Real-time monitoring of the hybridization reaction: application to the quantification of oligonucleotides in biological samples |
| |
Authors: | Boutet V Delaunay V De Oliveira M C Boquet D Grognet J M Grassi J Deverre J R |
| |
Affiliation: | CEA, Service de Pharmacologie et d'Immunologie, DRM, CEA Saclay, Gif-Sur Yvette Cedex, F-91191, France. |
| |
Abstract: | 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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|