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Osnabrück, Germany

Honigmann A.,University of Osnabruck | Walter C.,Ionovation GmbH | Erdmann F.,University of Osnabruck | Erdmann F.,University of Munster | And 2 more authors.
Biophysical Journal | Year: 2010

Artificial lipid membranes are widely used as a model system to study single ion channel activity using electrophysiological techniques. In this study, we characterize the properties of the artificial bilayer system with respect to its dynamics of lipid phase separation using single-molecule fluorescence fluctuation and electrophysiological techniques. We determined the rotational motions of fluorescently labeled lipids on the nanosecond timescale using confocal time-resolved anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigated by the lateral diffusion of the lipids using fluorescence correlation spectroscopy. Depending on the solvent used for membrane preparation, lateral diffusion coefficients in the range Dlat = 10-25 μm2/s and rotational diffusion coefficients ranging from Drot = 2.8 - 1.4 × 107 s-1 were measured in pure liquid-disordered (Ld) membranes. In ternary mixtures containing saturated and unsaturated phospholipids and cholesterol, liquid-ordered (L0) domains segregated from the Ld phase at 23°0C. The lateral mobility of lipids in L0 domains was around eightfold lower compared to those in the Ld phase, whereas the rotational mobility decreased by a factor of 1.5. Burstintegrated steady-state anisotropy histograms, as well as anisotropy imaging, were used to visualize the rotational mobility of lipid probes in phase-separated bilayers. These experiments and fluorescence correlation spectroscopy measurements at different focal diameters indicated a heterogeneous microenvironment in the L0 phase. Finally, we demonstrate the potential of the optoelectro setup to study the influence of lipid domains on the electrophysiological properties of ion channels. We found that the electrophysiological activity of gramicidin A (gA), a well-characterized ion channel-forming peptide, was related to lipid-domain partitioning. During liquid-liquid phase separation, gA was largely excluded from L0 domains. Simultaneously, the number of electrically active gA dimers increased due to the increased surface density of gA in the L d phase. © 2010 by the Biophysical Society.

Werz E.,University of Osnabruck | Werz E.,Ionovation GmbH | Rosemeyer H.,University of Osnabruck
Beilstein Journal of Organic Chemistry | Year: 2015

A series of six cyanine-5-labeled oligonucleotides (LONs 10-15), each terminally lipophilized with different nucleolipid head groups, were synthesized using the recently prepared phosphoramidites 4b-9b. The insertion of the LONs within an artificial lipid bilayer, composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine (POPE), was studied by single molecule fluorescence spectroscopy and microscopy with the help of an optically transparent microfluidic sample carrier with perfusion capabilities. The incorporation of the lipo-oligonucleotides into the bilayer was studied with respect to efficiency (maximal bilayer brightness) as well as stability against perfusion (final stable bilayer brightness). Attempts to correlate these parameters with the log P values of the corresponding nucleolipid head groups failed, a result which clearly demonstrates that not only the lipophilicity but mainly the chemical structure and topology of the head group is of decisive importance for the optimal interaction of a lipo-oligonucleotide with an artificial lipid bilayer. Moreover, fluorescence half-live and diffusion time values were measured to determine the diffusion coefficients of the lipo-oligonucleotides. © 2015 Werz and Rosemeyer; licensee Beilstein-Institut.

Werz E.,University of Osnabruck | Werz E.,Ionovation GmbH | Rosemeyer H.,University of Osnabruck
Beilstein Journal of Organic Chemistry | Year: 2014

The article describes the immobilization of different probe oligonucleotides (4 , 7, 10) carrying each a racemic mixture of 2,3- bis(hexadecyloxy)propan-1-ol ( 1a ) at the 5'-terminus on a stable artificial lipid bilayer composed of 1-palmitoyl-2-oleoyl- sn-glycero- 3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The bilayer separates two compartments (cis/trans channel) of an optical transparent microfluidic sample carrier with perfusion capabilities. Injection of unlabeled target DNA sequences (6, 8, or 9), differing in sequence and length, leads in the case of complementarity to the formation of stable DNA duplexes at the bilayer surface. This could be verified by Sybr Green I double strand staining, followed by incubation periods and thorough perfusions, and was visualized by single molecule fluorescence spectroscopy and microscopy. The different bilayer-immobilized complexes consisting of various DNA duplexes and the fluorescent dye were studied with respect to the kinetics of their formation as well as to their stability against perfusion. © 2014 Werz and Rosemeyer.

Malecki E.,University of Osnabruck | Knies C.,University of Osnabruck | Werz E.,University of Osnabruck | Werz E.,Ionovation GmbH | Rosemeyer H.,University of Osnabruck
Chemistry and Biodiversity | Year: 2013

The cancerostatic 5-fluorouridine (5-FUrd; 1) was sequentially sugar-protected by introduction of a 2′,3′-O-heptylidene ketal group (→2), followed by 5′-O-monomethoxytritylation (→3). This fully protected derivative was submitted to Mitsunobu reactions with either phytol ((Z and E)-isomer) or nerol ((Z)-isomer) to yield the nucleoterpenes 4a and 4b. Both were 5′-O-deprotected with 2% Cl2CHCOOH in CH 2Cl2 to yield compounds 5a and 5b, respectively. These were converted to the 5′-O-cyanoethyl phosphoramidites 6a and 6b, respectively. Moreover, the 2′,3′-O-(1-nonyldecylidene) derivative, 7a, of 5-fluorouridine was resynthesized and labelled at C(5′) with an Eterneon-480 fluorophor® (→7b). The resulting nucleolipid was studied with respect to its incorporation in an artificial bilayer, as well as to its aggregate formation. Additionally, two oligonucleotides carrying terminal phytol-alkylated 5-fluorouridine tags were prepared, one of which was studied concerning its incorporation in an artificial lipid bilayer. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

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