Temperature and Quenching Studies of Fluorescence Polarization Detection of DNA Hybridization

The effects of temperature and collisional quenching on fluorescence polarization detection of DNA hybridization were studied using measurements of fluorescence intensity and anisotropy and the dynamic decay of these properties. Three different tethers, 3, 6, and 12 carbons in length, were used to a...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 1997-02, Vol.69 (3), p.500-506
Main Authors: Kumke, Michael U, Shu, Luchuan, McGown, Linda B, Walker, G. Terrance, Pitner, J. Bruce, Linn, C. Preston
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Anisotropy
Biological and medical sciences
Chemistry
Deoxyribonucleic acid
DNA
DNA - analysis
Fluorescence
Fluorescence Polarization
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods
Nucleic Acid Hybridization
Nucleic acids
Temperature
Thermodynamics
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Summary:The effects of temperature and collisional quenching on fluorescence polarization detection of DNA hybridization were studied using measurements of fluorescence intensity and anisotropy and the dynamic decay of these properties. Three different tethers, 3, 6, and 12 carbons in length, were used to attach fluorescein label to the 5‘ end of the 33-base oligomers. Perrin plots showed that the effective rotating volume decreases with increasing tether length and approximately doubles upon hybridization. Hybridization increases the association between the tethered dye and the DNA for the shorter tethers but displaces the fluorescein on the 12C tether from the DNA, forcing it into greater contact with the bulk solution. The 6C tether appears to promote sequence-specific interaction between fluorescein label and the oligomer, which causes unexpectedly high anisotropy at higher temperatures and increased protection from collisional quenching. In all cases, there appear to exist several possible conformations for the tethered fluorescein. As temperature is increased, these conformations tend to collapse into a single, average or preferred, conformation. The results demonstrate the importance of the selection of tether, dye, and DNA probe in designing a polarization strategy for detection of DNA hybridization, particularly with respect to tether length and DNA probe sequence.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac9608230