Bulletin of the American Physical Society
2009 APS March Meeting
Volume 54, Number 1
Monday–Friday, March 16–20, 2009; Pittsburgh, Pennsylvania
Session D40: Nucleic Acids: Packaging, Ejection and Translocation |
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Sponsoring Units: DBP Chair: Xiangyun Qiu, National Institutes of Health Room: 412 |
Monday, March 16, 2009 2:30PM - 2:42PM |
D40.00001: Anomalous scaling of nano-pore translocation times of structured biomolecules Malcolm McCauley, Robert Forties, Ulrich Gerland, Ralf Bundschuh Translocation through a nano-pore is a new experimental technique to probe physical properties of biomolecules. A bulk of theoretical and computational work exists on how the main observable, the time to translocate a single molecule, depends on the length of the molecule for unstructured molecules. Here, we study the same problem but for RNA molecules for which the breaking of the secondary structure is the main barrier for translocation. To this end, we calculate the mean translocation time of single-stranded RNA through a nanopore of zero thickness and at zero voltage for many randomly chosen RNA sequences. We find the translocation time to depend on the length of the RNA molecule with a power law. The exponent changes as a function of temperature and exceeds the naively expected exponent of two for purely diffusive transport at all temperatures. [Preview Abstract] |
Monday, March 16, 2009 2:42PM - 2:54PM |
D40.00002: DNA Physical Mapping via the Controlled Translocation of Single Molecules through a 5-10nm Silicon Nitride Nanopore Derek Stein, Walter Reisner, Zhijun Jiang, Nick Hagerty, Charles Wood, Jason Chan The ability to map the binding position of sequence-specific markers, including transcription-factors, protein-nucleic acids (PNAs) or deactivated restriction enzymes, along a single DNA molecule in a nanofluidic device would be of key importance for the life-sciences. Such markers could give an indication of the active genes at particular stage in a cell's transcriptional cycle, pinpoint the location of mutations or even provide a DNA barcode that could aid in genomics applications. We have developed a setup consisting of a 5-10 nm nanopore in a 20nm thick silicon nitride film coupled to an optical tweezer setup. The translocation of DNA across the nanopore can be detected via blockades in the electrical current through the pore. By anchoring one end of the translocating DNA to an optically trapped microsphere, we hope to stretch out the molecule in the nanopore and control the translocation speed, enabling us to slowly scan across the genome and detect changes in the baseline current due to the presence of bound markers. [Preview Abstract] |
Monday, March 16, 2009 2:54PM - 3:06PM |
D40.00003: The Effects of Bio-functionalization on Solid-state Nanopore Transport -- Theory and Experiments on DNA Yaling Liu, Abhijit Ramachandran, Samir M. Iqbal Solid-state nanopore channels have been reported recently to show selectivity for various target bio-molecules. The surfaces of nanopore channels are functionalized to achieve such selectively. The organic molecule coatings alter the behavior of molecular transport as well as change surface energies, chemical and physical properties, and make these more bio-compatible. We present theoretical considerations of DNA-modified nanopore channels which treat the functional molecules on the surface as a combination of series of potential sites. The potential function depends on the physical interactions of two ssDNA molecules. The simulated DNA trajectories and translocation speeds under various test conditions are consistent with the reported experimental data. [Preview Abstract] |
Monday, March 16, 2009 3:06PM - 3:18PM |
D40.00004: Nanopores as a Single-Molecule Probe for Protein-DNA Complexes A.R. Hall, S.W. Kowalczyk, R.M.M. Smeets, N.H. Dekker, C. Dekker In recent years, solid state nanopores have emerged as a productive novel technique for molecular biophysics. The electrophoretic motion of single molecules through these small-scale structures can offer insights into both conformation and charge structure. Here, we apply the method to the RecA nucleoprotein filament - a conformation where proteins polymerize along the entire length of a double-stranded DNA. This offers a unique geometry and charge structure which we probe through a combination of translocation experiments and optical tweezer measurements. We discuss conductance blockade events that are notably larger (12 nS) than those measured for bare dsDNA (1 nS), and present force spectroscopy data showing a high level of charge screening in solution ($>$90{\%}). [Preview Abstract] |
Monday, March 16, 2009 3:18PM - 3:30PM |
D40.00005: Computational model of controlled translocation of DNA molecule through a nanopore membrane with tunable electrostatic potential Alexey Nikolaev, Maria Gracheva We present results of computational modeling of controllable DNA translocation through a nanopore in a thin electrically tunable membrane composed of two layers of n-type and p-type semiconductor materials. Membrane potential biases are used to obtain distinct electrostatic potential landscapes. The membrane-DNA system is immersed in a biased electrolyte solution under bias to induce DNA translocation. A simple charges-and-springs model is used to model polynucleotide molecule. We compare electrostatic potential landscapes of the membrane with one and more potential extrema and show how electrostatic potential landscape in the nanopore alters the control over the molecule translocation. In particular, we specify different conditions under which DNA nucleotides can be translocated through the nanopore one by one in both directions as well as paused in the nanopore. [Preview Abstract] |
Monday, March 16, 2009 3:30PM - 3:42PM |
D40.00006: Heterogeneity in Retroviral Nucleocapsid Protein Function Christy Landes Time-resolved single-molecule fluorescence spectroscopy was used to study the human T-cell lymphotropic virus type 1 (HTLV-1) nucleocapsid protein (NC) chaperone activity as compared to that of the HIV-1 NC protein. HTLV-1 NC contains two zinc fingers with each having a CCHC binding motif similar to HIV-1 NC. HIV-1 NC is required for recognition and packaging of the viral RNA and is also a nucleic acid chaperone protein that facilitates nucleic acid restructuring during reverse transcription. Because of similarities in structures between the two retroviruses, we have used single-molecule fluorescence energy transfer to investigate the chaperoning activity of HTLV-1 NC protein. The results indicate that HTLV-1 NC protein induces structural changes by opening the transactivation response (TAR)-DNA hairpin to an even greater extent than HIV-1 NC. However, unlike HIV-1 NC, HTLV-1 NC does not chaperone the strand-transfer reaction involving TAR-DNA. These results suggest that despite its effective destabilization capability, HTLV-1 NC is not as effective at overall chaperone function as is its HIV-1 counterpart. [Preview Abstract] |
Monday, March 16, 2009 3:42PM - 3:54PM |
D40.00007: Energetics of genome ejection from phage revealed by isothermal titration calorimetry Meerim Jeembaeva, Bengt Jonsson, Martin Castelnovo, Alex Evilevitch It has been experimentally shown that ejection of double-stranded DNA from phage is driven by internal pressure reaching tens of atmospheres. This internal pressure is partially responsible for delivery of DNA into the host cell. While several theoretical models and simulations nicely describe the experimental data of internal forces either resisting active packaging or equivalently favoring spontaneous ejection, there are no \textit{direct} energy measurements available that would help to verify how quantitative these theories are. We performed \textit{direct} measurements of the enthalpy responsible for DNA ejection from phage $\lambda $, using \textit{Isothermal Titration Calorimetry}. The phage capsids were ``opened'' \textit{in vitro} by titrating $\lambda $ into a solution with LamB receptor and the enthalpy of DNA ejection process was measured. In his way, enthalpy stored in $\lambda $ was determined as a function of packaged DNA length comparing wild-type phage $\lambda $ (48.5 kb) with a shorter $\lambda $-DNA length mutant (37.7 kb). The temperature dependence of the ejection enthalpy was also investigated. The values obtained were in good agreement with existing models and provide a better understanding of ds- DNA packaging and release mechanisms in motor-packaged viruses (e.g., tailed bacteriophages, Herpes Simplex, and adenoviruses). [Preview Abstract] |
Monday, March 16, 2009 3:54PM - 4:06PM |
D40.00008: Inhibition of DNA ejection from bacteriophage by Mg$^{+2}$ counterions Seil Lee, Cathy V. Tran, Toan T. Nguyen The problem of inhibiting viral DNA ejection from bacteriophages by multivalent counterions, especially Mg$^{+2}$ counterions, is studied. Experimentally, it is known that MgSO$_4$ salt has a strong and non-monotonic effect on the amount of DNA ejected. There exists an optimal concentration at which the least DNA is ejected from the virus. At lower or higher concentrations, more DNA is ejected from the capsid. We propose that this phenomenon is the result of DNA overcharging by Mg$^{+2}$ multivalent counterions. As Mg$^{+2}$ concentration increases from zero, DNA net charge changes from negative to positive. The optimal inhibition corresponds to the Mg$^{+2}$ concentration where DNA is neutral. At lower/higher concentrations, DNA genome is charged. It prefers to be in solution to lower its electrostatic self-energy, which consequently leads to an increase in DNA ejection. Our theory fits experimental data well. The strength of DNA-DNA short range attraction, mediated by Mg$^{+2}$, is found to be $-$0.003 $k_B$T per nucleotide base. [Preview Abstract] |
Monday, March 16, 2009 4:06PM - 4:18PM |
D40.00009: Osmotic pressure: resisting or promoting DNA ejection from phage? Internal capsid-pressure dependence of viral infection Alex Evilevitch, Meerim Jeembaeva, Sarah Koester, Martin Castelnovo, David Weitz Recent\textit{ in vitro }experiments have shown that DNA ejection from phage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I on the course of ejection. We argue in this work by combination of experimental techniques (UV absorbance, pulse-field electrophoresis, and cryo-EM) that intact genome ($i.e$. \textit{undigested}) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resisting it. While, in vitro, the ejection depends sensitively on internal pressure within the virus capsid, the effect of internal pressure on infection of bacteria is unknown. We use microfluidics to monitor individual cells and determine the distribution of lysis due to infection as the capsid pressure is varied. The lysis probability decreases markedly with decreased capsid pressure. [Preview Abstract] |
Monday, March 16, 2009 4:18PM - 4:30PM |
D40.00010: Charting the Structure and Energetics of Packaged DNA in Bacteriophages Xiangyun Qiu, Donald C. Rau, V. Adrian Parsegian, Li Tai Fang, Charles M. Knobler, William M. Gelbart Many bacterial viruses resort to pressure in order to infect bacteria, e.g., lambda phage stores its dsDNA genome at surprisingly high pressure and then uses this pressure to drive delivery of the genome. We report on a biophysical interrogation of the DNA configuration and pressure in lambda phage by combining structural and thermodynamic measurements with theoretical modeling. Changes in DNA organization in the capsid are monitored using solution small angle x-ray scattering (SAXS). We vary the DNA-DNA repulsion and DNA bending contributions to the capsid pressure by changing salt concentrations and packaged length, and augment SAXS data with osmotic stress measurements to elicit the evolving structure and energetics of the packaged DNA. [Preview Abstract] |
Monday, March 16, 2009 4:30PM - 4:42PM |
D40.00011: Stabilising the Herpes Simplex Virus capsid by DNA packaging Gijs Wuite, Kerstin Radtke, Beate Sodeik, Wouter Roos Three different types of Herpes Simplex Virus type 1 (HSV-1) nuclear capsids can be distinguished, A, B and C capsids. These capsids types are, respectively, empty, contain scaffold proteins, or hold DNA. We investigate the physical properties of these three capsids by combining biochemical and nanoindentation techniques. Atomic Force Microscopy (AFM) experiments show that A and C capsids are mechanically indistinguishable whereas B capsids already break at much lower forces. By extracting the pentamers with 2.0 M GuHCl or 6.0 M Urea we demonstrate an increased flexibility of all three capsid types. Remarkably, the breaking force of the B capsids without pentamers does not change, while the modified A and C capsids show a large drop in their breaking force to approximately the value of the B capsids. This result indicates that upon DNA packaging a structural change at or near the pentamers occurs which mechanically reinforces the capsids structure. The reported binding of proteins UL17/UL25 to the pentamers of the A and C capsids seems the most likely candidate for such capsids strengthening. Finally, the data supports the view that initiation of DNA packaging triggers the maturation of HSV-1 capsids. [Preview Abstract] |
Monday, March 16, 2009 4:42PM - 4:54PM |
D40.00012: Effect of ions on polymer ejection dynamics from viral capsids Issam Ali, Davide Marenduzzo, Julia Yeomans We present simulations investigating the impact of adding ions on the dynamics of semifexible (DNA-like) polymers ejecting from spherical viral capsids. We find that when the DNA charge is less screened, due to, for example, the addition of monovalent ions like Na+, the resulting electric interactions give rise to larger ejection forces, speeding up the ejection process. The results suggest that DNA ejection can be controlled by tuning the salt concentration in the environment, in agreement with recent experiments. We also observe that the DNA structure inside the capsid changes when electrical forces are present, tending to become more spool-like. [Preview Abstract] |
Monday, March 16, 2009 4:54PM - 5:06PM |
D40.00013: Investigating Nanopore Spatial Resolution Using Locally Coated RecA-dsDNA Filaments A.R. Hall, S.W. Kowalczyk, C. Dekker The translocation of molecules through nanometer-scale apertures has garnered much attention as a future sequencing method. Many challenges remain, including the high spatial and temporal resolution needed to do so. We examine the spatial limits of these measurements by translocating partially complexed~RecA nucleoprotein filaments. These are dsDNA polymerized with discrete RecA protein patches of random length, ranging from a few monomers to full coverage (average length $\sim $10 kbp). With these molecules, we use nanopores for the first time to map the location of features along the length of a single molecule. We show that resolution of less than 500 bp is achieved and discuss the implications on translocation measurements. [Preview Abstract] |
Monday, March 16, 2009 5:06PM - 5:18PM |
D40.00014: Electrically Gated Solid State Nanopores Zhijun Jiang, Walter Reisner, Derek Stein We are exploring the use of electrically functionalized solid-state nanopores for controlling the transport of ions and single DNA molecules in solution. We have integrated annular gate electrodes inside solid-state nanopores that can electrostatically adjust both the polarity and the density of the inner surface charge. An applied gate potential can thereby influence the density of mobile counter-ions inside a pore at low salt concentrations. Our theoretical calculations show that a 0.1 V change in the gate potential can change the pore conductance by more than a factor of 5, making the nanopore behavior similar to that of a transistor. Furthermore, the electrostatic interaction between the nanopore surface and negatively charged DNA molecules can be probed in the regime of Double-layer overlap. A negatively charged inner nanopore surface should repel DNA, and limit its possibility to insert into the nanopore. Positive surface charges, on the other hand, will attract DNA, and translocation should be favored. We seek to electrostatically control the translocation of DNA through the nanopore, and thereby mimic single-molecule regulatory capabilities of biological nanopores. [Preview Abstract] |
Monday, March 16, 2009 5:18PM - 5:30PM |
D40.00015: Structural transitions in packing of a semi-flexible chain confined in a sphere Artem Levandovsky, Leonid Pryadko, Roya Zandi We study phases and phase transitions (crossovers) between phases of a semi-flexible polymer chain confined in a spherical cavity. Such a problem is relevant to DNA or RNA packaging in viruses whose organization is characterized by both simplicity and economy. The confinement involves both energetic and entropic effects controlled by the stiffness of the chain, its length and diameter, and the sphere radius. Formation of different packing configurations and structural changes in these configurations is studied with a non-local ``cluster'' Monte Carlo method. We introduce several order parameters characterizing different packing symmetries and compute the corresponding probability distributions. This allows us to reconstruct the Landau free energies for these order parameters, and thus develop a simple theory of packing transitions. [Preview Abstract] |
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