Intracellular uptake of modified oligonucleotide studied by two fluorescence techniques

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-dT15 oligomer analogue containing isopolar, nonisosteric, phosphonate-based internucleotide linkages 3ʹ-O-P-CH2-O-5ʹ-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.