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Synthetic bioreporters for detection of environmental pollutants
New Biotechnology · Volume 31S · July 2014
SYMPOSIUM 9: BIOMARKERS AND DIAGNOSTIC TOOLS
Symposium 9: Biomarkers and diagnostic tools
recognize sequence epitopes that are difficult to target using conventional antibodies, and that these novel antibodies are potent inhibitors of protein aggregation.
O9-1
http://dx.doi.org/10.1016/j.nbt.2014.05.1693
Designing nanomaterials for ultrasensitive biosensing Molly Stevens
O9-3
Imperial College London, UK
Engineering cofactor specificity of methyltransferases Bio-responsive nanomaterials are of growing importance with potential applications including drug delivery, diagnostics and tissue engineering. This talk will provide an overview of our recent developments in the design of materials for ultrasensitive biosensing. Our recent simple conceptually novel approaches to real-time monitoring of protease, lipase and kinase enzyme action using modular peptide functionalized gold nanoparticles and quantum dots will be presented. Furthermore we have recently developed a new approach to ultrasensitive biosensing through plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth as well as a “Plasmonic ELISA” for the ultrasensitive detection of disease biomarkers with the naked eye. We are applying these biosensing approaches both in high throughput drug screening and to diagnose diseases ranging from cancer to global health applications. http://dx.doi.org/10.1016/j.nbt.2014.05.1692
O9-2 Antibodies by design Peter Tessier Rensselaer Polytechnic Institute, United States
The ability of antibodies to recognize target molecules (antigens) with high affinity and specificity is central to their widespread use in diagnostic and therapeutic applications. The binding activity of antibodies is encoded in up to six of their solvent-exposed peptide loops that directly contact antigens. Antibodies are generated by randomly varying the sequences of their antigen-binding loops and selecting rare variants that are complementary to target antigens. Due to the daunting number of possible antibody sequences with variation only within their ˆ variants), it seems unlikely that antigen-binding loops (>1030 the needles (antibodies with desired binding activity) in the haystack (all possible antibody variants) can be predicted instead of being selected. We have challenged this conventional wisdom by reducing the seemingly intractable problem of designing multiple antibody loops to cooperatively bind antigens to a tractable one in which we design individual antibody loops with binding activity. Using this simplified design strategy that is inspired by natural biological interactions, we find that antibody fragments can be readily engineered to recognize diverse aggregated proteins linked to neurodegenerative disorders (e.g., Alzheimer’s disease) by targeting unique structural features within such proteins. Our innovative approach generates single- and multidomain antibodies that recognize misfolded proteins not only based on their sequence, but also based on their conformation. We also find that our antibodies S36
www.elsevier.com/locate/nbt
Martin Tengg 1,∗ , Yu Zheng 2 , Mandana Gruber-Khadjawi 3 , Elmar Weinhold 1 1
Institute of Organic Chemistry, RWTH Aachen University, Germany New England Biolabs Inc., Ipswich, MA, USA 3 ACIB GmbH, Austrian Centre for Industrial Biotechnology, Graz, Austria 2
Biological methylations of various substrates occur in every living cell and are essential for cell survival. These transformations are catalyzed by methyltransferases (MTases) which generally transfer the activated methyl group from S-adenosyl-l-methionine (AdoMet) to DNA, RNA, proteins and small biomolecules. Recent studies demonstrated that many MTases can accept AdoMet analogues for transfer of extended carbon chains to their substrates [1,2]. The ability to accept a broad range of cofactor analogues is of great interest both in terms of DNA diagnostics and biocatalytic synthesis. In order to increase transfer rates of extended groups from AdoMet analogues protein engineering approaches are performed. High-throughput directed evolution of the DNA MTase M.SssI is performed by in vitro compartmentalization. This method links genotype and phenotype for an effective selection of active variants. The bacterial enzyme M.SssI is of particular interest because it performs the same reaction as mammalian DNA MTases by targeting 5 -CG-3 (CpG) DNA sequences. CpG modifications are key epigenetic signatures in transcriptional regulation and genome imprinting. The transfer of functional groups will provide new tools for DNA labelling as well as DNA modification detection. M.SssI evolution will also guide rational protein engineering of the homologous small molecule MTase NovO, for the synthesis of new fine chemicals as well as bioactive intermediates and products. References [1].Dalhoff C, Lukinavicius G, Klimas˘ auskas S, Weinhold E. Nat Chem Biol 2006;2:31–2. [2].Stecher H, Tengg M, Ueberbacher BJ, Remler P, Schwab H, Griengl H, Gruber-Khadjawi M. Angew Chem Int Ed 2009;48:9546–8.
http://dx.doi.org/10.1016/j.nbt.2014.05.1694
O9-4 Synthetic bioreporters for detection of environmental pollutants Jan Roelof van der Meer ∗ , Davide Merulla, Siham Beggah University of Lausanne, Switzerland
One of the main immediate application areas for synthetic biology are bioreporters, living cells with simple designed genetic
New Biotechnology · Volume 31S · July 2014
circuits that permit detection of a specific chemical or group of chemicals, under the concomitant production of an easily but accurately quantifiable reporter signal. Bioreporters have attracted considerable interest because they offer cheap alternatives for chemical analysis in remote areas where high-end instruments are unavailable. Such demands on bioreporters, however, require an almost fail-proof and robust technology that goes much beyond what traditional research “proof-of-principles” have been able to demonstrate. We will show on the example of a bioreporter for arsenic, how important improvements can be made in the circuit design and in the optimization of the assay technology. Arsenic is a recurring noxious contaminant of drinking water in large areas of our planet, and bioreporter technology can provide the means to rapidly quantify its presence in the concentration range of 1–10 g/L. First, we demonstrate how designing a feedback or an uncoupled circuit affects the signal output and detection sensitivity for arsenic. Secondly, we show a new system to better control residual background expression in the circuit while maintaining optimal induction, and we also provide experimental evidence to improve the circuit behaviour by avoiding cross-interference from the host. Finally, we present the use of micro-engineered structures that would permit continuous remote operation of a bioreporter sensor. http://dx.doi.org/10.1016/j.nbt.2014.05.1695
O9-5 A homogeneous quenching resonance energy transfer assay for H-Ras activation cycle monitoring and inhibitor screening Kari Kopra 1,∗ , Arjan van Aldrichem 2 , Markku Syrjänpää 1 , Stefan Veltel 3 , Pekka Hänninen 1 , Daniel Abankwa 4 , Urpo Lamminmäki 5 , Harri Härmä 1 1
Laboratory of Biophysics, University of Turku, Finland Institute for Molecular Medicine Finland, University of Helsinki, Finland 3 University Hospital Hamburg-Eppendorf, Finland 4 Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Finland 5 Department of Biotechnology, University of Turku, Finland 2
Background: Recently, we have developed a homogeneous single-label signaling technique, the Quenching Resonance Energy Transfer (QRET). In this study, the homogeneous QRET technology was applied to monitor GTPase activation cycle and activation cycle inhibition. Methods: The QRET system is based on the protection of target protein bound Eu3+ -GTP from a soluble quencher molecule. (1) The small GTPase cycle (nucleotide exchange and hydrolysis) can be monitored in the competitive assay by tracking GTP hydrolysis in the presence of nanomolar concentrations of H-RasWt , SOScat , and p120GAP. (2) The nucleotide exchange can be monitored using a second assay with H-RasWt and SOScat . The increased timeresolved luminescence (TRL) signal is monitored when either GTP hydrolysis (1) or Eu3+ -GTP association (2) occurs. Results: We have proven the suitability of the QRET system to monitor GTP hydrolysis (1) and nucleotide exchange (2). The
SYMPOSIUM 9: BIOMARKERS AND DIAGNOSTIC TOOLS
GTP hydrolysis assay was used to screen 1280 compound small molecule library whereof twelve inhibitors were found (average Zfactor 0.78). The GTP hydrolysis assay can simultaneously detect inhibitors affecting either nucleotide exchange or GTP hydrolysis. Therefore, same screening was performed by monitoring Eu3+ -GTP association to H-RasWt (average Z-factor 0.78). From the screening assays, seven same inhibitor hits were found. Additionally, five inhibitors were found only in GTP hydrolysis assay. Conclusions: These novel QRET assays for GTPase research can be performed using nanomolar protein concentrations. The presented QRET assays enable the study of whole small GTPase cycle by monitoring the guanine-nucleotide exchange factor (GEF) induced nucleotide exchange and/or GTPase-activating proteins (GAP) catalyzed GTP hydrolysis. http://dx.doi.org/10.1016/j.nbt.2014.05.1696
O9-6 Comparative large scale microRNA expression profiles of cynomolgus monkeys, rat and human reveal miR-182 associated with Type 2 diabetes Hongli Du ∗ , Jinghui Zhou, Yuhuan Meng, Xiaoning Wang South China University of Technology, China
Type 2 diabetes (T2D) is a prevalent disease that is present throughout the world, and is usually associated with insulin resistance. MicroRNAs (miRNAs) play important role in the suppression of gene expression and have been shown to be implicated in human diseases. We used a novel animal model, cynomolgus monkey fed with normal and high fatty diet (HFD) respectively, to analyze the miRNA expression profile in whole blood by deepsequencing. Finally in total 24 miRNAs with differential expression were filtered. Among them, mir-182 and mir-183, related to insulin resistance by modulating FOXO1 and PI3 K/AKT cascade, had the greatest copy number in the whole blood. Decrease of mir-182 in T2D cynomolgus individuals is completely consistent with the previous studies in human and rat. Integrating mir-182 tissue expression profile, target genes and copy number in blood revealed that mir-182 plays a key role in FOXO1 modulation that leads to potential hyperglycemia and modulates the insulin secretion. In addition, the possible miRNA regulation system of T2D under the influence of different diet conditions was also interpreted in the present study. The cholesterol content influences mir-182 and potentially other miRNAs expression level that causes the insulin resistance and the various miRNA regulation systems between normal diet and HFD. http://dx.doi.org/10.1016/j.nbt.2014.05.1697
www.elsevier.com/locate/nbt S37
SYMPOSIUM 9: BIOMARKERS AND DIAGNOSTIC TOOLS
Symposium 9: Biomarkers and diagnostic tools
recognize sequence epitopes that are difficult to target using conventional antibodies, and that these novel antibodies are potent inhibitors of protein aggregation.
O9-1
http://dx.doi.org/10.1016/j.nbt.2014.05.1693
Designing nanomaterials for ultrasensitive biosensing Molly Stevens
O9-3
Imperial College London, UK
Engineering cofactor specificity of methyltransferases Bio-responsive nanomaterials are of growing importance with potential applications including drug delivery, diagnostics and tissue engineering. This talk will provide an overview of our recent developments in the design of materials for ultrasensitive biosensing. Our recent simple conceptually novel approaches to real-time monitoring of protease, lipase and kinase enzyme action using modular peptide functionalized gold nanoparticles and quantum dots will be presented. Furthermore we have recently developed a new approach to ultrasensitive biosensing through plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth as well as a “Plasmonic ELISA” for the ultrasensitive detection of disease biomarkers with the naked eye. We are applying these biosensing approaches both in high throughput drug screening and to diagnose diseases ranging from cancer to global health applications. http://dx.doi.org/10.1016/j.nbt.2014.05.1692
O9-2 Antibodies by design Peter Tessier Rensselaer Polytechnic Institute, United States
The ability of antibodies to recognize target molecules (antigens) with high affinity and specificity is central to their widespread use in diagnostic and therapeutic applications. The binding activity of antibodies is encoded in up to six of their solvent-exposed peptide loops that directly contact antigens. Antibodies are generated by randomly varying the sequences of their antigen-binding loops and selecting rare variants that are complementary to target antigens. Due to the daunting number of possible antibody sequences with variation only within their ˆ variants), it seems unlikely that antigen-binding loops (>1030 the needles (antibodies with desired binding activity) in the haystack (all possible antibody variants) can be predicted instead of being selected. We have challenged this conventional wisdom by reducing the seemingly intractable problem of designing multiple antibody loops to cooperatively bind antigens to a tractable one in which we design individual antibody loops with binding activity. Using this simplified design strategy that is inspired by natural biological interactions, we find that antibody fragments can be readily engineered to recognize diverse aggregated proteins linked to neurodegenerative disorders (e.g., Alzheimer’s disease) by targeting unique structural features within such proteins. Our innovative approach generates single- and multidomain antibodies that recognize misfolded proteins not only based on their sequence, but also based on their conformation. We also find that our antibodies S36
www.elsevier.com/locate/nbt
Martin Tengg 1,∗ , Yu Zheng 2 , Mandana Gruber-Khadjawi 3 , Elmar Weinhold 1 1
Institute of Organic Chemistry, RWTH Aachen University, Germany New England Biolabs Inc., Ipswich, MA, USA 3 ACIB GmbH, Austrian Centre for Industrial Biotechnology, Graz, Austria 2
Biological methylations of various substrates occur in every living cell and are essential for cell survival. These transformations are catalyzed by methyltransferases (MTases) which generally transfer the activated methyl group from S-adenosyl-l-methionine (AdoMet) to DNA, RNA, proteins and small biomolecules. Recent studies demonstrated that many MTases can accept AdoMet analogues for transfer of extended carbon chains to their substrates [1,2]. The ability to accept a broad range of cofactor analogues is of great interest both in terms of DNA diagnostics and biocatalytic synthesis. In order to increase transfer rates of extended groups from AdoMet analogues protein engineering approaches are performed. High-throughput directed evolution of the DNA MTase M.SssI is performed by in vitro compartmentalization. This method links genotype and phenotype for an effective selection of active variants. The bacterial enzyme M.SssI is of particular interest because it performs the same reaction as mammalian DNA MTases by targeting 5 -CG-3 (CpG) DNA sequences. CpG modifications are key epigenetic signatures in transcriptional regulation and genome imprinting. The transfer of functional groups will provide new tools for DNA labelling as well as DNA modification detection. M.SssI evolution will also guide rational protein engineering of the homologous small molecule MTase NovO, for the synthesis of new fine chemicals as well as bioactive intermediates and products. References [1].Dalhoff C, Lukinavicius G, Klimas˘ auskas S, Weinhold E. Nat Chem Biol 2006;2:31–2. [2].Stecher H, Tengg M, Ueberbacher BJ, Remler P, Schwab H, Griengl H, Gruber-Khadjawi M. Angew Chem Int Ed 2009;48:9546–8.
http://dx.doi.org/10.1016/j.nbt.2014.05.1694
O9-4 Synthetic bioreporters for detection of environmental pollutants Jan Roelof van der Meer ∗ , Davide Merulla, Siham Beggah University of Lausanne, Switzerland
One of the main immediate application areas for synthetic biology are bioreporters, living cells with simple designed genetic
New Biotechnology · Volume 31S · July 2014
circuits that permit detection of a specific chemical or group of chemicals, under the concomitant production of an easily but accurately quantifiable reporter signal. Bioreporters have attracted considerable interest because they offer cheap alternatives for chemical analysis in remote areas where high-end instruments are unavailable. Such demands on bioreporters, however, require an almost fail-proof and robust technology that goes much beyond what traditional research “proof-of-principles” have been able to demonstrate. We will show on the example of a bioreporter for arsenic, how important improvements can be made in the circuit design and in the optimization of the assay technology. Arsenic is a recurring noxious contaminant of drinking water in large areas of our planet, and bioreporter technology can provide the means to rapidly quantify its presence in the concentration range of 1–10 g/L. First, we demonstrate how designing a feedback or an uncoupled circuit affects the signal output and detection sensitivity for arsenic. Secondly, we show a new system to better control residual background expression in the circuit while maintaining optimal induction, and we also provide experimental evidence to improve the circuit behaviour by avoiding cross-interference from the host. Finally, we present the use of micro-engineered structures that would permit continuous remote operation of a bioreporter sensor. http://dx.doi.org/10.1016/j.nbt.2014.05.1695
O9-5 A homogeneous quenching resonance energy transfer assay for H-Ras activation cycle monitoring and inhibitor screening Kari Kopra 1,∗ , Arjan van Aldrichem 2 , Markku Syrjänpää 1 , Stefan Veltel 3 , Pekka Hänninen 1 , Daniel Abankwa 4 , Urpo Lamminmäki 5 , Harri Härmä 1 1
Laboratory of Biophysics, University of Turku, Finland Institute for Molecular Medicine Finland, University of Helsinki, Finland 3 University Hospital Hamburg-Eppendorf, Finland 4 Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Finland 5 Department of Biotechnology, University of Turku, Finland 2
Background: Recently, we have developed a homogeneous single-label signaling technique, the Quenching Resonance Energy Transfer (QRET). In this study, the homogeneous QRET technology was applied to monitor GTPase activation cycle and activation cycle inhibition. Methods: The QRET system is based on the protection of target protein bound Eu3+ -GTP from a soluble quencher molecule. (1) The small GTPase cycle (nucleotide exchange and hydrolysis) can be monitored in the competitive assay by tracking GTP hydrolysis in the presence of nanomolar concentrations of H-RasWt , SOScat , and p120GAP. (2) The nucleotide exchange can be monitored using a second assay with H-RasWt and SOScat . The increased timeresolved luminescence (TRL) signal is monitored when either GTP hydrolysis (1) or Eu3+ -GTP association (2) occurs. Results: We have proven the suitability of the QRET system to monitor GTP hydrolysis (1) and nucleotide exchange (2). The
SYMPOSIUM 9: BIOMARKERS AND DIAGNOSTIC TOOLS
GTP hydrolysis assay was used to screen 1280 compound small molecule library whereof twelve inhibitors were found (average Zfactor 0.78). The GTP hydrolysis assay can simultaneously detect inhibitors affecting either nucleotide exchange or GTP hydrolysis. Therefore, same screening was performed by monitoring Eu3+ -GTP association to H-RasWt (average Z-factor 0.78). From the screening assays, seven same inhibitor hits were found. Additionally, five inhibitors were found only in GTP hydrolysis assay. Conclusions: These novel QRET assays for GTPase research can be performed using nanomolar protein concentrations. The presented QRET assays enable the study of whole small GTPase cycle by monitoring the guanine-nucleotide exchange factor (GEF) induced nucleotide exchange and/or GTPase-activating proteins (GAP) catalyzed GTP hydrolysis. http://dx.doi.org/10.1016/j.nbt.2014.05.1696
O9-6 Comparative large scale microRNA expression profiles of cynomolgus monkeys, rat and human reveal miR-182 associated with Type 2 diabetes Hongli Du ∗ , Jinghui Zhou, Yuhuan Meng, Xiaoning Wang South China University of Technology, China
Type 2 diabetes (T2D) is a prevalent disease that is present throughout the world, and is usually associated with insulin resistance. MicroRNAs (miRNAs) play important role in the suppression of gene expression and have been shown to be implicated in human diseases. We used a novel animal model, cynomolgus monkey fed with normal and high fatty diet (HFD) respectively, to analyze the miRNA expression profile in whole blood by deepsequencing. Finally in total 24 miRNAs with differential expression were filtered. Among them, mir-182 and mir-183, related to insulin resistance by modulating FOXO1 and PI3 K/AKT cascade, had the greatest copy number in the whole blood. Decrease of mir-182 in T2D cynomolgus individuals is completely consistent with the previous studies in human and rat. Integrating mir-182 tissue expression profile, target genes and copy number in blood revealed that mir-182 plays a key role in FOXO1 modulation that leads to potential hyperglycemia and modulates the insulin secretion. In addition, the possible miRNA regulation system of T2D under the influence of different diet conditions was also interpreted in the present study. The cholesterol content influences mir-182 and potentially other miRNAs expression level that causes the insulin resistance and the various miRNA regulation systems between normal diet and HFD. http://dx.doi.org/10.1016/j.nbt.2014.05.1697
www.elsevier.com/locate/nbt S37