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 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.
Boutet
V
V
CEA, Service de Pharmacologie et d'Immunologie, DRM, CEA Saclay, Gif-Sur Yvette Cedex, F-91191, France.
Delaunay
V
V
De Oliveira
M C
MC
Boquet
D
D
Grognet
J M
JM
Grassi
J
J
Deverre
J R
JR
D000818:Animals; D001483:Base Sequence; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D008297:Male; D051379:Mice; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D009841:Oligonucleotides / Q000032:analysis* / Q000235:genetics*; D013873:Thionucleotides / Q000032:analysis / Q000235:genetics; D014018:Tissue Distribution
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D013873:Thionucleotides