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A feasibility interlaboratory comparison (ILC) was organised by the European Commission’s (EC) Joint Research Centre (JRC) on the determination of the mass fraction of total cow’s milk protein in baked cookies. The ILC was organised to support Commission Regulation (EU) 1169/2011 on the provision of food information to consumers, including the mandatory allergen food labelling and the harmonisation of measurement procedures used for food allergen analysis. An incurred baked cookie was prepared at the JRC and samples were sent to participants for analysis. The laboratories were asked to report their results as “mass fraction of total cow’s milk protein in baked cookies”. This common well-defined measurand was used for the first time in such an ILC to assess the equivalence of measurement results. Homogeneity and stability of the test material were demonstrated to be adequate. An indicative assigned value was established using the JRC single-laboratory validated method based on liquid chromatography coupled to mass spectrometry (LC–MS), independently from the results reported by the participants. Twenty-three laboratories from the European Network of Food Allergen Detection Laboratories (ENFADL), representing 20 European Union (EU) Member States, reported results. Their laboratory performance was assessed using the percent difference Di score. The participating laboratories applied either enzyme-linked immunosorbent assays (ELISA) or LC–MS. Even though the majority of the laboratories used commercially available ELISA test kits, a significant scatter of all the reported results was observed. This indicates that further harmonisation is required for measurement procedures aiming to determine potentially allergenic constituents in food.

Background: Mixed solvents MD (MDmix) simulations have proved to be a useful and increasingly accepted technique with several applications in structure-based drug discovery. One of the assumptions behind the methodology is the transferability of free energy values from the simulated cosolvent molecules to larger drug-like molecules. However, the binding free energy maps (ΔGbind) calculated for the different moieties of the cosolvent molecules (e.g. a hydroxyl map for the ethanol) are largely influenced by the rest of the solvent molecule and do not reflect the intrinsic affinity of the moiety in question. As such, they are hardly transferable to different molecules. Method: To achieve transferable energies, we present here a method for decomposing the molecular binding free energy into accurate atomic contributions. Result: We demonstrate with two qualitative visual examples how the corrected energy maps better match known binding hotspots and how they can reveal hidden hotspots with actual drug design potential. Conclusion: Atomic decomposition of binding free energies derived from MDmix simulations provides transferable and quantitative binding free energy maps.

Highlights • The agricultural origin of carrots could be predicted. • Using yearly harvested samples allowed 100% correct classification of unknowns. • Metabolomic fingerprinting showed potential for organic food authentication., Increasing demand for organic products and their premium prices make them an attractive target for fraudulent malpractices. In this study, a large-scale comparative metabolomics approach was applied to investigate the effect of the agronomic production system on the metabolite composition of carrots and to build statistical models for prediction purposes. Orthogonal projections to latent structures-discriminant analysis (OPLS-DA) was applied successfully to predict the origin of the agricultural system of the harvested carrots on the basis of features determined by liquid chromatography–mass spectrometry. When the training set used to build the OPLS-DA models contained samples representative of each harvest year, the models were able to classify unknown samples correctly (100% correct classification). If a harvest year was left out of the training sets and used for predictions, the correct classification rates achieved ranged from 76% to 100%. The results therefore highlight the potential of metabolomic fingerprinting for organic food authentication purposes.

A method validation study for the determination of ochratoxin A in black and white pepper (Piper spp.), nutmeg (Myristica fragrans), spice mix (blend of ginger, turmeric, pepper, nutmeg, and chili), cocoa powder, and drinking chocolate was conducted according to the International Harmonized Protocol of the International Unionof Pure and Applied Chemistry. The method is based on the extraction of samples with aqueous methanol, followed by a cleanup of the extract with an immunoaffinity column. The determination is carried out by reversed-phase LC coupled with a fluorescence detector. The study involved 25 participants representing across-section of research, private, and official control laboratories from 12 European Union (EU) MemberStates, together with Turkey and Macedonia. Mean recoveries ranged from 71 to 85% for spices and from 85to 88% for cocoa and drinking chocolate. The RSDr values ranged from 5.6 to 16.7% for spices and from 4.5 to 18.7% for cocoa and drinking chocolate. The RSDR values ranged from 9.5 to 22.6% for spices and from 13.7 to 30.7% for cocoa and drinking chocolate. The resulting Horwitz ratios ranged from 0.4 to 1 for spices and from 0.6 to 1.4 forcocoa and drinking chocolate according to the Horwitz function modified by Thompson. The method showed acceptable within-laboratory and between-laboratory precision for each matrix, and it conforms to requirements set by current EU legislation.

The establishment of a reference method for the determination of the allergen protein content in a processed food material has been explored. An analytical approach was developed to enable the comparability of food allergen measurement results expressed in a decision-relevant manner. A proof of concept is here presented, resulting in quantity values for the common measurand, namely ‘mass of total allergen protein per mass of food’. The quantities are determined with SI traceability to enable the comparability of reported results. A method for the quantification of total milk protein content in an incurred baked food at a concentration level clinically relevant is presented. The strategy on how to obtain the final analytical result is outlined. Challenges associated with this method are discussed, in particular the optimal extraction of the marker proteins, the complete digestion and release of the peptides in an equimolar fashion, the use of conversion factors to translate the amount of measured proteins into total milk protein and the estimation of the uncertainty contributions as well as of the combined uncertainty of the final result. The implementation of such a reference method for the determination of the total allergen content in a processed food is an important step, which will provide comparable measurement data of relevance to risk assessors. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02959-0) contains supplementary material, which is available to authorized users.

Monitoring of tropane alkaloids is regulated in the European Union in cereal-based foods for infants and young children, tea and herbal infusions. The European Commission’s Joint Research Centre (JRC) developed an improved LC-MS/MS analytical method using a pentafluorophenyl column, validated it and conducted two proficiency tests targeting these food categories. A subset of the data gathered from laboratories that used the JRC method was additionally exploited to derive interlaboratory performance characteristics. The method showed fit-for-purpose figures of merit. The LOQs for atropine and scopolamine were around 0.4 and 1.2 µg/kg in cereal products, and in tea and herbal infusions, respectively. Uncertainties varied from 15 to 25%. The reproducibility varied from 11 to 38% for scopolamine and from 17 to 44% for atropine at levels ranging from 0.18 to 18.8 and 1.2–54.0 µg/kg, respectively. Recoveries ranged from 71 to 96%. These performance parameters render the method a good candidate for standardisation.

International audience; Intersex, the appearance of female characteristics in male gonads, has been identified in several aquatic species. It is a widespread phenomenon in populations of the bivalve, Scrobicularia plana, from the southwest coast of the U.K. Genes previously identified as differentially expressed (ferritin, testicular haploid expressed gene, THEG, proliferating cell nuclear antigen, PCNA; receptor activated protein kinase C, RACK; cytochrome B, CYB; and cytochrome c oxidase 1, COX1) in intersex clams relative to normal male clams, were selected for characterisation and an environmental survey of the Channel region. Transcripts were significantly differentially expressed at sites with varying intersex incidence and contaminant burdens. Significant correlations between specific gene expressions, key contaminants and sampling locations have been identified, though no single gene was associated with intersex incidence. The results highlight the difficulty in understanding the intersex phenomenon in molluscs where there is still a lack of knowledge on the control of normal reproduction.

European and global food policies require that food put on the market is authentic, which means that the label declaration matches the composition and provenance of the food item. During the last decades conventional approaches such as the determination of certain major and minor components by wet chemistry of chromatographic methods have been used for food authentication. Although many of the traditional methods are still used because they are part of product standards, new approaches that could complement existing methodologies, such as metabolomics are emerging in food authentication applications. Metabolomics aims to study multiple metabolites in a cell, a tissue or an organism. The main advantage of metabolomic applications in food authentication makes use of its untargeted nature, which can enable the detection of emerging frauds. The purpose of this review is to summarise and describe the recent metabolomic applications in the area of food authentication and to discuss its current limitations and future potential.

Marine mussels have been used as sentinel organisms to monitor exposure to a variety of chemical contaminants, including endocrine disrupting chemicals, in the aquatic environment. Although they are an important species for use in ecotoxicology investigations, information on their reproductive physiology and biochemistry is fragmentary. Mass spectrometry-based profiling techniques are increasingly being used to study how the metabolome of an organism changes as a result of tissue differentiation, disease or in response to environmental stressors. In this study, ultraperformance liquid chromatography-time-of-flight-mass spectrometry (UPLC-TOFMS) was used to investigate sex specific differences in the mussel metabolome in order to further investigate the reproductive physiology of this species. Using this method, a comparison of female and male mantle tissues containing mature gonad, revealed significant differences in glycerophosphatidylcholine (PC) and lysophosphatidylcholine (LPC) metabolites. A number of other unidentified metabolites, including those putatively identified as conjugated sterols, were also differentially expressed between male and female mantle/gonadal tissue.

The aim of this study is to develop a normalization method for real-time PCR data by analyzing the most stably expressed control genes in mussel (Mytilus edulis) reproductive tissue. To facilitate this, six candidate genes, including several commonly used in the literature, were investigated in mussels at different stages of gametogenesis and following experimental exposure to a model estrogen (17b-estradiol). GeNorm and NormFinder softwares were employed to assess the stability of the reference genes. Our results demonstrate that the most stable reference genes are not the same in mussels at different stages of gametogenesis and in experimentally E2-exposed mussels. Interestingly, HEL (helicase) and ACT (actin) mRNA expression levels were most affected by the stage of gametogenesis and yet, in molluscan studies, ACT is possibly the most frequently used reference gene. We demonstrate that the experimental results are highly dependent on the reference gene chosen and that statistically significant contrasting differences between sample groups are present or absent depending on the reference gene employed.

5-HT(2B) receptors have been reported to play an important role at cardiac, intestinal and central levels, as well as in bone marrow formation and growth. In the last decade, 5-HT(2B) receptors have also gained much attention as new targets in therapeutics, but also as off-targets because their activation along with the inhibition of serotonin transporters plays a significant role in the pathogenesis of 5-HT induced valvulopathy. Taking this into account, the present review focuses on the new therapeutic applications of 5-HT(2B) receptor ligands as well as on the potential concerns.

Based on a pharmacophore alignment on a 5-HT6 ligand applying 4SCan® technology, a new lead series was identified and further structurally investigated. Kis down to 8 nM were achieved.

The counterion distribution around a DNA dodecamer (5'-CGCGAATTCGCG-3') is analyzed using both standard and novel techniques based on state of the art molecular dynamics simulations. Specifically, we have explored the population of Na(+) in the minor groove of DNA duplex, and whether or not a string of Na(+) can replace the spine of hydration in the narrow AATT minor groove. The results suggest that the insertion of Na(+) in the minor groove is a very rare event, but that when once the ion finds specific sites deep inside the groove it can reside there for very long periods of time. According to our simulation the presence of Na(+) inside the groove does not have a dramatic influence in the structure or dynamics of the duplex DNA. The ability of current MD simulations to obtain equilibrated pictures of the counterion atmosphere around DNA is critically discussed.

The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCGGTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1 ′(chol)α or C1 ′(chol)β diastereoisomer form has been determined by using NMR and restrained molecular dynamics. This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the α anomer is repaired more efficiently than the β one. The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants. The structures have been refined with molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long range electrostatic interactions. These calculations converge to well defined structures whose conformation is intermediate between the A- and B-DNA families as judged by the root mean square deviation but with sugar puckerings and groove shapes corresponding to a distorted B-conformation. Both duplex adducts exhibit intercalation of the cholesterol group from the major groove of the helix and displacement of the guanine base opposite the modified nucleotide. Based on these structures and molecular dynamics calculations, we propose a tentative model for the recognition of damaged DNA substrates by the UvrB protein.

The structural, dynamical, and recognition properties of antiparallel DNA triplexes formed by the antiparallel d(G#G.C), d(A#A.T), and d(T#A.T) motifs (the pound sign and dot mean reverse-Hoogsteen and Watson-Crick hydrogen bonds, respectively) are studied by means of "state of the art" molecular dynamics simulations. Once the characteristics of the helix are defined, molecular dynamics and thermodynamic integration calculations are used to determine the expected stabilization of the antiparallel triplex caused by the introduction of 8-aminopurines. Finally, oligonucleotides containing 8-aminopurine derivatives are synthesized and tested experimentally using several approaches in a variety of systems. A very large stabilization of the triplex is found experimentally, as predicted by simulations. These results open the possibility for the use of oligonucleotides carrying 8-aminopurines to bind single-stranded nucleic acids by formation of antiparallel triplexes.

A perturbation approach based on the generalized molecular interaction potential with polarization (GMIPp) has been developed for combined quantum mechanical and molecular mechanical (QM/MM) simulations of solutions. A unique feature of the method is to avoid repeated self-consistent field calculations for each new solvent configuration during the fluid simulation. The results show that the GMIPp potential coupled to Monte Carlo simulations yields similar energetic and structural results for a series of 14 organic compounds in aqueous solution in comparison with the results obtained from full ab initio QM/MM simulations at the HF/6-31G(d) level. Importantly, the solute electronic polarization energy is reasonably estimated by the GMIPp method. The implementation of the GMIPp within the Monte Carlo simulation framework appears to be a promising computational strategy for carrying out QM/MM calculations for large molecules, which otherwise would be difficult by using standard QM/MM techniques.

The C–H⋯O interaction in the fluoroform–oxirane complex has recently been shown to exhibit features of anti-hydrogen bonding, which is characterized by a shortening of the C–H bond and a blue-shift of the C–H stretching frequency. We present here the results of a topological analysis of the electron density following the theory of atoms in molecules in order to gain insight into the nature of the anti-hydrogen bond. The study is focused on the two energetically most stable complexes, which correspond to a planar complex with a colinear C–H⋯O bond, and a non-planar structure with a bent C–H⋯O bond. The origin of the anti-H bond is discussed in terms of the electron density changes induced upon complexation.

A topological analysis of the electron density in cation−π complexes is presented. Calculations are performed for complexes involving the interaction of the Na+ ion with a variety of six- and five-membered aromatic rings, as well as for the interaction of benzene with diverse cations and hydrogen-bonded neutral molecules. The results reveal the existence of great differences in the topological features of the electron density, which reflect the profound influence of the substituents attached to the aromatic core or of the presence of heteroatoms and fused rings. Nevertheless, with exception of the complexes involving five-membered rings, there are strong relationships between the charge density at the cage critical points and both the energetic and geometrical parameters of the complexes. These relationships allow us to generalize the concept of bond order−bond length found for other intermolecular interactions.

We report the results of high-level ab initio calculations for the gas-phase interconversion of azido and tetrazole forms of thiazole[3,2-d]tetrazole. This study is supplemented with an analysis of the solvent effect on the isomerism using self-consistent reaction field (SCRF) and Monte Carlo-Free Energy Perturbation (MC-FEP) techniques in three different solvents: carbon tetrachloride, chloroform, and water. Finally, the influence exerted by the introduction of substituents on the relative population of isomers is also examined. The energy difference between azido and tetrazole species is found to be highly sensitive to the level of theory used to describe the gas-phase reaction. It also found that the free energies of solvation of azido and tetrazole species determined from SCRF or MC-FEP calculations allow us to predict the solvent-induced changes on the equilibrium. The results show that the azido form of thiazole[3,2-d]tetrazole is clearly disfavored as the solvent polarity increases. The influence ...

Intersex, the appearance of female characteristics in male gonads, has been identified in a wide range of aquatic species worldwide, yet the underpinning molecular etiology remains uncharacterized. The presence of intersex has been shown to be a widespread phenomenon in bivalve, S. plana, populations from the southwest coast of the U.K., as well as inducible in an experimental exposure regime using endocrine disrupting compounds (EDCs). Herein, we use the suppressive subtractive hybridization approach to isolate differentially expressed transcripts in S. plana males exhibiting intersex. Transcripts involved in cell signaling, cell cycle control, energy production/metabolism, microtubule assembly, and sperm physiology are all highlighted as differentially expressed in intersex male clams. These provide both an insight into the molecular mechanisms of action involved in the development of intersex, as well as facilitating potential molecular-level “early warning” biomarkers of the condition.

Members of the vertebrate CYP3A subfamily are involved in the metabolism of steroids and a wide range of xenobiotics. In this study two CYP3A-like mRNAs have been isolated from the mussel (Mytilus edulis), and their seasonal expression profile and modulation by estrogens examined. Sexual dimorphism of CYP3A-like mRNA expression was not observed in mussel gonads of individuals collected throughout a year. Nevertheless, natural variation in gonadal CYP3A-like mRNA expression was observed, with highest levels of CYP3A isoform1 and lowest levels of CYP3A isoform2 mRNA during the maturation and spawning season. Exposure to a 10% sewage treatment works extract did not result in any significant changes in mRNA expression of CYP3A-like. In contrast, exposure to E2 (200 ng/L) and TBT (100 ng/L) significantly down-regulated the expression of CYP3A-like isoform1 but not CYP3A-like isoform2 suggesting differential regulation.

We describe novel oligomers with a greater propensity to form triplexes than oligomers containing only natural bases. They consist of a polypyrimidine sequence linked head-to-head with a polypurine sequence carrying one or several 8-aminoadenine or 8-aminoguanines. The presence of 8-aminopurines also stabilised the parallel-stranded duplex structure. © 2001 Elsevier Science Ltd., The authors thank the Dirección General de Investigación Cientı́fica y Técnica (projects PB98-1222, PM99-0046 and BQU2000-0649), the Generalitat de Catalunya (project 2000-SGR-0018) and CyGene, Inc. for financial support

Mytilus edulis were exposed to 17beta-estradiol (E2) and the synthetic estrogens ethinyl estradiol (EE2) and estradiol benzoate (EB) for 10 days. Two exposures were performed to determine their effect on vitellogenin (VTG) and estrogen receptor 2 (ER2) mRNA expression at different stages of the reproductive cycle. Significant natural variation was not observed in VTG mRNA expression, though ER2 mRNA expression displayed significantly lower values during January, February and July compared with other times of the year. A significant increase in VTG and ER2 mRNA expression was observed in mussels exposed to estrogens at the early stage of gametogenesis. In contrast, mature mussels displayed no statistically significant change in the VTG or ER2 mRNA expression. The data presented suggests that the reproductive physiology of molluscs, in terms of VTG and ER2 mRNA expression, may be susceptible to damage by environmental estrogens at certain points in their gametogenesis process.

11 pages, 8 figures, 6 tables.-- PMID: 20798879[PubMed].-- PMCID: PMC2925217.-- Supplemental information available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925217/bin/763658.f1.pdf, The structure of G,T-parallel-stranded duplexes of DNA carrying similar amounts of adenine and guanine residues is studied by means of molecular dynamics (MD) simulations and UV- and CD spectroscopies. In addition the impact of the substitution of adenine by 8-aminoadenine and guanine by 8-aminoguanine is analyzed. The presence of 8-aminoadenine and 8-aminoguanine stabilizes the parallel duplex structure. Binding of these oligonucleotides to their target polypyrimidine sequences to form the corresponding G,T-parallel triplex was not observed. Instead, when unmodified parallel-stranded duplexes were mixed with their polypyrimidine target, an interstrand Watson-Crick duplex was formed. As predicted by theoretical calculations parallel-stranded duplexes carrying 8-aminopurines did not bind to their target. The preference for the parallel-duplex over the Watson-Crick antiparallel duplex is attributed to the strong stabilization of the parallel duplex produced by the 8-aminopurines. Theoretical studies show that the isomorphism of the triads is crucial for the stability of the parallel triplex., This study was supported by the Dirección General de Investigación Científica y Técnica (Grants BFU2007-63287, BIO2006-01602, CTQ2007-68014-C02-02, Consolider Escience Project), the Generalitat de Catalunya (2005/SGR/00693), the Fundación Marcelino Botín, and the Fundació LA CAIXA (BM04-52-0).

Estrogenic contaminants in the aquatic environment are associated with feminisation of male fish, however their effects on some invertebrate species, such as bivalve molluscs, have yet to be characterised. Gametogenesis represents a critical step in the reproductive process and is subjected to hormonal control by serotonin (5-HT), prostaglandins (synthesised by cyclooxygenases-COX) and steroids such as 17β-estradiol (E2). Here, we examine the responses of 5-HT receptor and COX mRNA expression in mussels, Mytilus edulis, exposed to estrogenic compounds during different stages of their reproductive cycle. In mature mussels, 5-HT receptor mRNA expression decreased following E2 exposure. The opposite trend was observed in mussels at early gametogenesis stages. COX mRNA expression levels at both stages were generally decreased by E2 exposure. Mussels at early gametogenesis stages were also exposed to ethynylestradiol (EE2) and estradiol benzoate (EB) and a significant increase in 5-HT receptor mRNA expression was observed with both xeno-estrogens. COX expression levels were increased with EB exposure but no significant effects were found with EE2 exposure. These results show that the natural estrogen, E2, as well as the synthetic estrogen, EE2, induce alterations, dependent on reproductive stage, in the mRNA expression levels of 5-HT receptor and/or COX in the marine bivalve M. edulis.

5-HT(2B) receptors have been reported to play an important role at cardiac, intestinal and central levels, as well as in bone marrow formation and growth. In the last decade, 5-HT(2B) receptors have also gained much attention as new targets in therapeutics, but also as off-targets because their activation along with the inhibition of serotonin transporters plays a significant role in the pathogenesis of 5-HT induced valvulopathy. Taking this into account, the present review focuses on the new therapeutic applications of 5-HT(2B) receptor ligands as well as on the potential concerns.

The chemistry of the tetrazole ring is gaining increasing attention due to its importance in a variety of synthetic and industrial processes.1 The balance between the deactivating effect of the three pyridine-type nitrogens and the influence of the single pyrrole-type nitrogen determines the reactivity of this heterocycle. Of particular interest is the azidoazomethine-tetrazole isomerism (Scheme 1), since this process is one of the main synthetic routes of tetrazole derivatives.1 The equilibrium between azido and tetrazole species strongly depends on the nature of atom X, the substituent groups R1 and R2, the temperature, and the solvent.2 We have recently studied by using theoretical methods the sensitivity of the azidoazomethine-tetrazole isomerism of thiazole [3,2-d] tetrazole to both solvent and substituents.3 Results showed the suitability of theoretical methods to predict the influence of these factors on the equilibrium. Here our purpose is to examine the effect of the temperature on the equilibrium by determining the enthalpy and entropy changes in solution in order to complete the thermochemical study of this reaction. Our interest is motivated by different reasons: (i) Even though theoretical results3 indicated that cleavage of the tetrazole ring is exothermic in the gas phase, it is known experimentally that this process is endothermic in condensed phases.4 (ii) Experimental values of the enthalpy (entropy) change for the azidoazomethine-tetrazole isomerism of thiazole [3,2-d] tetrazole are available, which allows a direct comparison.5 This makes possible to test the suitability of computational methods to determine the relevant thermochemical data in solution, beyond the calculation of free energy changes. Methods

Oligonucleotides can interact in a very specific manner with homopurine–homopyrimidine sequences of double-stranded DNA by forming triple helices (triplexes) that can be used to design sequence-specific DNA-binding molecules with diagnostic or therapeutic purposes. 2] Depending on the orientation of the third strand with respect to the central polypurine Watson–Crick strand, the triplexes are classified into two main categories: i) parallel and ii) antiparallel. Parallel triplexes require protonation of N3 of cytosine to form proper Hoogsteen bonding with N7 of guanine; this implies that its formation is strongly pH dependent. On the other hand, antiparallel triplexes require no protonation and exhibit largely pH-independent binding. 8-Amino-2’-deoxyguanosine (8AG) can be found in cellular DNA as a possible intermediate in the mutagenesis of nitroalkanes. Moreover, it has been stated that 8AG stabilizes parallel and antiparallel triplexes as well as parallel duplexes, causing an increase in the free energy of formation of 1– 4 kcalmol 1 per substitution. By using parallel and antiparallel clamps carrying the 8AG-containing strand linked to the Hoogsteen strand, it is possible to obtain DNA probes with very high affinity to single-stranded polypyrimidine targets. 8] Unfortunately, G-rich oligonucleotides easily form undesired intraor intermolecular G-tetrad structures. Thus, the ability of antiparallel clamps to form a triple-helical structure with its polypyrimidine target can be reduced by the formation of Gquartets. This has fueled the design of modified nucleobases that can destabilize the tetraplex; unfortunately, in most cases, these nucleobases also destabilize the triplex. Considering the large triplex-stabilizing effect of 8AG, we decided to test whether G tetrads were affected by the presence of 8AG residues. We selected the 15-base-long thrombin aptamer 5’GGTTGGTGTGGTTGG-3’ as a model for quadruplex-forming oligonucleotides (Scheme 1). It was shown that this oligonucleotide adopts a monomeric chair quadruplex structure in the presence of potassium and is characterized by a denaturation–renaturation profile that is reversible and can be observed by different techniques.

A series of d (AT)n oligonucleotides containing mixtures of normal B-type Watson-Crick and antiparallel Hoogsteen helices have been studied using molecular dynamics simulation techniques to analyze the structural and thermodynamic impact of the junction between Watson-Crick and antiparallel Hoogsteen structures. Analysis of molecular dynamics simulations strongly suggests that for all oligonucleotides studied the antiparallel Hoogsteen appears as a reasonable conformation, only slightly less stable than the canonical B-type Watson-Crick one. The junctions between the Watson-Crick and Hoogsteen structures introduces a priori a sharp discontinuity in the helix, because the properties of each type of conformation are very well preserved in the corresponding fragments. However, and quite counterintuitively, junctions do not largely distort the duplex in structural, dynamics or energetic terms. Our results strongly support the possibility that small fragments of antiparallel Hoogsteen duplex might be embedded into large fragments of B-type Watson-Crick helices, making possible protein-DNA interactions that are specific of the antiparallel Hoogsteen conformation.

Molecular dynamics and thermodynamic integration calculations have been carried out on a set of G-rich single-strand, duplex, triplex, and quadruplex DNAs to study the structural and stability changes connected with the guanine → 6-thioguanine (G → S) mutation. The presence of 6-thioguanine leads to a shift of the geometry from the B/A intermediate to the pure B-form in duplex DNA. The G → S mutation does not largely affect the structure of the antiparallel triplex when it is located at the reverse-Hoogsteen position, but leads to a non-negligible local distortion in the structure when it is located at the Watson−Crick position. The G → S mutation leads to destabilization of all studied structures: the lowest effect has been observed for the G → S mutation in the reverse-Hoogsteen strand of the triplex, a medium effect has been observed in the Watson−Crick strand of the triplex and duplex, and the highest influence of the G → S mutation has been found for the quadruplex structures.

The field of molecular dynamic simulations of nucleic acids is reviewed, with particular emphasis on recent methodological advances that have expanded the range of applicability and increased the accuracy of these techniques. Keywords: nucleic acids; molecular dynamics; molecular modeling; DNA; RNA

The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCGGTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1 '(chol)alpha or C1 '(chol)beta diastereoisomer form has been determined by using NMR and restrained molecular dynamics. This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the alpha anomer is repaired more efficiently than the beta one. The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants. The structures have been refined with molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long range electrostatic interactions. These calculations converge to well defined structures whose conformation is intermediate between the A- and B-DNA families as judged by the root mean square deviation but with sugar puckerings and groove shapes corresponding to a distorted B-conformation. Both duplex adducts exhibit intercalation of the cholesterol group from the major groove of the helix and displacement of the guanine base opposite the modified nucleotide. Based on these structures and molecular dynamics calculations, we propose a tentative model for the recognition of damaged DNA substrates by the UvrB protein.

The structure of a new form of duplex DNA, the antiparallel Hoogsteen duplex, is studied in polyd(AT) sequences by means of state-of-the-art molecular dynamics simulations in aqueous solution. The structure, which was found to be stable in all of the simulations, has many similarities with the standard Watson-Crick duplex in terms of general structure, flexibility, and molecular recognition patterns. Accurate MM-PB/SA (and MM-GB/SA) analysis shows that the new structure has an effective energy similar to that of the B-type duplex, while it is slightly disfavored by intramolecular entropic considerations. Overall, MD simulations strongly suggest that the antiparallel Hoogsteen duplex is an accessible structure for a polyd(AT) sequence, which might compete under proper experimental conditions with normal B-DNA. MD simulations also suggest that chimeras containing Watson-Crick duplex and Hoogsteen antiparallel helices might coexist in a common structure, but with the differential characteristics of both type of structures preserved.

Quantum mechanical and quantum mechanical/molecular mechanical calculations in conjunction with continuum solvation models have been used to analyze CH-pi interactions in model systems of aryl- and alkyl-aromatic interactions, as well as in a model folding system designed to study those interactions. High level calculations reproduced accurately the interaction of CH-pi interactions in both alkyl- and aryl-based model systems. Dispersion effects dominate the interaction, but the electrostatics term is also relevant for aryl CH-pi interactions. Theoretical calculations were also used to examine the influence of CH-pi interactions in determining the conformational flexibility of folding models. Finally, a critical comparison of the results obtained from high level calculations on model systems and the experimental data derived for folding models in apolar solvents was carried out, which allowed us to reconcile the apparent discrepancy found between both data.

The structure of parallel-stranded duplexes of DNA-containing a mixture of guanines (G) and adenines (A) is studied by means of molecular dynamics (MD) simulation, as well as NMR and circular dichroism (CD) spectroscopy. Results demonstrate that the structure is based on the Hoogsteen motif rather than on the reverse Watson-Crick one. Molecular dynamics coupled to thermodynamic integration (MD/TI) calculations and melting experiments allowed us to determine the effect of 8-amino derivatives of A and G and of 8-amino-2'-deoxyinosine on the stability of parallel-stranded duplexes. The large stabilization of the parallel-stranded helix upon 8-amino substitution agrees with a Hoogsteen pairing, confirming MD, NMR, and CD data, and suggests new methods to obtain DNA triplexes for antigene and antisense purposes.

Poly d(A:T) parallel-stranded DNA duplexes based on the Hoogsteen and reverse Watson-Crick hydrogen bond pairing are studied by means of extensive molecular dynamics (MD) simulations and molecular mechanics coupled to Poisson-Boltzmann (MM-PB/SA) calculations. The structural, flexibility, and reactivity characteristics of Hoogsteen and reverse Watson-Crick parallel duplexes are described from the analysis of the trajectories. Theoretical calculations show that the two parallel duplexes are less stable than the antiparallel Watson-Crick duplex. The difference in stability between antiparallel and parallel duplexes increases steadily as the length of the duplex increases. The reverse Watson-Crick arrangement is slightly more stable than the Hoogsteen duplex, the difference being also increased linearly with the length of the duplex. A subtle balance of intramolecular and solvation terms is responsible for the preference of a given helical structure.