Cellular uptake of modified oligonucleotides: fluorescence approach

Abstract: Cellular uptake and intracellular distribution of the synthetic antisense analogue of dT15 oligonucleotide (homogenously containing 3′-O–P–CH2–O-5′ internucleotide linkages and labeled with tetramethylrhodamine dye) was studied on B16 melanoma cell line by fluorescence micro-imaging and time-resolved microspectrofluorimetry. By using amphotericin B 3-dimethylaminopropyl amide as an enhancer molecule for the uptake process, homogenous staining of the cells with rather distinct nucleoli staining was achieved after 4h of incubation. Two spectral components of 2.7 and 1.3ns lifetime, respectively, were resolved in the emission collected from the cell nucleus. The way of staining and the long-lived component differed from our previous experiments demonstrating complexity of the intracellular oligonucleotide distribution and in particular of the binding inside the nucleus. [Copyright &y& Elsevier]