Fluorescence Properties of Fluorescein, Tetramethylrhodamine and Texas Red Linked to a DNA Aptamer¶

Abstract

We report the picosecond time-scale fluorescence dynamics of a dye-labeled DNA oligonucleotide or “aptamer” designed to bind specifically to Immunoglobulin E. Comparison of the photophysics of Texas Red (TR), fluorescein and 5′-carboxytetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for measurements of oligonucleotide structure and dynamics. The fluorescence decay of the TR-aptamer is a simple single exponential with a weak temperature dependence. The fluorescence decay of the fluorescein-aptamer (fl-aptamer) is pH dependent and displays a complex temperature dependence with significant changes on melting of the aptamer tertiary structure. Despite its similarities to TR, TAMRA is strongly quenched when conjugated to the aptamer and displays complex fluorescence kinetics best described by a distributed rate model. Using the maximum entropy method, we have discovered two highly temperature-dependent fluorescence lifetimes for the TAMRA-aptamer. One of these lifetimes is similar to that of free TAMRA and displays the same temperature dependence. The other lifetime is quenched and displays a temperature dependence characteristic of a charge transfer reaction. These data set TR apart as an attractive alternative to TAMRA and fluorescein for studies such as fluorescence polarization and fluorescence resonance energy transfer, where environmental sensitivity of the probe is not desired.