Intracellular Uptake of Modified Oligonucleotide Studied by Two Fluorescence Techniques Eva Koc ˇ is ˇ ova ´ 1 Petr Praus 1 Ivan Rosenberg 2 Olivier Seksek 3 Franck Sureau 3 Josef S ˇ te ˘ pa ´ nek 1 Pierre-Yves Turpin 3 1 Institute of Physics, Charles University, Ke Karlovu 5, 12116 Prague 2, Czech Republic 2 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo sq. 2, 16610 Prague 6, Czech Republic 3 Laboratoire de Physicochimie Biomole ´ culaire et Cellulaire (CNRS ESA 7033), Universite ´ P. et M. Curie, Case 138, 4 Place Jussieu, F-75252 Paris Cedex 05, France Received 1 September 2003; accepted 16 October 2003 Published online 13 April 2004 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20055 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 oligo- nucleotide– dT 15 oligomer analogue containing isopolar, nonisosteric, phosphonate-based inter- nucleotide 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 tech- nique 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. © 2004 Wiley Periodicals, Inc. Biopolymers 74: 110 –114, 2004 Keywords: cellular uptake; antisense oligonucleotide; fluorescence microimaging; fluorescence lifetime; phase/modulation spectroscopy INTRODUCTION Antisense, antigene, and aptamer strategies, devel- oped and researched in recent years, represent a new possible efficient molecular tool in efforts to modulate gene expression. These strategies involve specific sin- gle-stranded sequences of modified deoxyribo- or ri- bonucleotides to inhibit transcription of a specific Correspondence to: E. Koc ˇis ˇova ´ Biopolymers, Vol. 74, 110 –114 (2004) © 2004 Wiley Periodicals, Inc. 110