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High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA
Tuesday, February 14, 2017 1621-Plat Novel Local RYR1 Junctional Calcium Responses and Dynamics by Improved Calcium Sensor Catcher Jenny Yang1, Florence Reddish1, Cassie Miller1, Rakshya Gorkali1, Susan Treves2, Francesco Zorzato2. 1 Georgia State University, Atlanta, GA, USA, 2University of Ferrara, Ferrara, Italy. The precise spatio-temporal characteristics of Ca2þ transient are critical for the physiological regulation of Ca2þ-dependent signaling processes. Because determining Ca2þ dynamics occurring in the myoplasm and sarcoplasmic reticulum (SR) under normal conditions is essential in order to fully understand changes that may occur under stressful or pathological conditions, we designed a new generation of Ca2þ sensors. Here, we report the optimization of a genetically-encoded Ca2þ sensor called ‘‘CatchER’’ which exhibits rapid kinetics and specific targeting to the endoplasmic reticulum (ER). We also describe the characteristics of CatchER-T’, a sensor variant with significantly improved specificities, including enhanced fluorescence at 37 C and a greater calcium dependent dynamic range. CatchER-T’ is targeted to the ER via the signal peptide of calreticulin and the ER retention sequence KDEL. Differential drug induced Ca2þ releases and ER refilling are observed for various cell types. Furthermore in electroporated mouse flexor digitorum brevis (FDB) muscle fibers, targeting CatchER-T’ close to the RyR1 channel via a sequence obtained from the junctional SR protein JP-45, revealed that local Ca2þ release via electrical stimulation is significantly greater and faster than that occurring in the SR lumen as assessed using CatchER-T’, i.e. the probe targeted to the lumen of the longitudinal SR. The impact of local ER/SR calcium dynamics in controlling cellular signaling and excitation contraction coupling will be presented. 1622-Plat High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA Tory M. Schaaf1, Kurt C. Peterson2, Benjamin D. Grant2, Samantha L. Yuen1, Prachi Bawaskar1, Ji Li1, Joesph M. Muretta1, Gregory D. Gillispie2, David D. Thomas1. 1 Biochemsitry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA, 2Fluorescence Innovations, Minneapolis, MN, USA. A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral data also was applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentrationresponse curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS. This work was supported by NIH grants (GM27906, HL129814, AR07612, and DA037622). 1623-Plat Bright Monomeric Near-Infrared Fluorescent Proteins for Multiscale Imaging Daria Shcherbakova, Mikhail Baloban, Vladislav Verkhusha. Albert Einstein College of Medicine, Bronx, NY, USA. Monomeric near-infrared (NIR) fluorescent proteins (FPs) and biosensors are in high demand as additional colors for microscopy and, particularly, for deeptissue imaging. We engineered three bright and spectrally distinct monomeric NIR FPs, called miRFPs, miRFPs are 2-5-fold brighter in mammalian cells than the only available monomeric NIR FP and perform well in protein fusions, allowing multicolor structured illumination microscopy. Starting from miRFPs, we engineered the first monomeric multicolor fluorescence complementation reporters for protein-protein interactions and RNA labeling. We then designed NIR IkBa signaling reporter for canonical NF-kB signaling in both cells and animals. We further developed NIR cell cycle biosensor for detection of proliferation status of individual cells and cell populations. For the first time, miRFPs
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enable non-invasive visualization of biological processes at different scales, from super-resolution microscopy to in vivo imaging, using the same probes. 1624-Plat The Investigation of Dynamic Changes of the Particle Surface Charge with Resistive-Pulse Technique Yinghua Qiu1, Anna Dawid2, Preston Hinkle1, Yunfei Chen3, Zuzanna Siwy1. 1 Department of Physics and Astronomy, University of California, Irvine, CA, USA, 2College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Warsaw, Poland, 3School of Mechanical Engineering, Southeast University, Nanjing, China. In the resistive-pulse technique, individual particles passing through a pore cause transient changes of the transmembrane current, and the amplitude of the current change corresponds to the object size. This method has been applied to detect cells, viruses, molecules (e.g. DNA and proteins) and particles. With the special structure of track-etched polycarbonate pores, i.e. narrower entrances and cylindrical interior, the events of highly carboxylated charged polystyrene particles can show a current decrease when a particle enters the pore, a flat region in the center part, and a current increase when the particle exits the pore. The current increase results from concentration polarization as well as ionic sourcing effects, which depend on the particle surface charge density. Thus, the characteristics of the obtained resistive-pulse signal carry information not only on the size of the objects, but also on dynamic changes of the particle surface charge via protonation/deprotonation of surface carboxyl groups. Experiments were performed with a pH gradient set across the pore, so that the particles while translocating would change their effective surface charge due to the dependence of protonation of carboxyl groups on the local proton concentration. Analysis of the current increase at the end of the event revealed that the process of deprotonation/protonation required longer time than the transit time. This finding was unexpected, because the transit times were tens of milliseconds thus significantly longer then the chemical reaction time estimated based on proton diffusion. Our experiments also indicated that a longer pore and a higher pH gradient led to a more complete protonation/deprotonation in the pore during transit. Using our finding, we designed a protocol to trap individual particles in a pore for as long as 3 seconds. The experiments are important for developing new methods which can be used to in situ characterize and precisely control single molecules and particles. 1625-Plat Electrostatic Control of DNA Hydridization Kinetics Studied with the Single-Molecule Field-Effect Transistor Sefi Vernick, Scott M. Trocchia, Steven B. Warren, Erik F. Young, Delphine Bouilly, Ruben L. Gonzalez Jr., Colin Nuckolls, Kenneth L. Shepard. Columbia university, New York, NY, USA. Single-molecule studies generally rely on fluorescence-based reporting with signal levels limited by photon emission from single optical reporters to effective current levels in optical detectors of much less than 1 fA. Bioelectronic detection with a point-functionalized carbon nanotube transistor, known as the singlemolecule field-effect transistor (smFET), in contrast offers signal levels that are more than 106 times higher. In our case, point functionalization is achieved with a nano-confined diazonium attachment chemistry. We previously used smFETs to investigate DNA hybridization kinetics, yielding rate constants, melting curves and activation energies for different oligonucleotides. Temporal analysis of association and dissociation reaction rate constants with temperature allows both target cDNA concentrations and free energies for hybridization to be determined. Here we show that hybridization kinetics are strongly affected by bias between the smFET device and the surrounding electrolyte, allowing bias to act as a proxy for temperature. We identify various concentrations of 20mer target sequences from the Ebola Zaire nucleoprotein gene through smFET detection. Electrostatic modulation enables the detection of single base mismatches due to significantly altered kinetics under applied potential. 1626-Plat Locked Nucleic Acid Thymine Monomer Probe Identifies Four SingleNucleotide Variants by Melting Temperature Judy M. Obliosca1, Sara Y. Cheng2, Yu-An Chen1, Mariana F. Llanos3, Yen-Liang Liu1, Darren M. Imphean1, David Bell1, Jeffrey T. Petty3, Pengyu Ren1, Tim Yeh1. 1 Department of Biomedical Engineering, University of Texas at Austin, AUSTIN, TX, USA, 2Department of Physics, University of Texas at Austin, AUSTIN, TX, USA, 3Department of Chemistry, Furman University, Greenville, SC, USA. High-resolution melting (HRM) analysis of DNA is a closed-tube SNP detection method that has shown many advantages in clinical laboratory,
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enable non-invasive visualization of biological processes at different scales, from super-resolution microscopy to in vivo imaging, using the same probes. 1624-Plat The Investigation of Dynamic Changes of the Particle Surface Charge with Resistive-Pulse Technique Yinghua Qiu1, Anna Dawid2, Preston Hinkle1, Yunfei Chen3, Zuzanna Siwy1. 1 Department of Physics and Astronomy, University of California, Irvine, CA, USA, 2College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Warsaw, Poland, 3School of Mechanical Engineering, Southeast University, Nanjing, China. In the resistive-pulse technique, individual particles passing through a pore cause transient changes of the transmembrane current, and the amplitude of the current change corresponds to the object size. This method has been applied to detect cells, viruses, molecules (e.g. DNA and proteins) and particles. With the special structure of track-etched polycarbonate pores, i.e. narrower entrances and cylindrical interior, the events of highly carboxylated charged polystyrene particles can show a current decrease when a particle enters the pore, a flat region in the center part, and a current increase when the particle exits the pore. The current increase results from concentration polarization as well as ionic sourcing effects, which depend on the particle surface charge density. Thus, the characteristics of the obtained resistive-pulse signal carry information not only on the size of the objects, but also on dynamic changes of the particle surface charge via protonation/deprotonation of surface carboxyl groups. Experiments were performed with a pH gradient set across the pore, so that the particles while translocating would change their effective surface charge due to the dependence of protonation of carboxyl groups on the local proton concentration. Analysis of the current increase at the end of the event revealed that the process of deprotonation/protonation required longer time than the transit time. This finding was unexpected, because the transit times were tens of milliseconds thus significantly longer then the chemical reaction time estimated based on proton diffusion. Our experiments also indicated that a longer pore and a higher pH gradient led to a more complete protonation/deprotonation in the pore during transit. Using our finding, we designed a protocol to trap individual particles in a pore for as long as 3 seconds. The experiments are important for developing new methods which can be used to in situ characterize and precisely control single molecules and particles. 1625-Plat Electrostatic Control of DNA Hydridization Kinetics Studied with the Single-Molecule Field-Effect Transistor Sefi Vernick, Scott M. Trocchia, Steven B. Warren, Erik F. Young, Delphine Bouilly, Ruben L. Gonzalez Jr., Colin Nuckolls, Kenneth L. Shepard. Columbia university, New York, NY, USA. Single-molecule studies generally rely on fluorescence-based reporting with signal levels limited by photon emission from single optical reporters to effective current levels in optical detectors of much less than 1 fA. Bioelectronic detection with a point-functionalized carbon nanotube transistor, known as the singlemolecule field-effect transistor (smFET), in contrast offers signal levels that are more than 106 times higher. In our case, point functionalization is achieved with a nano-confined diazonium attachment chemistry. We previously used smFETs to investigate DNA hybridization kinetics, yielding rate constants, melting curves and activation energies for different oligonucleotides. Temporal analysis of association and dissociation reaction rate constants with temperature allows both target cDNA concentrations and free energies for hybridization to be determined. Here we show that hybridization kinetics are strongly affected by bias between the smFET device and the surrounding electrolyte, allowing bias to act as a proxy for temperature. We identify various concentrations of 20mer target sequences from the Ebola Zaire nucleoprotein gene through smFET detection. Electrostatic modulation enables the detection of single base mismatches due to significantly altered kinetics under applied potential. 1626-Plat Locked Nucleic Acid Thymine Monomer Probe Identifies Four SingleNucleotide Variants by Melting Temperature Judy M. Obliosca1, Sara Y. Cheng2, Yu-An Chen1, Mariana F. Llanos3, Yen-Liang Liu1, Darren M. Imphean1, David Bell1, Jeffrey T. Petty3, Pengyu Ren1, Tim Yeh1. 1 Department of Biomedical Engineering, University of Texas at Austin, AUSTIN, TX, USA, 2Department of Physics, University of Texas at Austin, AUSTIN, TX, USA, 3Department of Chemistry, Furman University, Greenville, SC, USA. High-resolution melting (HRM) analysis of DNA is a closed-tube SNP detection method that has shown many advantages in clinical laboratory,