Synthetic bioreporters for detection of environmental pollutants
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SYMPOSIUM 9: BIOMARKERS AND DIAGNOSTIC TOOLS New Biotechnology · Volume 31S · July 2014 Symposium 9: Biomarkers and diagnostic tools O9-1 Designing nanomaterials for ultrasensitive biosensing Molly Stevens Imperial College London, UK Bio-responsive nanomaterials are of growing importance with potential applications including drug delivery, diagnostics and tis- sue engineering. This talk will provide an overview of our recent developments in the design of materials for ultrasensitive biosens- ing. 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 apply- ing 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 (anti- gens) 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. Anti- bodies are generated by randomly varying the sequences of their antigen-binding loops and selecting rare variants that are com- plementary to target antigens. Due to the daunting number of possible antibody sequences with variation only within their antigen-binding loops (>10 ˆ 30 variants), it seems unlikely that 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 bio- logical interactions, we find that antibody fragments can be readily engineered to recognize diverse aggregated proteins linked to neu- rodegenerative disorders (e.g., Alzheimer’s disease) by targeting unique structural features within such proteins. Our innovative approach generates single- and multidomain antibodies that rec- ognize misfolded proteins not only based on their sequence, but also based on their conformation. We also find that our antibodies recognize sequence epitopes that are difficult to target using con- ventional antibodies, and that these novel antibodies are potent inhibitors of protein aggregation. http://dx.doi.org/10.1016/j.nbt.2014.05.1693 O9-3 Engineering cofactor specificity of methyltransferases Martin Tengg 1,∗ , Yu Zheng 2 , Mandana Gruber-Khadjawi 3 , Elmar Weinhold 1 1 Institute of Organic Chemistry, RWTH Aachen University, Germany 2 New England Biolabs Inc., Ipswich, MA, USA 3 ACIB GmbH, Austrian Centre for Industrial Biotechnology, Graz, Austria Biological methylations of various substrates occur in every living cell and are essential for cell survival. These transforma- tions 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 ana- logues 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 vari- ants. The bacterial enzyme M.SssI is of particular interest because it performs the same reaction as mammalian DNA MTases by tar- geting 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 S36 www.elsevier.com/locate/nbt
Transcript of Synthetic bioreporters for detection of environmental pollutants
SYMPOSIUM 9: BIOMARKERS ANDDIAGNOSTIC TOOLS New Biotechnology · Volume 31S · July 2014
Symposium 9: Biomarkers and diagnostictools
O9-1
Designing nanomaterials for ultrasensitive biosensing
Molly Stevens
Imperial College London, UK
Bio-responsive nanomaterials are of growing importance withpotential applications including drug delivery, diagnostics and tis-sue engineering. This talk will provide an overview of our recentdevelopments in the design of materials for ultrasensitive biosens-ing. Our recent simple conceptually novel approaches to real-timemonitoring of protease, lipase and kinase enzyme action usingmodular peptide functionalized gold nanoparticles and quantumdots will be presented. Furthermore we have recently developeda new approach to ultrasensitive biosensing through plasmonicnanosensors with inverse sensitivity by means of enzyme-guidedcrystal growth as well as a “Plasmonic ELISA” for the ultrasensitivedetection of disease biomarkers with the naked eye. We are apply-ing these biosensing approaches both in high throughput drugscreening and to diagnose diseases ranging from cancer to globalhealth 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 (anti-gens) with high affinity and specificity is central to theirwidespread use in diagnostic and therapeutic applications. Thebinding activity of antibodies is encoded in up to six of theirsolvent-exposed peptide loops that directly contact antigens. Anti-bodies are generated by randomly varying the sequences of theirantigen-binding loops and selecting rare variants that are com-plementary to target antigens. Due to the daunting number ofpossible antibody sequences with variation only within theirantigen-binding loops (>1030 variants), it seems unlikely thatthe needles (antibodies with desired binding activity) in thehaystack (all possible antibody variants) can be predicted instead ofbeing selected. We have challenged this conventional wisdom byreducing the seemingly intractable problem of designing multipleantibody loops to cooperatively bind antigens to a tractable one inwhich we design individual antibody loops with binding activity.Using this simplified design strategy that is inspired by natural bio-logical interactions, we find that antibody fragments can be readilyengineered to recognize diverse aggregated proteins linked to neu-rodegenerative disorders (e.g., Alzheimer’s disease) by targetingunique structural features within such proteins. Our innovativeapproach generates single- and multidomain antibodies that rec-ognize misfolded proteins not only based on their sequence, butalso based on their conformation. We also find that our antibodies
recognize sequence epitopes that are difficult to target using con-ventional antibodies, and that these novel antibodies are potentinhibitors of protein aggregation.
http://dx.doi.org/10.1016/j.nbt.2014.05.1693
O9-3
Engineering cofactor specificity of methyltransferases
Martin Tengg1,∗ , Yu Zheng2, Mandana Gruber-Khadjawi3, ElmarWeinhold1
1 Institute of Organic Chemistry, RWTH Aachen University, Germany2 New England Biolabs Inc., Ipswich, MA, USA3 ACIB GmbH, Austrian Centre for Industrial Biotechnology, Graz, Austria
Biological methylations of various substrates occur in everyliving cell and are essential for cell survival. These transforma-tions are catalyzed by methyltransferases (MTases) which generallytransfer the activated methyl group from S-adenosyl-l-methionine(AdoMet) to DNA, RNA, proteins and small biomolecules. Recentstudies demonstrated that many MTases can accept AdoMet ana-logues for transfer of extended carbon chains to their substrates[1,2]. The ability to accept a broad range of cofactor analogues isof great interest both in terms of DNA diagnostics and biocatalyticsynthesis.
In order to increase transfer rates of extended groups fromAdoMet analogues protein engineering approaches are performed.High-throughput directed evolution of the DNA MTase M.SssI isperformed by in vitro compartmentalization. This method linksgenotype and phenotype for an effective selection of active vari-ants. The bacterial enzyme M.SssI is of particular interest becauseit performs the same reaction as mammalian DNA MTases by tar-geting 5′-CG-3′ (CpG) DNA sequences. CpG modifications are keyepigenetic signatures in transcriptional regulation and genomeimprinting. The transfer of functional groups will provide newtools for DNA labelling as well as DNA modification detection.M.SssI evolution will also guide rational protein engineering of thehomologous small molecule MTase NovO, for the synthesis of newfine chemicals as well as bioactive intermediates and products.
References
[1].Dalhoff C, Lukinavicius G, Klimasauskas S, Weinhold E. Nat ChemBiol 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 environmentalpollutants
Jan Roelof van der Meer ∗ , DavideMerulla, Siham Beggah
University of Lausanne, Switzerland
One of the main immediate application areas for syntheticbiology are bioreporters, living cells with simple designed genetic
S36 www.elsevier.com/locate/nbt
New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 9: BIOMARKERS ANDDIAGNOSTIC TOOLS
circuits that permit detection of a specific chemical or group ofchemicals, under the concomitant production of an easily butaccurately quantifiable reporter signal. Bioreporters have attractedconsiderable interest because they offer cheap alternatives forchemical analysis in remote areas where high-end instruments areunavailable. Such demands on bioreporters, however, require analmost fail-proof and robust technology that goes much beyondwhat traditional research “proof-of-principles” have been able todemonstrate.
We will show on the example of a bioreporter for arsenic, howimportant improvements can be made in the circuit design andin the optimization of the assay technology. Arsenic is a recurringnoxious contaminant of drinking water in large areas of our planet,and bioreporter technology can provide the means to rapidlyquantify its presence in the concentration range of 1–10 �g/L. First,we demonstrate how designing a feedback or an uncoupled circuitaffects the signal output and detection sensitivity for arsenic. Sec-ondly, we show a new system to better control residual backgroundexpression in the circuit while maintaining optimal induction,and we also provide experimental evidence to improve the circuitbehaviour by avoiding cross-interference from the host. Finally, wepresent the use of micro-engineered structures that would permitcontinuous remote operation of a bioreporter sensor.
http://dx.doi.org/10.1016/j.nbt.2014.05.1695
O9-5
A homogeneous quenching resonance energy trans-fer assay for H-Ras activation cycle monitoring andinhibitor screening
Kari Kopra1,∗ , Arjan van Aldrichem2, Markku Syrjänpää1,Stefan Veltel 3, Pekka Hänninen1, Daniel Abankwa4, UrpoLamminmäki5, Harri Härmä1
1 Laboratory of Biophysics, University of Turku, Finland2 Institute for Molecular Medicine Finland, University of Helsinki, Finland3 University Hospital Hamburg-Eppendorf, Finland4 Turku Centre for Biotechnology, University of Turku and Åbo Akademi Univer-sity, Finland5 Department of Biotechnology, University of Turku, Finland
Background: Recently, we have developed a homogeneoussingle-label signaling technique, the Quenching Resonance EnergyTransfer (QRET). In this study, the homogeneous QRET technol-ogy was applied to monitor GTPase activation cycle and activationcycle inhibition.
Methods: The QRET system is based on the protection of tar-get protein bound Eu3+-GTP from a soluble quencher molecule. (1)The small GTPase cycle (nucleotide exchange and hydrolysis) canbe monitored in the competitive assay by tracking GTP hydrolysisin the presence of nanomolar concentrations of H-RasWt, SOScat,and p120GAP. (2) The nucleotide exchange can be monitoredusing a second assay with H-RasWt and SOScat. The increased time-resolved luminescence (TRL) signal is monitored when either GTPhydrolysis (1) or Eu3+-GTP association (2) occurs.
Results: We have proven the suitability of the QRET systemto monitor GTP hydrolysis (1) and nucleotide exchange (2). The
GTP hydrolysis assay was used to screen 1280 compound smallmolecule library whereof twelve inhibitors were found (average Z-factor 0.78). The GTP hydrolysis assay can simultaneously detectinhibitors affecting either nucleotide exchange or GTP hydrolysis.Therefore, same screening was performed by monitoring Eu3+-GTPassociation to H-RasWt (average Z-factor 0.78). From the screeningassays, seven same inhibitor hits were found. Additionally, fiveinhibitors were found only in GTP hydrolysis assay.
Conclusions: These novel QRET assays for GTPase researchcan be performed using nanomolar protein concentrations. Thepresented QRET assays enable the study of whole small GTPasecycle 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 profilesof cynomolgus monkeys, rat and human reveal miR-182associated with Type 2 diabetes
Hongli Du ∗ , Jinghui Zhou, YuhuanMeng, XiaoningWang
South China University of Technology, China
Type 2 diabetes (T2D) is a prevalent disease that is presentthroughout the world, and is usually associated with insulin resis-tance. MicroRNAs (miRNAs) play important role in the suppressionof gene expression and have been shown to be implicated inhuman diseases. We used a novel animal model, cynomolgus mon-key fed with normal and high fatty diet (HFD) respectively, toanalyze the miRNA expression profile in whole blood by deep-sequencing. Finally in total 24 miRNAs with differential expressionwere filtered. Among them, mir-182 and mir-183, related to insulinresistance by modulating FOXO1 and PI3 K/AKT cascade, had thegreatest copy number in the whole blood. Decrease of mir-182in T2D cynomolgus individuals is completely consistent with theprevious studies in human and rat. Integrating mir-182 tissueexpression profile, target genes and copy number in blood revealedthat mir-182 plays a key role in FOXO1 modulation that leadsto potential hyperglycemia and modulates the insulin secretion.In addition, the possible miRNA regulation system of T2D underthe influence of different diet conditions was also interpreted inthe present study. The cholesterol content influences mir-182 andpotentially other miRNAs expression level that causes the insulinresistance and the various miRNA regulation systems between nor-mal diet and HFD.
http://dx.doi.org/10.1016/j.nbt.2014.05.1697
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