Translocation of Structurally Defined Branched DNA through Nanopores

Sunday, February 12, 2017 widespread forms of DNA damage, mimics guanine with respect to hydrogen bonding, but perturbs base stacking, destabilizing the double helix. Other common forms of damage include mismatches, such at G-T and C-T base pairs, that are unable to form canonical hydrogen bonds. We have investigated the destabilizing effects of three abnormal base pairs (8-oxoG-C, G-T, and C-T) on the thermodynamic stability of DNA hairpins at the single molecule level. Each damaged hairpin shows a distinct pattern of unfolding that allows us to quantify hairpin destabilization. These data are combined with an mFold-based analysis to create energy landscapes of the unfolding pathway for perfectly matched, damaged, and mismatched hairpin duplexes. 350-Pos Board B115 Untwisting of Double-Stranded DNA and RNA Investigated by Molecular Dynamics Simulations Korbinian Liebl. Technical University Munich, Garching, Germany. The response of dsDNA and dsRNA to torsional stress influences many of their biological functions, including binding by proteins, transcription initiation and genome packaging. So far, the twist flexibility of DNA and RNA has been studied comparatively with single-molecule experiments. However, while these studies capture the global deformability of duplexes with a length of several thousand base pairs (bp), detailed insight into conformational changes on the base pair level remains elusive. We performed all-atom Molecular Dynamics (MD) simulations on 15 bp long DNA and RNA duplexes at femtosecond resolution. Employing an advanced sampling method based on a torsion-like restraining potential, we were able to untwist the molecules to induce localized melting. The simulations allowed us to determine the relative free energy and structural changes as a function of the mean twist. Suppression of local bending has a strong influence on the onset of DNA melting. Significant differences in the response were also observed for DNA and RNA. The results can have important implications for understanding the mechanism of replication and transcription of DNA the function of RNA molecules. 351-Pos Board B116 Translocation of Structurally Defined Branched DNA through Nanopores Philipp Karau, Kyle Briggs, Vincent Tabard-Cossa. University of Ottawa, Ottawa, ON, Canada. We use solid-state nanopores to study the translocation characteristics of different structurally defined DNA topologies. Site-specific modifications with non-natural nucleotides along the backbone of DNA fragments allow grafting of side branches at specific locations by ‘‘click’’ chemistry. We produce T- and pi-shaped DNA molecules with a 50bp double-stranded DNA backbone and either 25nt single-stranded or 25bp double stranded DNA branches. Nanopores ranging in size from 3 to 10nm are fabricated by controlled breakdown (CBD) in ultra thin 10-nm SiN membranes and used to electrophoretically translocate these short branched DNA molecules. We can distinguish the topologies of these DNA molecules through analysis of the ionic current blockages. Such structurally defined branched DNA molecules can be used for the development of multiplexed nanopore-based assays, and as position-controlled building blocks of much larger DNA polymers, to further our understanding of the fundamentals of molecular transport through nanopores by precisely measuring intra molecular velocity fluctuations. 352-Pos Board B117 Thermodynamic Linkage Analysis of pH-Induced Folding and Unfolding Transitions of I-Motifs Byul Kim, Tigran Chalikian. Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada. We describe the pH-induced folding/unfolding transitions of i-motifs by a linkage thermodynamics-based formalism in terms of three pKa’s of cytosines, namely, an apparent pKa in the unfolded conformation, pKau, and two apparent pKa’s in the folded state, pKaf1 and pKaf2. For the 50 -TTACCCACCCTACC CACCCTCA-3’ sequence from the human c-MYC oncogene promoter region, the values of pKau, pKaf1, and pKaf2 are 4.8, 6.0, and 3.6, respectively. With these pKa’s, we calculate the differential number of protons bound to the folded and unfolded states as a function of pH. Analysis along these lines offers an alternative interpretation to the experimentally observed shift in the pHinduced unfolded-to-i-motif transitions to neutral pH in the presence of cosolvents and crowders. Our simulations reveal that a significant increase in the transition midpoint pH can be achieved by an increase in the equilibrium constant between the folded and unfolded DNA conformations due to the excluded volume effect.

69a

353-Pos Board B118 Live Cell Imaging of Genomic Loci using Fluorescent RNA Aptamers Adam Cawte1, Sunny Jeng2, Peter Unrau2, David Rueda1. 1 MRC Clinical Science Centre, Imperial College London, London, United Kingdom, 2Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada. In recent years, there has been an explosion of SELEX-evolved fluorescent RNA aptamers, such as Spinach and Mango, with enhanced folding, fluorescence and a high affinity for their dyes. These aptamers have a drastically improved fluorescence contrast relative to EGFP and hold great promise for visualising vital cellular processes involving RNA molecules. However, the use of aptamers in live-cell imaging has experienced limited resolution and applicability due to the dynamic nature of RNA. We have recently developed a new method that combines CRISPR/Cas9-based nuclear localization with fluorescent RNA aptamer-based imaging. A major difference with existing approaches is that this system contains an engineered fluorescent RNA aptamer within the sgRNA scaffold in lieu of a fluorescent dCas9-EGFP fusion. This method enables the direct visualisation of genomic loci and their diffusion dynamics within live cells. We anticipate the development of this technology to improve our ability to target specific regions of the genome, as well as to develop multi-colour imaging using different fluorescent sgRNA constructs. 354-Pos Board B119 Elucidating the Role of Transcription in Shaping the 3D Structure of the Bacterial Genome Hugo Brandao1, Xindan Wang2, David Rudner2, Leonid Mirny3. 1 Harvard University, Cambridge, MA, USA, 2Harvard Medical School, Boston, MA, USA, 3Massachusetts Institute of Technology, Cambridge, MA, USA. Active transcription has been linked to several genome conformation changes in bacteria, including the recruitment of chromosomal DNA to the cell membrane and formation of nucleoid clusters. Using genomic and imaging data as input into mathematical models and polymer simulations, we sought to explore the extent to which bacterial 3D genome structure could be explained by 1D transcription tracks. Using B. subtilis as a model organism, we investigated via polymer simulations the role of loop extrusion and DNA supercoiling on the formation of interaction domains and other fine-scale features that are visible in chromosome conformation capture (Hi-C) data. We then explored the role of the condensin structural maintenance of chromosome complex on the alignment of chromosomal arms. A parameter-free transcription traffic model demonstrated that mean chromosomal arm alignment can be quantitatively explained, and the effects on arm alignment in genomically rearranged strains of B. subtilis were accurately predicted. 355-Pos Board B120 Investigation of the Melting Thermodynamics of a DNA 4-Way Junction: One Base at a Time Rachel E. Savage1, Wujie Wang2, Francis W. Starr2, Ishita Mukerji1. 1 Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, CT, USA, 2Physics Department, Wesleyan University, Middletown, CT, USA. DNA four-way junctions are branched structures that form between two homologous chromosomes. These junctions play key roles during several cellular processes, including meiosis and DNA double-stranded break repair, however their mechanism of formation and separation are not well understood, particularly with respect to branch migration. We have investigated the thermodynamic stability of the DNA four-way junction J3 using the fluorescent pteridine nucleoside analogues, 6-MAP and 6-MI, which provide site-specific information on the melting process. We have incorporated these probes at different locations throughout the junction to determine the influence of position on junction stability. Preliminary fluorescence data suggest that the central region of the fourway junction, a region under much torsional strain, is in fact more stable than previously hypothesized. We have also investigated the relative stability of the different arms, by incorporating probes on each arm approximately the same distance from the junction center. These results are compared with those predicted from coarse-grained simulations of the junction using the 3 sites per nucleotide (3SPN.2) model. Already, we have demonstrated the ability of this model to reproduce many experimentally determined aspects of DNA junction structure and stability, including the temperature dependence of melting on salt concentration, the bias between open and stacked conformations, the relative populations of conformers at high salt concentration, and the inter-duplex angle between arms. We are now using a replica-exchange molecular dynamics approach to evaluate the fraction of bonded bases along each arm of the junction over the temperature range associated with melting. This approach allows us to determine base-by-base the local melting temperature along the arm. We