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Single-Molecule Assay Development for Studying Human RNA Polymerase II Promoter-Proximal Pausing
Wednesday, March 2, 2016 cell and even approaching the single molecule scale. Here we present the capability of different light based techniques for biosensing. As the first example, surface enhanced Raman spectroscopy (SERS) is performed in onion using silver plasmonic nanostructures. Our studies detect different molecular compounds present in the plant based on their SERS signals. SERS imaging allows us to monitor the location of nanoparticles and to image chemical compounds within the target. Moreover, a pH-sensitive reporter molecule, pMBA attached to the silver nanoparticles, is used to infer pH values in the extracellular space of an onion layer. As a second example, we explore how a membrane protein may be used as an efficient sensor in an organic environment via a biomimetic membrane model. The combination of both biomimetic membranes and protein membranes as a signal transduction medium has interesting applications in biology and medicine. It is crucial that the matrix where a protein is embedded is optimal in order to maintain the concentration gradient. Moreover, curvature and mechanical forces in the membrane may also affect the protein function. In this work, by inducing chemical and mechanical changes of the matrix we optimize the system via measuring variations of the gradient through the membrane. 3151-Pos Board B528 Studying the Hsp90 Machinery in Living Cells by Single Molecule FRET Philipp Wortmann1, Fernando Aprile-Garcia2, Ritwick Sawarkar2, Thorsten Hugel1. 1 Physical Chemistry, University Freiburg, Freiburg, Germany, 2Max Planck Institute of Immunbiology and Epigenetics, Freiburg, Germany. The 90 kDa heat shock protein Hsp90 is an ubiquitous molecular chaperone regulated by cochaperones, overexpressed upon stress and involved in several cellular processes such as proliferation, signal transduction and transcription. The interaction with various oncoproteins makes Hsp90 an attractive drug target. It remains unclear, how exactly Hsp90 fulfills its various roles within a cell and how drugs affect Hsp90 inside a cell. Single molecule Fo¨rster Resonance Energy Transfer (smFRET) has been used to study the mechanism of Hsp90’s ATPase and chaperoning function. These experiments showed that the conformational dynamics of yeast Hsp90 is dominated by thermal fluctuations and not by nucleotides [1]. The presence of the cochaperone p23 increases the effect of nucleotides and introduces some directionality in Hsp90’s ATPase cycle [2]. However, until now more than 20 cochaperones have been identified in eukaryotes, which makes ‘‘bottom up’’ in vitro experiment very difficult. Here we therefore study the dynamics of Hsp90 and the associated heavily regulated complex machinery [3] by applying smFRET on it in living cells. We build on previous approaches [4,5] and decrease background noise and increase observation time. This allows us to study Hsp90 in its native environment and likely to directly observe drug effects on their target molecule in a cell. [1] Ratzk et al. ‘‘Hsp90’s mechano-chemical cycle is dominated by thermal fluctuations’’, PNAS,2012. [2] Ratzke, et al. ‘‘Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery’’, Nature Communications, 2014. [3] Taipale et al. ‘‘HSP90 at the hub of protein homeostasis: emerging mechanistic insights’’, Nature Reviews, 2010. [4] Crawford, Torella, Aigrain, Plochowietz et al. ‘‘Long-lived intracellular single-molecule fluorescence using electroporated molecules’’, Biophys J, 2013. [5] Ko¨nig et al. ‘‘Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells’’, Nature Methods, 2015. 3152-Pos Board B529 Diffusion and Biochemical Reactions in Inhomogeneous Crowded Fluids Olivia Stiehl, Matthias Weiss. University of Bayreuth, Bayreuth, Germany. Cellular fluids, e.g. the cytoplasm, are crowded with macromolecules at concentrations up to 400g/l. Macromolecular crowding influences transport processes and biochemical reactions [1]. A particular example for the latter is the opening and closing kinetics of DNA hairpins which we have studied by UV hyperchromicity and fluorescence correlation spectroscopy (FCS). Timing and steady state of this reaction are differentially altered by the crowders’ excluded volume and by a crowding-induced viscoelasticity of the fluid that also causes an anomalous diffusion of the hairpin. As a result, we observed an increased fraction of closed DNA hairpins in viscoelastic crowded fluids [2,3], which compares favorably to a simple statistical model that considers both facets of crowding.
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Going beyond these observations, we have also investigated the spatial heterogeneity of cellular and biomimetic crowded fluids using the molecular rotor dye DASPMI as a reporter. On mesoscale, FCS experiments revealed that both, cells and biomimetic solutions, exhibit a comparable spatial heterogeneity of diffusion times and anomalies. On nanoscale, however, fluorescent lifetime imaging microscopy (FLIM) highlighted a considerably increased inhomogeneity of cellular fluids as compared to artificial solutions. Hence, crowding in cellular fluids has certain facets that are not well captured by biomimetic fluids. [1] Weiss, Int. Rev. Cell Mol. Biol., 307, 383-417 (2014). [2] Stiehl, Weidner-Hertrampf & Weiss, New J. Phys. 15 113010 (2013). [3] Stiehl, Weidner-Hertrampf & Weiss, Phys. Rev. E 91, 012703 (2015). 3153-Pos Board B530 Single-Molecule Masspic Analysis of Short-Chain PEG Siyun Chen, Chan Cao, Yi-tao Long. Key Laboratory for Advanced Materials & Department of Chemistry, East China University of Science and Technology, Shanghai, China. Single-molecule masspic analysis is able to gain the mass information of a single molecule through nanopore technique. Moreover, a special conjugation of DNA hairpin structure to the poly(ethylene glycol) molecule is designed to enhance the temporal resolution of low molecular weight poly(ethylene glycol) in nanopore studies. With the merit of this design, the detection of an individual PEG with molecular weight as low as 140 Da is achieved at the single-molecule level in solution, which provides a novel strategy for characterization of an individual small molecule within a nanopore. 3154-Pos Board B531 Single-Molecule Assay Development for Studying Human RNA Polymerase II Promoter-Proximal Pausing Yazan K. Alhadid1,2, Benjamin L. Allen3, SangYoon Chung1,2, Dylan J. Taatjes3,4, Shimon Weiss1,2. 1 Chemistry & Biochemistry, UCLA, Los Angeles, CA, USA, 2Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA, 3 Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, CO, USA, 4Chemistry & Biochemistry, University of Colorado, Boulder, Boulder, CO, USA. Promoter-proximal RNA Polymerase II (Pol-II) pausing has been shown to play a significant role in transcription regulation of elongating Pol-II complexes in a large number of metazoan and mammalian genes (1). The traditional understanding of transcription regulation in mammals involved controlling Pol-II recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. Most works investigating promoter-proximal PolII pausing have employed chromatin immunoprecipitation followed by sequencing to determine Pol-II localization or in vitro transcriptional assays using nuclear extracts analyzed with radioactive gel electrophoresis. In order to gain greater mechanistic insight into the regulation of promoter-proximal Pol-II pausing, we use single molecule ALEX spectroscopy to monitor RNA transcripts production as function of composition and order of addition of transcription factors to an in vitro reconstituted human Pol-II system. The RNA transcripts are detected by complementary doubly dyelabeled single-stranded DNA (ssDNA) probes. The human gene HSPA1B for heat shock protein 70 (Hsp70) is used as a model system due to its extensive characterization in drosophila. Our approach provides a rapid, sensitive and robust avenue for screening protein factors regulating promoter-proximal Pol-II pausing. 1. H. Kwak, J. T. Lis, Control of transcriptional elongation. Annu. Rev. Genet. 47, 483-508 (2013). 3155-Pos Board B532 Design and Development of a Telomere Sensor Based on Fluorescence Energy Transfer Haitao Li1,2. 1 Department of Chemistry, University of Cambridge, Cambridge, United Kingdom, 2School of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou, China. A 200 nm in-diameter single-bead sensor for detecting single unlabeled DNA molecules in solution based on fluorescence resonance energy transfer is presented. Both bulk and single molecule fluorescence experiments were carried out at room temperature. The G-rich telomere forming a G-quadruplex structure in the presence of ZnTCPP showed two strong donor and acceptor signals.
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Going beyond these observations, we have also investigated the spatial heterogeneity of cellular and biomimetic crowded fluids using the molecular rotor dye DASPMI as a reporter. On mesoscale, FCS experiments revealed that both, cells and biomimetic solutions, exhibit a comparable spatial heterogeneity of diffusion times and anomalies. On nanoscale, however, fluorescent lifetime imaging microscopy (FLIM) highlighted a considerably increased inhomogeneity of cellular fluids as compared to artificial solutions. Hence, crowding in cellular fluids has certain facets that are not well captured by biomimetic fluids. [1] Weiss, Int. Rev. Cell Mol. Biol., 307, 383-417 (2014). [2] Stiehl, Weidner-Hertrampf & Weiss, New J. Phys. 15 113010 (2013). [3] Stiehl, Weidner-Hertrampf & Weiss, Phys. Rev. E 91, 012703 (2015). 3153-Pos Board B530 Single-Molecule Masspic Analysis of Short-Chain PEG Siyun Chen, Chan Cao, Yi-tao Long. Key Laboratory for Advanced Materials & Department of Chemistry, East China University of Science and Technology, Shanghai, China. Single-molecule masspic analysis is able to gain the mass information of a single molecule through nanopore technique. Moreover, a special conjugation of DNA hairpin structure to the poly(ethylene glycol) molecule is designed to enhance the temporal resolution of low molecular weight poly(ethylene glycol) in nanopore studies. With the merit of this design, the detection of an individual PEG with molecular weight as low as 140 Da is achieved at the single-molecule level in solution, which provides a novel strategy for characterization of an individual small molecule within a nanopore. 3154-Pos Board B531 Single-Molecule Assay Development for Studying Human RNA Polymerase II Promoter-Proximal Pausing Yazan K. Alhadid1,2, Benjamin L. Allen3, SangYoon Chung1,2, Dylan J. Taatjes3,4, Shimon Weiss1,2. 1 Chemistry & Biochemistry, UCLA, Los Angeles, CA, USA, 2Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA, 3 Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, CO, USA, 4Chemistry & Biochemistry, University of Colorado, Boulder, Boulder, CO, USA. Promoter-proximal RNA Polymerase II (Pol-II) pausing has been shown to play a significant role in transcription regulation of elongating Pol-II complexes in a large number of metazoan and mammalian genes (1). The traditional understanding of transcription regulation in mammals involved controlling Pol-II recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. Most works investigating promoter-proximal PolII pausing have employed chromatin immunoprecipitation followed by sequencing to determine Pol-II localization or in vitro transcriptional assays using nuclear extracts analyzed with radioactive gel electrophoresis. In order to gain greater mechanistic insight into the regulation of promoter-proximal Pol-II pausing, we use single molecule ALEX spectroscopy to monitor RNA transcripts production as function of composition and order of addition of transcription factors to an in vitro reconstituted human Pol-II system. The RNA transcripts are detected by complementary doubly dyelabeled single-stranded DNA (ssDNA) probes. The human gene HSPA1B for heat shock protein 70 (Hsp70) is used as a model system due to its extensive characterization in drosophila. Our approach provides a rapid, sensitive and robust avenue for screening protein factors regulating promoter-proximal Pol-II pausing. 1. H. Kwak, J. T. Lis, Control of transcriptional elongation. Annu. Rev. Genet. 47, 483-508 (2013). 3155-Pos Board B532 Design and Development of a Telomere Sensor Based on Fluorescence Energy Transfer Haitao Li1,2. 1 Department of Chemistry, University of Cambridge, Cambridge, United Kingdom, 2School of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou, China. A 200 nm in-diameter single-bead sensor for detecting single unlabeled DNA molecules in solution based on fluorescence resonance energy transfer is presented. Both bulk and single molecule fluorescence experiments were carried out at room temperature. The G-rich telomere forming a G-quadruplex structure in the presence of ZnTCPP showed two strong donor and acceptor signals.