Real-Time Monitoring of the Hybridization Reaction: Application to the Quantification of Oligonucleotides in Biological Samples

We describe here a competitive hybridization assay using TRACE technology which can be used for real-time monitoring of oligonucleotide hybridization. This assay quantifies all kinds of oligonucleotides in biological fluids without extraction. The assay makes use of two different probes and involves...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2000-02, Vol.268 (1), p.92-98
Main Authors: Boutet, Valérie, Delaunay, Valérie, De Oliveira, Monica Cristina, Boquet, Didier, Grognet, Jean-Marc, Grassi, Jacques, Deverre, Jean-Robert
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Evaluation Studies as Topic
Fluorescent Dyes
hybridization
Male
Mice
monitoring
Nucleic Acid Hybridization - methods
Oligonucleotide Probes - genetics
oligonucleotides
Oligonucleotides - analysis
Oligonucleotides - genetics
pharmacokinetic
phosphorothioate
Thionucleotides - analysis
Thionucleotides - genetics
Tissue Distribution
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Summary:We describe here a competitive hybridization assay using TRACE technology which can be used for real-time monitoring of oligonucleotide hybridization. This assay quantifies all kinds of oligonucleotides in biological fluids without extraction. The assay makes use of two different probes and involves a fluorescent transfer process. As fluorescence measurements are not destructive, they can be sequentially repeated, thereby allowing comparison of the hybridization kinetics and binding strength of chemically modified backbone oligonucleotides (>0.5 nM) in biological media. The assay was validated for pharmacokinetic analysis of phosphodiester and phosphorothioate oligonucleotides in plasma and in different organs (liver, kidneys, lungs, spleen) at low concentrations (0.4 mg/kg, corresponding to clinical doses). Respective sensitivities for phosphodiester and phosphorothioate were 0.2 and 0.8 pmol/ml in plasma and 2 and 8 pmol/g in tissues, which allow to recover intact phosphorothioate sequences in some organs even after 24 h.
ISSN:0006-291X
1090-2104
DOI:10.1006/bbrc.2000.2088