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.

A Multimode Optical Imaging System for Preclinical Applications In Vivo: Technology Development, Multiscale Imaging, and Chemotherapy Assessment

“…3c). Furthermore, this imaging mode can be used for monitoring tissue functional status in the vicinity of fluorophores [13, 17, 37]. Intravital confocal imaging has also demonstrated its usefulness for detecting structural and functional (including pathological) information in multimode optical imaging (Fig.…”

Highly efficient cellular cloning using Ferro-core Micropallet Arrays

“…However, we would argue that traditional cloning methods also do not readily accommodate this when limited numbers of cells reside within the presumed clonal population. Given the extensive development of non-lethal reporter constructs and increasing use of fluorophore labeled aptamers and oligonucleotide probes as intracellular probes in intact and viable cells 50–56 , the necessity of cell lethal screening modalities is not as critical as in past generations of molecular biology studies.…”

Magnetron-Sputtered Polytetrafluoroethylene-Stabilized Silver Nanoisland Surface for Surface-Enhanced Fluorescence

“…A confocal microspectrofluorimeter adapted for time-resolved fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain the SEF spectra and to determine the fluorescence lifetimes [29][30][31][32]. Its simplified block diagram is shown in Figure S1.…”

“…The fluorescence lifetime was determined simultaneously for all emission wavelengths by acquiring several even phase-shifted spectra of the modulated excitation laser beam in relation to the fixed modulation phase shift of the detector gain. The fluorescence lifetime was calculated from the frequency-dependent phase shift and the intensity demodulation [29][30][31][32] for the modulation frequencies evenly covering a 5-200 MHz interval. A laser diode module (Omicron LDM 442.50.A350, Rodgau-Dudenhofen, Germany) with a sinusoidal intensity modulation (50 mW peak output, attenuated to one to tens of µW at the sample) was used for the excitation at a 445 nm wavelength.…”