Some like it hot: Biomolecule Analytics using Microscale Thermophoresis

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1 Some like it hot: Biomolecule Analytics using Microscale Thermophoresis NanoTemper Technologies GmbH Flößergasse 4, München, Germany.

2 Thermophoresis Electrophoresis Electric Field Thermophoresis Temperature-Gradient + - hot cold charge, (size) charge, size and hydration shell Boehringer-Ingelheim # 21 German-Russian Forum Munich

3 Optical-Thermophoresis Basic setup of the instrument LED excitation Fluorescence detector Temperature field induced by IR-Laser IR -Laser 23 C 20 C capillary 1K /10µm temperature increase corresponds to 1000K/cm - huge gradient on microscale area - biomolecule compatible temprature increase:2-6 K - low volume :4µl of sample per titration point sec measurement per titration point 2

4 Thermophoresis DNA Positive -Thermophoresis Microbeads Negative -Thermophoresis Boehringer-Ingelheim # 34

5 Theory c hot /c cold = exp (-S T ΔT) ionic shielding entropy Duhr and Braun PNAS 103, (2006) Duhr and Braun PRL 96, (2006) size charge 2 hydration shell hydration entropy Boehringer-Ingelheim # 54

6 Typical Thermophoretic Signal # 6 Boehringer-Ingelheim 5

7 Simulation of Optical-Thermophoresis sample heating < 1sec temperature gradient Thermophoresis ~30sec convection > 1sec Jerabek-Willemsen et al,

8 Bio-molecule Interactions baseline saturation Boehringer-Ingelheim # 87

9 Approved MST applications protein-protein protein-dna/rna nucleic acid nucleic acid protein-small molecule, peptides, ions protein or small molecule HMW complexes (e.g. ribosome) protein or peptide-liposome/vesicle covalent modifications of nucleic acids, proteins etc. enzymatic activity 8

10 Depletion (%) Ligand Receptor binding KD = 1.8 nm +/- 0.3 nm 1,5 FolR1 ΔFnorm (% ) 1,0 0,5 0, conc FolR1 (nm). Material was kindly provided by Dr. Carolyn Simpson, Covidien, St. Louis, USA Mikrogen GmbH # 109

11 Fragments and Small Molecules binding p38 p38 Material was kindly provided by Krishna Saxena, University Frankfurt, Germany Boehringer-Ingelheim # 11 10

12 Competitive Approach - Tracer Tr p38 C1 C1 p38 Tr - Site Specific - Fixed Response Amplitude (Cut-off) - Strong Response - Functional Assay Material was kindly provided by Dr.Krishna Saxena (University Frankfurt) Boehringer-Ingelheim # 12 11

13 NanoTemper response (a.u.) NanoTemper response (a.u.) NanoTemper response (a.u.) NanoTemper response (a.u.) Binding Kinetics p38 ΔFnorm (% ) 4,0 3,5 3,0 2,5 2,0 1,5 1,0 0 min, KD = 439 nm +/- 127 nm 30 min, KD = 31 nm +/- 8 nm 3,0 0 min. 30 min. ΔFnorm (% ) 2,5 2,0 1,5 1,0 0,5 0,5 0,0 0,0-0, conc BIRB-796 (nm) conc BIRB-796 (nm) ΔFnorm (% ) 3,0 2,5 2,0 1,5 1,0 0,5 0,0 120 min, KD = 6,3 nm +/- 2,3 nm 60 min, KD =12 nm +/- 3 nm 2,5 60 min. 120 min. ΔFnorm (% ) 2,0 1,5 1,0 0,5 0, conc BIRB-796 (nm) conc BIRB-796 (nm) Material was kindly provided by Dr.Krishna Saxena (University Frankfurt) Boehringer-Ingelheim # 13 12

14 Ca2+-Ion Binding to Calmodulin Ca2+ Ca 2+ Mg2+ Conformational change of calmodulin from Ca 2+ unbound to bound Wienken et al., nature communication, Sandoz # 14 13

15 Protein Protein Interactions Boehringer-Ingelheim # 15 14

16 Binding to High MW Complexes I Binding of a multimeric ribosomal interactor to the ribosome multimeric ribosomal interactor Ribosome Results were kindly provided by Julian Deeng, Genzentrum LMU München, Germany Boehringer-Ingelheim # 16 15

17 Binding to High MW Complexes II Binding of erythromycin to the 50S ribosome ΔFnorm (% ) Negative control: negamycin ΔFnorm (% ) Mikrogen GmbH # 17 15

18 Interaction in Bioliquids PKA-quercetin binding in human serum PKA-quercetin binding in HSA-containing buffer KD=0.13µM KD=6µM KD=50µM KD=8µM KD=43µM w/o Serum 5% Serum 30% Serum Direct Assay HSA-quercetin binding KD=9µM Wienken et al., Nature Communications, Boehringer-Ingelheim # 18 16

19 Membrane Systems Fnorm (% ) Fnorm (% ) nm vesicle containing coiled-coil forming petides Material was kindly provided by Prof. Andreas Janshoff, University of Göttingen, Dept. of Physical Chemistry. Labeled Liposom contains SNAP-25, Syntaxin-1A and labeled syb2-peptid vs. Unlabeled liposom contains SNARE protein synaptobrevin-2 Material was kindly provided by Karsten Meyenberg, Geert van den Bogaart, Reinhard Jahn and Ulf Diederichsen, University of Göttingen, Dept. of Physical Chemistry Boehringer-Ingelheim # 19 17

20 Direct analysis of DNA adducts IF based assay NTX 20 µm CisPt Detection in cells Time intensive High background/unspecific signal Low throghput DAPI Cy3 MST MST-Signal (%) Detection on DNA level ( > ng DNA) Short sample processing (30 Minutes) Almost no background high throghput possible (30 Sec / sample) Concentration cisplatin (µm) # 20 19

21 Enzymatic Activity Assays ΔFnorm (% ) Boehringer-Ingelheim # 21 20

22 Compound Induced Aggregation/ ph shift Boehringer-Ingelheim # 22 21

23 Benefits User friendly: Easy to Handle Rapid: < 45 seconds Maintenance-Free Low costs per experiment Mix-and-Read Low sample consumption (< 4 µl, 1nM of Sample) No surface immobilization; No gel-matrix No change in molecule size or mass needed for the analysis Close-to-in-vivo conditions (measurements in pure serum and cell lysate) Dynamic Range: nm to mm (molecule concentration and dissociation constants) same sensitivity for all kinds of interaction (e.g. protein-protein or protein smallmolecule) FRET compatible Multiplexing Boehringer-Ingelheim # 23

24 NanoTemper Instrument # 24 Roche 24

25 NanoTemper Instrument Academic Institutions: University of Göttingen, Prof.Wintermeyer Pettenkofer Institute, Munich, Prof. Baiker University of Munich, Prof. Imhof Genecenter LMU, Munich Prof. Förstemann University of Göttingen, Prof. Diederichsen / Janshoff Forschungszentrum Jülich, Prof. Willbold FMP Berlin, Dr. Haseloff University of Marburg, Prof. Klebe University of Frankfurt, Dr. Krishna Saxena University of Regensburg, Prof. Längst University of Hannover, Prof. Manstein University of Berlin (FU), Prof. Heberle University of Munich, Prof. Rädler University of Bayreuth, Prof. Steegborn University of Wuerzburg, Prof. Mueller University of Bonn, Prof. Famulok Helmholtz Center Munich, PD Kamyar Hadian MPI for Immunbiology, Freiburg, Prof. Jenuwein MPI for Biophysical Chemistry, Göttingen, Prof. Rodnina TU Eindhoven, Netherlands, Prof. Brunsveld Cincinnati Childrens Hospital, USA, Prof. Zheng NIH, USA, Prof. Jim Inglese University of Texas, Prof. John Ladbury University Aarhus, Denmark, Prof. Stougaard Marie-Curie Institute, France, Prof. Houdusse Industry: Novartis, Switzerland Sandoz (Hexal), Germany Boehringer-Ingelheim, Germany Glenmark Pharmaceuticals, Switzerland Aptares AG, Germany Crelux GmbH, Germany Qiagen, Germany Roche, Germany Sanofi-Aventis, France Roche # 25

26 Thanks for your Attention! NanoTemper is supported by: Center for Nanoscience, University Munich Ludwig-Maximilians-University Munich Nano-Initiative-Munich Federal-Ministry for Science and Education, Germany Bavarian-Ministry for Science and Education, Germany # 26