FLUORESCENT LABELING OF UNMODIFIED PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDES - SYNTHESIS AND CHARACTERIZATION

In this paper we describe the preparation and characterization of phos phorothioate oligodeoxynucleotides (pt-odn) substituted with a fluores cein molecule linked to the non-bridging sulfur of the internucleotidi c linkage. This technique is original in that the starting material is a perphosphorothioate oligonucleotide. The phosphorothioate oligonucl eotides, after reaction at pH 8.0 and 50 degrees C with iodoacetamido- fluorescein, yielded a fluorescent derivative, termed F-pt-odn. The tw o F-pt-odn used in this report contained 1.6 and 3.4 fluorescein per o ligonucleotide and were found to be respectively 1.5 and 2.2 times mor e fluorescent than an alkylamidothiocarbamyl-fluoresceinyl-pt-odn (F-N H-pt-odn) containing a single reporter group. We examined a number of the properties of these oligonucleotides to assess their utility in ph armacokinetic studies. The endocytosis, cellular distribution, and ant isense biological activity of these F-pt-odn were similar to those of the starting material (GEM91, complementary to the AUG region of the H IVgag gene) and a F-NH-pt-odn. The F-pt-odn hybridize to their complem entary sequence at temperatures up to their Tm of 47.5 degrees C, whic h is 7 degrees C lower than unmodified GEM91. F-pt-odn are sensitive t o alkaline pH and temperature. However, under experimental conditions (pH 7.4) F-pt-odn are more than 70% unchanged after 15 days. After int ravenous injection into mice, fluorescent oligonucleotides were easily detectable, in most organs, by HPLC and spectrofluorometry. The tissu e distribution of F-pt-odn was found to be similar to that previously reported. We believe that these fluorescent oligonucleotides are of va lue 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.