Biomimetic Nanopores for Studying Yeast Nuclear Pore Transport

Monday, February 29, 2016 homemade two-nanopipette system has been developed. One nanopipette is used for nanodosing of protein aggregates of beta amyloid, the protein associated with Alzheimer’s disease, from the tip of a nanopippete. A second nanopipette is used as an adenosine triphosphate (ATP) sensor, fabricated at the tip of a double-barrel carbon-filled nanopipette. The operational principle of this ATP sensor is based on a Hexokinase-modified electrolyte-gated organic field-effect-transistor (EGOFET), where polypyrrole is used as the biocompatible active material, and its conductivity is increased by protonation due to ATP reactions mediated by hexokinases. Using this instrument the transient ATP release from a single astrocyte or neuron, resulting from controlled dosing of oligomers of beta amyloid to the cell, can be recorded. This should allow us to obtain new insights into the molecular mechanism of cellular damage. 1651-Pos Board B628 Live Cell Imaging of Cytosolic NADH/NADD Ratio in Hepatocytes using the Fluorescent Sensor Peredox Ricard Masia1,2, William J. McCarty3, Carolina Lahmann2, Jay Luther4, Raymond T. Chung4, Martin L. Yarmush3, Gary Yellen2. 1 Pathology, Massachusetts General Hospital, Boston, MA, USA, 2 Neurobiology, Harvard Medical School, Boston, MA, USA, 3Surgery, Massachusetts General Hospital, Boston, MA, USA, 4Gastroenterology Unit, Massachusetts General Hospital, Boston, MA, USA. Fatty liver disease (FLD) is the most common chronic liver disease worldwide. FLD may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NADþ ratio, but it remains unresolved whether the increased ratio is important for development of FLD. Our ability to examine this is hindered by limitations of available methods to measure cytosolic NADH/NADþ ratio. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NADþ ratio in living hepatocytes. Peredox was expressed in cultured rat hepatocytes by transfection and in acute mouse liver slices by tail vein injection of AAV-packaged sensor. Under control conditions (glucose present), cultured hepatocytes and hepatocytes in liver slices exhibit a relatively low (oxidized) cytosolic NADH/NADþ ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH), as expected for mammalian cells: lactate increases it, while pyruvate decreases it. Ethanol causes a robust dose-dependent increase in cytosolic NADH/NADþ ratio which is prevented by the ADH inhibitor 4-methylpyrazole (4-MP). Substrates of sorbitol dehydrogenase (SDH, expressed at high levels in hepatocytes) also alter cytosolic NADH/NADþ ratio in a predictable manner: sorbitol and xylitol increase it, while fructose decreases it. The effects of ethanol on cytosolic NADH/NADþ ratio are mitigated by the presence of NADþ-generating substrates of LDH (pyruvate) or SDH (fructose). Live cell imaging with Peredox is a promising approach to examine the role of increased cytosolic NADH/NADþ ratio in the pathogenesis of FLD. The ability to perform these experiments in liver slices is particularly attractive because, unlike dissociated hepatocytes, they allow preservation of liver microanatomy and metabolic zonation of hepatocytes. 1652-Pos Board B629 Synonymous Modification Enables High Fidelity Expression of Biosensors and Probes with Repetitive Protein and Nucleotide Sequences Bin Wu, Veronika Miskolci, Louis Hodgson, Robert H. Singer. Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA. Repetitive nucleotide or amino acid sequences are often engineered into biosensors and probes to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously difficult to construct and maintain for stable expression. They are easily deleted or truncated, which occurs randomly and is not a priori predictable, resulting in aberrant expression and impacting the ability to correctly detect and to interpret biological functions. Here, we introduce a simple and generally applicable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them non-repetitive but still functional (‘‘synonymous’’). We first demonstrate the procedure by designing a cassette of synonymous MS2 RNA aptamers, and tandem coat proteins for RNA imaging and show a dramatic improvement in signal and reproducibility in single RNA detection in live cells. The same approach is extended to enhance the stability of engineered fluorescent biosensors containing FRET-pair of fluorescent proteins, upon which a great majority of systems thus far in the field are based. Using the synonymous modified FRET biosensors, we achieve correct expression of full-length sensors, eliminating the aberrant truncation products that were often assumed to be due to non-specific proteolytic cleavages. Importantly,

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the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus the method we describe here should be routinely employed in generation of probes and sensors containing multiple, repetitive motifs to achieve correct expression these state-of-the-art tools. 1653-Pos Board B630 Biomimetic Nanopores for Studying Yeast Nuclear Pore Transport Adithya N. Ananth1, Roderick Versloot2, Aravind Dwarkasing1, Steffen Frey3, Dirk Goerlich3, Cees Dekker1. 1 Bionanoscience, TU Delft, Delft, Netherlands, 2TU Delft, Delft, Netherlands, 3Max Planck Institute for Biophysical Chemistry, Goettingen, Germany. Nuclear pore complexes (NPC) acts as the gatekeeper to and from the nucleus in eukaryotic cells. NPCs function by creating a selective diffusion barrier for molecules bigger than 30kD (~5 nm), where all proteins are barred from transport, except importer proteins that can freely pass. The mechanism underlying this intriguing selectivity of nuclear pore complexes is still under debate. A crucial element are the intrinsically disordered proteins with phenylalanine (F) and glycine (G) repeats, known as FG-NUPs, which form the centre of the NPC channel. Here, we fabricate and characterize biomimetic nanopores that mimic the NPC, with the aim to address the question of selective transport. We coat solid-state nanopores with key FG NUPs to create a biomimetic nanopores as a single-molecule detector. Yeast NSP1 (wild type or mutated) proteins were coated onto nanopore. The yeast importer protein KAP95 was found to be able to translocate across the biomimetic nanopores, unlike control protein tCherry which did not. We find the translocation times of KAP95 to be higher in FG-NUP-coated pores compared to uncoated nanopores. In mutated FG-NUP-NSP1 hydrophobic amino acid residues (F, I, L and V) were replaced with serine, thus making the NUPs hydrophilic. The translocation times of both KAP95 and tCherry through such pores are comparable with those for uncoated nanopores. We thus show that hydrophobic amino acid residues are essential for the cohesiveness and the selective behavior of FG-NUPs based nanopores. The lack of cohesiveness in mutated NSP1 coated nanopores is presumably caused by the lack of inter-repeat interactions. These findings explain why mutated-Nsp1-modified nanopores do not exhibit selectivity and contribute to the understanding of the mechanism of selectivity in the NPC at the simplest level. Key words: Nanopores, Biomimetics, Nuclear pore complex and FG NUPs. 1654-Pos Board B631 Theoretical Simulation and Experimental Investigation for the Identification and Analysis of Biphasic Surface Plasmon Resonance Data Purushottam Tiwari1, Yesim Darici2, Jin He2, Xuewen Wang2, Aykut Uren1. 1 Georgetown University, Washington, DC, USA, 2Florida International University, Miami, FL, USA. Surface plasmon resonance (SPR) is a label-free widely used biophysical technique to investigate biochemical processes, including protein-protein, proteinDNA, and protein-small molecule interactions. SPR data are not always governed by the simplest 1:1 interaction mechanism. In many cases biphasic mechanisms are employed for the interpretation of the SPR data. An accurate identification of the underlying biphasic mechanism is therefore crucial for the correct interpretation. Unlike traditional method of model identification based on the comparison of errors of SPR data fitting to different biphasic models, our method can help uniquely identify and analyze the underlying biphasic mechanism. The same biphasic SPR data can actually be fitted with two different biphasic models with comparable fitting errors. We have performed theoretical simulation and experimental investigation in order to support our method. 1655-Pos Board B632 Optical Detection of Biological Activity, One Molecule at a Time Markita P. Landry1, Jingqing Zhang2, Paul W. Barone2, Jong-Ho Kim2, Michael S. Strano2. 1 Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, CA, USA, 2Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. Molecular recognition is central to the design of therapeutics, chemical catalysis and sensor platforms, with the most common mechanisms involving biological structures such as protein antibodies. However, identifying and isolating an antibody for a particular biological molecule is often costly and time-consuming, and their use is limited to physiological conditions in which protein antibodies remain stable. Furthermore, most metabolites lack molecular recognition elements. Therefore, the biggest limitation to metabolite detection