Osnabrück, Germany
Osnabrück, Germany

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Honigmann A.,University of Osnabrück | Walter C.,Ionovation GmbH | Erdmann F.,University of Osnabrück | 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 Osnabrück | Werz E.,Ionovation GmbH | Rosemeyer H.,University of Osnabrück
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.


Bartsch P.,University of Osnabrück | Bartsch P.,Ionovation GmbH | Walter C.,University of Osnabrück | Walter C.,Ionovation GmbH | And 4 more authors.
Materials | Year: 2012

Artificial bilayer containing reconstituted ion channels, transporters and pumps serve as a well-defined model system for electrophysiological investigations of membrane protein structure-function relationship. Appropriately constructed microchips containing horizontally oriented bilayers with easy solution access to both sides provide, in addition, the possibility to investigate these model bilayer membranes and the membrane proteins therein with high resolution fluorescence techniques up to the single-molecule level. Here, we describe a bilayer microchip system in which long-term stable horizontal free-standing and hydrogel-supported bilayers can be formed and demonstrate its prospects particularly for single-molecule fluorescence spectroscopy and high resolution fluorescence microscopy in probing the physicochemical properties like phase behavior of the bilayer-forming lipids, as well as in functional studies of membrane proteins. © 2012 by the authors.


Werz E.,University of Osnabrück | Werz E.,Ionovation GmbH | Rosemeyer H.,University of Osnabrück
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 Osnabrück | Knies C.,University of Osnabrück | Werz E.,University of Osnabrück | Werz E.,Ionovation GmbH | Rosemeyer H.,University of Osnabrück
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.


Werz E.,University of Osnabrück | Korneev S.,University of Osnabrück | Montilla-Martinez M.,University of Osnabrück | Wagner R.,University of Osnabrück | And 5 more authors.
Chemistry and Biodiversity | Year: 2012

A novel technique is described which comprises a base-specific DNA duplex formation at a lipid bilayer-H 2O-phase boundary layer. Two different probes of oligonucleotides both carrying a double-tailed lipid at the 5′-terminus were incorporated into stable artificial lipid bilayers separating two compartments (cis/trans-channel) of an optically transparent microfluidic sample carrier with perfusion capabilities. Both the cis- and trans-channels are filled with saline buffer. Injection of a cyanine-5-labeled target DNA sequence, which is complementary to only one of the oligonucleotide probes, into the cis-channel, followed by a thorough perfusion, leads to an immobilization of the labeled complementary oligonucleotide on the membrane as detected by single-molecule fluorescence spectroscopy and microscopy. In the case of fluorescent but non-complementary DNA sequences, no immobilized fluorescent oligonucleotide duplex could be detected on the membrane. This clearly verifies a specific duplex formation at the membrane interface. © 2012 Verlag Helvetica Chimica Acta AG, Zürich.


The present invention relates to a method for the isolation and/or identification of known or unknown sequences of nucleic acids (target sequences) optionally marked with reporter groups by base specific hybridation with, essentially, complementary sequences (in the following referred to as sample oligo-nucleotides, sample sequences or sample nucleic acids), which belong to a library of sequences. Further, the invention relates to nucleolipids used in the method of the invention and a process for the preparation of said nucleolipids. In addition, the invention refers to a pharmaceutical composition comprising said nucleolipids.


The present invention relates to a method for the isolation and/or identification of known or unknown sequences of nucleic acids (target sequences) optionally marked with reporter groups by base specific hybridation with, essentially, complementary sequences (in the following referred to as sample oligonucleotides, sample sequences or sample nucleic acids), which belong to a library of sequences. Further, the invention relates to nucleolipids used in the method of the invention and a process for the preparation of said nucleolipids.


Grant
Agency: Cordis | Branch: H2020 | Program: SME-1 | Phase: NMP-25-2015-1 | Award Amount: 71.43K | Year: 2015

Many drugs are designed to target molecules inside cells. Due to the hydrophobic nature of the cell membranes however not all types of drugs can enter the cells in sufficient quantities. Thus large amounts of drug need to be administered which can lead to highly toxic systemic effects for the patient. Furthermore, new classes of therapeutics such as RNA- and DNA-based drugs require advanced delivery techniques to reach their intracellular targets. Ionovations proprietary IonoChem technology will provide a novel and highly effective solution to this problem by adding lipohilic tails directly to drug molecules at a one-on-one ratio. These tails allow drugs to easily pass through the cell membrane. As part of a service offer Ionovation will work with clients to identify the most effective tail for a particular molecule, and will also perform the lipophilization. The IonoChem platform will enable Ionovation GmbH (a manufacturer of experimental set-ups for the analysis of biological membranes) to enter the market for drug delivery technologies, thus creating a new line of business for the company. IonoChem will target the market for drug delivery systems, which was valued at US$151 billion in 2013. Market segments for initial introduction of the technology include RNAi drug delivery (a market worth US $11,7 billion in 2013) and drugs withdrawn from the market (eg Vioxx with 2.5 billion US $ annual global sales). Objectives of the phase I project include analysis of customer needs (pricing, expected turn-around times etc.), refinement of business model (licensing/fee for use model for contract manufacturers vs. service offer with production facilities at Ionovations head quarters); search for a suitable contract manufacturer for the phase II activities and the elaboration of a business plan. Phase II objectives include upscaling of the production under GMP conditions, establishment of contact to customers and the qualification of IonoChem in a preclinical setting.


A device is disclosed which contains one or more pores 10. The pores 10 in turn contain one or more translocase proteins which from a translocation system 20. At the pores 10, two zones, the cis zone 50 and the trans zone 60, are separated from one another on a support body 90 by means of translocation systems 20 in such a way that only those proteins that are recognized by the translocation systems 20 due to specific molecular signals can exclusively pass over from the cis zone 50 into the trans zone 60.

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