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The Structural and Mechanistic Origins of Catalysis in Nucleolytic Ribozymes
26a
Sunday, February 12, 2017
DNA origami. First, we present a fully autonomous algorithm to produce a single-stranded DNA scaffold and complementary staple strands, which fold into nearly arbitrary target 3D objects, from Platonic solids to non-spherical topologies, with near quantitative synthetic yield, demonstrated for a dozen structures experimentally (Veneziano, Ratanalert, et al., Science, 2016). Second, we present a powerful approach that eliminates staple strands entirely, offering the ability to program a single DNA molecule to fold into these arbitrary 3D shapes on its own, similar to natural RNA assemblies. We examine the folding pathways of each of these design modalities using quantitative PCR and present a thermodynamic model to optimize sequence design, folding temperature, and yield (Ratanalert et al., in prep, 2016). Together, these algorithms solve a longstanding challenge of synthetic structural biology to program nearly arbitrary 3D geometries using synthetic nucleic acids. 133-Plat pH-Responsive Reversible Regulation of Enzyme Activity by DNA-Based Nanostructure Seong Ho Kim1,2, So Yeon Kim1,2. 1 University of Science and Technology, Seoul, Korea, Republic of, 2Korea Institute of Science and technology, Seoul, Korea, Republic of. Here, we present a novel method for reversibly regulating enzyme activity by caging an enzyme into a pH-responsive DNA-based tetrahedron nanostructure. pH-dependent opening/closing of the DNA cage was verified by measuring fluorescence resonance energy transfer between two vertex corners of the tetrahedron. The position of the covalent enzyme attachment in DNA was carefully chosen such that the attached enzyme faced inward the DNA cage. Both proteinase K protection assay and single-molecule based pull-down assay showed that the encapsulated enzyme were exposed to either proteinase K or target antibody by pH-dependent opening of DNA cage. Remarkably, we found that the caging/uncaging process were reversible, implying that enzyme activity toward relatively larger substrates than DNA cage can be reversibly regulated. Considering that the DNA cage is widely used as a delivery carrier, our method can be further extended to reversibly regulate cell function by pH-dependent activity control of delivered enzyme. 134-Plat Eco-Friendly Processing for Engineering Bio-Safe Quantum Dots and their Interaction with Biological Systems Marta d’Amora1, Marina Rodio1, Alberto Diaspro1,2, Romuald Intartaglia1. 1 Istituto Italiano di Tecnologia, genova, Italy, 2Department of Physics, University of Genoa, Genova, Italy. Inorganic nanomaterials have gained attention for delivery vehicles, gene detection systems, labeling and therapeutic applications. Many efforts have been reported in the synthesis of heavy metal quantum dots (QDs), for longterm, real-time cell labeling applications. Exposure to these QDs in living tissue endanger several issues due to their chemical composition, artificial ligand and/or the employed solution routes. In particular, surface coating/stabilization of nanomaterials by chemical organic molecules, such as citrate have shown to trigger different interaction at cellular level [1]. However, it is still difficult to draw a definite conclusion. Therefore, other alternatives, taking into account the nanoparticles fabrication strategy and the minimum toxicity of the carrier itself, are crucial for potential success of nanomaterials in the clinical setting. [2,3] Owing to its biocompatibility and biodegrability, silicon based nanomaterials are ideal candidates for in vivo applications [4]. Here, we will report a safe engineering approach based on liquid phase pulsed laser ablation technique enabling the generation of photoemissive, highly pure (i.e. free of chemical ligand at the surface) silicon quantum dots. Furthermore, we will present the physicochemical interactions of these non-toxic nanotools having unique surface chemistry with biological systems. (1) Mu et al., Chem Rev., 114, 7740. (2014) (2) Intartaglia et al., Nanoscale, 4, 1271, (2012) (3) Rodio et al., J. Colloid Interface Sci. 465, 242 (2016) (4) Park et al, Nature Materials, 8, 331 (2009)
Symposium: RNA Structures and Dynamics 135-Symp Cleaving Fast and Slow: Strategies for Self-Assembly of Catalytic RNA Sarah A. Woodson1, Subrata Panja1, Boyang Hua2, Krishnarjun Sarkar1, Taekjip Ha3. 1 Dept Biophysics, Johns Hopkins Univ, Baltimore, MD, USA, 2Dept Biophysics & Biochemistry, Johns Hopkins Medical School, Baltimore, MD, USA, 3Dept Biophysics & Biochemistry, HHMI & Johns Hopkins Medical School, Baltimore, MD, USA. Ribozyme RNA motifs are widespread in nature and their adaptation to varied growth conditions has been sparsely investigated. Ribozymes fold
into specific three-dimensional structures to promote self-cleavage of specific phosphodiester bonds. Metal cations stabilize the folded RNA, which usually correlates with biochemical activity. We used ensemble and single molecule FRET to compare the folding dynamics of a 195 nt bacterial group I ribozyme and a 54 nt Twister ribozyme from Oryza sativa. Whereas the group I ribozyme remains stably folded in 2 mM MgCl2 that is sufficient for its activity, Twister ribozyme folds and unfolds on the second timescale even in 100 mM MgCl2. Nevertheless, Twister still self-cleaves in 50 mM MgCl2, although visits to the folded state are transient and infrequent. Surprisingly, transition metals activated Twister ribozyme even more efficiently than Mg2þ. Ongoing efforts to understand how folding dynamics tune RNA activity will be discussed. 136-Symp Adventures with RNA Graphs Tamar Schlick. Courant Inst, New York Univ, HHMI, New York, NY, USA. RNA’s modular, hierarchical and versatile structure makes possible diverse, essential regulatory and catalytic roles in the cell. It also invites systematic modeling and simulation approaches. Among the diverse computational and theoretical approaches to model RNA structures, graph theory has been applied in various contexts to study RNA structure and function. I will present an overview of recent graph theoretical approaches for predicting and designing RNA topologies using graphical representations of RNA secondary structure, datamining tools for junction topology prediction, and hierarchical sampling of graphs based on statistical potentials. As evident from the work of many groups in the mathematical and biological sciences, graph theoretical approaches offer a fruitful avenue for designing novel RNA topologies and predicting tertiary structures from given secondary structures. Of possible interest - H.H. Gan, S. Pasquali and T. Schlick, Nucl. Acids Res. 31:2926 (2003) - N. Kim et al., J. Mol. Biol. 341:1129 (2004) - G. Quarta and K. Sin and T. Schlick, PLoS Comput. Biol. 8: e1002368 (2012). - C. Laing, S. Jung, N. Kim, S. Elmetwaly, M. Zharan, and T. Schlick, PLOS One 8(8): e71947 (2013). - N. Kim, C. Laing, S. Elmetwaly, S. Jung, J. Curuksu, and T. Schlick, Proc. Natl. Acad. Sci. USA 111: 4079 (2014). - M. Zharan, C. S. Bayrak, S. Elmetwaly, and T. Schlick, Nuc. Acids Res. 43: 9474 (2015). - N. Baba, S. Elmetwaly, N. Kim, and T. Schlick, J. Mol. Biol. 428: 811 (2016). - L. Hua, Y. Song, N. Kim, C. Laing, J. T. L. Wang, and T. Schlick, PlOS One 11: e0147097 (2016). 137-Symp The Structural and Mechanistic Origins of Catalysis in Nucleolytic Ribozymes David M. Lilley, Timothy J. Wilson, Yijin Liu. Life Sciences, University of Dundee, Dundee, United Kingdom. The nucleolytic ribozymes are a structurally diverse and widespread group of catalytic RNA species. They accelerate transesterification reactions around a million fold, resulting in the site-specific cleavage or ligation of RNA. The potential entities that can participate are the nucleobases, 20 -hydroxyl groups and hydrated metal ions. Probable catalytic strategies are the facilitation of in-line attack, stabilization of the phosphorane transition state, deprotonation of the nucleophile and protonation of the oxyanion leaving group (these last two being general base-acid catalysis). While much of the above was studied in ribozymes such as hammerhead, hairpin and VS, these principles are well illustrated by newer ribozymes. We have solved the crystal structure of the twister ribozyme, which adopts a double pseudoknot fold with a central active site. This well illustrates the four strategies summarized above. We have recently solved the structure of a new ribozyme, that appears mechanistically very different, with a key role for a bound metal ion where an inner-sphere water molecule acts as a general base.
Platform: Membrane Receptors and Signal Transduction I 138-Plat Oligomerization of the Epidermal Growth Factor Receptor Organizes Kinase-Active Dimers into Competent Signaling Platforms Sarah R. Needham1, Laura C. Zanetti-Domigues1, Anton Arkhipov2, Venkatesh P. Mysore3, Dimitrios Korovesis1, Selene K. Roberts1, Christopher J. Tynan1, Daniel J. Rolfe1, Michael Hirsch1, Alireza Lajevardipour4, Andrew H.A Clayton4, Peter J. Parker5,6, Yibing Shan3, David E. Shaw3,7, Marisa L. Martin-Fernandez1.
Sunday, February 12, 2017
DNA origami. First, we present a fully autonomous algorithm to produce a single-stranded DNA scaffold and complementary staple strands, which fold into nearly arbitrary target 3D objects, from Platonic solids to non-spherical topologies, with near quantitative synthetic yield, demonstrated for a dozen structures experimentally (Veneziano, Ratanalert, et al., Science, 2016). Second, we present a powerful approach that eliminates staple strands entirely, offering the ability to program a single DNA molecule to fold into these arbitrary 3D shapes on its own, similar to natural RNA assemblies. We examine the folding pathways of each of these design modalities using quantitative PCR and present a thermodynamic model to optimize sequence design, folding temperature, and yield (Ratanalert et al., in prep, 2016). Together, these algorithms solve a longstanding challenge of synthetic structural biology to program nearly arbitrary 3D geometries using synthetic nucleic acids. 133-Plat pH-Responsive Reversible Regulation of Enzyme Activity by DNA-Based Nanostructure Seong Ho Kim1,2, So Yeon Kim1,2. 1 University of Science and Technology, Seoul, Korea, Republic of, 2Korea Institute of Science and technology, Seoul, Korea, Republic of. Here, we present a novel method for reversibly regulating enzyme activity by caging an enzyme into a pH-responsive DNA-based tetrahedron nanostructure. pH-dependent opening/closing of the DNA cage was verified by measuring fluorescence resonance energy transfer between two vertex corners of the tetrahedron. The position of the covalent enzyme attachment in DNA was carefully chosen such that the attached enzyme faced inward the DNA cage. Both proteinase K protection assay and single-molecule based pull-down assay showed that the encapsulated enzyme were exposed to either proteinase K or target antibody by pH-dependent opening of DNA cage. Remarkably, we found that the caging/uncaging process were reversible, implying that enzyme activity toward relatively larger substrates than DNA cage can be reversibly regulated. Considering that the DNA cage is widely used as a delivery carrier, our method can be further extended to reversibly regulate cell function by pH-dependent activity control of delivered enzyme. 134-Plat Eco-Friendly Processing for Engineering Bio-Safe Quantum Dots and their Interaction with Biological Systems Marta d’Amora1, Marina Rodio1, Alberto Diaspro1,2, Romuald Intartaglia1. 1 Istituto Italiano di Tecnologia, genova, Italy, 2Department of Physics, University of Genoa, Genova, Italy. Inorganic nanomaterials have gained attention for delivery vehicles, gene detection systems, labeling and therapeutic applications. Many efforts have been reported in the synthesis of heavy metal quantum dots (QDs), for longterm, real-time cell labeling applications. Exposure to these QDs in living tissue endanger several issues due to their chemical composition, artificial ligand and/or the employed solution routes. In particular, surface coating/stabilization of nanomaterials by chemical organic molecules, such as citrate have shown to trigger different interaction at cellular level [1]. However, it is still difficult to draw a definite conclusion. Therefore, other alternatives, taking into account the nanoparticles fabrication strategy and the minimum toxicity of the carrier itself, are crucial for potential success of nanomaterials in the clinical setting. [2,3] Owing to its biocompatibility and biodegrability, silicon based nanomaterials are ideal candidates for in vivo applications [4]. Here, we will report a safe engineering approach based on liquid phase pulsed laser ablation technique enabling the generation of photoemissive, highly pure (i.e. free of chemical ligand at the surface) silicon quantum dots. Furthermore, we will present the physicochemical interactions of these non-toxic nanotools having unique surface chemistry with biological systems. (1) Mu et al., Chem Rev., 114, 7740. (2014) (2) Intartaglia et al., Nanoscale, 4, 1271, (2012) (3) Rodio et al., J. Colloid Interface Sci. 465, 242 (2016) (4) Park et al, Nature Materials, 8, 331 (2009)
Symposium: RNA Structures and Dynamics 135-Symp Cleaving Fast and Slow: Strategies for Self-Assembly of Catalytic RNA Sarah A. Woodson1, Subrata Panja1, Boyang Hua2, Krishnarjun Sarkar1, Taekjip Ha3. 1 Dept Biophysics, Johns Hopkins Univ, Baltimore, MD, USA, 2Dept Biophysics & Biochemistry, Johns Hopkins Medical School, Baltimore, MD, USA, 3Dept Biophysics & Biochemistry, HHMI & Johns Hopkins Medical School, Baltimore, MD, USA. Ribozyme RNA motifs are widespread in nature and their adaptation to varied growth conditions has been sparsely investigated. Ribozymes fold
into specific three-dimensional structures to promote self-cleavage of specific phosphodiester bonds. Metal cations stabilize the folded RNA, which usually correlates with biochemical activity. We used ensemble and single molecule FRET to compare the folding dynamics of a 195 nt bacterial group I ribozyme and a 54 nt Twister ribozyme from Oryza sativa. Whereas the group I ribozyme remains stably folded in 2 mM MgCl2 that is sufficient for its activity, Twister ribozyme folds and unfolds on the second timescale even in 100 mM MgCl2. Nevertheless, Twister still self-cleaves in 50 mM MgCl2, although visits to the folded state are transient and infrequent. Surprisingly, transition metals activated Twister ribozyme even more efficiently than Mg2þ. Ongoing efforts to understand how folding dynamics tune RNA activity will be discussed. 136-Symp Adventures with RNA Graphs Tamar Schlick. Courant Inst, New York Univ, HHMI, New York, NY, USA. RNA’s modular, hierarchical and versatile structure makes possible diverse, essential regulatory and catalytic roles in the cell. It also invites systematic modeling and simulation approaches. Among the diverse computational and theoretical approaches to model RNA structures, graph theory has been applied in various contexts to study RNA structure and function. I will present an overview of recent graph theoretical approaches for predicting and designing RNA topologies using graphical representations of RNA secondary structure, datamining tools for junction topology prediction, and hierarchical sampling of graphs based on statistical potentials. As evident from the work of many groups in the mathematical and biological sciences, graph theoretical approaches offer a fruitful avenue for designing novel RNA topologies and predicting tertiary structures from given secondary structures. Of possible interest - H.H. Gan, S. Pasquali and T. Schlick, Nucl. Acids Res. 31:2926 (2003) - N. Kim et al., J. Mol. Biol. 341:1129 (2004) - G. Quarta and K. Sin and T. Schlick, PLoS Comput. Biol. 8: e1002368 (2012). - C. Laing, S. Jung, N. Kim, S. Elmetwaly, M. Zharan, and T. Schlick, PLOS One 8(8): e71947 (2013). - N. Kim, C. Laing, S. Elmetwaly, S. Jung, J. Curuksu, and T. Schlick, Proc. Natl. Acad. Sci. USA 111: 4079 (2014). - M. Zharan, C. S. Bayrak, S. Elmetwaly, and T. Schlick, Nuc. Acids Res. 43: 9474 (2015). - N. Baba, S. Elmetwaly, N. Kim, and T. Schlick, J. Mol. Biol. 428: 811 (2016). - L. Hua, Y. Song, N. Kim, C. Laing, J. T. L. Wang, and T. Schlick, PlOS One 11: e0147097 (2016). 137-Symp The Structural and Mechanistic Origins of Catalysis in Nucleolytic Ribozymes David M. Lilley, Timothy J. Wilson, Yijin Liu. Life Sciences, University of Dundee, Dundee, United Kingdom. The nucleolytic ribozymes are a structurally diverse and widespread group of catalytic RNA species. They accelerate transesterification reactions around a million fold, resulting in the site-specific cleavage or ligation of RNA. The potential entities that can participate are the nucleobases, 20 -hydroxyl groups and hydrated metal ions. Probable catalytic strategies are the facilitation of in-line attack, stabilization of the phosphorane transition state, deprotonation of the nucleophile and protonation of the oxyanion leaving group (these last two being general base-acid catalysis). While much of the above was studied in ribozymes such as hammerhead, hairpin and VS, these principles are well illustrated by newer ribozymes. We have solved the crystal structure of the twister ribozyme, which adopts a double pseudoknot fold with a central active site. This well illustrates the four strategies summarized above. We have recently solved the structure of a new ribozyme, that appears mechanistically very different, with a key role for a bound metal ion where an inner-sphere water molecule acts as a general base.
Platform: Membrane Receptors and Signal Transduction I 138-Plat Oligomerization of the Epidermal Growth Factor Receptor Organizes Kinase-Active Dimers into Competent Signaling Platforms Sarah R. Needham1, Laura C. Zanetti-Domigues1, Anton Arkhipov2, Venkatesh P. Mysore3, Dimitrios Korovesis1, Selene K. Roberts1, Christopher J. Tynan1, Daniel J. Rolfe1, Michael Hirsch1, Alireza Lajevardipour4, Andrew H.A Clayton4, Peter J. Parker5,6, Yibing Shan3, David E. Shaw3,7, Marisa L. Martin-Fernandez1.