Bulletin of the American Physical Society
APS March Meeting 2017
Volume 62, Number 4
Monday–Friday, March 13–17, 2017; New Orleans, Louisiana
Session X14: Knotted BiomoleculesFocus
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Sponsoring Units: DBIO Chair: Jaynath Banavar, University of Maryland Room: 273 |
Friday, March 17, 2017 8:00AM - 8:12AM |
X14.00001: Dynamics of Knot Relaxation in Stretched DNA Alexander Klotz, Vivek Narsimhan, Beatrice Soh, Patrick Doyle Knots occur naturally in biological DNA and have been shown to be relevant for next-generation sequencing applications. Knots and other topological constraints in bulk polymer systems have been shown to influence the overall dynamical behavior of aggregate materials, but it is an open question as to the role that individual knots play in polymer dynamics. Here, we investigated the dynamics of polymer knot relaxation by stretching knotted DNA with an extensional field in a microfluidic device and allowing it to relax to its coiled state, measuring the growth rate of the knot using fluorescence microscopy. We find that knots swell during relaxation with a timescale comparable to that of the end-to-end relaxation. The knot growth timescale in insensitive to differences in the perceived topological complexity of the knot and increases with polymer chain length with the same scaling as the end-to-end relaxation timescale. These findings suggest that the timescale governing the swelling of knots in initially stretched chains is subject to global rather than local polymer dynamics. [Preview Abstract] |
Friday, March 17, 2017 8:12AM - 8:24AM |
X14.00002: Knotting of long DNA molecules confined to nanochannels close to the persistence length Kevin Dorfman, Aashish Jain Understanding the confinement of long DNA within a nanochannel close to its circa 50 nm persistence length is important for genome mapping, an emerging genomics method that complements next-generation sequencing. Knotting of the DNA inside a nanochannel poses a challenge to genome mapping, since the topological complexity of the chain can scramble the labels used to identify the genomic sequence of the DNA. To date, simulations of the knotting of DNA in nanochannels have tended to focus on short chains, likely due to the computational costs to simulate and analyze long chains in narrow channels. However, practical applications involve long DNA, typically in excess of 150 kilobase pairs, confined in narrow channels with sizes less than 50 nm. We have studied the equilibrium ensemble of such chains using pruned-enriched Rosenbluth method (PERM) simulations of a DNA model confined in channels between 30 nm and 50 nm in width. We will present results on the location and size of knots in confined DNA as a function of channel size and knot type. Our results provide new insights into the complexity of knotting of very long DNA, including the presence of multiple knots on single chains and very complex knots. We will also provide a comparison with experiments. [Preview Abstract] |
Friday, March 17, 2017 8:24AM - 8:36AM |
X14.00003: Spatial variation of knotting free energy and knot interactions in a knot factory on chip Susan Amin, Ahmed Khorshid, Lili Zeng, Philip Zimny, Walter Reisner We demonstrate that DNA molecules can be knotted following hydrodynamic compression against nanofabricated barriers in nanochannels. In particular, we measure the probability of forming single or multiple knots on a chain as a function of compression and waiting time in the compressed state. We observe that knotting probability increases as the chain is compressed, with multiple knot states dominating for the highest compression achieved. In addition, we observe that knot formation probability increases with waiting time, enabling direct measurement of knot formation kinetics. Using a free energy derived from scaling arguments that incorporates full details regarding the non-uniform compressed ramp-like concentration profile, we show that the enhanced knotting probability at high compression arises by avoiding the free energy cost of high self-exclusion interactions due to contour stored in the knot. In addition, the knot spatial distribution along the chain suggests that multiple knots exhibit single-file diffusion in the channel, resulting in an increased knot interaction free energy. [Preview Abstract] |
Friday, March 17, 2017 8:36AM - 9:12AM |
X14.00004: Pierced Lasso Proteins Invited Speaker: Patricia Jennings Entanglement and knots are naturally occurring, where, in the microscopic world, knots in DNA and homopolymers are well characterized. The most complex knots are observed in proteins which are harder to investigate, as proteins are heteropolymers composed of a combination of 20 different amino acids with different individual biophysical properties. As new-knotted topologies and new proteins containing knots continue to be discovered and characterized, the investigation of knots in proteins has gained intense interest. Thus far, the principle focus has been on the evolutionary origin of tying a knot, with questions of how a protein chain `self-ties' into a knot, what the mechanism(s) are that contribute to threading, and the biological relevance and functional implication of a knotted topology in vivo gaining the most insight. Efforts to study the fully untied and unfolded chain indicate that the knot is highly stable, remaining intact in the unfolded state orders of magnitude longer than first anticipated. The persistence of ``stable'' knots in the unfolded state, together with the challenge of defining an unfolded and untied chain from an unfolded and knotted chain, complicates the study of fully untied protein in vitro. Our discovery of a new class of knotted proteins, the Pierced Lassos (PL) loop topology, simplifies the knotting approach. While PLs are not easily recognizable by the naked eye, they have now been identified in many proteins in the PDB through the use of computation tools. PL topologies are diverse proteins found in all kingdoms of life, performing a large variety of biological responses such as cell signaling, immune responses, transporters and inhibitors (http://lassoprot.cent.uw.edu.pl/). Many of these PL topologies are secreted proteins, extracellular proteins, as well as, redox sensors, enzymes and metal and co-factor binding proteins; all of which provide a favorable environment for the formation of the disulphide bridge. In the PL topologies, the threaded topology is formed by a covalent loop where part of the polypeptide chain is threaded through, forming what we term a PL. The advantage of a PL topology for fundamental studies, compared to other knotted proteins, is that the threaded topology can easily be manipulated to yield an unknotted state. Exploiting the oxidative state of the cysteines, the building blocks that form the disulphide bridge generating the covalent loop, through altering the chemical environment, and thereby controlling the formation of the covalent loop, easily generates unknotted protein. The biological advantage, we have found, is that the PL can exert allosteric control through this on/off mechanism in a target protein. Most significantly, as the disulphide bridge acts as an on/off switch in knotting, the biophysical investigation of PL topologies can provide a new tool to steer folding and function in proteins, as disulphide bridges are commonly used in protein engineering and therapeutics. [Preview Abstract] |
Friday, March 17, 2017 9:12AM - 9:24AM |
X14.00005: Understanding of knots in polymers by a unified theory Liang Dai, Patrick Doyle Knots can occur in DNA and other polymers, in particular, for long polymers. Our research addresses two questions: what are the probabilities of random knots, and what are the typical knot sizes? In statistical physics, these two questions are related to the free energy cost of knot formation as a function of knot size. Using computer simulation as well as a unified theoretical framework based on the free energy expression of knot formation, we investigated knots under various conditions: semiflexible chains, flexible chains, polymers in confinement, and polymers with various intra-chain interactions. Several counterintuitive phenomena were obtained and explained, e.g. existence of metastable knots, knot shrinking by intra-chain repulsion. These predictions can be validated by experiments and may have impacts on DNA behaviors in vivo. [Preview Abstract] |
Friday, March 17, 2017 9:24AM - 9:36AM |
X14.00006: RACER a Coarse-Grained RNA Model for Capturing Folding Free Energy in Molecular Dynamics Simulations Sara Cheng, David Bell, Pengyu Ren RACER is a coarse-grained RNA model that can be used in molecular dynamics simulations to predict native structures and sequence-specific variation of free energy of various RNA structures. RACER is capable of accurate prediction of native structures of duplexes and hairpins (average RMSD of 4.15 angstroms), and RACER can capture sequence-specific variation of free energy in excellent agreement with experimentally measured stabilities (r-squared $=$0.98). The RACER model implements a new effective non-bonded potential and re-parameterization of hydrogen bond and Debye-Huckel potentials. Insights from the RACER model include the importance of treating pairing and stacking interactions separately in order to distinguish folded an unfolded states and identification of hydrogen-bonding, base stacking, and electrostatic interactions as essential driving forces for RNA folding. Future applications of the RACER model include predicting free energy landscapes of more complex RNA structures and use of RACER for multiscale simulations. [Preview Abstract] |
Friday, March 17, 2017 9:36AM - 9:48AM |
X14.00007: Resolving DNA-ligand intercalation in the entropic stretching regime Ali A. Almaqwashi Single molecule studies of DNA intercalation are typically conducted by applying stretching forces to obtain force-dependent DNA elongation measurements. The zero-force properties of DNA intercalation are determined by equilibrium and kinetic force-analysis. However, the applied stretching forces that are above the entropic regime (\textgreater 5 pN) prevent DNA-DNA contact which may eliminate competitive DNA-ligand interactions. In particular, it is noted that cationic mono-intercalators investigated by single molecule force spectroscopy are mostly found to intercalate DNA with single rate, while bulk studies reported additional slower rates. Here, a proposed framework quantifies DNA intercalation by cationic ligands in competition with relatively rapid kinetic DNA-ligand aggregation. At a constant applied force in the entropic stretching regime, the analysis illustrates that DNA intercalation would be measurably optimized only within a narrow range of low ligand concentrations. As DNA intercalators are considered for potential DNA-targeted therapeutics, this analysis provides insights in tuning ligand concertation to maximize therapeutics efficiency. [Preview Abstract] |
Friday, March 17, 2017 9:48AM - 10:00AM |
X14.00008: Dynamic Coarse-Graining of DNA Melting Kinetics: Enthalpic and Entropic Effects of Cooperative Base-Pair Dynamics Sebastian Sensale, Zhangli Peng, Hsueh-Chia Chang An MD dynamic coarse-graining approach is developed to analyze the correct melting kinetics of short DNA sequences. Existing models described well the thermodynamic melting temperature and near-equilibrium phenomena such as bubble formation and sharp phase transitions. However, the predicted kinetic rates were off by several orders of magnitude because they do not capture non-equilibrium coupled dynamics near the transition state like cooperative separation due to base stacking, hydration cage vibration and unwinding of the helix. By projecting onto the one-dimensional cooperative reaction coordinate, based on time-scale separation, we decipher the sequential triggering of several key events that determine the successive changes in enthalpy, vibrational entropy and configurational entropy towards the melting barrier. This results in a Fick-Jacobs type funnel transition state theory which allows us to use all-atomic simulations, Principal Component Analyses and elastic homogenization theory to identify the enthalpies and entropies at strategic locations along the reaction coordinate and to correctly estimate the melting rate. [Preview Abstract] |
Friday, March 17, 2017 10:00AM - 10:12AM |
X14.00009: Design Of Novel Magnetic Tweezers And Its Use For Studying DNA-Compacting Proteins Roberto Fabian, Christopher Tyson, Anneliese Striz, Pamela Tuma, Ian Pegg, Abhijit Sarkar We developed a novel transverse magnetic tweezers that can apply force to single DNA molecules in the horizontal plane. We use a $\lambda $- DNA attached to a 2.8 $\mu $m superparamagnetic bead on both ends. We describe the tweezers in detail and present data validating its performance. We show that using a simple design complemented with image processing techniques, we can reliably measure changes in the DNA's extension suitable for studying the binding of proteins. We conclude with a discussion of our experiments on the binding mechanism of the protein mIHF that plays an important role in the infection pathway of tuberculosis. [Preview Abstract] |
Friday, March 17, 2017 10:12AM - 10:24AM |
X14.00010: DNA -- peptide polyelectrolyte complexes: Phase control by hybridization Jeffrey Vieregg, Michael Lueckheide, Amanda Marciel, Lorraine Leon, Matthew Tirrell DNA is one of the most highly-charged molecules known, and interacts strongly with charged molecules in the cell. Condensation of long double-stranded DNA is one of the classic problems of biophysics, but the polyelectrolyte behavior of short and/or single-stranded nucleic acids has attracted far less study despite its importance for both biological and engineered systems. We report here studies of DNA oligonucleotides complexed with cationic peptides and polyamines. As seen previously for longer sequences, double-stranded oligonucleotides form solid precipitates, but single-stranded oligonucleotides instead undergo liquid-liquid phase separation to form coacervate droplets. Complexed oligonucleotides remain competent for hybridization, and display sequence-dependent environmental response. We observe similar behavior for RNA oligonucleotides, and methylphosphonate substitution of the DNA backbone indicates that nucleic acid charge density controls whether liquid or solid complexes are formed. Liquid-liquid phase separations of this type have been implicated in formation of membraneless organelles in vivo, and have been suggested as protocells in early life scenarios; oligonucleotides offer an excellent method to probe the physics controlling these phenomena. [Preview Abstract] |
Friday, March 17, 2017 10:24AM - 10:36AM |
X14.00011: Genomic Mapping of Human DNA provides Evidence of Difference in Stretch between AT and GC rich regions Jeffrey Reifenberger, Kevin Dorfman, Han Cao Human DNA is a not a polymer consisting of a uniform distribution of all 4 nucleic acids, but rather contains regions of high AT and high GC content. When confined, these regions could have different stretch due to the extra hydrogen bond present in the GC basepair. To measure this potential difference, human genomic DNA was nicked with NtBspQI, labeled with a cy3 like fluorophore at the nick site, stained with YOYO, loaded into a device containing an array of nanochannels, and imaged. Over 473,000 individual molecules of DNA, corresponding to roughly 30x coverage of a human genome, were collected and aligned to the human reference. Based on the known AT/GC content between aligned pairs of labels, the stretch was measured for regions of similar size but different AT/GC content. We found that regions of high GC content were consistently more stretched than regions of high AT content between pairs of labels varying in size between 2.5 kbp and 500 kbp. We measured that for every 1{\%} increase in GC content there was roughly a 0.06{\%} increase in stretch. While this effect is small, it is important to take into account differences in stretch between AT and GC rich regions to improve the sensitivity of detection of structural variations from genomic variations. [Preview Abstract] |
Friday, March 17, 2017 10:36AM - 10:48AM |
X14.00012: Droplet Microfluidics for Compartmentalized Cell Lysis and Extension of DNA from Single-Cells Philip Zimny, David Juncker, Walter Reisner Current single cell DNA analysis methods suffer from (i) bias introduced by the need for molecular amplification and (ii) limited ability to sequence repetitive elements, resulting in (iii) an inability to obtain information regarding long range genomic features. Recent efforts to circumvent these limitations rely on techniques for sensing single molecules of DNA extracted from single-cells. Here we demonstrate a droplet microfluidic approach for encapsulation and biochemical processing of single-cells inside alginate microparticles. In our approach, single-cells are first packaged inside the alginate microparticles followed by cell lysis, DNA purification, and labeling steps performed off-chip inside this microparticle system. The alginate microparticles are then introduced inside a micro/nanofluidic system where the alginate is broken down via a chelating buffer, releasing long DNA molecules which are then extended inside nanofluidic channels for analysis via standard mapping protocols. [Preview Abstract] |
Friday, March 17, 2017 10:48AM - 11:00AM |
X14.00013: The Extended Core Coax: A novel nanoarchitecture for lab-on-a-chip electrochemical diagnostics Amy E. Valera, Luke D'Imperio, Michael J. Burns, Michael J. Naughton, Thomas C. Chiles We report a novel nanoarchitecture, the Extended Core Coax (ECC) that has applicability for the detection of biomarkers in lab-on-a-chip diagnostic devices. ECC is capable of providing accessible, highly sensitive, and specific disease diagnosis at point-of-care. The architecture represents a vertically oriented nanocoax comprised of a gold inner metal core that extends \textasciitilde 200nm above a chrome outer metal shield, separated by a dielectric annulus. Each ECC chip contains 7 discrete sensing arrays, 0.49 mm$^{\mathrm{2}}$ in size, containing \textasciitilde 35,000 nanoscale coaxes wired in parallel. Previous non-extended nanocoaxial architectures have demonstrated a limit of detection (LOD) of 2 ng/mL of cholera toxin using an off-chip setup [1]. This sensitivity compares favorably to the standard optical ELISA used in clinical settings. The ECC matches this LOD, and additionally offers the benefit of specific and reliable biofunctionalization on the extended gold core. Thus, the ECC is an attractive candidate for development as a full lab-on-a-chip biosensor for detection of infectious disease biomarkers, such as cholera toxin, through tethering of biomarker recognition proteins, such as antibodies, directly on the device. [1] M.M. Archibald, et al., Biosens. Bioelectron. (2015) [Preview Abstract] |
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