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Energetic Contributions of Plectoneme Tips and Tails
70a
Sunday, February 12, 2017
specifically compare the patterns of melting observed experimentally with those obtained from the coarse-grained simulations.
Through a combination of simulations and control experiments we demonstrate these limits, and present a collection of best-practices.
356-Pos Board B121 Role of Watson-Crick-Like Mismatches in DNA Replication Fidelity Eric S. Szymanski1, Isaac J. Kimsey2, Hashim M. Al-Hashimi1. 1 Dept. of Biochemistry, Duke University, Durham, NC, USA, 2Nymirium, Ann Arbor, MI, USA. DNA replication, transcription, and translation rely on the strict Watson-Crick base pairing rules to ensure faithful transmission of genetic information. The Watson-Crick pairing rules are determined by the predominant neutral tautomeric forms of the nucleic acid bases. Incorrect base pairing during replication, if left unrepaired, leads to transition or transversion point mutations. Spontaneous mutagenesis from replication errors is believed to be a prominent source of base substitution errors in tumor suppressor genes in multiple forms of cancer. Rare tautomeric and ionized nucleotide bases can form mismatches that conform to the Watson-Crick like geometry, subverting proof reading mechanisms. These tautomeric and anionic mismatches have long been suspected to contribute to spontaneous replication errors; they have proved difficult to visualize as the conformational changes are subtle and involve the rearmament of protons. Nuclear magnetic resonance relaxation dispersion techniques have allowed for the characterization of a highly sequencedependent kinetic network connecting the wobble dG$dT mismatch to multiple Watson-Crick-like tautomeric and anionic dG$dT mismatch ‘excited states’. We have obtained evidence in support of a kinetic model for misincorporation which introduces a rate-limiting on-pathway tautomerization or ionization step that leads to Watson-Crick-like mismatches prior to incorporation through the canonical synthesis pathway. This kinetic model can account for i) the three orders of magnitude difference seen in vitro between rates of correct and incorrect nucleotide incorporations, ii) nucleotide selectivity fidelity as low as 10 6, and iii) the poorly understood sequence dependence of polymerization errors.
359-Pos Board B124 Small Molecule Aptamers for Biosensing Gregory Wiedman, Yunan Zhao, David Perlin. Public Health Research Institute, Rutgers New Jersey Medical School, Newark, NJ, USA. In this work, we used modified Synthetic Evolution of Ligands through Exponential Enrichment (SELEX) to discover a DNA aptamer recognizing azole class antifungal drugs. This aptamer undergoes a secondary structural change upon binding its target molecule as shown through fluorescence anisotropy based binding measurements. Using circular dichroism spectroscopy, we found a unique double G-quadruplex structure that was essential for binding to the target: azole antifungal drugs. This type of aptamer has the potential to be used as a small molecule captor component of a device for therapeutic drug monitoring.
357-Pos Board B122 Direct Observation of Single Biopolymer Folding and Unfolding Process by Solid-State Nanopore Xin Shi, Rui Gao, Shao-Chuang Liu, Qiao Li, Yi-Tao Long. East China University of Science and Technology, Shanghai, China. Biomolecular conformation and their transition play a crucial role in various in vivo or in vitro system. The most of the practical techniques for resolving the secondary structures of biomolecules could provide quite precise structural information for their solid-state or steady state, even at atomic resolution. For example, Cryo-EM determines high-resolution structures for the frozenhydrated specimens of biomolecules. polymers, but it is still challenging to resolve the dynamic process of multiple functional conformational states for biomolecules at single-molecular scale. Here, we direct observed DNA folding and unfolding process in real-time by using sub-5 nm solid-state nanopores. In our experiments, a single-stranded DNA adhered to single monovalent streptavidin could be reversibly trapped in a solid-state nanopore. Then, the fluctuations of the blockade current could be recorded, which reveals the dynamic structural transitions among DNA secondary structures. For example, after trapping the cytosine-rich DNA strains in slightly alkaline solution, the formation of multiple unstable and semi-folded i-motif structures could be observed. More important, well time-resolved transitions between these structures could be obtained. When using slightly acidic solution, the stable structures with stable blockade current could be found. With this new approach, we can directly observe the dynamic conformational change of biomolecules at single-molecular scale, which would be of great help for resolving single molecule interactions, designing single-molecule machine and understanding the working process of biomolecular in biological system. 358-Pos Board B123 Tuning Up Tethered Particle Motion Daniel T. Kovari, Eric Weeks, David Dunlap, Laura Finzi. Physics, Emory University, Atlanta, GA, USA. Tethered Particle Motions assays are a simple but powerful tools for monitoring the effective length of individual DNA strands and other linear bio-polymers in real-time. The technique has been employed in various capacities including characterization of DNA topology, transcription factor - DNA interactions, and monitoring progress of enzymes that translocate along DNA. At its core the technique is relatively simple to implement, only requiring a researchgrade microscope and a video camera; however, it is important to note that optical resolution, frame rate, exposure time, particle size, and solution viscosity all affect the ability to discriminate different tether lengths and detect changes.
360-Pos Board B125 Improved Sampling in Molecular Dynamics Studies of DNA and the B To Z[WC] To Z-DNA Transition Lam T. Nguyen, Ashutosh Rai, Micaela E. Bush, Alma Gracic, Ahsan A. Khoja, Jinhee Kim, Sunil Pun, Alexander K. Seewald, Benjamin L. Yee, Michael G. Lerner. Physics and Astronomy, Earlham College, Richmond, IN, USA. Although DNA is most commonly found in the right-handed B-DNA structure, it is known that biologically active systems also contain left-handed ZIIDNA. We used both steered and targeted molecular dynamics in combination with umbrella sampling to produce potentials of mean force for the B to ZII transition along both the direct B-ZII pathway as well as the B-Z[WC]-ZII pathway. Full pathway sampling is not feasible on smaller computer clusters, so we used Hamiltonian Replica Exchange to relax individual portions of the pathway. This technique is generalizable to larger systems and larger computer clusters. 361-Pos Board B126 Energetic Contributions of Plectoneme Tips and Tails Andrew Dittmore, Keir C. Neuman. NIH, Bethesda, MD, USA. Global DNA topology is sensed locally by enzymes that act on plectonemes in supercoiled DNA. Here we report that the formation and diffusion of plectonemes are determined by the energetic contributions of their tips and tails. First, to systematically vary the geometry and formation energy of plectoneme endloops, we introduced base-pair defect regions of variable size (1-16 bp) using a cassette based single-strand nicking template generated by PCR. Direct manipulation measurements with magnetic tweezers revealed that even a single mismatch or abasic site is sufficient to nucleate formation of a plectoneme. Presentation of the defect precisely at an extruded plectoneme tip potentially serves as a damage-sensing mechanism and may facilitate the search process of repair enzymes. Second, our measurements unexpectedly revealed that after twisted DNA abruptly buckles into an initial plectoneme loop, further plectoneme extrusion occurs through a cascade of additional buckling steps in which the torque changes by roughly half of the initial overshoot value. These discrete steps do not match any obvious scale of the system but are consistent with discontinuous feed-in of curving plectoneme tails. In light of these results, theoretical models of plectonemes should include their overall structure, including the often neglected tips and tails. 362-Pos Board B127 Multi-Scale Structure and Conformational Dynamics of Scaffolded DNA Origami Nanoparticles William Bricker, Keyao Pan, Mark Bathe. Massachusetts Institute of Technology, Cambridge, MA, USA. Synthetic DNA can be programmed into self-assembled 3D nanoparticles using a DX design motif and the principle of scaffolded DNA origami. A topdown design procedure (DAEDALUS) (Veneziano, Ratanalert, et al., Science, 2016) facilitates the design of arbitrary DNA nanoparticle geometries on the 5 to 100 nanometer scale, which we investigate in detail here using multi-scale structural modeling. While coarse-grained modeling is useful for generating equilibrium structures of DNA nanoparticles (Pan et al., Nat. Comm., 2014), only all-atom models reveal fine structural details and mechanical properties that contribute to overall structure and conformational dynamics. Here, we first use all-atom molecular dynamics (MD) to simulate two 0.5 1.0 MDa DNA polyhedral nanoparticles: a tetrahedron with 63 base pair
Sunday, February 12, 2017
specifically compare the patterns of melting observed experimentally with those obtained from the coarse-grained simulations.
Through a combination of simulations and control experiments we demonstrate these limits, and present a collection of best-practices.
356-Pos Board B121 Role of Watson-Crick-Like Mismatches in DNA Replication Fidelity Eric S. Szymanski1, Isaac J. Kimsey2, Hashim M. Al-Hashimi1. 1 Dept. of Biochemistry, Duke University, Durham, NC, USA, 2Nymirium, Ann Arbor, MI, USA. DNA replication, transcription, and translation rely on the strict Watson-Crick base pairing rules to ensure faithful transmission of genetic information. The Watson-Crick pairing rules are determined by the predominant neutral tautomeric forms of the nucleic acid bases. Incorrect base pairing during replication, if left unrepaired, leads to transition or transversion point mutations. Spontaneous mutagenesis from replication errors is believed to be a prominent source of base substitution errors in tumor suppressor genes in multiple forms of cancer. Rare tautomeric and ionized nucleotide bases can form mismatches that conform to the Watson-Crick like geometry, subverting proof reading mechanisms. These tautomeric and anionic mismatches have long been suspected to contribute to spontaneous replication errors; they have proved difficult to visualize as the conformational changes are subtle and involve the rearmament of protons. Nuclear magnetic resonance relaxation dispersion techniques have allowed for the characterization of a highly sequencedependent kinetic network connecting the wobble dG$dT mismatch to multiple Watson-Crick-like tautomeric and anionic dG$dT mismatch ‘excited states’. We have obtained evidence in support of a kinetic model for misincorporation which introduces a rate-limiting on-pathway tautomerization or ionization step that leads to Watson-Crick-like mismatches prior to incorporation through the canonical synthesis pathway. This kinetic model can account for i) the three orders of magnitude difference seen in vitro between rates of correct and incorrect nucleotide incorporations, ii) nucleotide selectivity fidelity as low as 10 6, and iii) the poorly understood sequence dependence of polymerization errors.
359-Pos Board B124 Small Molecule Aptamers for Biosensing Gregory Wiedman, Yunan Zhao, David Perlin. Public Health Research Institute, Rutgers New Jersey Medical School, Newark, NJ, USA. In this work, we used modified Synthetic Evolution of Ligands through Exponential Enrichment (SELEX) to discover a DNA aptamer recognizing azole class antifungal drugs. This aptamer undergoes a secondary structural change upon binding its target molecule as shown through fluorescence anisotropy based binding measurements. Using circular dichroism spectroscopy, we found a unique double G-quadruplex structure that was essential for binding to the target: azole antifungal drugs. This type of aptamer has the potential to be used as a small molecule captor component of a device for therapeutic drug monitoring.
357-Pos Board B122 Direct Observation of Single Biopolymer Folding and Unfolding Process by Solid-State Nanopore Xin Shi, Rui Gao, Shao-Chuang Liu, Qiao Li, Yi-Tao Long. East China University of Science and Technology, Shanghai, China. Biomolecular conformation and their transition play a crucial role in various in vivo or in vitro system. The most of the practical techniques for resolving the secondary structures of biomolecules could provide quite precise structural information for their solid-state or steady state, even at atomic resolution. For example, Cryo-EM determines high-resolution structures for the frozenhydrated specimens of biomolecules. polymers, but it is still challenging to resolve the dynamic process of multiple functional conformational states for biomolecules at single-molecular scale. Here, we direct observed DNA folding and unfolding process in real-time by using sub-5 nm solid-state nanopores. In our experiments, a single-stranded DNA adhered to single monovalent streptavidin could be reversibly trapped in a solid-state nanopore. Then, the fluctuations of the blockade current could be recorded, which reveals the dynamic structural transitions among DNA secondary structures. For example, after trapping the cytosine-rich DNA strains in slightly alkaline solution, the formation of multiple unstable and semi-folded i-motif structures could be observed. More important, well time-resolved transitions between these structures could be obtained. When using slightly acidic solution, the stable structures with stable blockade current could be found. With this new approach, we can directly observe the dynamic conformational change of biomolecules at single-molecular scale, which would be of great help for resolving single molecule interactions, designing single-molecule machine and understanding the working process of biomolecular in biological system. 358-Pos Board B123 Tuning Up Tethered Particle Motion Daniel T. Kovari, Eric Weeks, David Dunlap, Laura Finzi. Physics, Emory University, Atlanta, GA, USA. Tethered Particle Motions assays are a simple but powerful tools for monitoring the effective length of individual DNA strands and other linear bio-polymers in real-time. The technique has been employed in various capacities including characterization of DNA topology, transcription factor - DNA interactions, and monitoring progress of enzymes that translocate along DNA. At its core the technique is relatively simple to implement, only requiring a researchgrade microscope and a video camera; however, it is important to note that optical resolution, frame rate, exposure time, particle size, and solution viscosity all affect the ability to discriminate different tether lengths and detect changes.
360-Pos Board B125 Improved Sampling in Molecular Dynamics Studies of DNA and the B To Z[WC] To Z-DNA Transition Lam T. Nguyen, Ashutosh Rai, Micaela E. Bush, Alma Gracic, Ahsan A. Khoja, Jinhee Kim, Sunil Pun, Alexander K. Seewald, Benjamin L. Yee, Michael G. Lerner. Physics and Astronomy, Earlham College, Richmond, IN, USA. Although DNA is most commonly found in the right-handed B-DNA structure, it is known that biologically active systems also contain left-handed ZIIDNA. We used both steered and targeted molecular dynamics in combination with umbrella sampling to produce potentials of mean force for the B to ZII transition along both the direct B-ZII pathway as well as the B-Z[WC]-ZII pathway. Full pathway sampling is not feasible on smaller computer clusters, so we used Hamiltonian Replica Exchange to relax individual portions of the pathway. This technique is generalizable to larger systems and larger computer clusters. 361-Pos Board B126 Energetic Contributions of Plectoneme Tips and Tails Andrew Dittmore, Keir C. Neuman. NIH, Bethesda, MD, USA. Global DNA topology is sensed locally by enzymes that act on plectonemes in supercoiled DNA. Here we report that the formation and diffusion of plectonemes are determined by the energetic contributions of their tips and tails. First, to systematically vary the geometry and formation energy of plectoneme endloops, we introduced base-pair defect regions of variable size (1-16 bp) using a cassette based single-strand nicking template generated by PCR. Direct manipulation measurements with magnetic tweezers revealed that even a single mismatch or abasic site is sufficient to nucleate formation of a plectoneme. Presentation of the defect precisely at an extruded plectoneme tip potentially serves as a damage-sensing mechanism and may facilitate the search process of repair enzymes. Second, our measurements unexpectedly revealed that after twisted DNA abruptly buckles into an initial plectoneme loop, further plectoneme extrusion occurs through a cascade of additional buckling steps in which the torque changes by roughly half of the initial overshoot value. These discrete steps do not match any obvious scale of the system but are consistent with discontinuous feed-in of curving plectoneme tails. In light of these results, theoretical models of plectonemes should include their overall structure, including the often neglected tips and tails. 362-Pos Board B127 Multi-Scale Structure and Conformational Dynamics of Scaffolded DNA Origami Nanoparticles William Bricker, Keyao Pan, Mark Bathe. Massachusetts Institute of Technology, Cambridge, MA, USA. Synthetic DNA can be programmed into self-assembled 3D nanoparticles using a DX design motif and the principle of scaffolded DNA origami. A topdown design procedure (DAEDALUS) (Veneziano, Ratanalert, et al., Science, 2016) facilitates the design of arbitrary DNA nanoparticle geometries on the 5 to 100 nanometer scale, which we investigate in detail here using multi-scale structural modeling. While coarse-grained modeling is useful for generating equilibrium structures of DNA nanoparticles (Pan et al., Nat. Comm., 2014), only all-atom models reveal fine structural details and mechanical properties that contribute to overall structure and conformational dynamics. Here, we first use all-atom molecular dynamics (MD) to simulate two 0.5 1.0 MDa DNA polyhedral nanoparticles: a tetrahedron with 63 base pair