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 w hich can be used for realtime monitoring of oligonucleotide hybridization. This assay quantifies all kinds of oligonucleotides in biological fluids wi thout extraction. The assay makes use of two different probes and involves a fluorescent transfer process. As fluorescence measurements are not destru ctive, they can be sequentially repeated, thereby allowing comparison of th e hybridization kinetics and binding strength of chemically modified backbo ne oligonucleotides (>0.5 nM) in biological media. The assay was validated for pharmacokinetic analysis of phosphodiester and phosphorothioate oligonu cleotides in plasma and in different organs (liver, kidneys, lungs, spleen) at low concentrations (0.4 mg/kg, corresponding to clinical doses). Respec tive 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 in tact phosphorothioate sequences in some organs even after 24 h. (C) 2000 Ac ademic Press.