Orientational Dynamics and Dye-DNA Interactions in a Dye- Labeled DNA Aptamer

¹ University of Kansas

^* To whom correspondence should be addressed. E-mail: ckjohnson{at}ku.edu.

Submitted on October 6, 2004
Revised on November 9, 2004
Accepted on 26 January 2005

	Abstract

We report the picosecond and nanosecond time-scale rotational dynamics of a dye-labeled DNA oligonucleotide or "aptamer" designed to bind specifically to Immunoglobulin E. Rotational dynamics in combination with fluorescence lifetime measurements provide information about dye-DNA interactions. Comparison of Texas Red (TR), fluorescein, and tetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for biophysical studies employing such conjugates. Time-resolved anisotropy studies demonstrate that the TR- and TAMRA-aptamer anisotropy decays are dominated by the overall rotation of the aptamer, whereas the fluorescein-aptamer (fl-aptamer) anisotropy decay displays a sub-nanosecond rotational correlation time much shorter than that expected for the overall rotation of the aptamer. Docking and molecular dynamics simulations suggest that the low mobility of TR is a result of binding in the groove of the DNA helix. Additionally, associated anisotropy analysis of the TAMRA-aptamer reveals both quenched and unquenched states that experience significant coupling to the DNA motion. Therefore, quenching of TAMRA by guanosine must depend on the configuration of the dye bound to the DNA. The strong coupling of TR to the rotational dynamics of the DNA aptamer, together with the absence of quenching of its fluorescence by DNA, makes it a good probe of DNA orientational dynamics. The understanding of the nature of dye-DNA interactions provides the basis for the development of bioconjugates optimized for specific biophysical measurements and is important for the sensitivity of anisotropy-based DNA-protein interaction studies employing such conjugates.

Key Words: Texas Red, anisotropy decay, aptamer, dye-DNA interaction, fluorescence polarization, immunoglobulin E

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