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<strong>International</strong> <strong>Carl</strong> <strong>Zeiss</strong> <strong>Workshop</strong> on Fluorescence Correlation Spectroscopy and Related Methods March, 30 to March 31, Jena / Germany Fluorescence Correlation Spectroscopy for THE characterisation OF drug delivery systems F. Delie 1 , R. Gurny 1 and A. Zimmer 2 (1) Department of Pharmceutical Technology and Biopharmaceutics, 1211 Geneva, Switzerland, (2)Biocenter, Johann Wolfgang Goethe-Universität, Institut for Pharmaceutical Technology, 60439 Frankfurt (Main), Germany Fluorescence Correlation Spectroscopy (FCS) is a unique and very sensitive technique to dynamically characterise ligand-receptor interaction. The FCS principle is based on a time resolved analysis of the fluorescence intensity fluctuations. The sensitivity of such a system allows a single molecule detection. This technique has been used for drug characterisation, high-throughput screening in pharmaceutical drug discovery and, more recently, to measure the diffusion coefficient of drugs within living cells (1). Part of the effort in pharmaceutical development is based on modulating drug availability by the use of carriers that will either controlled the release of the compound or target the drug to the active site. The most commonly used systems are entrapment in liposomes or polymeric particles, and complexation with macromolecules. The nature of the association of the active compounds to the carriers is rather critical to improve the systems and to predict in vivo release kinetics. By providing a real time in situ characterisation, FCS may assist the design of new drug delivery systems. The utility of this method in the field of drug delivery systems was investigated with a special attention to oligonucleotide delivery systems. In the present study, we measured in a time resolved manner the interactions between a fluorescently labelled oligonucleotide and cationically charged peptides and polymeric nanoparticles instantaneously in solution. - 29 -
<strong>International</strong> <strong>Carl</strong> <strong>Zeiss</strong> <strong>Workshop</strong> on Fluorescence Correlation Spectroscopy and Related Methods March, 30 to March 31, Jena / Germany Chymotrypsin-catalyzed fluorescence-labeling of ribonuclease T1 Antje Hascher, Kerstin Korn, Hans-Heinrich Foerster, Ulrich Hahn and Frank Bordusa Institut fuer Biochemie, Fakultaet fuer Biowissenschaften, Pharmazie und Psychologie Universitaet Leipzig, Germany Since proteins play an essential role in numerous biological processes, many attempts have been made to study their interaction with their environment. In this context, FCS becomes more and more important. Usually proteins are detectable by fluorescence spectroscopy, via the amino acids Trp and Tyr or after labeling with a fluorescent dye. One main problem appears using conventional labeling procedures due to the unselective coupling (e. g. lysine-moities) of the dye to the protein molecules. In the approach described here specific enzymes were used to link one dye per protein selectively to the N-terminus of the target. Since we used the back reaction of chymotrypsin for selective labeling, the target protein had to show an N-terminal Arg for specific substrate recognition [1]. The target protein - in our case ribonuclease T1 – was modified first by recombinant techniques by adding an Arg at the N-terminus yielding the variant RG- RNase T1. For labeling, the fluorescent dye 6-carboxy-tetramethylrhodamine (TAMRA) was chemically linked to the tripeptide ester Gly-Pro-Tyr-OMe resulting in TAMRA-GPY- OMe. The fluorescent dye labeled tripeptide ester was then enzymatically ligated to the N-terminal Arg of the RG-RNase T1 variant. Purified TAMRA-GPY-RG-RNase T1 could be proven by FCS measurements. Furthermore the selective N-terminal labeling of RNase T1 could be verified by the protease mediated cleavage of the fluorescent dye from the labeled protein. [1] Schellenberger, V., Turck, C. W., Hedstrom, L., and Rutter, W. J. (1993) Mapping the S´-Subsites of Serine Protease Using Acyl Transfer to Mixtures of Peptide Nucleophiles. Biochemistry 32, 4349-4353. - 30 -
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