Abstract
Quantitative description of the interaction between doxorubicin (DOX), a broadly used anticancer drug, and DNA is the key to understand the action mechanism and side effects of its clinical use. However, the reported equilibrium constants of DOX-DNA interaction obtained using a range of different analytical methods vary even by several orders of magnitude. Herein, we propose a novel application of a single-molecule technique - fluorescence correlation spectroscopy (FCS) - to probe the interaction between DOX and two types of DNA (pUC19 and calf thymus DNA), taking advantage of intrinsic self-fluorescence of DOX. We provide an analytical formula for autocorrelation analysis to determine the equilibrium constant of DOX-DNA complex-formation, where binding of multiple DOX molecules to a DNA chain is included in the reaction-diffusion model. Our FCS-based method not only quantitatively revealed the values of equilibrium constant, but also implied that the stability of DOX-DNA complex is related to the types of base pair rather than the length or structure of the DNA. This work opens a promising pathway toward quantitative determination of molecular interactions in complex systems such as living cells or organisms at single-molecule level.
Highlights
Fluorescence correlation spectroscopy (FCS), first introduced by Magde et al in the early 1970s,16–18 is an ideal experiment tool that can be used for investigations of molecular interactions with single molecule sensitivity.[19,20,21] fluorescence correlation spectroscopy (FCS) provides more precise values in the determination of molecular interactions in comparison with classical methods, since bigger errors may be introduced to the system in the latter case where much larger amount of reactants are required
Considering that the sizes of DNA molecule we used are much larger than the DOX, we describe the formation of DOX–DNA complexes using the ligands–macromolecule interaction model:[31] consecutive binding of DOX ligands to the multiple active site in a double-stranded DNA (dsDNA) chain until saturation of the chain achieves
We observed a second, slow components emerging at the long lag time region of experimental autocorrelation function (ACF) in the case of DOX diffusing in the solutions of pUC19 and calf thymus DNA (ctDNA)
Summary
Fluorescence correlation spectroscopy (FCS), first introduced by Magde et al in the early 1970s,16–18 is an ideal experiment tool that can be used for investigations of molecular interactions with single molecule sensitivity.[19,20,21] FCS provides more precise values in the determination of molecular interactions in comparison with classical methods, since bigger errors may be introduced to the system in the latter case where much larger amount (several orders of magnitude) of reactants are required. FCS records the fluctuations in the fluorescence intensity of probes diffusing within a femtoliter focal volume (0.2 f L, FV) created by the excitation laser beam. Autocorrelation of such signal reveals the characteristic time-scales of fluorescence fluctuations, which correspond to the time of residence of probes inside the focal volume. Application of an appropriate theoretical model to the resulting autocorrelation curve allows to retrieve a variety of physicochemical information on the system of interest, such as diffusion coefficients, conformational changes, 1572 | Phys.
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