Rapid and sensitive ethidium bromide fluorescence quenching assay of polyamine conjugate–DNA interactions for the analysis of lipoplex formation in gene therapy

A rapid and sensitive fluorescent assay method is reported for assessing polyamine conjugate–calf thymus DNA binding affinity using cholesterol polyamine carbamates with ethidium bromide as a probe. A reproducible method has been developed with an optimal excitation wavelength. Salt concentration is...

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Bibliographic Details
Published in:Journal of pharmaceutical and biomedical analysis 2000-06, Vol.22 (5), p.849-859
Main Authors: Geall, Andrew J, Blagbrough, Ian S
Format: Article
Language:English
Subjects:
Analysis
Biological and medical sciences
Cholesterol polyamine carbamates
DNA - chemistry
DNA binding affinity and condensation
Ethidium - chemistry
Ethidium bromide
Fluorescence quenching
General pharmacology
Genetic Therapy
Intercalation
Lipoplexes in gene therapy
Medical sciences
Pharmacology. Drug treatments
Polyamines - chemistry
Reproducibility of Results
Spectrometry, Fluorescence
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Summary:A rapid and sensitive fluorescent assay method is reported for assessing polyamine conjugate–calf thymus DNA binding affinity using cholesterol polyamine carbamates with ethidium bromide as a probe. A reproducible method has been developed with an optimal excitation wavelength. Salt concentration is shown to be a critical parameter for both the observed fluorescence intensity of ethidium intercalated in DNA, and also for the binding of positively charged polyammonium ions to DNA, effecting charge neutralisation. This charge neutralisation precedes DNA condensation, a key first step in gene therapy.
ISSN:0731-7085
1873-264X
DOI:10.1016/S0731-7085(00)00250-8