Bulletin of the American Physical Society
APS March Meeting 2014
Volume 59, Number 1
Monday–Friday, March 3–7, 2014; Denver, Colorado
Session Q11: Nucleic Acids: Structure, Analysis, and Interaction with Proteins |
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Sponsoring Units: DBIO Room: 203 |
Wednesday, March 5, 2014 2:30PM - 2:42PM |
Q11.00001: The effect of long-range interactions in DNA melting Aaron Santos, William Klein` A theoretical understanding of the DNA melting transition may provide insight into the biological mechanisms of transcription and replication. If this process occurs via nucleation, it should exhibit several key features: metastability, rapid spontaneous growth, and droplet formation. In this talk, I describe the results of recent computational and theoretical studies on nearest-neighbor and long-range DNA models. While the models exhibit some characteristics of classical nucleation when the interaction range is short, they may undergo spinodal nucleation when the interaction range is long. In contrast to classical nucleation droplets, which are compact, spinodal critical droplets are diffuse, fractal-like, and similar to the metastable state. These results have clear implications for transcription and replication in biological DNA. [Preview Abstract] |
Wednesday, March 5, 2014 2:42PM - 2:54PM |
Q11.00002: Correlated local bending of DNA and its effect on DNA flexibility Xinliang Xu, Jianshu Cao The flexibility of long DNA chains can be well described by the worm-like chain model (WLC) as a semi-flexible polymer with all local details coarse grained into one parameter, the persistence length l$_{\mathrm{p}}$ (approximately 150 base pairs). Recent experimental studies of DNA in the sub persistence length regime have shown a dramatic departure from WLC and suggested a length dependent DNA flexibility. Here we report an improved model of DNA flexibility with explicit considerations of a new length scale l$_{\mathrm{D}}$ (approximately 10 base pairs), over which DNA local bend angles are correlated (arxiv.org/abs/1309.7515). In this correlated worm-like chain (C-WLC) model, a finite length correction term is analytically derived and the persistence length is found to be contour length dependent. While our model reduces to the traditional worm-like chain model when treating long DNA at length scales much larger than l$_{\mathrm{p}}$, it predicts that DNA becomes much more flexible at shorter sizes, in good agreement with recent cyclization measurements of short DNA fragments around 100 base pairs. [Preview Abstract] |
Wednesday, March 5, 2014 2:54PM - 3:06PM |
Q11.00003: Nucleosome phasing -- new insights Razvan Chereji Eukaryotic genomes are organized into arrays of nucleosomes, in which stretches of 147 base-pairs of DNA are wrapped around octameric histones. Recently, a new method of mapping nucleosome positions was developed, which gives a much higher accuracy than the typical MNase-seq method. I present a statistical mechanics model which is able to reproduce the high-resolution nucleosome positioning data. I show that the DNA sequence is not the main cause of the nucleosome phasing which is observed genome-wide, and I present the major nucleosome phasing elements. The statistical mechanics framework is general enough to be useful in explaining different experimental observations, and I present a few results of this model. [Preview Abstract] |
Wednesday, March 5, 2014 3:06PM - 3:18PM |
Q11.00004: The DNA mismatch repair protein MutS forms a one-dimensional Tonks gas on DNA Ralf Bundschuh, Piotr Klajner, Jeungphill Hanne, Brooke M. Britton, Jianquan Liu, Jonghyun Park, Jong-Bong Lee, Richard Fishel MutS is a protein involved in DNA mismatch repair. It recognizes the mismatch, forms a sliding clamp around the DNA, and displaces other proteins bound to the DNA prior to the actual repair process. Here, we present a quantitative model of an ensemble of MutS molecules on a short strand of DNA with one mismatch. We model the ensemble as a Tonks gas of passively diffusing one-dimensional particles of finite extension and include clamp formation at the mismatch and random detachment. The distributions of MutS number bound to the DNA for different mismatch positions and different MutS concentrations in solution fit very well with distributions determined by single molecule experiments, thereby establishing the Tonks gas as an excellent model of MutS action on DNA. [Preview Abstract] |
Wednesday, March 5, 2014 3:18PM - 3:30PM |
Q11.00005: Fluctuation and fidelity control of a non-proofreading polymerase Jin Yu Polymerases catalyze gene replication and transcription. They modulate activation barriers of nucleotide incorporation and amplify maximal free energy differentiation between the right and wrong nucleotides. It is essential for the polymerases to achieve sufficiently high fidelity at sufficiently high speed. We had noticed a small free energy bias in the translocation of T7 RNA polymerase (RNAP) that aids nucleotide selection. We investigated further how polymerases select against wrong nucleotides efficiently with given kinetics for the right, and with controlled differentiation capacities. We found that early selections on the reaction path outperform the late ones in error reduction. In particular, initial screening seems indispensable for lowering error rates without lowering much the speed. To see how exactly the nucleotide selection proceeds, we studied T7 RNAP also in atomistic simulations. We found that substantial nucleotide selection happens early, prior to full insertion of the nucleotide for complete Watson-Crick base pairing. A highly conserved residue brings up the small translocation energy bias by marginally blocking the active site. The residue senses the nucleotide species upon the nucleotide pre-insertion, and selectively `gates' the nucleotide during insertion. Our studies thus provide a kinetic survey of the nucleotide selection system along with underlying molecular mechanisms. [Preview Abstract] |
Wednesday, March 5, 2014 3:30PM - 3:42PM |
Q11.00006: Loop cost in RNA secondary structures and the long-range cooperativity between RNA-binding proteins Yi-Hsuan Lin, Ralf Bundschuh The interactions between RNAs and RNA-binding proteins (RBPs) are significant in post-transcriptional regulation, and thus ensure that messenger RNAs can perform appropriate biological functions. Typically, in post-transcriptional regulation a single RNA is bound by multiple RBPs, which are likely to work together, resulting in ``cooperativity.'' This cooperativity can be a consequence of a mechanism mediated by RNA secondary structures, without assuming any direct interaction between the RBPs. The basic idea is that a bound RBP prohibits the nucleobases in its footprint from forming base pair bonds with other bases, thus changing the ensemble of RNA secondary structures, resulting in a shift on the binding probability of the other RBPs on the same RNA. We focus on the simplest RNA-protein complex: one RNA with two RBP binding sites. We study this effect analytically in the simplest model of RNA secondary structure formation, the molten RNA model. We measure the cooperativity as the correlation function between the RBPs and demonstrate that an algebraic correlation function occurs, implying that the cooperativity is long-range, and that a free energy cost for loop formation in the RNA secondary structures is the crucial ingredient that generates this cooperativity. [Preview Abstract] |
Wednesday, March 5, 2014 3:42PM - 3:54PM |
Q11.00007: Single Molecule Observation of the Cyclization of Short DNA Duplex Teckla Akinyi, I-Ren Lee, Taekjip Ha In the presented work, a single molecule DNA cyclization assay was used to follow the looping kinetics of single DNA 83 bp molecules, utilizing single molecule fluorescence energy transfer (smFRET) technique. The assay was first prepared in a Na$^{+}$ free condition and the majority of the DNA was in its unlooped form. A sudden Na$^{+}$ jump was introduced at different concentrations (0.05-1.75M) and finally yielded DNA in its looping state by annealing the complementary single-strand overhangs of the assay. Looping and unlooping rates were obtained from the kinetic measurements. The result shows a positive and negative linear dependence of the Na$^{+}$ concentration to the looping and unlooping rate, respectively, until they reach a plateau at 500 mM. The plateau persists until about 1M. For concentrations beyond 1M, an immoderate increase in looping rate is noticed while the unlooping rate does so gradually. Above 1M Na$^{+}$ there is a preference of looping events that is attributed to the increase of the annealing rate of the overhangs rather than increased flexibility, consistent with earlier studies by Ibrahim Cisse \textit{et al. }(2012). A protein mediated cyclization assay was also used in experiments with HU protein in which a dramatic increase in the looping rate is noticeable. However in high HU concentration, looping is prohibited implying filament formation. [Preview Abstract] |
Wednesday, March 5, 2014 3:54PM - 4:06PM |
Q11.00008: Structure and Dynamics of the tRNA-like Structure Domain of Brome Mosaic Virus Mario Vieweger, David Nesbitt Conformational switching is widely accepted as regulatory mechanism in gene expression in bacterial systems. More recently, similar regulation mechanisms are emerging for viral systems. One of the most abundant and best studied systems is the tRNA-like structure domain that is found in a number of plant viruses across eight genera. In this work, the folding dynamics of the tRNA-like structure domain of Brome Mosaic Virus are investigated using single-molecule Fluorescence Resonance Energy Transfer techniques. In particular, Burst fluorescence is applied to observe metal-ion induced folding in freely diffusing RNA constructs resembling the 3'-terminal 169nt of BMV RNA3. Histograms of E$_{\mathrm{FRET}}$ probabilities reveal a complex equilibrium of three distinct populations. A step-wise kinetic model for TLS folding is developed in accord with the evolution of conformational populations and structural information in the literature. In this mechanism, formation of functional TLS domains from unfolded RNAs requires two consecutive steps; 1) hybridization of a long-range stem interaction followed by 2) formation of a 3' pseudoknot. This three-state equilibrium is well described by step-wise dissociation constants $K_{1}$\textit{ (328(30) }$\mu M)$ and $K_{2}$\textit{ (1092(183) }$\mu M) $for [Mg$^{\mathrm{2+}}$] and $K_{1}$\textit{ (74(6) mM)} and $K_{2}$\textit{ (243(52) mM)} for [Na$^{\mathrm{+}}$]-induced folding. The kinetic model is validated by oligo competition with the STEM interaction. Implications of this conformational folding mechanism are discussed in regards to regulation of virus replication. [Preview Abstract] |
Wednesday, March 5, 2014 4:06PM - 4:18PM |
Q11.00009: Re-sensitizing drug-resistant bacteria to antibiotics by designing Antisense Therapeutics Colleen Courtney, Anushree Chatterjee ``Super-bugs'' or ``multi-drug resistant organisms'' are a serious international health problem, with devastating consequences to patient health care. The Center for Disease Control has identified antibiotic resistance as one of the world's most pressing public health problems as a significant fraction of bacterial infections contracted are drug resistant. Typically, antibiotic resistance is encoded by ``resistance-genes'' which express proteins that carryout the resistance causing functions inside the bacterium. We present a RNA based therapeutic strategy for designing antimicrobials capable of re-sensitizing resistant bacteria to antibiotics by targeting labile regions of messenger RNAs encoding for resistance-causing proteins. We perform \textit{in silico} RNA secondary structure modeling to identify labile target regions in an mRNA of interest. A synthetic biology approach is then used to administer antisense nucleic acids to our model system of ampicillin resistant \textit{Escherichia coli}. Our results show a prolonged lag phase and decrease in viability of drug-resistant~\textit{E. coli~}treated with antisense molecules. The antisense strategy can be applied to alter expression of other genes in antibiotic resistance pathways or other pathways of interest. [Preview Abstract] |
Wednesday, March 5, 2014 4:18PM - 4:30PM |
Q11.00010: RNA secondary structure critical exponents of random sequences near the glass transition William Baez, Ralf Bundschuh RNA forms elaborate secondary structures through intramolecular base pairing. These structures are important for the RNA's biological function but, due to the availability of a polynomial algorithm to calculate the partition function, they are also a model system for the study of statistical physics of disordered systems. In this context, it is known that below the denaturation temperature random RNA secondary structures can exist in one of two phases: a strongly disordered, low-temperature glass phase and a weakly disordered, high-temperature molten phase. The probability of two bases pairing in these phases have been shown to scale with the distance between the two bases as -3/2 and -1.33 in the molten and glass phases, respectively. In this study, we attempt to answer the question as to the value and behavior of this scaling exponent at and around the transition temperature. We present a precise determination of the location of the critical point and then use several methods to measure the exponent at this critical point including a comparison of different analytical models to describe finite-size effects developed within both phases. [Preview Abstract] |
Wednesday, March 5, 2014 4:30PM - 4:42PM |
Q11.00011: Probe DNA-Cisplatin Interaction with Solid-State Nanopores Zhi Zhou, Ying Hu, Wei Li, Zhi Xu, Pengye Wang, Xuedong Bai, Xinyan Shan, Xinghua Lu Understanding the mechanism of DNA-cisplatin interaction is essential for clinical application and novel drug design. As an emerging single-molecule technology, solid-state nanopore has been employed in biomolecule detection and probing DNA-molecule interactions. Herein, we reported a real-time monitoring of DNA-cisplatin interaction by employing solid-state SiN nanopores. The DNA-cisplatin interacting process is clearly classified into three stages by measuring the capture rate of DNA-cisplatin adducts. In the first stage, the negative charged DNA molecules were partially discharged due to the bonding of positive charged cisplatin and forming of mono-adducts. In the second stage, forming of DNA-cisplatin di-adducts with the adjacent bases results in DNA bending and softening. The capture rate increases since the softened bi-adducts experience a lower barrier to thread into the nanopores. In the third stage, complex structures, such as micro-loop, are formed and the DNA-cisplatin adducts are aggregated. The capture rate decreases to zero as the aggregated adduct grows to the size of the pore. The characteristic time of this stage was found to be linear with the diameter of the nanopore and this dynamic process can be described with a second-order reaction model. [Preview Abstract] |
Wednesday, March 5, 2014 4:42PM - 4:54PM |
Q11.00012: Entropic trapping of single DNA molecules emerging from a nanopore Xu Liu, Mirna Mihovilovic, Derek Stein We developed nanostructures with a cavity that receives and entropically traps a single DNA molecule after it translocates a nanopore in the cavity wall. The 1.5 $\mu $m-high, 2.2 $\mu $m-wide cavity has a 200 nm-wide opening across from the nanopore that is too large to affect the electrical resistance of the structure in solution, but small enough to confine $\lambda$ DNA. A voltage bias drew a DNA molecule through the nanopore, resulting in a blockage of the ionic current. 2 ms after the end of the translocation was detected, the bias was removed. A predetermined pause time, $t_p$, elapsed before a bias of the opposite polarity was applied. The current was monitored to detect the recapture of the same molecule. We found that the mean interval between the voltage reversal and the molecule's recapture, $t_r$, increased with $t_p$ until $t_p= 700$ ms, where it saturated at $t_r\approx 250$ ms. The molecules were recaptured with nearly unit efficiency for all $t_p$ tested, up to $t_p=50$ s. By contrast, when DNA emerged from a nanopore into an open reservoir with no cavity, $t_r$ increased continuously with $t_p$, and the probability of recapturing the molecule within 5 s of the voltage reversal dropped precipitously for $t_p>1$ s. [Preview Abstract] |
Wednesday, March 5, 2014 4:54PM - 5:06PM |
Q11.00013: Mechanism of DNA Trapping in Nanoporous Structures during Asymmetric Pulsed-Field Electrophoresis Ya Zhou, D. Jed Harrison DNA molecules (\textgreater 100kbp) are trapped in separation sieves when high electric fields are applied in pulsed field electrophoresis, seriously limiting the speed of separation. Using crystalline particle arrays, to generate interstitial pores for molecular sieving, allows higher electric fields than in gels, (e.g 40 vs 5 V/cm), however trapping still limits the field strength. Using reverse pulses, which release DNA from being fully-stretched, allows higher fields (140 V/cm). We investigate the trapping mechanism of individual DNA molecules in ordered nanoporous structures. Two prerequisites for trapping are revealed by the dynamics of single trapped DNA, hernia formation and fully-stretched U/J shapes. Fully stretched DNA has longer unhooking times than expected by simple models. We propose a dielectrophoretic (DEP) force reduces the mobility of segments at the apex of the U or J, where field gradients are highest, based on simulations. A modified model for unhooking time is obtained after the DEP force is introduced. The new model explains the unhooking time data by predicting an infinite trapping time when the ratio of arm length differences (of the U or J) to molecule length $\Delta x/L<\beta $. $\beta $ is a DEP parameter that is found to strongly increase with electric field. [Preview Abstract] |
Wednesday, March 5, 2014 5:06PM - 5:18PM |
Q11.00014: DNA transport and conformation in confined environments: novel separation mechanism using hydrodynamics and electrophoresis Hubert Ranchon, Qihao He, Joris Lacroix, Aur\'elien Bancaud Nanofluidics has gained popularity because it offered new solutions for single molecule manipulation and for the separation of biomolecules [1]. In addition to confinement, which enables to induce the elongation of DNA through steric repulsion, we recently showed that the degree of spreading of single molecules could be monitored by tuning the flow in nanochannels [2]. In this report we investigate the concomitant flow actuation with hydrodynamics and electrophoresis to transport DNA molecules in confined slit-like channels. We demonstrate that DNA size separation can be performed with no separation matrix, and we prove that our approach outperforms conventional separation methods, e.g. gel electrophoresis, in terms of separation performances, because we report power scaling dependence of up to -3 for the DNA mobility \textit{vs}. size response. We also describe the physics of DNA migration by single molecule microscopy and provide a mechanistic model of the separation.\\[4pt] [1] Dorfman, \textit{AIChE} 59, 346 (2013).\\[0pt] [2] He, \textit{Macromolecules} 46, 6195 (2013). [Preview Abstract] |
Wednesday, March 5, 2014 5:18PM - 5:30PM |
Q11.00015: High-throughput DNA Stretching in Continuous Elongational Flow for Genome Sequence Scanning Robert Meltzer, Joshua Griffis, Mikhail Safranovitch, Gene Malkin, Douglas Cameron Genome Sequence Scanning (GSS) identifies and compares bacterial genomes by stretching long (60 -- 300 kb) genomic DNA restriction fragments and scanning for site-selective fluorescent probes. Practical application of GSS requires: 1) high throughput data acquisition, 2) efficient DNA stretching, 3) reproducible DNA elasticity in the presence of intercalating fluorescent dyes. GSS utilizes a pseudo-two-dimensional micron-scale funnel with convergent sheathing flows to stretch one molecule at a time in continuous elongational flow and center the DNA stream over diffraction-limited confocal laser excitation spots. Funnel geometry has been optimized to maximize throughput of DNA within the desired length range (\textgreater 10 million nucleobases per second). A constant-strain detection channel maximizes stretching efficiency by applying a constant parabolic tension profile to each molecule, minimizing relaxation and flow-induced tumbling. The effect of intercalator on DNA elasticity is experimentally controlled by reacting one molecule of DNA at a time in convergent sheathing flows of the dye. Derivations of accelerating flow and non-linear tension distribution permit alignment of detected fluorescence traces to theoretical templates derived from whole-genome sequence data. [Preview Abstract] |
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