Bulletin of the American Physical Society
APS March Meeting 2016
Volume 61, Number 2
Monday–Friday, March 14–18, 2016; Baltimore, Maryland
Session X39: Method in Molecular Biophysics: DBIO Doctoral Thesis AwardFocus
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Sponsoring Units: DBIO Chair: Henrik Flyvbjerg, Danish Technical University Room: 342 |
Friday, March 18, 2016 8:00AM - 8:12AM |
X39.00001: How to determine local stretching and tension in a flow-stretched DNA molecule Jonas N. Pedersen, Rodolphe Marie, Anders Kristensen, Henrik Flyvbjerg We determine the nonuniform stretching of and tension in a Mbp-long fragment of DNA that is flow-stretched in a nanofluidic chip. We use no markers, do not know the contour length of the DNA, and do not have the full DNA molecule inside our field-of-view. Instead we analyze the transverse thermal motion of the DNA. Tension at the center of the DNA adds up to 16 pN, giving almost fully stretched DNA. Fitted parameters agree well with simplified expressions, where the DNA is modeled as a cylinder in a parallel flow. [Preview Abstract] |
Friday, March 18, 2016 8:12AM - 8:24AM |
X39.00002: Magnetic Actuation of Self-Assembled DNA Hinges S. Lauback, K. Mattioli, M. Armstrong, C. Miller, C. Pease, C. Castro, R. Sooryakumar DNA nanotechnology offers a broad range of applications spanning from the creation of nanoscale devices, motors and nanoparticle templates to the development of precise drug delivery systems. Central to advancing this technology is the ability to actuate or reconfigure structures in real time, which is currently achieved primarily by DNA strand displacement yielding slow actuation times (about 1-10min). Here we exploit superparamagnetic beads to magnetically actuate DNA structures which also provides a system to measure forces associated with molecular interactions. DNA nanodevices are folded using DNA origami, whereby a long single-stranded DNA is folded into a precise compact geometry using hundreds of short oligonucleotides. Our DNA nanodevice is a nanohinge from which rod shaped DNA nanostructures are polymerized into micron-scale filaments forming handles for actuation. By functionalizing one arm of the hinge and the filament ends, the hinge can be attached to a surface while still allowing an arm to rotate and the filaments can be labeled with magnetic beads enabling the hinge to be actuated almost instantaneously by external magnetic fields. These results lay the groundwork to establish real-time manipulation and direct force application of DNA constructs. [Preview Abstract] |
Friday, March 18, 2016 8:24AM - 8:36AM |
X39.00003: Nanopore DNA sequencing using kinetic proofreading Xinsheng Ling We propose a method of DNA sequencing by combining the physical method of nanopore electrical measurements and Southern's sequencing-by-hybridization. The new key ingredient, essential to both lowering the costs and increasing the precision, is an asymmetric nanopore sandwich device capable of measuring the DNA hybridization probe twice separated by a designed waiting time. Those incorrect probes appearing only once in nanopore ionic current traces are discriminated from the correct ones that appear twice. This method of discrimination is similar to the principle of kinetic proofreading proposed by Hopfield and Ninio in gene transcription and translation processes. An error analysis is of this nanopore kinetic proofreading (nKP) technique for DNA sequencing is carried out in comparison with the most precise 3' dideoxy termination method developed by Sanger. [Preview Abstract] |
Friday, March 18, 2016 8:36AM - 8:48AM |
X39.00004: Genetic Assessment of the Space Environment using MEMS Technologies$\backslash $f1 Dilip Jana, Dileon Saint Jean, Siyovush Abdurakhimov, Varun Kopparthy, Gergana Nestorova, Nabamita Pal, Nam Nguyen, Pedro Derosa, Lee Sawyer, Niel Crews, Mark DeCoster h $-abstract-$\backslash $pard For decades, researchers have studied the damage to DNA by high-energy radiation. Radiation induced damage include DNA strand breaks, base damage and base substitution. Currently, though, scientists are discovering that it is, in fact, the non-irradiated cells adjacent to the irradiated cells are the primary source of carcinogenesis. To address these ``bystander effects'', we developed a radiation detector using multi-clad scintillating fibers and silicon pixel arrays to study the effect of radiation on gene expression changes using Microelectromechanical systems (MEMS) technology. The efficiency of proton energy deposition on each of the different layers of the radiation tracking detector has been simulated using GEANT4 toolkit and tested experimentally using the detector. The position of the proton beam was determined from the intensity of the output signal from orthogonal planes of the tracking detector. We have developed and tested an instrument that automates the extraction and quantification of RNA from living cells that automates the collection, purification, and reverse transcription (RT) of RNA from a precisely-defined area of the biological sample. $\backslash $pard-/abstract-$\backslash $\tex [Preview Abstract] |
Friday, March 18, 2016 8:48AM - 9:00AM |
X39.00005: Fabrication and characterization of nanopore sandwich devices for DNA kinetic proofreading studies Zhishan Yuan, Jiajia Ye, Hongwen Wu, Xiao Xie, Qianjin Wang, Jingjie Sha, Yunfei Chen, Zhonghua Ni, Xinsheng Ling It has been proposed [1] that solid-state nanopores can be used as a kinetic proofreading mechanism for oligonucleotide hybridization on ssDNA molecules. We describe the first generation of nanopore sandwich structures consisted of two nanopores of different thicknesses of Si$_{3}$N$_{4}$ separated by a SiO$_{2}$ cavity. We will discuss the results of helium ion-beam and Ga FIB drilling and TEM characterization of the nanopore sandwiches devices. This work was supported by the National 1000-People Plan of China and Jiangsu-985 Fund, and NSFC grant no.51302037. [1] X.S. Ling, ``Methods of sequencing nucleic acids using nanopores and active kinetic proofreading'', WO/2013/119784, International Application No.: PCT/US2013/025106. [Preview Abstract] |
Friday, March 18, 2016 9:00AM - 9:12AM |
X39.00006: Confinement-induced Molecular Templating and Controlled Ligation Daniel Berard, Marjan Shayegan, François Michaud, Gil Henkin, Shane Scott, Jason Leith, Sabrina Leslie Loading and manipulating long DNA molecules within sub-50 nm cross-section nanostructures for genomic and biochemical analyses, while retaining their structural integrity, present key technological challenges to the biotechnology sector, such as device clogging and molecular breakage. We overcome these challenges by using Convex Lens-induced Confinement (CLiC) technology to gently load DNA into nanogrooves from above. Here, we demonstrate single-fluorophore visualization of custom DNA barcodes as well as efficient top-loading of DNA into sub-50 nm nanogrooves of variable topographies. We study confinement-enhanced self-ligation of polymers loaded in circular nanogrooves. Further, we use concentric, circular nanogrooves to eliminate confinement gradient-induced drift of stretched DNA. [Preview Abstract] |
Friday, March 18, 2016 9:12AM - 9:24AM |
X39.00007: Computational and Experimental Characterization of Ribosomal DNA and RNA G-Quadruplexes Samuel Cho DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in controlling the growth of cancer cells. Recent studies strongly suggest that guanine (G)-rich genes encoding pre-ribosomal RNA (pre-rRNA) are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis. However, the structures of ribosomal G-quadruplexes at atomic resolution are unknown, and very little biophysical characterization has been performed on them to date. Here, we have modeled two putative rDNA G-quadruplex structures, NUC 19P and NUC 23P, which we observe via circular dichroism (CD) spectroscopy to adopt a predominantly parallel topology, and their counterpart rRNA. To validate and refine the putative ribosomal G-quadruplex structures, we performed all-atom molecular dynamics (MD) simulations using the CHARMM36 force field in the presence and absence of stabilizing K$+$ or Na$+$ ions. We optimized the CHARMM36 force field K$+$ parameters to be more consistent with quantum mechanical calculations (and the polarizable Drude model force field) so that the K$+$ ion is predominantly in the G-quadruplex channel. Our MD simulations show that the rDNA G-quadruplex have more well-defined, predominantly parallel-topology structures than rRNA and NUC 19P is more structured than NUC 23P, which features extended loops. Our study demonstrates that they are both potential targets for the design of novel chemotherapeutics. [Preview Abstract] |
Friday, March 18, 2016 9:24AM - 9:36AM |
X39.00008: Observation of an angular change in the structure of an RNA complex using Fluorescence Resonance Energy Transfer Sheema Rahmanseresht, Peker Milas, Louis Parrot, Lori S. Goldner Single-molecular-pair FRET is often used to study distance fluctuations of single molecules. It is harder to capture angular changes using FRET, because rotational motion of the dyes tends to wash out the angular sensitivity. Using a dye labeling scheme that minimizes the rotational motion of the dyes with respect to the RNA, we use spFRET to measure an angular change in structure of an RNA kissing complex upon protein binding. The model system studied here, R1inv-R2inv, is derived from the RNAI-RNAII complex in \textit{E.coli}. RNA II is a primer for replication of the ColE1 plasmid; its function is modulated by interaction with RNA I, Rop protein is known to stabilize the bent R1inv-R2inv kissing complex against dissociation. The effect, if any, of Rop protein on the conformation of the kissing complex is not known. The eight minimized-energy NMR structures reported for R1inv-R2inv show a small difference in end-to-end distances and much larger differences in twist and bend angles. We compare a first-principles model with spFRET data to determine if the observed change in FRET is consistent with an angular change in structure, as suggested by the model. [Preview Abstract] |
Friday, March 18, 2016 9:36AM - 9:48AM |
X39.00009: Two-color spectroscopy of UV excited ssDNA complex with a single-wall nanotube (SWNT) probe: Fast nucleobase autoionization mechanism Slava V Rotkin, Tetyana Ignatova, Alexander Balaeff, Ming Zheng, Michael Blades, Peter Stoeckl DNA autoionization is a fundamental process wherein UV-photoexcited nucleobases dissipate energy to the environment without undergoing chemical damage. SWNT is shown to serve as a photoluminescent reporter for studying the mechanism and rates of DNA autoionization. Two-color photoluminescence (PL) spectroscopy revealed a strong SWNT PL quenching when the UV pump is resonant with the DNA absorption [Nano Research, 2015]. Semiempirical calculations of the DNA-SWNT electronic structure, combined with a Green's function theory for charge transfer, show a 20 fs autoionization rate, dominated by the hole transfer. Rate-equation analysis of the spectroscopy data confirms that the quenching rate is limited by the thermalization of the free charge carriers transferred to the nanotube reservoir. The developed approach has a great potential for monitoring DNA excitation, autoionization, and chemical damage both {\it in vivo} and {\it in vitro}. [Preview Abstract] |
Friday, March 18, 2016 9:48AM - 10:00AM |
X39.00010: Mapping DNA methylation by transverse current sequencing: Reduction of noise from neighboring nucleotides Jose Alvarez, Steven Massey, Alan Kalitsov, Julian Velev Nanopore sequencing via transverse current has emerged as a competitive candidate for mapping DNA methylation without needed bisulfite-treatment, fluorescent tag, or PCR amplification. By eliminating the error producing amplification step, long read lengths become feasible, which greatly simplifies the assembly process and reduces the time and the cost inherent in current technologies. However, due to the large error rates of nanopore sequencing, single base resolution has not been reached. A very important source of noise is the intrinsic structural noise in the electric signature of the nucleotide arising from the influence of neighboring nucleotides. In this work we perform calculations of the tunneling current through DNA molecules in nanopores using the non-equilibrium electron transport method within an effective multi-orbital tight-binding model derived from first-principles calculations. We develop a base-calling algorithm accounting for the correlations of the current through neighboring bases, which in principle can reduce the error rate below any desired precision. Using this method we show that we can clearly distinguish DNA methylation and other base modifications based on the reading of the tunneling current. [Preview Abstract] |
Friday, March 18, 2016 10:00AM - 10:12AM |
X39.00011: Amylin Detection with a Miniature Optical-Fiber Based Sensor Zhaowen Liu, Matsko Ann, Adam Hughes, Mark Reeves We present results of a biosensor based on shifts in the localized surface plasmon resonance of gold nanoparticles self-assembled on the end of an optical fiber. This system allows for detection of protein expression in low sensing volumes and for scanning in cell cultures and tissue samples. Positive and negative controls were done using biotin/avidin and the BSA/Anti-BSA system. These demonstrate that detection is specific and sensitive to nanomolar levels. Sensing of amylin, an important protein for pancreatic function, was performed with polyclonal and monoclonal antibodies. The measured data demonstrates the difference in sensitivity to the two types of antibodies, and titration experiments establish the sensitivity of the sensor. Further experiments demonstrate that the sensor can be regenerated and then reused. [Preview Abstract] |
Friday, March 18, 2016 10:12AM - 10:24AM |
X39.00012: A Nanocoaxial-Based Electrochemical Sensor for the Detection of Cholera Toxin Michelle Archibald, Binod Rizal, Timothy Connolly, Michael J. Burns, Michael J. Naughton, Thomas C. Chiles We report a nanocoax-based electrochemical sensor for the detection of bacterial toxins using an electrochemical enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV). The device architecture is composed of vertically-oriented, nanoscale coaxial electrodes, with coax cores and shields serving as integrated working and counter electrodes, respectively. Proof-of-concept was demonstrated for the detection of cholera toxin (CT), with a linear dynamic range of detection was 10 ng/ml - 1 \textmu g/ml, and a limit of detection (LOD) of 2 ng/ml. This level of sensitivity is comparable to the standard optical ELISA used widely in clinical applications. The nanocoax array thus matches the detection profile of the standard ELISA while providing a simple electrochemical readout and a miniaturized platform with multiplexing capabilities, toward point-of-care (POC) implementation. In addition, next generation nanocoax devices with extended cores are currently under development, which would provide a POC platform amenable for biofunctionalization of ELISA receptor proteins directly onto the device. [Preview Abstract] |
Friday, March 18, 2016 10:24AM - 11:00AM |
X39.00013: DBIO Doctoral Thesis Award: Enabling multivariate investigation of single-molecule dynamics in solution by counteracting Brownian motion Invited Speaker: Quan Wang |
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