Intracellular fate of oligodeoxynucleotides: A microspectro FRET study

Abstract

Fluorescence resonance energy transfer (FRET) under confocal microscopy allows direct intracellular measurements of both localisation and interactions of tagged molecules to be performed. In the antigene and antisense strategies, short oligonucleotide sequences are used to inhibit RNA translation in cell. Numerous techniques (Crook, R.M. “In Vitro Cellular Uptake, Distribution, and Metabolism of Oligonucleotides” Handbook of Experimental Pharmacology, Vol. 131 Antisense Research and Application : 103-140 (1998) were used to follow oligonucleotide penetration and distribution in cultured cells, but no in situ study of the oligonucleotide stability was performed. Therefore, microspectroFRET was chosen as a non destructive method to study in differents cell lines the fate of short oligonucleotides, fluorescently labeled at both ends. When an oligonucleotide is intact, FRET occurs, so, in this study, intramolecular FRET permits us to know the ratio of intact oligonucleotides during time, i. e. the ratio of potentially active oligonucleotides ; single fluorescent labeling is not sufficient : the observed fluorescence may be due to fluorophores alone or still grafted on the oligonucleotide. The oligonucleotides, labeled with fluorescein at the 3’ end and rhodamine at the 5’ end, were added to the cells and subcellular microvolumes (-10-18 cubic meter) were analysed with the confocal microspectrofluorometer (Vigny, P. Amirand, C. Ballini, J.P. Chinsky, L. Duquesne, M. Laigle, A. Manfait, A. Sureau, F. Turpin, P.Y. “Microspectrofluorometry of Single Living Cells” Spectroscopy of Biological Molecules - State of the Art : 345-348 (1989) developed in the laboratory. Different wavelengths of an Argon LASER were used for excitation of the samples. The spectra were recorded on an optical multichannels analyser after separation of the different emission wavelengths by a monochromator. After FFT smoothing and background subtraction, comparisons of the spectra were achieved and analysed in term of : Stability of the oligonucleotides, which is directly related to the transfer efficiency, as a function of time Quantitative localisation of the oligonucleotides, as fluorescence intensity is proportional to concentration Surrounding of the oligonucleotides, as the quatum yield of fluorescein is pH dependent. As an example of granulocytic cell model, we used K562 cells. But these cells have a large nucleus, preventing to distinguish subcellular compartments. So we also used Hela cells as an example of epithelial-like cells with a real differentiation between cytoplasm and nucleus. 3e Colloque SFp, Paris-Sud ‘99 icole Polytechnique, Palaiseau, 28 juin-2 juillet 1999