Blinking and Bleaching of Tetramethylrhodamine on DNA Induced by Paramagnetic Cations

Abstract

Certain proteins require metal cation cofactors to function. The conformations and dynamics of such proteins are often studied using single molecule fluorescence techniques. Blinking of fluorophores can make analysis of single molecule fluorescence data difficult. During the investigation of a manganese-dependent enzyme that binds DNA, we observed fluorescence fluctuations of tetramethylrhodamine (TAMRA) when in the presence of manganese, but not magnesium. This project seeks to understand the origin of fluorescent fluctuations when in presence of certain cations and why they occur.A DNA strand with an internally labeled TAMRA was studied while in the presence of divalent calcium, magnesium, manganese, cobalt, nickel, and zinc. Using fluorescence correlation spectroscopy, the fluctuations of TAMRA were analyzed. When in the presence of calcium, magnesium, and zinc, the correlation of TAMRA can be described by diffusion only. However, a process that occurs in the micro-second timescale is present when manganese, cobalt, and nickel are present. Flash photolysis experiments were done to characterize the nature of the excited state species that causes fluorescence fluctuations. The results indicate that a triplet forms while in the presence of manganese but not magnesium, and only in the presence of DNA. Manganese, cobalt, and nickel are paramagnetic cations and can induce triplet formation in dyes, while diamagnetic cations, like calcium, magnesium, and zinc, do not. This conclusion is supported through lifetime and photodegradation experiments. When DNA is present, the cations can induce a triplet at much lower concentrations than when DNA is not present. This can be explained by the fact that when cations bind to DNA, they are in closer proximity to the dye.