The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch

The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with Ssubstitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T) as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening

Quadruplex DNA: sequence, topology and structure.

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes

Mechanism of MicroRNA-Target Interaction: Molecular Dynamics Simulations and Thermodynamics Analysis

MicroRNAs (miRNAs) are endogenously produced ∼21-nt riboregulators that associate with Argonaute (Ago) proteins to direct mRNA cleavage or repress the translation of complementary RNAs. Capturing the molecular mechanisms of miRNA interacting with its target will not only reinforce the understanding of underlying RNA interference but also fuel the design of more effective small-interfering RNA strands. To address this, in the present work the RNA-bound (Ago-miRNA, Ago-miRNA-target) and RNA-free Ago forms were analyzed by performing both molecular dynamics simulations and thermodynamic analysis. Based on the principal component analysis results of the simulation trajectories as well as the correlation analysis in fluctuations of residues, we discover that: 1) three important (PAZ, Mid and PIWI) domains exist in Argonaute which define the global dynamics of the protein; 2) the interdomain correlated movements are so crucial for the interaction of Ago-RNAs that they not only facilitate the relaxation of the interactions between residues surrounding the RNA binding channel but also induce certain conformational changes; and 3) it is just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release. In addition, by thermodynamic analysis we also discover that for both the guide RNA 5′-end recognition and the facilitated site-specific cleavage of the target, the presence of two metal ions (of Mg2+) plays a predominant role, and this conclusion is consistent with the observed enzyme catalytic cleavage activity in the ternary complex (Ago-miRNA-mRNA). Our results find that it is the set of arginine amino acids concentrated in the nucleotide-binding channel in Ago, instead of the conventionally-deemed seed base-paring, that makes greater contributions in stabilizing the binding of the nucleic acids to Ago

Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations

Alkali (Li+, Na+, K+, Rb+, and Cs+) and halide (F−, Cl−, Br−, and I−) ions play an important role in many biological phenomena, roles that range from stabilization of biomolecular structure, to influence on biomolecular dynamics, to key physiological influence on homeostasis and signaling. To properly model ionic interaction and stability in atomistic simulations of biomolecular structure, dynamics, folding, catalysis, and function, an accurate model or representation of the monovalent ions is critically necessary. A good model needs to simultaneously reproduce many properties of ions, including their structure, dynamics, solvation, and moreover both the interactions of these ions with each other in the crystal and in solution and the interactions of ions with other molecules. At present, the best force fields for biomolecules employ a simple additive, nonpolarizable, and pairwise potential for atomic interaction. In this work, we describe our efforts to build better models of the monovalent ions within the pairwise Coulombic and 6-12 Lennard-Jones framework, where the models are tuned to balance crystal and solution properties in Ewald simulations with specific choices of well-known water models. Although it has been clearly demonstrated that truly accurate treatments of ions will require inclusion of nonadditivity and polarizability (particularly with the anions) and ultimately even a quantum mechanical treatment, our goal was to simply push the limits of the additive treatments to see if a balanced model could be created. The applied methodology is general and can be extended to other ions and to polarizable force-field models. Our starting point centered on observations from long simulations of biomolecules in salt solution with the AMBER force fields where salt crystals formed well below their solubility limit. The likely cause of the artifact in the AMBER parameters relates to the naive mixing of the Smith and Dang chloride parameters with AMBER-adapted Åqvist cation parameters. To provide a more appropriate balance, we reoptimized the parameters of the Lennard-Jones potential for the ions and specific choices of water models. To validate and optimize the parameters, we calculated hydration free energies of the solvated ions and also lattice energies (LE) and lattice constants (LC) of alkali halide salt crystals. This is the first effort that systematically scans across the Lennard-Jones space (well depth and radius) while balancing ion properties like LE and LC across all pair combinations of the alkali ions and halide ions. The optimization across the entire monovalent series avoids systematic deviations. The ion parameters developed, optimized, and characterized were targeted for use with some of the most commonly used rigid and nonpolarizable water models, specifically TIP3P, TIP4PEW, and SPC/E. In addition to well reproducing the solution and crystal properties, the new ion parameters well reproduce binding energies of the ions to water and the radii of the first hydration shells

NMR cross-correlated relaxation rates reveal ion coordination sites in DNA

In this work, a novel NMR method for the identification of preferential coordination sites between physiologically relevant counterions and nucleic acid bases is demonstrated. In this approach, the NMR cross-correlated relaxation rates between the aromatic carbon chemical shift anisotropy and the proton–carbon dipolar interaction are monitored as a function of increasing Na+, K+, and Mg2+ concentrations. Increasing the counterion concentration modulates the residence times of the counterions at specific sites around the nucleic acid bases. It is demonstrated that the modulation of the counterion concentration leads to sizable variations of the cross-correlated relaxation rates, which can be used to probe the site-specific counterion coordination. In parallel, the very same measurements report on the rotational tumbling of DNA, which, as shown here, depends on the nature of the ion and its concentration. This methodology is highly sensitive and easily implemented. The method can be used to cross-validate and/or complement direct but artifact-prone experimental techniques such as X-ray diffraction, NMR analysis with substitutionary ions, and molecular dynamics simulations. The feasibility of this technique is demonstrated on the extraordinarily stable DNA mini-hairpin d(GCGAAGC)

NMR cross-correlated relaxation rates reveal ion coordination sites in DNA

In this work, a novel NMR method for the identification of preferential coordination sites between physiologically relevant counterions and nucleic acid bases is demonstrated. In this approach, the NMR cross-correlated relaxation rates between the aromatic carbon chemical shift anisotropy and the proton–carbon dipolar interaction are monitored as a function of increasing Na+, K+, and Mg2+ concentrations. Increasing the counterion concentration modulates the residence times of the counterions at specific sites around the nucleic acid bases. It is demonstrated that the modulation of the counterion concentration leads to sizable variations of the cross-correlated relaxation rates, which can be used to probe the site-specific counterion coordination. In parallel, the very same measurements report on the rotational tumbling of DNA, which, as shown here, depends on the nature of the ion and its concentration. This methodology is highly sensitive and easily implemented. The method can be used to cross-validate and/or complement direct but artifact-prone experimental techniques such as X-ray diffraction, NMR analysis with substitutionary ions, and molecular dynamics simulations. The feasibility of this technique is demonstrated on the extraordinarily stable DNA mini-hairpin d(GCGAAGC)

Analyte transport to micro-and nano-plasmonic structures

The study of optical affinity biosensors based on plasmonic nanostructures has received significant attention in recent years. The sensing surfaces of these biosensors have complex architectures, often composed of localized regions of high sensitivity (electromagnetic hot spots) dispersed along a dielectric substrate having little to no sensitivity. Under conditions such that the sensitive regions are selectively functionalized and the remaining regions passivated, the rate of analyte capture (and thus the sensing performance) will have a strong dependence on the nanoplasmonic architecture. Outside of a few recent studies, there has been little discussion on how changes to a nanoplasmonic architecture will affect the rate of analyte transport. We recently proposed an analytical model to predict transport to such complex architectures; however, those results were based on numerical simulation and to date, have only been partially verified. In this study we measure the characteristics of analyte transport across a wide range of plasmonic structures, varying both in the composition of their base plasmonic element (microwires, nanodisks, and nanorods) and the packing density of such elements. We functionalized each structure with nucleic acid-based bioreceptors, where for each structure we used analyte/receptor sequences as to maintain a Damk\uf6hler number close to unity. This method allows to extract both kinetic (in the form of association and dissociation constants) and analyte transport parameters (in the form of a mass transfer coefficient) from sensorgrams taken from each substrate. We show that, despite having large differences in optical characteristics, measured rates of analyte transport for all plasmonic structures match very well to predictions using our previously proposed model. These results highlight that, along with optical characteristics, analyte transport plays a large role in the overall sensing performance of a nanoplasmonic biosensor