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Article Dans Une Revue New Journal of Chemistry Année : 1997

Fluorescent labelling of unmodified phosphorothioate oligodeoxynucleotides: Synthesis and characterization

A.J. Stewart
  • Fonction : Auteur
Chantal Pichon
Patrick Midoux
Michel Monsigny
  • Fonction : Auteur
  • PersonId : 875218
A.M. Aubertin
  • Fonction : Auteur

Résumé

In this paper we describe the preparation and characterization of phosphorothioate oligodeoxynucleotides (pt-odn) substituted with a fluorescein molecule linked to the non-bridging sulfur of the internucleotidic linkage. This technique is original in that the starting material is a perphosphorothioate oligonucleotide. The phosphorothioate oligonucleotides, after reaction at pH 8.0 and 50°C with iodoacetamido-fluorescein, yielded a fluorescent derivative, termed F-pt-odn. The two F-pt-odn used in this report contained 1.6 and 3.4 fluorescein per oligonucleotide and were found to be respectively 1.5 and 2.2 times more fluorescent than an alkylamidothiocarbamyl-fluoresceinyl-pt-odn (F-NH-pt-odn) containing a single reporter group. We examined a number of the properties of these oligonucleotides to assess their utility in pharmacokinetic studies. The endocytosis, cellular distribution, and antisense biological activity of these F-pt-odn were similar to those of the starting material (GEM91, complementary to the AUG region of the HIVgag gene) and a F-NH-pt-odn. The F-pt-odn hybridize to their complementary sequence at temperatures up to their Tm of 47.5°C, which is 7°C lower than unmodified GEM91. F-pt-odn are sensitive to alkaline pH and temperature. However, under experimental conditions (pH 7.4) F-pt-odn are more than 70% unchanged after 15 days. After intravenous injection into mice, fluorescent oligonucleotides were easily detectable, in most organs, by HPLC and spectrofluorometry. The tissue distribution of F-pt-odn was found to be similar to that previously reported. We believe that these fluorescent oligonucleotides are of value due to their easy preparation and their high specific fluorescence. In addition, their unaltered cellular uptake and biological activity make them ideal tools for use in pharmacokinetic studies.
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Dates et versions

hal-02161369 , version 1 (20-06-2019)

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  • HAL Id : hal-02161369 , version 1

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A.J. Stewart, Chantal Pichon, Patrick Midoux, R. Mayer, Michel Monsigny, et al.. Fluorescent labelling of unmodified phosphorothioate oligodeoxynucleotides: Synthesis and characterization. New Journal of Chemistry, 1997, 21 (1), pp.87-98. ⟨hal-02161369⟩
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