with previous research.,

Figure 2

QCM-D fifth overtone frequency and dissipation shifts of (A) the creation of a calf thymus dsDNA surface and (B) its subsequent exposure to 0.1mM CuSO₄ then 0.1mM quercetin with 0.1mM CuSO₄. (A) shows the creation of a mostly dsDNA surface by allowing ssDNA to hybridize with its complementary strand on a PEI surface as previously described. Significant shifts in ΔF and ΔD are labeled and we have discussed elsewhere the significance of these shifts as well as the lag time observed between the ssDNA deposition and hybridization. ΔF for the total DNA surface corresponds to a calculated areal mass of 170 ng/cm² and a density of 1.70 g/cm³ using the Sauerbrey equation with an estimated thickness of 1.0 nm from ellipsometric thickness data (not shown). (B) shows the exposure of that same DNA surface to Cu(II) and then quercetin/Cu(II). Gray shading indicates an ultra-pure water rinse.

Figure 3

QCM-D fifth overtone frequency and dissipation shifts of DNA surfaces exposed to (A) 0.1mM quercetin then 0.1mM quercetin with 0.1mM CuSO₄ or (B) 0.1mM CuSO₄, an ultra-pure water rinse, and then 0.1mM quercetin alone. Each figure represents a zoom on the data collected after the formation of the respective DNA surfaces. Periods of exposure of the DNA surface to CuSO₄ and/or quercetin are labeled. Gray shading indicates an ultra-pure water rinse.