Cellular uptake of modified oligonucleotides enhanced by porphyrins studied by time-resolved microspectrofluorimetry and fluorescence imaging techniques
Abstract
Fluorescence microimaging and homodyne phase-resolved confocal microspectrofluorimetry were used to monitor the transport of antisense oligonucleotide into 3T3 living cells and its subsequent intracellular distribution. Phosphorothioate analog of 15-mer oligothymidylate labeled by ATTO 425 was complexed with 5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin (H2TMPyP4) as an uptake-mediating agent.
High frequency (up to 180 MHz) analog modulation of both exciting diode laser and the detector image intensifier gain was used to record time-resolved fluorescence spectra. Fluorescence lifetime data within a broad spectral range have revealed preservation of oligonucleotide/porphyrin complex integrity and binding properties of both components inside the cell.