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Unformatted text preview: Time-resolved Microspectrofluorometry and Fluorescence Imaging Techniques Study of Porphyrin-mediated Cellular Uptake of Oligonucleotides P ETR P RAUS , a E VA K O ˇ CI ˇ SOV ´ A , a P ETER M OJZE ˇ S , a J OSEF ˇ S T ˇ EP ´ ANEK , a O LIVIER S EKSEK , b F RANCK S UREAU , b AND P IERRE-Y VES T URPIN b a Charles University in Prague, Faculty of Mathematics and Physics, Prague, Czech Republic b BioMoCeTi (Laboratoire de Biophysique Mol´eculaire, Cellulaire et Tissulaire), Paris, France Time-resolved confocal microspectrofluorometry and fluorescence microscopy imaging were ap- plied to monitor the cellular uptake of fluorescent-labeled oligonucleotides (ONs) delivered by a porphyrin molecule. The fate of porphyrin–ON complexes inside living cells has also been monitored. Due to intrinsic fluorescence of the porphyrin and sensitivity of its characteristics to microenvironment, multicomponent analysis of time-resolved fluorescence provides unique information about stability of the porphyrin–ON complexes, ON interactions with their target se- quences, and ON and porphyrin distributions after delivery inside the cells. Time-resolved confo- cal microspectrofluorometry indeed delivers additional information compared with fluorescence confocal microscopy imaging widely employed to study ON uptake. Key words: oligonucleotide; porphyrin; antisense; cellular uptake; fluorescence lifetime; spec- trofluorometry; microscopy; fluorescence imaging Oligonucleotide Therapy and Drug Delivery Oligonucleotide (ON) strategies represent exciting possibilities to modulate protein expression inside liv- ing cells and thus to act effectively against persis- tent viral, bacterial, or gene diseases. 1 In the pro- cesses of genetic information transfer from replication to translation via transcription, almost each step can theoretically be affected by the introduction of spe- cific deoxyribo-, ribo-, or modified oligonucleotides interacting with definite target sites on DNA or RNA molecules. Currently considered mechanisms include antisense binding, 2 triplex formation, 3 aptamer in- teraction, 4 chimeric RNA/DNA, 5 small interfering RNA, 6 ribozymes, 7 DNA enzymes, 8 exon skipping, 9 and alternative splicing. 10 One of the drawbacks of using ONs for therapeu- tic purposes is their low ability to penetrate cellular Address for correspondence: Petr Praus, Charles University in Prague, Faculty of Mathematics and Physics, Ke Karlovu 3, Prague 2, CZ-121 16, Czech Republic. [email protected] membranes; indeed the phospholipid bilayer acts as a strong barrier for diffusion of large, anionic, and hydrophilic ONs. To overcome this problem, vari- ous delivery techniques (e.g., microinjection, electro- poration, nano-encapsulation, or nanosphere carriers) were applied, and numerous synthetic systems me- diating the ON uptake (on lipid, cyclodextrin, den- drimer, and peptide basis) were developed....
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This note was uploaded on 07/11/2010 for the course SPECTOGRAP 545 taught by Professor Gdf during the Spring '10 term at AIB College of Business.
- Spring '10