Session L28: Nucleic Acids: Structure & Function

Measurement of the elastic energy of sharply bent ds DNA

We present measurements of the elastic energy of short (30 bp), sharply bent, ds DNA molecules. The measurements are obtained by two independent methods: one is based on the monomer-dimer equilibrium of an appropriate configuration where the elastic energy stored in the bent strands drives dimer formation; the other is based on melting curves analysis. We find that, for example, the elastic energy of a sharply bent 30 bp double stranded DNA molecule with a nick at the center does not exceed 10 kBT.   

Partitioning of the elastic energy in protein-DNA chimeras

We synthesize Protein-DNA chimeras where a DNA molecular spring mechanically perturbs the conformation of the protein. We measured the elastic energy stored in one such molecule, consisting of the enzyme Guanylate Kinase coupled to a 60 bp DNA spring. From these measurements, the response of the protein in terms of its enzymatic activity, and a mechanical model of the DNA spring we deduce that, in this case, most of the elastic energy of the molecule is stored in the DNA spring. Thus the DNA spring is ``softer'' than the protein.   

A lattice-based, coarse-grain tertiary structure model for RNA folding: predictive power and impact of excluded volume

We present a semi-quantitative lattice model of RNA folding which is able to reproduce complex folded structures like multi-loops and pseudoknots without relying on the frequently employed ad hoc generalization of the Jacobson-Stockmayer loop entropy. We derive the model parameters from the Turner description of simple secondary structural elements and pay particular attention to the unification of border and non- local loop parameters, resulting in a reduced, unified parameter set for simple loops of arbitrary type and size. For elementary structures, the predictive power of the model is comparable to standard secondary structure approaches. For complex structures, our approach offers a systematic treatment of generic effects of chain connectivity as well as of excluded volume interactions between and within all elements of the secondary structure.   

A coarse-grained model of the 10-23 DNAzyme

DNAzymes are single-stranded DNA that catalyze nucleic acid biochemistry. We have adopted a simple bead-spring model that captures, in a coarse-grained manner, the diffusive motion and self-interactions of single-stranded DNA. We used this model to investigate the structure and dynamics of the RNA cleaving 10-23 DNAzyme over long time scales via Brownian dynamics simulations. We start from an unfolded state where the DNAzyme is bound to its substrate and allow the system to relax. The structural data thus obtained agree well with FRET measurements and provide a connection between the proposed structure of the DNAzyme and chemical rate data appearing in the literature. We have also investigated the changes in the structure of the DNAzyme/substrate complex following the cleavage of the substrate. In agreement with single-molecule FRET data, we find that the DNAzyme rapidly unwinds into an extended structure.   

Supercoil formation in DNA denaturation

We generalize the Poland-Scheraga model to the case of a circular DNA, taking into account the winding of the two strains around each other. Guided by recent single-molecule experiments on DNA strands, we assume that the torsional stress induced by denaturation enforces the formation of supercoils whose writhe absorbs the linking number expelled by the loops. Our model predicts that when the entropy parameter of a loop satisfies $c\le 2$, a thermodynamic denaturation transition does not exist. On the other hand, for $c>2$, a first-order denaturation transition is consistent with our model and may take place in the actual system, as in the case with no supercoils. These results are in contrast with other treatments of melting circular DNA where denaturation is assumed to be accompanied by an increase in twist rather than writhe on the bound segments.   

Direct Observation of Multiple Overstretching Pathways of poly dA by Atomic Force Microscopy

During DNA interactions, single-stranded DNA (ssDNA) is often stretched and stabilized by coupling with ssDNA binding proteins to serve as an intermediate state. The conformational and energetic changes of stretched DNA are of great interest because of their relevance in biological functions. Poly dA has been shown to have unique over-stretching transitions among single-stranded DNA. We used atomic force microscope to stretch poly dA mechanically to study their phase transition under external force. In addition to the equilibrium pathway showing two pronounced plateaus indicating conformational transitions, multiple high energy pathways above the plateau were observed. We constructed a two states model and suggest the nonequilibrium pathways are kinetically trapped metastable states of the backbone conformation. Moreover, we have observed hopping between two states with constant force measurements. The finding may have implications in detailed mechanisms in DNA replication and transcription, and other protein-DNA interactions.   

Ethanol Induced Shortening of DNA in Nanochannels

The confinement of DNA in nanochannels has greatly facilitated the study of DNA polymer physics and holds promise as a powerful tool for genomic sequencing. Ethanol precipitation of DNA is a common tool in molecular biology, typically in $>$70{\%} [EtOH]. Even at lower ethanol concentrations, however, DNA transforms from B-form to A-form, a shorter yet slightly less twisted conformation. Accordingly, we isolated individual YOYO-1 labeled $\lambda $-DNA molecules in 100nm$\times $100nm channels in 0, 20, 40 and 60{\%} [EtOH]. We observed a dramatic shortening in the mean measured lengths with increasing [EtOH] and a broadening of the distribution of measured lengths at the intermediate concentrations. These observed lengths are less than those expected from fully A-form $\lambda $-DNA, suggesting that poor solvency effects are involved. Also, substantial spatial variations in intensity in a small number of molecules at the higher [EtOH] suggest the presence of higher order DNA conformations, in accord with the observation that the effective persistence length of DNA has been greatly reduced.   

DNA dynamics near modified solid state nanopore

Solid state nanopore is a promising method for rapid single DNA characterization. Translocation of the DNA through the nanopore provides information of the length and the folding configuration by measuring the ionic currents which flows through the nanopore. Recently, the surface modifications of nanopore for better sensing of ionic currents or the electrode fabrication near nanopore for current measuring are proposed and tested by several groups. Previously we have estimated the electric field profile using the Langevin equation near nanopore by tracing the dynamics of DNA's before translocation. Fluorescence microscopy is used to observe DNA's directly. We found that bias voltage above 1V applied across the nanopore produced prominent electric field extended up to 8 micrometer away from nanopore and induced DNA clogging into nanopore. To advance our research, modified nanopores are fabricated in two ways, 1. evaporating Au film, 2. depositing organosilanes around nanopore as we followed the recent development of the nanopore based DNA analysis. We will discuss how these modifications influence the electric field profile near nanopore.   

Effect of mismatched base pair on electrical current through homogeneous DNA molecule

The fact that GA mispair causes only small geometrical changes, makes GA mispair recognition a formidable task; and , on the other hand, local, though relatively significant changes on the transfer integral, makes electrical characteristic a favorable tool. We have investigated the possibility of utilizing electrical properties, through I-V characteristic, in recognizing the presence of GA mispair in homogeneous DNA strand. Ab-into calculations were performed to find transfer integrals and onsite energies. Then within the tight binding model the I-V characteristics of DNA molecule with mispair were calculated numerically. The changes in electrical current due to mispair are visible for DNA molecule with upto 90 base pairs.   

Validating a DNA Simulation Model through Hairpin Experiments

We will present a comparison between large-scale melting experiments of DNA hairpins and the predictions of a coarse-grained BD computation model. Most computational efforts to date take a physical approach to modeling ssDNA. While this is effective for understanding the fundamental properties of self-interacting polymers, such studies are rarely related to laboratory experiments at biological conditions. To address this need, we have investigated DNA hairpins. The system emphasizes the role of stacking and hydrogen bonding energies, characteristics of DNA, rather than backbone bending, stiffness, and excluded volume interactions, which are generic characteristics of semi-flexible polymers. In conjunction with our melting curve experiments, we varied the temperature, hydrogen bonding, and stacking parameters in our model. The comparison of our simulation and experimental results provides a strong test of our model's suitability for capturing the sensitive behavior of simple hairpin systems. Furthermore, our approach and experimental data can be used to validate other similar coarse-grained simulation models.   

A Generalized Theory of DNA Looping and Cyclization

We have developed a semi-analytic method for calculating the Stockmayer Jacobson J-factor for protein mediated DNA loops. The formation of DNA loops on the order of a few persistence lengths is a key component in many biological regulatory functions. The binding of LacI protein within the Lac Operon of E.coli serves as the canonical example for loop regulated transcription. We use a non-linear rod model to determine the equilibrium shape of the inter-operator DNA loop under prescribed binding constraints while taking sequence-dependent curvature and elasticity into account. Then we construct a Hamiltonian that describes thermal fluctuations about the open and looped equilibrium states, yielding the entropic and enthalpic costs of loop formation. Our work demonstrates that even for short sequences of the order one persistence length, entropic terms contribute substantially to the J factor. We also show that entropic considerations are able to determine the most favorable binding topology. The J factor can be used to compare the relative loop lifetimes of various DNA sequences, making it a useful tool in sequence design. A corollary of this work is the computation of an effective torsional persistence length, which demonstrates how torsion bending coupling in a constrained geometry affects the conversion of writhe to twist.   

Proposed Physical Mechanism of Chromosome Segregation in Caulobacter crescentus

Chromosome segregation is a fundamental process for all cells, but the force-generating mechanisms that drive chromosome movements in bacteria are especially unclear. In Caulobacter crescentus, recent work has demonstrated that a structure made up of the ParA protein elongates from one cell pole and interacts with ParB, a protein binding to the chromosome near the origin of replication (ori). ParB disassembles ParA, causing ParA to pull ParB, and thus, the ori to the opposite end of the cell. We performed Brownian dynamics simulations of this system in order to uncover the physical mechanism of this motion. We find that motion of the ori is robust to several variations of the model as long as a steady-state concentration gradient of ParA is established in the moving frame of the ParB-decorated chromosome. We suggest that the mechanism is ``self-diffusiophoretic'': by disassembling ParA, ParB creates a concentration gradient of ParA so that the ParA concentration is higher in front of the chromosome than behind it. Since the chromosome is attracted to ParA via ParB, it moves up the gradient in the desired direction.   

Nucleation at the DNA supercoiling transition

When overtwisted, DNA forms the same wound coils that are familiar from phone cords and water hoses, creating coiled structures known as plectonemes. Experiments that twist single molecules of DNA have shown that the nucleation of plectonemes is thermally activated, with hopping near the supercoiling transition between states with and without a plectoneme. Theoretical estimates of the energy barrier of $\sim 6 k_B T$ naively seem incompatible with the experimental rates of $\sim$ 1 Hz. Combining techniques from polymer physics and transition state theory, we use a dilute-gas path integral calculation to explain why the measured rate of hopping is so slow.   

A Bayesian Approach to Detecting Amino Acid Covariance in Multiple Sequence Alignments

Determining which residues of a protein control its biological function is a classical question in molecular biology. In particular, proteins can change their structure or function by mutating just a small set of residues. An attractive idea is that distinct sets of residues are responsible for different phenotypic properties, so that one property can be changed while another is not. Members of such a set mutate at similar points in a multiple sequence alignment and so are correlated. It has long been proposed that analysis of correlations in the mutation patterns of protein sequences may provide an important means of extracting functional information about proteins from sequence alignments. Here, we propose a methodology for incorporating functional and structural annotations of the sequences analyzed to improve the efficacy of algorithms at detecting such residue sets. We provide a Bayesian framework in which known biological properties of the sequences are used to define a prior probability that quantifies our belief that sequence positions with different conservation levels are associated with the phenotype of interest. Recent experimental data is used to demonstrate that applying these principles results in improved detection ability, allowing us to distinguish between pairs that demonstrate similar levels of correlation but are not of equal relevance to the phenotypic purpose being addressed.