title: Intracellular uptake of modified oligonucleotide studied by two fluorescence techniques.
abstract: Interaction, i.e., cellular uptake and intracellular distribution, of synthetic modified antisense oligonucleotide with the B16 melanoma cell line was studied using cationic polyene antibiotic, amphotericin B 3-dimethylaminopropyl amide, as a carrier vector. The antisense oligonucleotide--dT(15) oligomer analogue containing isopolar, nonisosteric, phosphonate-based internucleotide linkages 3'-O-P-CH(2)-O-5'--was labeled with fluorescent tetramethylrhodamine marker. The oligonucleotide itinerancy across the cell membrane and its distribution inside the cell was visualized using fluorescence microimaging. During the first several hours a strong preference staining of the cell nucleus was found. Fluorescence lifetime measurements from the intracellular environment (confocal laser microspectrofluorimeter, frequency domain phase/modulation technique in 1 to 200 MHz frequency region) yielded two spectral components of 4.9 and 1.4 ns lifetime, respectively. While the former component correlates with the previously characterized effect of the fluorophore binding to biomolecular targets in membranes and/or cytoplasm, the latter component is newly observed and its possible origin is discussed.
authors.affiliation: Institute of Physics, Charles University, Ke Karlovu 5, 12116 Prague 2, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Kocisová
authors.forename: Petr
authors.initials: P
authors.lastname: Praus
authors.forename: Ivan
authors.initials: I
authors.lastname: Rosenberg
authors.forename: Olivier
authors.initials: O
authors.lastname: Seksek
authors.forename: Franck
authors.initials: F
authors.lastname: Sureau
authors.forename: Josef
authors.initials: J
authors.lastname: Stĕpánek
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Turpin
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D007834:Lasers; D008546:Melanoma, Experimental; D051379:Mice; D018613:Microscopy, Confocal; D009841:Oligonucleotides / Q000493:pharmacokinetics*; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D002412:Cations; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C408366:amphotericin B3-(N'-dimethylamino)propylamide; D000666:Amphotericin B