DNA Phosphate Research Articles

Internal Motions of Basic Side Chains of the Antennapedia Homeodomain in the Free and DNA-Bound States.

Basic side chains play crucial roles in protein-DNA interactions. In this study, using NMR spectroscopy, we investigated the dynamics of Arg and Lys side chains of the fruit fly Antennapedia homeodomain in the free state and in the complex with target DNA. We measured 15N relaxation for Arg and Lys side chains at two magnetic fields, from which generalized order parameters for the cationic groups were determined. Mobility of the R5 side chain, which makes hydrogen bonds with a thymine base in the DNA minor groove, was greatly dampened. Several Lys and Arg side chains that form intermolecular ion pairs with DNA phosphates were found to retain high mobility with the order parameter being <0.6 in the DNA-bound state. Interestingly, some of the interfacial cationic groups in the complex were more mobile than in the free protein. The retained or enhanced mobility of the Arg and Lys side chains in the complex should mitigate the overall loss of conformational entropy in the protein-DNA association and allow dynamic molecular recognition.

Electrochemical Quantitation of a Repeat Expansion Sequence (GGGGCC/CCCCGG) in Length-Mismatched Duplexes

Repeat expansion disorders are a growing class of neurological diseases that are currently incurable and estimated to be 1 in 2,000 individuals worldwide. c9FTD/ALS is one of such classes, caused by a GGGGCC/CCCCGG hexanucleotide repeat expansion in the first intron of a gene called C9ORF72. Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are thought to be members of c9FTD/ALS [1]. Laboratory testing may take several days for diagnosis, therefore a cost-effective and rapid detection method is urgently needed at early signs of neurological decline. Electrochemical methods have been extensively employed as rapid and sensitive tool to detect DNA hybridization for matched and mismatched sequences. Surface density of DNA on electrode surface plays a critical role in a sensor’s sensitivity, which can be affected by length mismatch between a target and its probe sequence. This situation may occur while detecting repeat expansions in a target RNA/DNA sequence. To best of our knowledge, the sensitivity of solution-based versus surface-bound DNA hybridization between a short target and a longer probe sequences has not been investigated by electrochemical methods before. Here, we report chronocoulometric study to compare solution versus surface hybridization sensitivity on gold electrode surfaces. Specifically, for solution hybridization method, an 18-mer ss-DNA (5′-HS-C6H12 -ATT AGA CCC CGG AGA TTA-3′) was hybridized with a hexanucleotide GC-rich complementary sequence (5′-CCG GGG-3′) in buffer solution followed by surface immobilization of the ds-DNA helix on gold macroelectrodes. For surface hybridization method, gold macroelectrodes were modified with single-stranded probe DNA (ss- DNA) followed by hybridization on surface by incubating the modified electrodes with the complementary sequence. Then, ds-DNA surface densities were measured using chronocoulometry in absence and presence of redox probe, [Ru(NH3)6]3+. Based on previous report [2], the number of phosphate residues and therefore the surface density of the oligonucleotide strands can be determined by electrostatic binding of the redox probe to the anionic DNA phosphate backbone. The DNA surface density was determined to be (3.84 ± 1.1)×1012 molecules/cm2 for surface hybridized ds-DNA versus (2.15 ± 1.1)×1012 molecules/cm2 for solution hybridized ds-DNA. The higher surface density by surface-bound hybridization was also confirmed by cyclic voltammetry that show lower current response for surface-bound hybridization compared to solution-hybridized immobilization. In conclusion, even though solution hybridization has been reported efficient compared to surface-bound hybridization, however, surface-bound hybridization favors higher ds-DNA surface density that may lead to higher sensitivity at very low sample concentrations. 1. Bible, E., Neurodegenerative disease: Researchers identify the protein in c9FTD/ALS inclusions. Nat Rev Neurol, 2013. 9(4): p. 183-183. 2. Steel, A.B., Herne, T.M., and Tarlov, M.J., Electrochemical Quantitation of DNA Immobilized on Gold. Analytical Chemistry, 1998. 70(22): p. 4670-4677.

Copper complexes as artificial DNA metallonucleases: From Sigman’s reagent to next generation anti-cancer agent?

Abstract Platinum drugs continue to dominate medical oncology treatments but they have shortcomings associated with their use including dose-limiting toxic side effects and acquired or intrinsic drug resistance. Copper complexes have been extensively investigated as an alternative class of cancer therapeutic. In contrast to Pt drugs which covalently bind DNA nucleobases, Cu derivatives predominantly form non-covalent interactions with DNA – either via intercalation, electrostatic forces of attraction and/or major or minor groove binding. Sigman et al. pioneered research in the late 1970s into Cu complexes as potential chemical nuclease agents. Copper complexes have since been shown to induce DNA cleavage via nucleobase oxidation, hydrolysis of DNA phosphate esters and/or deoxyribose sugar oxidation. The scope of this review covers the period 2014 – present, with a distinct focus on Cu(II) systems which have been investigated as chemical nuclease agents. Specifically we describe how Cu complexes exert their chemical nuclease activity prior to presenting an overview of Cu complexes incorporating Schiff bases, amino acids, peptides, azoles, terpyridines or polypyridyls as ligands as well as including a section on dinuclear Cu complexes and Cu complexes incorporating natural products or bioactive ligands.

Stability and binding affinity of DNA/chitosan complexes by polyanion competition

The stability of DNA/chitosan complexes upon exposure to hyaluronic acid, chondroitin sulfate, and heparin, was assessed by fluorescence spectroscopy to quantify DNA release. Only the highly charged heparin was found to release DNA from the complexes. Complex stability upon exposure to heparin increased with the degree of deacetylation and molecular weight of chitosan and with the ratio of chitosan amino groups to DNA phosphate groups (N/P ratio) in the complexes. Isothermal titration microcalorimetry revealed that among polyanions tested, only heparin has a binding affinity to chitosan approaching that of DNA and can therefore release DNA from the complexes. These results also indicate that anionic components with sufficiently high charge density can induce extracellular or intracellular release of DNA, the former negatively affecting delivery efficiency while the latter is required for gene transfer to occur. Our findings also suggest that increased N/P ratio of the complexes can play an important role in preventing premature dissociation of DNA/polycation complexes upon interaction with anionic components in extracellular milieu.

Aluminum Nanocrystals: A Sustainable Substrate for Quantitative SERS-Based DNA Detection.

Since its discovery in the 1970s, surface-enhanced Raman scattering (SERS) has been primarily associated with substrates composed of nanostructured noble metals. Here we investigate chemically synthesized nanocrystal aggregates of aluminum, an inexpensive, highly abundant, and sustainable metal, as SERS substrates. Al nanocrystal aggregates are capable of substantial near-infrared SERS enhancements, similar to Au nanoparticles. The intrinsic nanoscale surface oxide of Al nanocrystals supports molecule-substrate interactions that differ dramatically from noble metal substrates. The preferential affinity of the single-stranded DNA (ssDNA) phosphate backbone for the Al oxide surface preserves both the spectral features and nucleic acid cross sections relative to conventional Raman spectroscopy, enabling quantitative ssDNA detection and analysis.

DNA conformational transitions inferred from re-evaluation of m|Fo| - D|Fc| electron-density maps.

Conformational flexibility of DNA plays important roles in biological processes such as transcriptional regulation and DNA packaging etc. To understand the mechanisms of these processes, it is important to analyse when, where and how DNA shows conformational variations. Recent analyses have indicated that conventional refinement methods do not always provide accurate models of crystallographic heterogeneities and that some information on polymorphism has been overlooked in previous crystallographic studies. In the present study, the m|Fo| - D|Fc| electron-density maps of double-helical DNA crystal structures were calculated at a resolution equal to or better than 1.5 Å and potential conformational transitions were found in 27% of DNA phosphates. Detailed analyses of the m|Fo| - D|Fc| peaks indicated that some of these unassigned densities correspond to ZI ↔ ZII or A/B → BI conformational transitions. A relationship was also found between ZI/ZII transitions and metal coordination in Z-DNA from the detected peaks. The present study highlights that frequent transitions of phosphate backbones occur even in crystals and that some of these transitions are affected by the local molecular environment.

Selective detection of Mg2+ ions via enhanced fluorescence emission using Au-DNA nanocomposites.

The biophysical properties of DNA-modified Au nanoparticles (AuNPs) have attracted a great deal of research interest for various applications in biosensing. AuNPs have strong binding capability to the phosphate and sugar groups in DNA, rendering unique physicochemical properties for detection of metal ions. The formation of Au–DNA nanocomposites is evident from the observed changes in the optical absorption, plasmon band, zeta potential, DLS particle size distribution, as well as TEM and AFM surface morphology analysis. Circular dichroism studies also revealed that DNA-functionalized AuNP binding caused a conformational change in the DNA structure. Due to the size and shape dependent plasmonic interactions of AuNPs (33–78 nm) with DNA, the resultant Au–DNA nanocomposites (NCs) exhibit superior fluorescence emission due to chemical binding with Ca2+, Fe2+ and Mg2+ ions. A significant increase in fluorescence emission (λex = 260 nm) of Au–DNA NCs was observed after selectively binding with Mg2+ ions (20–800 ppm) in an aqueous solution where a minimum of 100 ppm Mg2+ ions was detected based on the linearity of concentration versus fluorescence intensity curve (λem = 400 nm). The effectiveness of Au–DNA nanocomposites was further verified by comparing the known concentration (50–120 ppm) of Mg2+ ions in synthetic tap water and a real life sample of Gelusil (300–360 ppm Mg2+), a widely used antacid medicine. Therefore, this method could be a sensitive tool for the estimation of water hardness after careful preparation of a suitably designed Au–DNA nanostructure.

New Possibilities for Magnetic Control of Chemical and Biochemical Reactions.

Chemistry is controlled by Coulomb energy; magnetic energy is lower by many orders of magnitude and may be confidently ignored in the energy balance of chemical reactions. The situation becomes less clear, however, when reaction rates are considered. In this case, magnetic perturbations of nearly degenerate energy surface crossings may produce observable, and sometimes even dramatic, effects on reactions rates, product yields, and spectroscopic transitions. A case in point that has been studied for nearly five decades is electron spin-selective chemistry via the intermediacy of radical pairs. Magnetic fields, external (permanent or oscillating) and the internal magnetic fields of magnetic nuclei, have been shown to overcome electron spin selection rules for pairs of reactive paramagnetic intermediates, catalyzing or inhibiting chemical reaction pathways. The accelerating effects of magnetic stimulation may therefore be considered to be magnetic catalysis. This type of catalysis is most commonly observed for reactions of a relatively long-lived radical pair containing two weakly interacting electron spins formed by dissociation of molecules or by electron transfer. The pair may exist in singlet (total electron spin is zero) or triplet (total spin is unity) spin states. In virtually all cases, only the singlet state yields stable reaction products. Magnetic interactions with nuclear spins or applied fields may therefore affect the reactivity of radical pairs by changing the angular momentum of the pairs. Magnetic catalysis, first detected via its effect on spin state populations in nuclear and electron spin resonance, has been shown to function in a great variety of well-characterized reactions of organic free radicals. Considerably less well studied are examples suggesting that the basic mechanism may also explain magnetic effects that stimulate ATP synthesis, eliminating ATP deficiency in cardiac diseases, control cell proliferation, killing cancer cells, and control transcranial magnetic stimulation against cognitive deceases. Magnetic control has also been observed for some processes of importance in materials science and earth and environmental science and may play a role in animal navigation. In this Account, the radical pair mechanism is applied as a consistent explanation for several intriguing new magnetic phenomena. Specific examples include acceleration of solid state reactions of silicon by the magnetic isotope 29Si, enrichment of 17O during thermal decomposition of metal carbonates and magnetic effects on crystal plasticity. In each of these cases, the results are consistent with an initial one-electron transfer to generate a radical pair. Similar processes can account for mass-independent fractionation of isotopes of mercury, sulfur, germanium, tin, iron, and uranium in both naturally occurring samples and laboratory experiments. In the area of biochemistry, catalysis by magnetic isotopes has now been reported in several reactions of DNA and high energy phosphate. Possible medical applications of these observations are pointed out.

DNA Deformation-Coupled Recognition of 8-Oxoguanine: Conformational Kinetic Gating in Human DNA Glycosylase.

8-Oxoguanine (8-oxoG), a mutagenic DNA lesion generated under oxidative stress, differs from its precursor guanine by only two substitutions (O8 and H7). Human 8-oxoguanine glycosylase 1 (OGG1) can locate and remove 8-oxoG through extrusion and excision. To date, it remains unclear how OGG1 efficiently distinguishes 8-oxoG from a large excess of undamaged DNA bases. We recently showed that formamidopyrimidine-DNA glycosylase (Fpg), a bacterial functional analog of OGG1, can selectively facilitate eversion of oxoG by stabilizing several intermediate states, and it is intriguing whether OGG1 also employs a similar mechanism in lesion recognition. Here, we use molecular dynamics simulations to explore the mechanism by which OGG1 discriminates between 8-oxoG and guanine along the base-eversion pathway. The MD results suggest an important role for kinking of the DNA by the glycosylase, which positions DNA phosphates in a way that assists lesion recognition during base eversion. The computational predictions were validated through experimental enzyme assays on phosphorothioate substrate analogs. Our simulations suggest that OGG1 distinguishes between 8-oxoG and G using their chemical dissimilarities not only at the active site but also at earlier stages during base eversion, and this mechanism is at least partially conserved in Fpg despite a lack of structural homology. The similarity also suggests that lesion recognition through multiple gating steps may be a common theme in DNA repair. Our results provide new insight into how enzymes can exploit kinetics and DNA conformational changes to probe the chemical modifications present in DNA lesions.

Biophysical characterization of quaternary pyridinium functionalized polynorbornenes for DNA complexation and their cellular interactions.

Cationic polymers with hydrophobic side chains have gained great interest as DNA carriers since they form a compact complex with negatively charged DNA phosphate groups and interact with the cell membrane. Amphiphilic polyoxanorbornenes with different quaternary alkyl pyridinium side chains with ethyl-p(OPy2) and hexyl units-p(OPy6) bearing 10 kDa MWT were synthesized by living Ring-Opening Metathesis Polymerization method. The physicochemical characteristics: critical micellar concentration, size distribution, surface charge, and condensation of polymer/DNA complex were investigated. Morphology of complexes was monitored by Atomic force microscopy. Cytotoxicity and interaction of these complexes with model lipid vesicles mimicking the cell membrane were examined. These polymers were enabled to form small sized complexes of DNA, which interact with model membrane vesicles. It was found that the nature of hydrophobicity of the homopolymers significantly impacts rates of DNA complexation and the surface charge of the resulting complexes. These results highlight the prospect of the further examinations of these polymers as gene carriers.

Precipitation of DNA with Ethanol.

DNA can be precipitated out of solution for the removal of salts and/or for resuspension in an alternative buffer. Either ethanol or isopropanol can be used to achieve this purpose; however, the use of ethanol is generally preferred. Cations, provided as salts, are typically included to neutralize the negative charge of the DNA phosphate backbone. This method describes ethanol precipitation of DNA in microcentrifuge tubes.

Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein-DNA Complex.

Metal ion cofactors can alter the energetics and specificity of sequence specific protein–DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein–DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg2+ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu3+ or two La3+ cations bind, and two new crystal structures confirm that Lu3+ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for 1H–15N amide resonances, for 1H–13C Ile-δ-CH3 resonances, and for stereospecifically assigned Leu-δ-CH3 and Val-γ-CH3 resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex.

NMR Scalar Couplings across Intermolecular Hydrogen Bonds between Zinc-Finger Histidine Side Chains and DNA Phosphate Groups.

NMR scalar couplings across hydrogen bonds represent direct evidence for the partial covalent nature of hydrogen bonds and provide structural and dynamic information on hydrogen bonding. In this article, we report heteronuclear 15N-31P and 1H-31P scalar couplings across the intermolecular hydrogen bonds between protein histidine (His) imidazole and DNA phosphate groups. These hydrogen-bond scalar couplings were observed for the Egr-1 zinc-finger-DNA complex. Although His side-chain NH protons are typically undetectable in heteronuclear 1H-15N correlation spectra due to rapid hydrogen exchange, this complex exhibited two His side-chain NH signals around 1H 14.3 ppm and 15N 178 ppm at 35 °C. Through various heteronuclear multidimensional NMR experiments, these signals were assigned to two zinc-coordinating His side chains in contact with DNA phosphate groups. The data show that the Nδ1 atoms of these His side chains are protonated and exhibit the 1H-15N cross-peaks. Using heteronuclear 1H, 15N, and 31P NMR experiments, we observed the hydrogen-bond scalar couplings between the His 15Nδ1/1Hδ1 and DNA phosphate 31P nuclei. These results demonstrate the direct involvement of the zinc-coordinating His side chains in the recognition of DNA by the Cys2His2-class zinc fingers in solution.

Binding energy and biophysical properties of ionic liquid-DNA complex: Understanding the role of hydrophobic interactions

The properties of DNA in ionic liquids (ILs) have been well studied. However, it is not widely known about the quantitative analysis of ILs such as the binding profile, which could offer a detailed insight of their binding characteristic. In this study, the binding energy of alkylimidazolium-based ILs ([Cnbim]Br where n=2, 4 and 6) to calf thymus DNA was determined using fluorescence spectroscopy. It was found that the binding constant increased while the values of binding Gibbs energy decreased linearly with increase of alkyl chain length of the ILs. Based on these data, the contribution of each methylene group in the tail of ILs in IL-DNA complex was determined. Moreover, low quenching constant was observed from the competitive displacement of intercalation of ethidium bromide to DNA, hence confirming that ILs are not intercalator. The length of alkyl chain contributed to the increase in the thermal stability of DNA as observed from UV–visible spectra while the CD results indicated that DNA retained its B-form without significant change in the conformation of its base pairs. The findings show that although the stability of DNA is mainly due to the electrostatic attraction between cationic head charge group of imidazolium cation and the DNA phosphate groups, the contribution from the length of alkyl chains also plays a significant role in IL-DNA complex.

A new dual-signalling electrochemical aptasensor with the integration of “signal on/off” and “labeling/label-free” strategies

The integration of “signal on/off” and “labeling/label-free” strategies was applied to the fabrication of a sensitive and selective dual-signalling electrochemical aptasensor. Ferrocene (Fc), being labeled on single stranded DNA as one signal indicator, could provide “turn on” signal through the DNA conformational changes resulted from target binding with aptamer. [Ru(NH3)6]3+ (RuHex), as another signal indicator, could provide “turn off” signal through the electrostatic interaction between the anionic DNA phosphate backbones and the cationic RuHex, which is a label-free signal. Cyclic voltammetry (CV) and electrochemical impedance spectrum (EIS) were employed for monitoring the stepwise fabrication process of the biosensor. Under the optimized conditions, the dual signal changes were quantified using square wave voltammetry (SWV). Based on the superposition of the dual signal changes (|DIRuHex|+ΔIFc), the sensitivity of this aptasensor for lysozyme detection was improved compared to individual signal. The results indicated that lysozyme could be detected in a wide linear range (1.0×10−11–1.0×10−7M) with a low detection limit down to 0.8 pM. Moreover, the resulting biosensor exhibited good specificity, stability and reproducibility, indicating that the present strategy was promising for broad potential application in clinic assay.

Cr(3+) Binding to DNA Backbone Phosphate and Bases: Slow Ligand Exchange Rates and Metal Hydrolysis.

The interaction between chromium ions and DNA is of great interest in inorganic chemistry, toxicology, and analytical chemistry. Most previous studies focused on in situ reduction of Cr(VI), producing Cr(3+) for DNA binding. Recently, Cr(3+) was reported to activate the Ce13d DNAzyme for RNA cleavage. Herein, the Ce13d is used to study two types of Cr(3+) and DNA interactions. First, Cr(3+) binds to the DNA phosphate backbone weakly through reversible electrostatic interactions, which is weakened by adding competing inorganic phosphate. However, Cr(3+) coordinates with DNA nucleobases forming stable cross-links that can survive denaturing gel electrophoresis condition. The binding of Cr(3+) to different nucleobases was further studied in terms of binding kinetics and affinity by exploiting carboxyfluorescein-labeled DNA homopolymers. Once binding takes place, the stable Cr(3+)/DNA complex cannot be dissociated by EDTA, attributable to the ultraslow ligand exchange rate of Cr(3+). The binding rate follows the order of G > C > T ≈ A. Finally, Cr(3+) gradually loses its DNA binding ability after being stored at neutral or high pH, attributable to hydrolysis. This hydrolysis can be reversed by lowering the pH. This work provides a deeper insight into the bioinorganic chemistry of Cr(3+) coordination with DNA, clarifies some inconsistency in the previous literature, and offers practically useful information for generating reproducible results.

Science Rewritten to Accommodate its Origin

A ferroelectric phase transition in ice at ~72 K releases latent energy as λ ~ 4 μ infrared laser light, ‘ice light’, energizing selected targets. Polarized by multiple reflection[22], it photophosphorylated deoxynucleotides, turning tropical waters into a noodle soup of chiral DNA. tRNA analogues ‘transport DNAs’, tDNAs embedded in Alexander Oparin’s lightning-charged coacervates[23] formed H-bond lined pores. Absorbed ice light created an electric field, driving a ratchet mechanism which actively imported charged carrier-substrate complexes.&#x0D; All life descends from the first cell to replicate a tDNA and depends on the trace element requirements of 64 tDNA variants. They constitute life’s atomic alphabet, their substrate complexes its molecular vocabulary. The inviolable rules of active transport predate protein synthesis and enzyme catalysis. Medical, veterinary and pharmaceutical data are consistent with nine independent metabolic pathways. My account of water transport contradicts Peter Mitchell’s chemiosmotic hypothesis.[24] Trace element deficiencies disabling active transport cause most mental and physical maladies, supplements could prevent them.&#x0D; ‘Differentiation DNAs’, dDNAs selecting tDNAs control cells’ substrate uptake, c.f. mRNAs encode protein synthesis by selecting tRNAs. Barrels of α-helices[25] supplement tDNAs and tDNA genetics governs familial inheritance of metabolic disorders, a minion-based data base would facilitate their diagnosis. Trace element supplements would prevent heart attacks, strokes, cancers, diabetes, Alzheimer’s dementia and rarer conditions, improve health and reduce NHS expenditure.&#x0D; The 189*18 arrays of proton ordered H-bonds connecting amino acid ω-amines to DNA phosphates on ‘minions’ control metabolism, serve as biological clocks, memory stores and personality biased brain chips and are basis for reinterpreting scientific dogma. Nine parallel metabolic pathways reflecting their 9-fold symmetry control metabolism. Oscillating H-bonds accelerate protons along minion tunnels, driving molecular scale nuclear fusion. Trapping the γ-rays released at source could solve current energy and pollution crises. Computers and data-bases modelled on minions promise human-friendly artificial intelligence satisfying Turing’s criteria.[26]&#x0D; Comparing cybernetics, psychology and traditional wisdom suggested ‘minions’, the name connotes subservience. They comprise 189 anti-parallel β-sheet hairpins[27] with alternate neutral and basic amino acid residues and proline forming an asymmetric U-bend. They hold nine DNA base pairs flat, forming a nine-coil abacus. Evolved to pack DNA on chromosomes for efficient replication, they underpin philosophy and science, account for human intelligence and resolve many questions. My mutually consistent arguments contradict accepted dogma.

Improved model of hydrated calcium ion for molecular dynamics simulations using classical biomolecular force fields.

Calcium ions (Ca(2+) ) play key roles in various fundamental biological processes such as cell signaling and brain function. Molecular dynamics (MD) simulations have been used to study such interactions, however, the accuracy of the Ca(2+) models provided by the standard MD force fields has not been rigorously tested. Here, we assess the performance of the Ca(2+) models from the most popular classical force fields AMBER and CHARMM by computing the osmotic pressure of model compounds and the free energy of DNA-DNA interactions. In the simulations performed using the two standard models, Ca(2+) ions are seen to form artificial clusters with chloride, acetate, and phosphate species; the osmotic pressure of CaAc2 and CaCl2 solutions is a small fraction of the experimental values for both force fields. Using the standard parameterization of Ca(2+) ions in the simulations of Ca(2+) -mediated DNA-DNA interactions leads to qualitatively wrong outcomes: both AMBER and CHARMM simulations suggest strong inter-DNA attraction whereas, in experiment, DNA molecules repel one another. The artificial attraction of Ca(2+) to DNA phosphate is strong enough to affect the direction of the electric field-driven translocation of DNA through a solid-state nanopore. To address these shortcomings of the standard Ca(2+) model, we introduce a custom model of a hydrated Ca(2+) ion and show that using our model brings the results of the above MD simulations in quantitative agreement with experiment. Our improved model of Ca(2+) can be readily applied to MD simulations of various biomolecular systems, including nucleic acids, proteins and lipid bilayer membranes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 752-763, 2016.

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 OP1 and OP2 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 OP1 or OP2 atom in DNA phosphate at the Lys57 interaction site of the Antennapedia homeodomain-DNA complex. Using crystallography, we obtained structural information on the RP and SP diastereomers of the phosphoromonothioate and their interaction with Lys57. Using fluorescence-based assays, we found significant affinity enhancement upon sulfur substitution of the OP2 atom. Using NMR spectroscopy, we found significant mobilization of the Lys57 side-chain NH3 (+) group upon sulfur substitution of the OP2 atom. These data provide further mechanistic insights into the affinity enhancement by oxygen-to-sulfur substitution in DNA phosphate.

Changes in conformational dynamics of basic side chains upon protein-DNA association.

Basic side chains play major roles in recognition of nucleic acids by proteins. However, dynamic properties of these positively charged side chains are not well understood. In this work, we studied changes in conformational dynamics of basic side chains upon protein–DNA association for the zinc-finger protein Egr-1. By nuclear magnetic resonance (NMR) spectroscopy, we characterized the dynamics of all side-chain cationic groups in the free protein and in the complex with target DNA. Our NMR order parameters indicate that the arginine guanidino groups interacting with DNA bases are strongly immobilized, forming rigid interfaces. Despite the strong short-range electrostatic interactions, the majority of the basic side chains interacting with the DNA phosphates exhibited high mobility, forming dynamic interfaces. In particular, the lysine side-chain amino groups exhibited only small changes in the order parameters upon DNA-binding. We found a similar trend in the molecular dynamics (MD) simulations for the free Egr-1 and the Egr-1–DNA complex. Using the MD trajectories, we also analyzed side-chain conformational entropy. The interfacial arginine side chains exhibited substantial entropic loss upon binding to DNA, whereas the interfacial lysine side chains showed relatively small changes in conformational entropy. These data illustrate different dynamic characteristics of the interfacial arginine and lysine side chains.