Session C64: Biopolymers II (DNA, RNA, Biocompatible, Gels)

The Mechanism of DNA Junction Melting

Four-way Holliday junctions play vital roles in genetic recombination, DNA damage repair, and other mechanisms of chromosomal rearrangement. Here we examine the melting of Holliday junctions through a combination of FRET experiments and MD simulations. We show that melting of this junction initiates at specific locations. At physiological salt concentrations, the junction “folds” into a stacked conformation consisting of two crossing pseudo-duplexes, and our results indicate that melting proceeds as a quasi-independent process along these pseudo-duplexes, rather than as a uniform process throughout the junction. We interrogate the melting locally by measuring FRET from fluorescent nucleotide analogs strategically placed at several locations in the junction. To help interpret the FRET data, we use the coarse-grained 3SPN.2 model to simulate the junction melting. By analyzing an ensemble of simulations, we describe approaches to characterize junction conformation and the dynamics of melting. Our results demonstrate the ability of the 3SPN.2 model to predict structural and dynamic aspects of Holliday junctions and offer a two-step mechanism for junction melting.   

Dynamic conversion of RNA in crowded environments: implication of significance of micro-viscosity against molecular confinement

The conformational behavior of RNA in densely packed intracellular conditions is a long-standing area of interest and is critical for understanding biologically important dynamics in a living cell. The effect of viscosity at the solvent-accessible surface area on the concerted conformational motions of tRNA in crowded conditions must be considered. We have examined the effect of PEG-induced crowding on the internal dynamics of unfolded and folded tRNA using ps ~ ns Quasi-Elastic Neutron Scattering spectroscopy (QENS) to probe the role of the local micro-viscosity as a function of the molecular confinement effects from crowding. At temperatures lower than water crystallization (T_c) and higher than dynamic glass transition temperature (T_d) of tRNA, the conformational flexibility becomes larger with more confinement, and it is suppressed more strongly at higher temperatures. The dynamic conversion across T_c indicates the significance of the micro-viscosity versus molecular confinement, both of which can orthogonally vary with crowding conditions. Comparison of dynamics from QENS with the aid of molecular dynamics simulations provides insight into the physical origin of this dynamic conversion and its coupling with the local structure of water at the macromolecular surface.   

Diffusion and Conformational Dynamics of Linear and Circular DNA in Crosslinked Cytoskeleton Composites

In order to carry out key cellular processes, DNA molecules must diffuse through the crowded cytoskeletal network, comprised of thin semiflexible actin filaments and thick rigid microtubules. Each of these cytoskeletal filaments can also be crosslinked to varying degrees, altering the network structure and dynamics and hence the impact on DNA diffusion and conformational dynamics. Here, we use single-molecule conformational tracking to investigate the effect of cytoskeleton crosslinking on the transport properties and time-varying conformations of diffusing DNA molecules. Specifically, we track the center-of-mass, size, and shape of single linear and circular DNA molecules diffusing in crosslinked composites of actin and microtubules. We quantify transport coefficients, anomalous diffusion scaling exponents, lengths and timescales of intramolecular fluctuations, and shape and size dynamics of DNA. We determine the role of DNA topology (circular vs linear) as well as cytoskeleton crosslinking motif on DNA dynamics and conformations. Our results shed light on how macromolecular topology and network structure impact macromolecular mobility in crowded cell-like environments.   

Binding of oligos to DNA secondary structures

RNAs are molecules that rely on their 3D structure to regulate cell processes, such as delivering molecules or making proteins. Therefore, disrupting the structure of RNA could help us better understand or even modulate these functions. In principle, one way to do this is to bind DNA oligonucleotides to the RNA to change its conformation so that it no longer works. In practice, this is a difficult task as the complex secondary and tertiary structures of RNA often prevent DNA from binding stably. We aim to understand the kinetic and thermodynamic reasons why oligonucleotides fail to bind to secondary structures. In this talk, I will describe a series of experiments investigating the binding of short DNA oligonucleotides to nucleic acids with prescribed secondary structures. We design a set of hairpins, bulge loops, and internal loops with various loop and toehold lengths, which should result in different opening rates when the oligo binds. A fluorescent probe binds to all these structures, allowing us to make measurements of the amount of unfolded molecules. Preliminary results show that opening rates of different secondary structures vary by orders of magnitude, with hairpins being fastest, and internal loops being slowest.   

Molecular Dynamics Simulation of Supercoiled DNA with Mismatched Base Pair-Probing the Role of Structural Defect on Plectoneme Pinning

Mismatch repair (MMR) proteins correct mismatches in DNA. The exact mechanism by which MMR proteins recognize mismatch is still not well understood, but it is believed that the process involves introducing a sharp bend in the DNA and flipping out the mismatched base. Recent, single-molecule magnetic tweezers-based DNA supercoiling studies, conducted at salt concentrations of 0.5 M NaCl and higher, have shown that the mismatched base pair will always localizes at the end of the plectoneme loop and this may facilitate mismatch detection. Theoretical studies have found that the localization of mismatched base pair in the plectoneme end loop, at 0.2 M NaCl, becomes probabilistic. However, these studies were limited to positively supercoiled DNA. Here, we carry out molecular dynamics simulation to study supercoiling of DNA in presence of mismatched base pairs. We study both positively and negatively supercoiled DNA at salt concentration of 0.2 M NaCl using the oxDNA model. We simulate a DNA molecule with 0, 2, 4 and 6 consecutive G:T type mismatches. We find that the plectoneme localization at the mismatched base pair becomes probabilistic. We will present the details of the relation between the number of mismatched base pairs and the probability of plectoneme localization.
  

Mechanical Properties of DNA Replication

Successful DNA replication requires significant mathematical and physical constraints to be met. The helical structure of DNA results in interwound replicated strands of DNA causing over twisting in the unreplicated portion of DNA presenting a significant topological barrier to cellular division. Though the basic conceptual elements of this process have long been known the necessary physics has just recently been developed. In this work, we outline the basic mathematical and physical properties of an idealized DNA replication process. The resulting framework makes predictions on the relative concatenation of replicated DNA and supercoiled unreplicated DNA generated during replication. These elements are central in understanding the mechanical nature of the replication machinery as well as in transcription replication conflicts.   

Quantifying free energy pathways of competitive DNA-ligand interactions

DNA intercalators are considered for potential DNA targeted therapeutics. The kinetics of simple DNA intercalators are well-modeled and characterized in single molecules studies. However, DNA-ligand that have multi-binding modes including intercalation are often studied in equilibrium approach, which does not address the governing free energy pathways. It is crucial to map the transition pathways, in particular, for competitive DNA-ligand interactions that alter DNA extension, which provides insights for rational drug design. While intercalation elongates DNA, other binding modes can be distinguished by distinct rates of increase or decrease of DNA extension. For practical experimental conditions, a frame work is proposed here to analyze the kinetics of DNA-ligand multi-binding modes probed by time-dependent and force-dependent DNA extension. This enables characterizing and quantifying the free energy pathways in order to identify and further optimize the molecular binding mechanism.   

Looping and Clustering: a statistical physics approach to protein-DNA complexes in bacteria

The DNA shows a high degree of spatial and dynamical organization over a broad range of length scales. It interacts with different populations of proteins and can form protein-DNA complexes that underlie various biological processes, including chromosome segregation. A prominent example is the large ParB-DNA complex, an essential component of a widely spread mechanism for DNA segregation in bacteria. Recent studies suggest that DNA-bound ParB proteins interact with each other and condense into large clusters with multiple extruding DNA-loops.

In my talk, I present the Looping and Clustering model [1], a simple statistical physics approach to describe how proteins assemble into a protein-DNA cluster with multiple loops. Our analytic model predicts binding profiles of ParB proteins in good agreement with data from high precision ChIP-sequencing – a biochemical technique to analyze the interaction between DNA and proteins at the level of the genome. The Looping and Clustering framework provides a quantitative tool that could be exploited to interpret further experimental results of ParB-like protein complexes and gain some new insights into the organization of DNA.

[1] Walter, J.-C., Walliser, N.-O., ... & Broedersz, C. P., New J. Phys. 20, 035002 (2018).   

Coarse-grained molecular simulation studies of effect of solvent quality on melting thermodynamics of oligonucleic acids (ONA) and ONA-polymer conjugates

Hybridization or melting thermodynamics of oligomers of nucleic acids (e.g., DNA, RNA, PNA) duplexes is dependent on oligonucleic acid (ONA) backbone chemistry, length, base sequence and composition, and solvent chemistry. Understanding how solvent quality affects ONA stability, in free state and when conjugated to polymers is important for use of ONA in nucleotide-based bio- and nano- technologies. In this talk, I will present our recent molecular simulation work using a coarse-grained model capable of capturing the specific and directional H-bonds between complementary bases in ONA, in implicit solvents. We found that, as the solvent quality worsens for the polymers conjugated to neutral and flexible ONAs (such as PNA), the ONA melting temperature increases for all the ONA sequences, G-C content and polymer length studied. For negatively charged and semi-flexible ONAs (DNA-like), the conjugation of longer solvophobic polymer (as compared to ONA length) decreases the ONA melting temperatures while conjugation of relatively shorter solvophobic polymer, similar to ONA length, does not affect melting temperatures for all the ONA sequences and G-C content studied.   

Terahertz Spectroscopic Study on Protein Myoglobin: Polymer-Like Behavior of Vibrational Density of States

We performed terahertz time-domain spectroscopy on protein myoglobin to investigate the boson peak dynamics which universally appears in glassy materials and fracton which is expected to universally appear in polymer glasses. The obtained spectral shape of log ε'' vs log frequency [ε'': the imaginary part of complex dielectric constants] indicates that the fracton region appears above the boson peak frequency. The fractal and fracton dimensions were evaluated from the obtained spectra.   

Dynamics of supercoiled knotted DNA: largescale rearrangements and persistent multistrand interlocking

Catalytic processes in bacterial plasmid introduce knots and supercoiling. The effect of the latter has been separately and extensively studied, however much less is known about their concurrent action. Thus, to study the kinetic and metric changes introduced by complex knots and supercoiling in 2kbp-long DNA rings, we use molecular dynamics simulation and oxDNA, a mesoscopic DNA model, finding several unexpected results. First, two distinct states dominate the conformational ensemble, they differ in branchedness and knot size; secondly, fluctuations between these states are as fast as the metric relaxation of unknotted rings. Nevertheless, certain boundaries of knotted and plectonemically wound regions can persist over much longer timescales. These regions involve multiple strands that are interlocked by the cooperative action of topological and supercoiling constraints. Their long liver character may be relevant for the simplifying action of topoisomerases.   

Fast combinatorial nanofluidic device for the dynamic study of biomolecular interactions

We present a nanofluidic device for fast combinatorial exposure of stretched and localized DNA molecules to reagents, such as DNA biding proteins, salts, and restriction enzymes. Nanofluidic devices with channel sizes close to the persistence length of DNA have long been used to stretch, visualize, and study biomolecular interactions that modify the configuration of DNA. However, there is a temporal limitation on how long you can study the interaction dynamics before the probing light photocleaves DNA and fluorescence dyes. In addition, study of the interactions among DNA and multiple reagents in different sequences needs the rapid exchange of buffers. Such studies in the nanochannels with traditional passive exchange of buffers via diffusion process is not feasible. Our device allows the simultaneous or the sequential exposure of multiple reagents with active flow of buffers. The buffers could be exchanged under 20 sec while DNA molecule still confined in the field of view. To illustrate the concept of the device, we will show the conformational change of DNA in the varying ionic concentration, restriction enzymes and DNA binding proteins.   

Multiscale Modeling of DNA Translocation through Multiple Nanopores

We report preliminary results for a multiscale modeling scheme to decipher signals recorded for a translocating DNA through a series of nanopores. We propose to relate the time series of the cross-correlation current from different pores calculated from ab initio studies [1] to the velocity correlation obtained by carrying out Brownian dynamics simulation on a coarse-grained (CG) model of a DNA as well as the nano-pores [2]. By suitable choices of different pore-poymer interactions we propose to relate the results obtained from a simple CG model to more refined but computationaly expensive models.


[1] Towfiq Ahmed, Jason T Haraldsen, John J Rehr, Massimiliano Di Ventra, Ivan Schuller and Alexander V Balatsky, Nanotechnology 25, 125705 (2014).
[2] Kaifu Luo, Tapio Ala-Nissila, See-Chen Ying, and Aniket Bhattacharya, Phys. Rev Lett. 100, 058101 (2008).
  

DNA-based molecular force sensors in reconstituted actin networks

Actin, a major cytoskeletal biopolymer of eukaryotic cells self-assembles into networks of crosslinked filaments and bundles and is largely responsible for cellular shape and mechanical stability. Actin assemblies are also responsible for active cellular processes ranging from migration, division and intracellular transport to morphogenesis. Crucial for such processes is the spatial and temporal regulation of the structure and dynamics of the networks and the generation of force, mostly by myosin motors. To measure forces in cytoskeletal networks, we have developed a FRET based molecular force sensor consisting of a DNA hairpin loop, which can be cross-linked into actin networks. We characterized the force sensor via fluorescence lifetime imaging.