Characterization of Nucleosomes using DNA Origami

Tuesday, March 1, 2016 increasing speed but by focusing measurements on the interesting areas based on the incoming data. Here we describe work on combining this approach with novel, high speed-actuators to yield ultra-high speed imaging. A dualstage actuator is used to drive the tip around a small-radius circle and the measurements used in real time to both center that circle on the biopolymer and to scan the circle along the sample. A proof-of-concept instrument is expected to hit 30 frames per second with the final design aiming for 100 frames per second. 2469-Pos Board B613 High Resolution Magnetic Tweezers to Probe Single Molecule Dynamics Bob M. Lansdorp, Omar A. Saleh. Materials, UCSB, Santa Barbara, CA, USA. The magnetic tweezer is a single molecule manipulation instrument ideally suited to measuring biophysical systems at a constant applied force. The development of a magnetic tweezers with a high-speed camera and GPU-accelerated particle tracking has allowed for the measurement of molecular events at the millisecond time scale. However, as the spatial resolution of the instrument is improved, previously neglected sources of noise start to become limiting, such as: mechanical stability of the sample stage, and coherent light artifacts such as speckle. Here, we isolate the various sources of noise in an attempt to determine the fundamental limit to magnetic tweezer resolution. We use a state-of-the-art high spatial and temporal resolution magnetic tweezer to measure the dynamics of model systems such as DNA hairpins. 2470-Pos Board B614 Multiplexed Mechanochemistry Assay - A Tool for Multiplexed Single Molecule Bond Rupture Force Studies Bhavik Nathwani1,2, Darren Yang3, Wesley Wong2,4, William M. Shih1,2. 1 Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA, 2 Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA, 3Harvard University, Cambridge, MA, USA, 4 Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA. Studies in mechanochemistry have greatly benefited from advances in single molecule force spectroscopy (SMFS) techniques. Despite success, conventional SMFS techniques are inherently low throughput. For example, Atomic Force Microscopy (AFM) and optical tweezers are serial techniques. In recent years, considerable effort has been made to develop massively parallel force spectroscopy methods such as the centrifugal force microscope (CFM), which applies centrifugal force to tethered beads. The capability to apply centrifugal force on a large number of beads opens new multiplexing possibilities. Here, we report the development of a novel technique — Multiplexed Mechanochemistry Assay (MMA) — enabling large-scale study of single molecule chemical bond rupture events. Briefly, in a bead surface assay configuration, we designed a centrifugal system for the application of pre-calibrated forces on up to 320 samples simultaneously. Our system enables two-dimensional multiplexing, (1) number of beads being studied simultaneously, and (2) number of experimental conditions being probed. The force range afforded by our equipment ranges from a fraction of a pN to ~30 nN. This unprecedented level of ultraplexing afforded by our approach, hundreds of experimental conditions tested against orders of magnitude variation in force, provides a dramatic improvement in throughput of individual experiments over conventional SMFS techniques. As system validation, we report a case study on studying bond rupture of covalent bonds. We believe this simple tool will greatly increase the rate of progress in probing the relation between force-lifetime and chemistry in single molecular bonds. 2471-Pos Board B615 Label-Free Intramolecular Chemical Microscopy of a Protein-RNA Complex Duckhoe Kim1, Zhenghan Gao2, Ozgur Sahin1,2. 1 biological sciences, Columbia University, New York, NY, USA, 2Physics, Columbia University, New York, NY, USA. Obtaining structural information from single biomolecules using imaging methods is challenging due to resolution limitations and difficulties in labelling specific sites of biomolecules. Atomic force microscopy based imaging of interaction forces between a specially designed probe molecule and a biomolecular complex of interest can, in principle, allow determining the locations of various regions within the complex, if the probe is designed to exhibit affinity to these regions. Here we demonstrate this concept with protein-RNA complex by targeting specific segments of the RNA with a DNA probe having sequence complementarity with these segments. To

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map the interaction forces between the probe and the sample, we used T-shaped cantilevers that allow mapping interaction forces during the tapping mode imaging process [1]. We worked with stem loop binding proteins in complex with RNA [2] and we targeted the flanking RNA regions adjacent to the stem loop. We show that locations and sequence identities of the RNA segments can be imaged with this approach. Repeated measurements of the same protein-RNA complex showed the consistency of the images. Our results show that it is possible obtain chemically-specific structural information from single biomolecules in physiologically relevant conditions without using labels. 1. Kim, D., Sahin, O. Nature Nanotechnology 10, 264-269 (2015). 2. Tan, D., Mazluff, W. F., Dominski, Z., Tong, L. Science 339, 318-321 (2013). 2472-Pos Board B616 Tuning the Music: Acoustic Force Spectroscopy (AFS) 2.0 Douwe Kamsma1, Ramon Creyghton2, Gerrit Sitters1, Erwin J.G. Peterman1, Gijs J.L. Wuite1. 1 VU Vrije Universiteit, Amsterdam, Netherlands, 2UvA university of Amsterdam, Amsterdam, Netherlands. AFS is a novel technique that uses an acoustic standing wave in a microfluidic chip to apply forces on single tethers to study the structural and mechanochemical properties of biomolecules. It distinguishes itself by a high experimental throughput, a wide force range and an unmatched range of force loading rates. In its original implementation the method has several limitations: it makes use of an opaque piezo element to generate the acoustic force, the force profile is not constant over the fluid layer and it is not possible to apply forces close to the coverslip side. Here we present innovative solutions to tackle these shortcomings, making AFS a more valuable technique. The use of a transparent piezo element allows transillumination of the sample, greatly improving optical performance and improving compatibility with existing microscopes. We also have developed a model to calculate forces in the sample, which is used to optimize the dimensions of the system, creating the possibility to overstretch a DNA molecule tethered to a 1 mm in diameter silica beads at the coverslip side. In addition, a superposition of standing ultrasound waves can be applied to the sample, which allow modification of the force profile, for example to generate a more constant force across the fluid channel. This approach can also be used to create highly distance-dependent force profiles, transforming AFS in a distance clamp. Finally, we show that AFS is compatible with high NA water or oil immersion objectives, although less high forces can be achieved. AFS already distinguishes itself by its relative simplicity, low cost and compactness, which allow straightforward implementation in lab-on-a-chip devices and these new developments make AFS an even more accessible technique, with a wider field of applications that can be integrated in almost every microscope.

Micro- and Nanotechnology I 2473-Pos Board B617 Characterization of Nucleosomes using DNA Origami Jenny V. Le1,2, Yi Luo1,3, Christopher R. Lucas2, Michael G. Poirier1,3, Carlos E. Castro1,2. 1 Biophysics, The Ohio State University, Columbus, OH, USA, 2Mechanical and Aerospace Engineering, The Ohio State University, Columbus, OH, USA, 3Physics, The Ohio State University, Columbus, OH, USA. While biophysical tools such as single molecule fluorescence and force spectroscopy have been used to study the structural dynamics of individual nucleosomes or large chromatin assemblies, it is challenging to probe conformational dynamics of gene regulation in the 10-100nm range. This is the length scale of genomic critical features, including promoter regions that span several nucleosomes. Our work aims to develop DNA origami tools to probe these mesoscale structure and dynamics of nucleosome arrays and chromatin. DNA origami itself is an emerging nanotechnology that enables the selfassembly of precisely designed molecular-scale structures. Here, we implement a DNA origami hinge device with the length scale of ~100nm as a nanocaliper to study the structure and dynamics of a single nucleosome as proof-of-concept. The measurement capabilities of the device were calibrated by integrating DNA duplexes of varying lengths between the hinge arms to demonstrate the use the hinge angular distribution as a readout for the sample size attached between the arms. We further integrated single nucleosomes with varying but symmetric lengths of DNA linkers (i.e. DNA extending out after wrapping around the histone core) to verify our ability to detect structural

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Tuesday, March 1, 2016

changes in nucleosomes. Our immediate goal is to measure the structural changes in single nucleosomes and nucleosome arrays in response to transcription factor (TF) binding. Initial experiments using the nucleosomecaliper construct with GAL4-VP16, a hybrid transcription factor capable of highly-efficient transcription activation, shows that the angular distribution broadens as a result of increasing TF concentrations. Our measurements revealed a dissociation constant in the range of 1-10nM, which agrees with bulk measurements. These results demonstrate the potential of a DNA origami device probing chromatin structure and function in vitro, in particular, the ability to measure structural changes in the range of 10-100nm. 2474-Pos Board B618 Therapeutic Enhancement with Nuclear Targeted Gold Nanoparticles Celina J. Yang, Devika B. Chithrani. Biomedical Physics, Ryerson University, Toronto, ON, Canada. Nanoparticles have been used as a platform to improve therapeutic results in medical research. Gold nanoparticles (GNPs) have been extensively studied among other nanoparticles in cancer research due to its property being radiosensitizing by producing more secondary electrons in response to irradiation. It is predicted that closer the GNPs can get to the DNA, the extra secondary electrons will cause more breaks in the DNA. However, the regular uptake pathway of GNPs into the cell is known to be through receptor mediated endocytosis, where GNPs follow the endo-lyso pathway. An effective design of GNP-peptide complex for nuclear targeting will be presented, where three different peptide sequences are conjugated onto 10 nm sized spherical GNPs. An effective design of a GNP-peptide complex for nuclear targeting will be presented, where three different peptide sequences are conjugated onto 10 nm sized spherical GNPs. Each of the peptides is used for enhancing intracellular retention, inducing nuclear delivery and stabilization. When in vitro cells were irradiated with 2 Gy of radiation, the cells incubated with peptidemodified GNPs had an improved therapeutic result, not only compared to the control sample (no incubation of GNP) but to cells incubated with unmodified GNPs. This research will provide insight to a more successful NP-based platform for combining treatment modalities that could eventually lead to a more effective approach in the treatment of cancer. 2475-Pos Board B619 Microcarrier-Guided Nanopore Dielectrophoresis for Selective Nucleic Acid Detection Kai Tian1, Karl Decker2, Aleksei Aksimentiev2, Liqun Gu1. 1 Biological Engineering, University of Missouri, Columbia, MO, USA, 2 Physics, University of Illinois at Urbana-Champaign, Champaign, IL, USA. Dielectrophoresis (DEP) is the motion of a polarizable particle in a nonuniform electric field due to an unbalanced electrostatic force on the particle’s induced dipole. The DEP mechanism has been extensively utilized for manipulation of biological particles, from cancer cells and viruses to biomolecules such as DNAs and proteins, for their concentration, separation, sorting, and transport. However, current DEP approaches to molecular manipulation are not selective, as DEP is not sensitive enough to discriminate among the induced dipoles of different molecules. Here we explore a novel single-molecule DEP mechanism, carrier-guided nanopore dielectrophoresis, for selective nucleic acid sequence detection. Rather than rely on a target’s native polarizability, we designed a polycationic carrier to impart a tunable synthetic dipole to the target nucleic acid molecule; carrier sensitivity and selectivity are both programmable. Such synthetic dipoles can be captured in an engineered nanopore, which acts as an ideal foulfree point source to generate an extremely high field gradient (DE~107 V$m-1 per nanometer or ~1016 V$m-2) for molecular dipole manipulation. Non-target nucleic acids do not bind the carriers and hence are repelled from the pore by electrophoresis. To elucidate the mechanism of the dipole capture by the nanopore, we took an all-atom molecular dynamics (MD) simulation approach to observe the movements of and forces on the dipole. Simulation results predicted significantly increased force attracting the probe into the engineered nanopore as opposed to the wild type, consistent with the increased capture rates observed in experiment. Most strikingly of all, we find that a carrier with only a few positive charges can drive any length of DNA or RNA with equal capture efficiency. Development of nanopore dielectrophoretic detection thus offers ready medical applications of nanopore technology.

2476-Pos Board B620 Dipole Effects on Ion Transport Demonstrated in Aprotic Solvents Timothy S. Plett1, Wenqing Shi2, Yuhan Zeng2, William Mann1, Ivan Vlassiouk3, Lane Baker2, Zuzanna S. Siwy1. 1 Physics, University of California, Irvine, Irvine, CA, USA, 2Chemistry, Indiana University, Bloomington, IN, USA, 3Oak Ridge National Laboratory, Oak Ridge, TN, USA. Dipole interactions play a significant role in biological systems, influencing key functions such as protein folding and selective transport of ions through channels, among many others. Possible importance of the presence of dipoles in systems of synthetic nanopores has not yet been explored. Here, we report experiments and modeling of ionic current through nanopores in polymer films as well as glass nanopipettes. In order to probe importance of dipoles for ionic transport, experiments were performed with aprotic solvents characterized with dipole moments between 5D and 1.7D. LiClO4 was used as the electrolyte due to its high solubility in a wide range of solvents. The conical nanopores used in the experiments rectify the curent with the direction and degree being very sensitive to the surface properties of the pore walls. Current-voltage curves in aprotic solvents with high dipole moments provided evidence that the solvent and possible cation adsorption caused formation of an effectively positive surface potential, which was further confirmed by scanning ion conductance microscopy. Continuum modeling of ion current using models developed for biological systems confirmed that presence of dipoles on pore walls can indeed modify local ionic concentrations and the recorded current. 2477-Pos Board B621 A Novel Multi-Layer Microfluidic Pipette Aspiration Device for Studying Mechanosensitive Vesicles Lap Man Lee1, Danielle Chase2, Allen Liu1. 1 Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA, 2 Mechanical Engineering, University of Minnesota, Minneapolis, MN, USA. Mechanosensitive channel of large conductance (MscL) is a prokaryotic channel that opens due to increased membrane tension. It has been reconstituted in vesicles to study how various biophysical factors influence their mechanical gating properties. In recent years, several theoretical and computational models based on molecular dynamics and continuum mechanics have been developed to understand the underlying mechanisms for the activation of MscLs in vesicles. Experimental approaches for MscL gating studies have focused on cell-by-cell techniques, such as pressure patch clamp, on reconstituted MscL vesicles. Due to the limitation of throughput, such studies are time intensive with low sample numbers. Recently, a microfluidic pipette aspiration array device, based on PDMS multi-layer soft-lithography technique, has been developed in our group to trap and apply mechanical perturbation to single cells in a parallel manner1. This device was used to study the stiffness of human breast cancer cell lines and mechanical gating threshold of reconstituted MscL on infected mammalian cell lines. In this work, we have developed a new device that incorporates a pressure valve and a smaller micropipette dimension to increase trapping efficiency of vesicles. Using this device, we demonstrate stable trapping of single vesicles and expand our efforts to study mechanical gating threshold of reconstituted MscL in vesicles formed by electroformation. Development of novel microtechnology tools that can trap single vesicles and exert tension will have numerous applications to the study of other mechanosensitive channels. [1] Lee & Liu, Lab Chip, 2015, 15, 264-273. 2478-Pos Board B622 An Ion-Specific Effect on Polymer-Protein Interaction Enhances Resolution of Nanopore-Based Detection Aleksandra Dylewska-Chaumeil1, Gerhard Baaken2, Jan C. Behrends1. 1 Physiology, University of Freiburg, Freiburg, Germany, 2Ionera Technologies GmbH, Freiburg, Germany. Poly(ethyleneglycol) (PEG) oligomers partition into acqueous transmembrane channels formed by alpha-Hemolysin (aHL) and Aerolysin (AeL) proteins and transiently bind the pore to induce reductions in conductance, i.e. resistive pulses, the amplitude of which is strongly related to polymer chain length. Histograms of such data for PEG yield mass spectrograms with single monomer resolution between approximately 20 and 60 repeat units (r.u.). For chain lengths <20 the duration of resistive pulses becomes too short (e.g.<50 ms) to be fully resolved using state-of-the art electrophysiological recording technology. Recently, computational reconstruction of unresolved events has revealed the presence in a sample of PEG species down to