Construction of T‐Motif‐Based DNA Nanostructures through Enzymatic Reactions

The most common way to fabricate DNA nanostructures is to mix individually synthesized DNA oligomers in one pot. However, if DNA nanostructures could be produced through enzymatic reactions, they could be applied in various environments, including in vivo. Herein, an enzymatic method developed to construct a DNA nanostructure from a simple motif called a T‐motif is reported. A long, repeated structure was replicated from a circular template by rolling circle amplification and then cleaved into T‐motif segments by restriction enzymes. These motifs have been successfully assembled into a ladder‐like nanostructure without purification or controlled annealing. This approach is widely applicable to constructing a variety of DNA nanostructures through enzymatic reactions.     

Base Flip in DNA Studied by Molecular Dynamics Simulations of Differently-Oxidized Forms of Methyl-Cytosine

Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme’s active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition.     

Numerical study on electrophoretical stretching dynamics of DNA in microcontraction

Brownian dynamics simulations are used to characterize the electrophoretical stretching process of long T4 DNA in microchannels. When DNA is forced to move through the microchannels, the pure elongational flow generated by electric field gradients in hyperbolic contraction will unravel the molecules of DNA. The effects of hydrodynamic interactions, the strain rate, the Brownian fluctuation, and the initial states of molecules on the stretching dynamics are analyzed in this paper. The computational results show us the weak dependence of polymer dynamics on hydrodynamic interactions in microcontractions. In the case of low Deborah number, the stretching process of a molecule depends on the Brownian fluctuation. However, in the case of high Deborah number, the individualistic stretching behavior can be traced to variations in the starting conformation.     

Electrophoretical stretching of DNA in a hybrid microchannel

The electrophoretical stretching of DNA in a hybrid microchannel is analyzed in this paper. The channel comprises a large insulating cylinder and a hyperbolic contraction that can cause the DNA deformation. Brownian dynamics simulation is used to characterize the dynamical stretching process of a long T4 DNA in hybrid microchannels. The computational results show us the larger average extension of DNA in cylinder-hybridized microchannels than that in the single microcontractions due to the prestretched effect of cylinder on DNA. Moreover, the location and the radius of the insulating cylinder in hybrid microchannels have great effects on the stretching behavior of DNA.     

Molecular Modeling and Molecular Dynamics Studies of Hydralazine with Human DNA Methyltransferase 1

A series of DNA methyltransferase 1 (DNMT1) inhibitors were modeled by docking and molecular dynamics studies to rationalize their activity. Our findings will be valuable in guiding research efforts toward the rational design and virtual screening of novel DNMT inhibitors.

     

Simulations of scalar dispersion in fluidized solid–liquid suspensions

Direct, particle‐resolved simulations of solid–liquid fluidization with the aim of quantifying dispersion have been performed. In addition to simulating the multiphase flow dynamics (that is dealt with by a lattice‐Boltzmann method coupled to an event‐driven hard‐sphere algorithm), a transport equation of a passive scalar in the liquid phase has been solved by means of a finite‐volume approach. The spreading of the scalar—as a consequence of the motion of the fluidized, monosized spherical particles that agitate the liquid—is quantified through dispersion coefficients. Particle self‐diffusivities have also been determined. Solids volume fractions were in the range 0.2–0.5, whereas single‐sphere settling Reynolds numbers varied between approximately 3 and 20. The dispersion processes are highly anisotropic with lateral spreading much slower (by one order of magnitude) than vertical spreading. Scalar dispersion coefficients are of the same order of magnitude as particle self‐diffusivities. © 2014 American Institute of Chemical Engineers AIChE J, 60: 1880–1890, 2014     

Stereospecific Effects of Oxygen‐to‐Sulfur Substitution in DNA Phosphate on Ion Pair Dynamics and Protein–DNA Affinity

Oxygen‐to‐sulfur substitutions in DNA phosphate often enhance affinity for DNA‐binding proteins. Our previous studies have suggested that this effect of sulfur substitution of both O_P1 and O_P2 atoms is due to an entropic gain associated with enhanced ion pair dynamics. In this work, we studied stereospecific effects of single sulfur substitution of either the O_P1 or O_P2 atom in DNA phosphate at the Lys57 interaction site of the Antennapedia homeodomain–DNA complex. Using crystallography, we obtained structural information on the R_P and S_P diastereomers of the phosphoromonothioate and their interaction with Lys57. Using fluorescence‐based assays, we found significant affinity enhancement upon sulfur substitution of the O_P2 atom. Using NMR spectroscopy, we found significant mobilization of the Lys57 side‐chain NH₃⁺ group upon sulfur substitution of the O_P2 atom. These data provide further mechanistic insights into the affinity enhancement by oxygen‐to‐sulfur substitution in DNA phosphate.     

Single‐Molecule Unzipping Force Analysis of HU–DNA Complexes

The genome of bacteria is organized and compacted by the action of nucleoid‐associated proteins. These proteins are often present in tens of thousands of copies and bind with low specificity along the genome. DNA‐bound proteins thus potentially act as roadblocks to the progression of machinery that moves along the DNA. In this study, we have investigated the effect of histone‐like protein from strain U93 (HU), one of the key proteins involved in shaping the bacterial nucleoid, on DNA helix stability by mechanically unzipping single dsDNA molecules. Our study demonstrates that individually bound HU proteins have no observable effect on DNA helix stability, whereas HU proteins bound side‐by‐side within filaments increase DNA helix stability. As the stabilizing effect is small compared to the power of DNA‐based motor enzymes, our results suggest that HU alone does not provide substantial hindrance to the motor's progression in vivo.     

The role of DNA in PANI–DNA hybrid: Template and dopant

We exposed a novel method by using DNA as the dopant as well as template at the same time to prepare PANI–DNA hybrid micro/nanowires with conductivity as high as 10⁻² S cm⁻¹. The high conductivity is due to the co-doping function of DNA with HCl produced by FeCl₃. It is found that the morphology and conductivity of the PANI–DNA hybrids are affected by the [DNA]/[AN] ratio due to the co-operation and competition of DAN's dopant and template function, and the role of DNA in PANI–DNA hybrid varies with the changing of [DNA/[AN] ratios.     

Simulations of vapor–liquid phase equilibrium and interfacial tension in the CO2–H2O–NaCl system

Direct interfacial molecular dynamics simulations are used to obtain the phase behavior and interfacial tension of CO₂–H₂O–NaCl mixtures over a broad temperature and pressure range (50°C ≤ T ≤ 250°C, 0 ≤ P ≤ 600 bar) and NaCl concentrations (1–4 mol/kg H₂O). The predictive ability of several existing water (SPC and TIP4P2005), carbon dioxide (EPM2 and TraPPE), and sodium chloride (SD and DRVH) models is studied and compared, using conventional Lorentz–Berthelot combining rules for the unlike‐pair parameters. Under conditions of moderate NaCl molality (～1 mol/kg H₂O), the predictions of the CO₂ solubility in the water‐rich and CO₂‐rich phase resemble those in the CO₂–H₂O system [Liu et al., J Phys Chem B. 2011;115:6629–6635]. Consistent with our previous work, the TraPPE/TIP4P2005 model combination gives the best overall performance in predicting coexistence composition and pressure in the water‐rich phase. Critical assessments are also made on the ranges of temperature and pressure where particular model combinations work better. The dependence of the interfacial tension on temperature and pressure is better predicted by the TraPPE/TIP4P2005 and EPM2/SPC models, whereas the EPM2/TIP4P2005 model overestimates this property by 10–20%, possibly due to the inadequacy of the combining rules. It is also found that the interfacial tension increases with salt concentration, consistent with experimental observations. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3514–3522, 2013     

Enhanced Heat Stability of α‐Chymotrypsin through Single‐Enzyme Confinement in Attoliter Liposomes

The entrapment of α‐chymotrypsin (α‐CT) within 70–140 nm liposomes formed from POPC (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) leads to an unexpected and remarkable increase in the thermal stability of the enzyme. This finding is based on the observation that heating aqueous suspensions of α‐CT‐containing POPC liposomes to 80 °C for 30 minutes resulted in partial enzyme inactivation, whereas the same treatment of aqueous solutions of free α‐CT inactivated the enzyme completely. The stabilizing effect of enzyme confinement in the attoliter volumes of the liposomes was found to increase with decreasing numbers of α‐CT molecules per liposome. Single‐enzyme confinement was particularly effective, as intermolecular interactions between heat‐denatured α‐CT molecules (causing irreversible inactivation) are not possible.     

Optimizing the Kinetics and Thermodynamics of DNA i‐Motif Folding

Under slightly acidic conditions, single cytidine‐rich DNA strands can form four‐stranded structures called i‐motifs. The stability of the i‐motif structure is based on the intercalation of hemiprotonated C–C⁺ base pairs. In addition, the stability of these structures is influenced by pH, temperature, salt concentration, number of cytidines per C‐rich stretch, and length of sequence; it also depends on the nucleotides in the connecting loop regions. Here, we investigated the influence of the loop nucleotides on i‐motif stability, structure, and kinetics of folding, in five structures with the same loop‐size but different adenosine and thymidine residues within the loop. The stabilities of the i‐motif structures were determined by CD melting, and structure and kinetics of folding were studied by static and time‐resolved NMR experiments.     

Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics Simulations

Hydration is a major determinant of activity and selectivity of enzymes in organic solvents or in gas phase. The molecular mechanism of the hydration of Candida antarctica lipase B (CALB) and its dependence on the thermodynamic activity of water (a_w) was studied by molecular dynamics simulations and compared to experimentally determined water sorption isotherms. Hydration occurred in two phases. At low water activity, single water molecules bound to specific water binding sites at the protein surface. As the water activity increased, water networks gradually developed. The number of protein‐bound water molecules increased linearly with a_w, until at a_w=0.5 a spanning water network was formed consisting of 311 water molecules, which covered the hydrophilic surface of CALB, with the exception of the hydrophobic substrate‐binding site. At higher water activity, the thickness of the hydration shell increased up to 10 Å close to a_w=1. Above a limit of 1600 protein‐bound water molecules the hydration shell becomes unstable and the formation of pure water droplets occurs in these oversaturated simulation conditions. While the structure and the overall flexibility of CALB was independent of the hydration state, the flexibility of individual loops was sensitive to hydration: some loops, such as those part of the substrate‐binding site, became more flexible, while other parts of the protein became more rigid upon hydration. However, the molecular mechanism of how flexibility is related to activity and selectivity is still elusive.     

Experimental and computational study of the bed dynamics of semi‐cylindrical gas–solid fluidized bed

With computational fluid dynamics (CFD) it is possible to get a detailed view of the flow behaviour of the fluidized beds. A profound and fundamental understanding of bed dynamics such as bed pressure drop, bed expansion ratio, bed fluctuation ratio, and minimum fluidization velocity of homogeneous binary mixtures has been made in a semi‐cylindrical fluidized column for gas–solid systems, resulting in a predictive model for fluidized beds. In the present work attempt has been made to study the effect of different system parameters (viz., size and density of the bed materials and initial static bed height) on the bed dynamics. The correlations for the bed expansion and bed fluctuations have been developed on the basis of dimensional analysis using these system parameters. Computational study has also been carried out using a commercial CFD package Fluent (Fluent, Inc.). A multifluid Eulerian model incorporating the kinetic theory for solid particles was applied in order to simulate the gas–solid flow. CFD simulated bed pressure drop has been compared with the experimental bed pressure drops under different conditions for which the results show good agreements.     

A Versatile Endoribonuclease Mimic Made of DNA: Characteristics and Applications of the 8–17 RNA‐Cleaving DNAzyme

Enzymes play a crucial role in all living organisms by accelerating the rates of a myriad of biochemical reactions that are necessary to sustain life. Although the vast majority of known enzymes are made of protein, in recent years it has become increasingly apparent that other molecular formats, like nucleic acids, can also serve in this capacity. DNAzymes (also known as deoxyribozymes) are synthetic enzymes made of short, single strands of deoxyribonucleic acid. These DNA‐based enzymes offer the prospect of significant commercial utility, because they are exceptionally stable and can be produced very easily and inexpensively. The study of one particular DNAzyme, known as “8–17”, has enhanced our understanding of DNAzyme‐mediated catalysis. Moreover, the function of 8–17 has been regarded with special importance because it can catalyze sequence‐specific cleavage of RNA, a reaction that has broad implications in biotechnology and biomedical fields. In this review, we explore the creation, characterization, and application of the 8–17 RNA‐cleaving DNAzyme.     

Kinetic Analysis of the Interaction of Mos1 Transposase with its Inverted Terminal Repeats Reveals New Insight into the Protein–DNA Complex Assembly

Transposases are specific DNA‐binding proteins that promote the mobility of discrete DNA segments. We used a combination of physicochemical approaches to describe the association of MOS1 (an eukaryotic transposase) with its specific target DNA, an event corresponding to the first steps of the transposition cycle. Because the kinetic constants of the reaction are still unknown, we aimed to determine them by using quartz crystal microbalance on two sources of recombinant MOS1: one produced in insect cells and the other produced in bacteria. The prokaryotic‐expressed MOS1 showed no cooperativity and displayed a K_d of about 300 nM . In contrast, the eukaryotic‐expressed MOS1 generated a cooperative system, with a lower K_d (～2 nm) . The origins of these differences were investigated by IR spectroscopy and AFM imaging. Both support the conclusion that prokaryotic‐ and eukaryotic‐expressed MOS1 are not similarly folded, thereby resulting in differences in the early steps of transposition.     

Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot

Enantioselective bond making and breaking is a hallmark of enzyme action, yet switching the enantioselectivity of the reaction is a difficult undertaking, and typically requires extensive screening of mutant libraries and multiple mutations. Here, we demonstrate that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase gives access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in the case of acetoin even starting from the unselective wild‐type protein. Protein crystallography was used to rationalize these findings and to propose a mechanistic model of how enantioselectivity is controlled. In a broader context, our studies highlight the efficiency of mechanism‐inspired and structure‐guided rational protein design for enhancing and switching enantioselectivity of enzymatic reactions, by systematically exploring the biocatalytic potential of a single hot spot.     

Progress and Prospects of Pyrrole‐Imidazole Polyamide–Fluorophore Conjugates as Sequence‐Selective DNA Probes

Recently, the versatility of N‐methylpyrrole (Py)‐N‐methylimidazole (Im) polyamide conjugates, which have been developed from the DNA‐binding antibiotics distamycin A and netropsin, has been shown. These synthetic small molecules can permeate cells to bind with duplex DNA in a sequence‐specific manner, and hence can influence gene expression in vivo. Accordingly, several reports demonstrating the sequence specificity and biological activity of Py‐Im polyamides have accumulated. However, the benefits of Py‐Im polyamides, in particular those conjugated with fluorophores, has been overlooked. Moreover, clear directions for the employment of these attractive artificial small molecules have not yet been shown. Here, we present a detailed overview of the current and prospective applications of Py‐Im polyamide–fluorophore conjugates, including sequence‐specific recognition with fluorescence emission properties, and their potential roles in biological imaging.