Digital frequency domain fluorometry and the study of Hoechst 33258 DYE-DNA interactions
Feddersen, Brett Andrew
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Permalink
https://hdl.handle.net/2142/28678
Description
Title
Digital frequency domain fluorometry and the study of Hoechst 33258 DYE-DNA interactions
Author(s)
Feddersen, Brett Andrew
Issue Date
1993
Doctoral Committee Chair(s)
Gratton, E.
Department of Study
Physics
Discipline
Physics
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
fluorescence
hoechst 33258 dye
Deoxyribonucleic Acid (DNA)
Language
en
Abstract
"Fluorescence is a powerful tool for the study of chemical and biological processes. The typical decay times of fluorescence are ideal to study events in the pico to nanosecond range. On these time scales, the motions of many biological processes can be studied. The use of frequency domain fluorometry to measure the lifetime of the excited state has been used for many years. Previously, its use was limited by the constraints of analog electronics. These electronics restricted the type of detector used, and because of the relatively large time constant in the electronics, also limited the types of experiments that could be done. These limitations were removed with the development of an acquisition system based on modem digital techniques. The digital acquisition method has opened the door to different types of experiments that previously were either too time consuming or could not be done. The use of digital techniques and the development of a method to modulate an image intensifier made it possible to incorporate linear and matrix detectors in frequency domain fluorometry. The extension of time-resolved fluorescence
measurements to linear arrays has made it possible to follow the time evolution of the emission spectra while the use of matrix detectors has permitted the measurement of the lifetime at every ""pixel"" of an image.
The dye Hoechst 33258 has been used for many years in the study of DNA and DNA binding. However, the fluorescent properties of Hoechst 33258 are not well understood. The dye is highly quenched in aqueous solutions and becomes brightly fluorescent when bound to A·T rich sequences of DNA or placed in non-aqueous solutions. The fluorescence of Hoechst 33258 seems to arise from two different solvation states of the molecule. When Hoechst 33258 binds to calf thymus DNA or poly[d(A•T)], the molecule becomes highly fluorescent, yet the two states can still be distinguished. The two states are attributed to different binding modalities of the dye. The loose binding allows access of water molecules which results in different emission properties. On the other hand, when Hoechst binds onto d(CGCGAATTCGCG) only one lifetime is observed. The single lifetime has been attributed to strong binding of the Hoechst molecule onto the AATT site. The tight binding of Hoechst 33258 to AATT sites
excludes water molecules from interacting with the dye, resulting in only one lifetime
component."
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