Session V35: DNA/RNA in Vitro

UV exposed electronically activated damage and photoreactivation repair

An investigation of the possible physics underlying the damage caused to DNA by UV radiation and its subsequent repair via a photoreactivation mechanism is presented in this study. An electronic pathway starting from the initial damage to the final repair process is proposed. UV radiation is absorbed to create a hole-excited thymine or other pyrimidine that subsequently is responsible for the formation of the thymine dimer. The negative-ion of the cofactor riboflavin, FADH-, formed by the exposure of the photolyase protein to visible light interacts with the hole-excited electronic orbital of the thymine dimer inducing a photon-less Auger transition, which restores the two thymines to the ground state, thereby detaching the lesion and repairing the DNA. Due to energy balance, the process has to involve an electronic excited state (s). The mechanism involves the least amount of energy dissipation and is charge neutral. It also avoids radiation damage in the repair process, that is, is a radiationless process.   

Mean-Field Analysis of Recursive Entropic Segmentation of Biological Sequences

Horizontal gene transfer in bacteria results in genomic sequences which are mosaic in nature. An important first step in the analysis of a bacterial genome would thus be to model the statistically nonstationary nucleotide or protein sequence with a collection of $P$ stationary Markov chains, and partition the sequence of length $N$ into $M$ statistically stationary segments/domains. This can be done for Markov chains of order $K = 0$ using a recursive segmentation scheme based on the Jensen-Shannon divergence, where the unknown parameters $P$ and $M$ are estimated from a hypothesis testing/model selection process. In this talk, we describe how the Jensen-Shannon divergence can be generalized to Markov chains of order $K > 0$, as well as an algorithm optimizing the positions of a fixed number of domain walls. We then describe a mean field analysis of the generalized recursive Jensen-Shannon segmentation scheme, and show how most domain walls appear as local maxima in the divergence spectrum of the sequence, before highlighting the main problem associated with the recursive segmentation scheme, i.e. the strengths of the domain walls selected recursively do not decrease monotonically. This problem is especially severe in repetitive sequences, whose statistical signatures we will also discuss.   

Exploring Threaded Intercalation Using Optical Tweezers

Dumbbell-shaped binuclear ruthenium complexes are of interest due to their potential for use in selective chemotherapy. In bulk experiments, these complexes exhibit extremely slow binding kinetics. In contrast, single molecule studies use optical tweezers to stretch the DNA and induce much more rapid intercalation. The observed DNA force-extension curves clearly indicate an increase in DNA melting force and elongation of the DNA molecule upon drug binding, which is evidence of stabilization of the DNA and intercalation of the binuclear ruthenium complex. Hysteresis in the stretching-relaxation curves implies very slow dissociation of these molecules due to threaded intercalation. The concentration profile suggests unusually strong DNA binding affinity for the binuclear complexes compared to simple intercalators.   

A simple method to deposit elongated DNA onto fused-silica surfaces for single molecule studies of protein-DNA interactions

In order to study facilitated diffusion of proteins along DNA using single molecule fluorescence imaging methods, it is necessary to deposit elongated DNA molecules along fused-silica surfaces [1]. Here we have developed a simple method to deposit elongated DNA molecules onto fused-silica surfaces with high yield. We attached the ends of DNA molecules to streptavidin coated quantum dots and then deposited the end-labeled DNA onto fused-silica surfaces. The flow created by a cover slip is adequate to generate arrays of elongated and suspended DNA anchored by the two ends of each molecule, ideal for protein-DNA interaction studies. Interactions of LacI with these elongated DNA molecules will also be discussed. \newline \newline [1] Y. M. Wang, E. C. Cox and R. Austin, ``Single molecule measurements of repressor proteins 1D diffusion on DNA,'' Phys. Rev. Lett., 97, 048302, (2006).   

Strain dependent twist-stretch elasticity in elastic filaments

Structural chirality (i.e. handedness) often results in large mechanical couplings which modify the conformation and expression of natural and synthetic filamentous aggregates. Twist-stretch elasticity of double stranded DNA is vital during chromatin organization, transcription regulation and protein binding. Engineering such couplings in structurally robust and multifunctional nanowires and nanotubes offers an elegant route for fabrication of nanoscale motors, oscillators and switches. In instances where the device operation relies upon mechanical coupling, twist-stretch elasticity, eliminating the need for an externally actuated rotational degree of freedom. Recent results on single-walled carbon nanotubes and DNA reveal a reversal in the sign of the twist-stretch coupling at large strains. Here, we present a simple non-linear theory that captures the behavior macroscopically. Model simulations reveal that the higher order coefficients are sensitive functions of the microscopic deformation energetics. Such dynamic couplings already exist in nature, a general design principle that remains to be exploited for mechanically coupled self-actuation in nanoscale devices and biomimetic strategies.   

Condensation of liquid crystals of complementary nDNA duplexes from a solution of mixed oligomers

We have investigated the phase behavior of concentrated mixtures of: (i) the complementary oligonucleotides CCTCAAAACTCC (``oligoA'') + GGAGTTTTGAGG (``oligoB'') and (ii) the self complementary oligomer CGCGAAAATTTTCGCG (``oligoSelf'') with mixed random 20-22bp non-complementary single stranded oligomers (``oligoMix''). We find that upon cooling from above the duplex unbinding temperature, sub-picoliter liquid crystal domains of complementary oligomers condense out from the isotropic mixture of non-complementary sequences. This phenomenon is observed in 300-600 mg/ml oligomer solutions and for mixtures with the ratio of complementary/non-complementary sequences down to [oligoA]/[oligoB] = 1/15 and [oligoSelf]/[oligoMix] = 1/5. Comparison of condensated volumes and complementary/non-complementary weight ratios indicates that the segregation is strong, as also suggested by the columnar ordering on the condensed domains. We interpret these findings in terms of depletion forces acting on mixtures of flexible+rigid solutes. The spontaneous condensation of well paired sequences into microdroplets where the duplexes face each other at their endings opens new possibilities to prebiotic scenarios for the formation of biopolymers. Work was supported by NSF Grant DMR 0606528 and NSF MRSEC Grant No. DMR 0213918.   

Structural Analysis of D- and L-RNA by UV-Resonance Raman Spectroscopy

Chirality is a fundamental aspect of chemical biology. Nucleic molecules naturally only exist in D- but not in L-configuration. However, the origins of this homochirality are not understood. Here we show that there are differences between the Raman spectra of D-RNA and L-RNA at different photon energies. We have analyzed the L and the D enantiomer of an RNA molecule with the sequence (r(CUGGGCGG)\textbf{.}r(CCGCCUGG)) by Raman spectroscopy at different wavelengths. The bases of nucleic acids as well as aromatic amino acids and peptide bonds show electronic transitions in the deep UV. As the oscillation modes depend on conformation and surrounding of a protein, Raman Spectroscopy can be used for structural analysis.$^{ }$When subtracting the Raman spectra of D- and L-RNA from each other, the resulting Raman Difference Spectra indicates that both forms have slightly different Raman tensors. Differences in the D- and L-RNA spectra for different incident photon energies can be explained when assuming that the electronic states in both configurations are slightly shifted with respect to each other. Our results therefore reveal new insights into the nature of chirality in nucleic acids.   

Anomalous small-angle x-ray scattering (ASAXS) study of multivalent ion-DNA interactions

Multivalent ion-DNA interactions are important for biological function. The condensation and aggregation of DNA by multivalent ions has been extensively studied theoretically and (to a lesser extent) experimentally. We report on the related, but largely unexplored, interactions between DNA and multivalent ions below the critical concentration for condensation/aggregation. Using ASAXS, a technique used for previous studies of monovalent and divalent atmospheres around DNA, we have investigated the competition of monovalent and trivalent ions around the biopolymer. These data should prove vital for modeling DNA-trivalent ion interactions and the mechanisms of DNA condensation and aggregation.   

Study of Electronic Structures of Nucleobases and Associated Nuclear Quandrupole Interactions for $^{14}$N, $^{17}$O and $^{2}$H in A-DNA and B-DNA

As part of a research program for first-principles investigation of electronic structures of A-DNA and B-DNA systems we have previously carried out studies of the magnetic hyperfine interactions for the spin-label[1] muonium attached to A-DNA and B-DNA. The present work involves the nuclear quadrupole interactions (NQI) of $^{14}$N, $^{17}$O and $^{2}$H in these two systems. We will present the results of our investigations of the NQI properties using the Hartree-Fock-Roothaan procedure with many-electron correlations included using many-body perturbation theory. For the A-DNA and B-DNA systems we are using available structural data for the four nucleobases. For the free nucleobases, the geometry from the energy optimization procedure is being employed. Comparisons will be made with available experimental NQI data and planned future improvements will be discussed. [1] R.H. Scheicher, E. Torikai, F.L. Pratt, K Nagamine, and T.P. Das, Hyperfine Interactions,158, 53 (2004); Physica B, Physics of Condensed Matter, 374, 448 (2006).   

Experimental studies of the relationship between DNA structure and chemical modification, and its charge transport properties

We experimentally investigate the influence of the physico-chemical properties of DNA molecules on its charge transport capabilities. By performing comparative rather than absolute charge transport measurements, we probe the effect of chemical modifications on the electronic properties of the molecule. Modifications include the introduction of phosphodiester bond breaks, and intercalation of metal cations, as probes to ascertain the relationship between DNA structure and electronic properties. Furthermore, we perform comparative measurements between double strand and single strand DNA molecules, to probe the importance of DNA duplex structure on its electronic properties. Our comparative current-voltage measurements yield distinct curves associated with specific modifications to the DNA molecule. We also investigate different lengths of lambda DNA. AFM images confirm the presence of DNA molecules between the lithographic measurement electrodes. (NSF DMR 0103034).   

Density Functional Analysis of Stabilizing Effects of Stacking Interactions in Nucleic Acid Base Pair Steps

Base pair stacking interactions contribute significantly to the stability of DNA. In addition, numerous studies highlight the stabilizing effect of thymine within DNA. Electrostatic, van der Waals (vdW) and hydrophobic interactions all contribute to these stacking interactions, but their relative contributions are unclear. In this paper, we use the newly developed vdW density functional\footnote{Dion, Rydberg, Schr\"{o}der, Langreth, Lundqvist, PRL \textbf{{92}}, 246401 (2004)} to investigate the importance of vdW interactions to stacking interactions between Watson-Crick DNA base pairs. Our results indicate that these interactions are essential for defining both the base pair step distance and the helical twist angle of DNA. Furthermore, we show that the stability gained from the presence of thymine is due to vdW interactions between the methyl group of the thymine with neighboring bases.   

Molecular Simulations of DNA Hybridization in Solution and in Microarrays.

Nucleic acid hybridization describes a thermodynamic transition in which a single-stranded DNA molecule associates with its complementary sequence. A comprehensive understanding of the thermodynamic behavior of this process can be achieved by computer simulation. However, the collective behavior of DNA hybridization in solution and on grafted surfaces exhibits disparate time and length scales that make atomistic simulations technically unfeasible. We propose a coarse-grained model where DNA strands are described by the single-site bond-fluctuation model on a cubic lattice. Our approach incorporates physically relevant features such as the sequence and orientation dependence of base-stacking and base-pairing interactions. We perform parallel tempering Monte Carlo simulations of DNA oligomers in the canonical ensemble. We explore how chain length, interaction heterogeneity, chain stiffness, and surface density alter the location of the melting temperature and the width of the transition. The model allows the determination of the free energy change associated with the grafting of probe chains onto the array surface with respect to the free probes in solution. Overall, the thermodynamic behavior predicted is in qualitative agreement with experimental observations both in solution and in microarrays.   

Interactions between Counterions and Brushes of ssDNA

We investigate interactions between counterions and brushes of single-stranded DNA (ssDNA) using x-ray photoelectron (XPS) and near-edge x-ray absorption fine structure (NEXAFS) spectroscopies. Monolayers of thiol-modified thymine homo-oligonucleotides on gold are convenient model systems because for these ssDNA films the interpretation of the spectroscopic data is simplified and therefore quantitative analysis of the surface density, conformation, and composition is possible. A series of experiments was designed to quantify residual counterions retained in ssDNA brushes after common rinsing procedures. We find that while the residual amount of divalent Ca cations is essentially unaffected by rinsing, the monovalent K cations can be effectively removed by a rinse under flowing deionized water. Our results demonstrate that ex situ surface spectroscopies can be effectively used to systematically investigate interactions between ssDNA and counterions.   

Single DNA electrophoresis in Pluronic F127 in a real-time fluorescence microscopy

Electrophoresis is the separation of bio-molecules in a sieving medium by applying an electric field. The Pluronic F127 gel was introduced as a new sieving medium for electrophoresis. The mobility of DNA in this gel is not fully explained by conventional reptation theories. Here, in our work, the migration of single DNA molecule pre-stained was studied on the gel electrophoresis by real-time fluorescence microscopy. Separations were performed on dsDNA fragments ranging in length from 200 base pairs (bp) to 2500 bp in pluronic gel in various concentrations. Evidence is presented that in some cases DNA fragments electrophorese along gel crystallite grain boundaries and in other cases directly through gel crystallites. This is direct observation of DNA migration through the pluronic gel on a microscopic scale.   

Shear unzipping of DNA: A semi-microscopic approach

The denaturation force of double stranded DNA in shear mode is observed to be much higher than the force required to unzip individual base pairs. We present an analysis of this problem using a nonlinear generalization of a model of shear unzipping first considered by deGennes. We find that the strain on the DNA is localized over a small region on either side of the chain. The nonlinear springs of length $\kappa^{-1}$ acting in parallel on either side of the chain make the chain stiffer. The competition between this length scale $\kappa^{-1}$ and the system size $L$ gives rise to a system size dependent rupture force. While for small systems, the force scales as $F_{c} \approx f_0 L$, where $f_0$ is the rupture force of a single bond, it saturates to a value $F_{c} \approx 2 \kappa^{-1} f_0$ for large systems. We explore the role of temperature and sequence heterogeneity on the unzipping process and discuss its implications in biology and material science.