Biochemical and Biophysical Research Communications, 2005
We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethan... more We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethanolamine with high affinity down to the low nanomolar range. These aptamers were selected for their ability to bind to ethanolamine immobilised on magnetic beads, from an 96mer library of initially about 1 x 10(16) randomised ssDNA molecules. The dissociation constants of these aptamers range between K(D)=6 and K(D)=19 nmol L(-1). The aim of the development of ethanolamine aptamers is their use for the detection of this substance in clinical and environmental analysis. Ethanolamine is associated with several diseases. Moreover, ethanolamine and its derivatives di- and tri-ethanolamine are used in chemical and cosmetic industries. The use of biosensors with ethanolamine aptamer as new molecular recognition element could be an innovative method for an easy and fast detection of ethanolamine.
Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affini... more Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor ...
Aptamers are promising recognition elements for sensitive and specific detection of small molecul... more Aptamers are promising recognition elements for sensitive and specific detection of small molecules. We have previously selected ssDNA aptamers for ethanolamine, one of the smallest aptamer targets so far. The work presented here focuses on the determination of the binding region within the aptamer structure and its exploitation for the development of an aptamer-based assay for detection of ethanolamine. Sequence analysis of the aptamers resulted in the identification of a G-rich consensus sequence, which was able to fold in a typical two- or three-layered G-quartet structure. Experiments with stepwise truncated variants of the aptamers revealed that the consensus sequence is responsible and sufficient for binding to the target. On the basis of the knowledge of the aptamers binding site, we developed an aptamer-based microarray assay relying on competition between ethanolamine and an oligonucleotide complementary to the consensus sequence. Competitive binding of ethanolamine and fluorescently labeled complementary oligonucleotides resulted in fluorescence intensities dependent on ethanolamine concentration with a limit of detection of 10 pM. This method enables detection of small molecules without any labeling of analytes. The competitive assay could potentially be transferred to other aptamers and thus provides a promising system for aptamer-based detection of diverse small molecules.
ABSTRACT S-layer proteins provide a biocompatible environment with different kinds of functional ... more ABSTRACT S-layer proteins provide a biocompatible environment with different kinds of functional groups, perfect for the sequential coupling of any kind of biofunctional molecule. In addition, their nanostructure and their ability to crystallize on surfaces in a nanometer-thick monolayer ensures a regular arrangement of these molecules on solid supports. In this work, a thrombin-binding aptamer and an ofloxacin-binding aptamer were coupled with different chemical crosslinkers to S-layer proteins using them for defined immobilization. S-layer protein monomers and paracrystalline S-layers were successfully modified with the thrombin-binding aptamer. However, S-layer protein monomers were not able to crystallize after aptamer modification and showed no thrombin binding during random surface attachment. In contrast, aptamers linked to an intact S-layer in suspension or an S-layer coating were still functional. The modification rate of S-layers with the thrombin-binding aptamer was estimated with one aptamer to two unit cells (unit cell = four monomers). Verification of the functionality of both aptamers through target-binding after S-layer-immobilization on solid supports was proven by laser-induced fluorescence spectroscopy (LIFS), resonant mirror sensor (IAsys) and quartz crystal microbalance with dissipation monitoring (QCM-D), respectively. Hence, this study presents S-layer proteins as an interesting alternative to existing immobilization matrices for recognition biomolecules.This article is protected by copyright. All rights reserved
Page 1. CHAPTER 2 SELEX AND ITS RECENT OPTIMIZATIONS BEATE STREHLITZ and REGINA STOLTENBURG 2.1 I... more Page 1. CHAPTER 2 SELEX AND ITS RECENT OPTIMIZATIONS BEATE STREHLITZ and REGINA STOLTENBURG 2.1 INTRODUCTION Nucleic acids are very attractive compounds for combinatorial chemistry. They are able ...
ABSTRACT S-layer proteins provide a biocompatible environment with different kinds of functional ... more ABSTRACT S-layer proteins provide a biocompatible environment with different kinds of functional groups, perfect for the sequential coupling of any kind of biofunctional molecule. In addition, their nanostructure and their ability to crystallize on surfaces in a nanometer-thick monolayer ensures a regular arrangement of these molecules on solid supports. In this work, a thrombin-binding aptamer and an ofloxacin-binding aptamer were coupled with different chemical crosslinkers to S-layer proteins using them for defined immobilization. S-layer protein monomers and paracrystalline S-layers were successfully modified with the thrombin-binding aptamer. However, S-layer protein monomers were not able to crystallize after aptamer modification and showed no thrombin binding during random surface attachment. In contrast, aptamers linked to an intact S-layer in suspension or an S-layer coating were still functional. The modification rate of S-layers with the thrombin-binding aptamer was estimated with one aptamer to two unit cells (unit cell = four monomers). Verification of the functionality of both aptamers through target-binding after S-layer-immobilization on solid supports was proven by laser-induced fluorescence spectroscopy (LIFS), resonant mirror sensor (IAsys) and quartz crystal microbalance with dissipation monitoring (QCM-D), respectively. Hence, this study presents S-layer proteins as an interesting alternative to existing immobilization matrices for recognition biomolecules.This article is protected by copyright. All rights reserved
... Electrocatalytic oxidation of NADH at mediator-modified electrode surfaces Wolfgang Schuhmann... more ... Electrocatalytic oxidation of NADH at mediator-modified electrode surfaces Wolfgang Schuhmann a, Johanna Huber a, Heidi Wohlschl~iger a ... Quinones (Cenas et al., 1984), phenazines (Torstensson and Gorton, 1981), phenoxazine derivatives (Huck, 1982; Huck et al., 1984 ...
... Electrocatalytic oxidation of NADH at mediator-modified electrode surfaces Wolfgang Schuhmann... more ... Electrocatalytic oxidation of NADH at mediator-modified electrode surfaces Wolfgang Schuhmann a, Johanna Huber a, Heidi Wohlschl~iger a ... Quinones (Cenas et al., 1984), phenazines (Torstensson and Gorton, 1981), phenoxazine derivatives (Huck, 1982; Huck et al., 1984 ...
ABSTRACT The genetically modified yeast strain Yarrowia lipolytica H222-S4(p67ICL1)T5 is able to ... more ABSTRACT The genetically modified yeast strain Yarrowia lipolytica H222-S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild-type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose-peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose-peptone. Furthermore, two process strategies (fed-batch and repeated fed-batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed-batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.
Biochemical and Biophysical Research Communications, 2005
We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethan... more We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethanolamine with high affinity down to the low nanomolar range. These aptamers were selected for their ability to bind to ethanolamine immobilised on magnetic beads, from an 96mer library of initially about 1 x 10(16) randomised ssDNA molecules. The dissociation constants of these aptamers range between K(D)=6 and K(D)=19 nmol L(-1). The aim of the development of ethanolamine aptamers is their use for the detection of this substance in clinical and environmental analysis. Ethanolamine is associated with several diseases. Moreover, ethanolamine and its derivatives di- and tri-ethanolamine are used in chemical and cosmetic industries. The use of biosensors with ethanolamine aptamer as new molecular recognition element could be an innovative method for an easy and fast detection of ethanolamine.
Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affini... more Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor ...
Aptamers are promising recognition elements for sensitive and specific detection of small molecul... more Aptamers are promising recognition elements for sensitive and specific detection of small molecules. We have previously selected ssDNA aptamers for ethanolamine, one of the smallest aptamer targets so far. The work presented here focuses on the determination of the binding region within the aptamer structure and its exploitation for the development of an aptamer-based assay for detection of ethanolamine. Sequence analysis of the aptamers resulted in the identification of a G-rich consensus sequence, which was able to fold in a typical two- or three-layered G-quartet structure. Experiments with stepwise truncated variants of the aptamers revealed that the consensus sequence is responsible and sufficient for binding to the target. On the basis of the knowledge of the aptamers binding site, we developed an aptamer-based microarray assay relying on competition between ethanolamine and an oligonucleotide complementary to the consensus sequence. Competitive binding of ethanolamine and fluorescently labeled complementary oligonucleotides resulted in fluorescence intensities dependent on ethanolamine concentration with a limit of detection of 10 pM. This method enables detection of small molecules without any labeling of analytes. The competitive assay could potentially be transferred to other aptamers and thus provides a promising system for aptamer-based detection of diverse small molecules.
ABSTRACT S-layer proteins provide a biocompatible environment with different kinds of functional ... more ABSTRACT S-layer proteins provide a biocompatible environment with different kinds of functional groups, perfect for the sequential coupling of any kind of biofunctional molecule. In addition, their nanostructure and their ability to crystallize on surfaces in a nanometer-thick monolayer ensures a regular arrangement of these molecules on solid supports. In this work, a thrombin-binding aptamer and an ofloxacin-binding aptamer were coupled with different chemical crosslinkers to S-layer proteins using them for defined immobilization. S-layer protein monomers and paracrystalline S-layers were successfully modified with the thrombin-binding aptamer. However, S-layer protein monomers were not able to crystallize after aptamer modification and showed no thrombin binding during random surface attachment. In contrast, aptamers linked to an intact S-layer in suspension or an S-layer coating were still functional. The modification rate of S-layers with the thrombin-binding aptamer was estimated with one aptamer to two unit cells (unit cell = four monomers). Verification of the functionality of both aptamers through target-binding after S-layer-immobilization on solid supports was proven by laser-induced fluorescence spectroscopy (LIFS), resonant mirror sensor (IAsys) and quartz crystal microbalance with dissipation monitoring (QCM-D), respectively. Hence, this study presents S-layer proteins as an interesting alternative to existing immobilization matrices for recognition biomolecules.This article is protected by copyright. All rights reserved
Page 1. CHAPTER 2 SELEX AND ITS RECENT OPTIMIZATIONS BEATE STREHLITZ and REGINA STOLTENBURG 2.1 I... more Page 1. CHAPTER 2 SELEX AND ITS RECENT OPTIMIZATIONS BEATE STREHLITZ and REGINA STOLTENBURG 2.1 INTRODUCTION Nucleic acids are very attractive compounds for combinatorial chemistry. They are able ...
ABSTRACT S-layer proteins provide a biocompatible environment with different kinds of functional ... more ABSTRACT S-layer proteins provide a biocompatible environment with different kinds of functional groups, perfect for the sequential coupling of any kind of biofunctional molecule. In addition, their nanostructure and their ability to crystallize on surfaces in a nanometer-thick monolayer ensures a regular arrangement of these molecules on solid supports. In this work, a thrombin-binding aptamer and an ofloxacin-binding aptamer were coupled with different chemical crosslinkers to S-layer proteins using them for defined immobilization. S-layer protein monomers and paracrystalline S-layers were successfully modified with the thrombin-binding aptamer. However, S-layer protein monomers were not able to crystallize after aptamer modification and showed no thrombin binding during random surface attachment. In contrast, aptamers linked to an intact S-layer in suspension or an S-layer coating were still functional. The modification rate of S-layers with the thrombin-binding aptamer was estimated with one aptamer to two unit cells (unit cell = four monomers). Verification of the functionality of both aptamers through target-binding after S-layer-immobilization on solid supports was proven by laser-induced fluorescence spectroscopy (LIFS), resonant mirror sensor (IAsys) and quartz crystal microbalance with dissipation monitoring (QCM-D), respectively. Hence, this study presents S-layer proteins as an interesting alternative to existing immobilization matrices for recognition biomolecules.This article is protected by copyright. All rights reserved
... Electrocatalytic oxidation of NADH at mediator-modified electrode surfaces Wolfgang Schuhmann... more ... Electrocatalytic oxidation of NADH at mediator-modified electrode surfaces Wolfgang Schuhmann a, Johanna Huber a, Heidi Wohlschl~iger a ... Quinones (Cenas et al., 1984), phenazines (Torstensson and Gorton, 1981), phenoxazine derivatives (Huck, 1982; Huck et al., 1984 ...
... Electrocatalytic oxidation of NADH at mediator-modified electrode surfaces Wolfgang Schuhmann... more ... Electrocatalytic oxidation of NADH at mediator-modified electrode surfaces Wolfgang Schuhmann a, Johanna Huber a, Heidi Wohlschl~iger a ... Quinones (Cenas et al., 1984), phenazines (Torstensson and Gorton, 1981), phenoxazine derivatives (Huck, 1982; Huck et al., 1984 ...
ABSTRACT The genetically modified yeast strain Yarrowia lipolytica H222-S4(p67ICL1)T5 is able to ... more ABSTRACT The genetically modified yeast strain Yarrowia lipolytica H222-S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild-type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose-peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose-peptone. Furthermore, two process strategies (fed-batch and repeated fed-batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed-batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.
Uploads
Papers by Beate Strehlitz