Synthesis and properties of isobicyclo-DNA

We present the synthesis of the isobicyclo-DNA building blocks with the nucleobases A, C, G and T, as well as biophysical and biological properties of oligonucleotides derived thereof. The synthesis of the sugar part was achieved in 5 steps starting from a known intermediate of the tricyclo-DNA synthesis. Dodecamers containing single isobicyclo-thymidine incorporations, fully modified A- and T-containing sequences, and fully modified oligonucleotides containing all four bases were synthesized and characterized. Isobicyclo-DNA forms stable duplexes with natural nucleic acids with a pronounced preference for DNA over RNA as complements. The most stable duplexes, however, arise by self-pairing. Isobicyclo-DNA forms preferentially B-DNA-like duplexes with DNA and A-like duplexes with complementary RNA as determined by circular dichroism (CD) spectroscopy. Self-paired duplexes show a yet unknown structure, as judged from CD spectroscopy. Biochemical tests revealed that isobicyclo-DNA is stable in fetal bovine serum and does not elicit RNaseH activity.

Base pairing and miscoding properties of 1,N(6)-ethenoadenine- and 3,N(4)-ethenocytosine-containing RNA oligonucleotides

Two RNA phosphoramidites containing the bases 1,N(6)-ethenoadenine (εA) and 3,N(4)-ethenocytosine (εC) were synthesized. These building blocks were incorporated into two 12-mer oligoribonucleotides for evaluation of the base pairing properties of these base lesions by UV melting curve (Tm) and circular dichroism measurements. The Tm data of the resulting duplexes with the etheno modifications opposing all natural bases showed a substantial destabilization compared to the corresponding natural duplexes, confirming their inability to form base pairs. The coding properties of these lesions were further investigated by introducing them into 31-mer oligonucleotides and assessing their ability to serve as templates in primer extension reactions with HIV, AMV, and MMLV reverse transcriptases (RT). Primer extension reactions showed complete arrest of the incorporation process using MMLV RT and AMV RT, while HIV RT preferentially incorporates dAMP opposite εA and dAMP as well as dTMP opposite εC. The properties of these RNA lesions are discussed in the context of its putative biological role.

EEG microstate duration and syntax in acute, medication-naive, first-episode schizophrenia: a multi-center study

In young, first-episode, productive, medication-naive patients with schizophrenia, EEG microstates (building blocks of mentation) tend to be shortened. Koenig et al. [Koenig, T., Lehmann, D., Merlo, M., Kochi, K., Hell, D., Koukkou, M., 1999. A deviant EEG brain microstate in acute, neuroleptic-naïve schizophrenics at rest. European Archives of Psychiatry and Clinical Neuroscience 249, 205–211] suggested that shortening concerned specific microstate classes. Sequence rules (microstate concatenations, syntax) conceivably might also be affected. In 27 patients of the above type and 27 controls, from three centers, multichannel resting EEG was analyzed into microstates using k-means clustering of momentary potential topographies into four microstate classes (A–D). In patients, microstates were shortened in classes B and D (from 80 to 70 ms and from 94 to 82 ms, respectively), occurred more frequently in classes A and C, and covered more time in A and less in B. Topography differed only in class B where LORETA tomography predominantly showed stronger left and anterior activity in patients. Microstate concatenation (syntax) generally were disturbed in patients; specifically, the class sequence A→C→D→A predominated in controls, but was reversed in patients (A→D→C→A). In schizophrenia, information processing in certain classes of mental operations might deviate because of precocious termination. The intermittent occurrence might account for Bleuler's “double bookkeeping.” The disturbed microstate syntax opens a novel physiological comparison of mental operations between patients and controls.

OMA and OPA—Software-Supported Mass Spectra Analysis of Native and Modified Nucleic Acids

The platform-independent software package consisting of the oligonucleotide mass assembler (OMA) and the oligonucleotide peak analyzer (OPA) was created to support the analysis of oligonucleotide mass spectra. It calculates all theoretically possible fragments of a given input sequence and annotates it to an experimental spectrum, thus, saving a large amount of manual processing time. The software performs analysis of precursor and product ion spectra of oligonucleotides and their analogues comprising user-defined modifications of the backbone, the nucleobases, or the sugar moiety, as well as adducts with metal ions or drugs. The ability to expand the library of building blocks and to implement individual structural variations makes it extremely useful for supporting the analysis of therapeutically active compounds. The functionality of the software tool is demonstrated on the examples of a platinated doublestranded oligonucleotide and a modified RNA sequence. Experiments also reveal the unique dissociation behavior of platinated higher-order DNA structures.

Lipid synthesis in protozoan parasites: a comparison between kinetoplastids and apicomplexans.

Lipid metabolism is of crucial importance for pathogens. Lipids serve as cellular building blocks, signalling molecules, energy stores, posttranslational modifiers, and pathogenesis factors. Parasites rely on a complex system of uptake and synthesis mechanisms to satisfy their lipid needs. The parameters of this system change dramatically as the parasite transits through the various stages of its life cycle. Here we discuss the tremendous recent advances that have been made in the understanding of the synthesis and uptake pathways for fatty acids and phospholipids in apicomplexan and kinetoplastid parasites, including Plasmodium, Toxoplasma, Cryptosporidium, Trypanosoma and Leishmania. Lipid synthesis differs in significant ways between parasites from both phyla and the human host. Parasites have acquired novel pathways through endosymbiosis, as in the case of the apicoplast, have dramatically reshaped substrate and product profiles, and have evolved specialized lipids to interact with or manipulate the host. These differences potentially provide opportunities for drug development. We outline the lipid pathways for key species in detail as they progress through the developmental cycle and highlight those that are of particular importance to the biology of the pathogens and/or are the most promising targets for parasite-specific treatment.

The ins and outs of phosphatidylethanolamine synthesis in Trypanosoma brucei.

Phospholipids are not only major building blocks of biological membranes but fulfill a wide range of critical functions that are often widely unrecognized. In this review, we focus on phosphatidylethanolamine, a major glycerophospholipid class in eukaryotes and bacteria, which is involved in many unexpected biological processes. We describe (i) the ins, i.e. the substrate sources and biochemical reactions involved in phosphatidylethanolamine synthesis, and (ii) the outs, i.e. the different roles of phosphatidylethanolamine and its involvement in various cellular events. We discuss how the protozoan parasite, Trypanosoma brucei, has contributed and may contribute in the future as eukaryotic model organism to our understanding of phosphatidylethanolamine homeostasis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

Observation of the rare chrysene excimer

Formation of the so far elusive chrysene excimer in solution is achieved by using DNA as a supramolecular scaffold. Oligonucleotides possessing one or two chrysene building blocks have been synthesized. Chrysene excimer fluorescence has been unambiguously observed in DNA double strands, as well as in single strands containing two neighbouring chrysenes.

REE and Hf distribution among mineral phases in the CV-CK clan : a way to explain present-day Hf isotopic variations in chondrites

Chondrites are among the most primitive objects in the Solar System and constitute the main building blocks of telluric planets. Among the radiochronometers currently used for dating geological events, Sm–Nd and Lu–Hf are both composed of refractory, lithophile element. They are thought to behave similarly as the parent elements (Sm and Lu) are generally less incompatible than the daughter elements (Nd and Hf) during geological processes. As such, their respective average isotopic compositions for the solar system should be well defined by the average of chondrites, called Chondritic Uniform Reservoir (CHUR). However, while the Sm–Nd isotopic system shows an actual spread of less than 4% in the average chondritic record, the Lu–Hf system shows a larger variation range of 28% [Bouvier A., Vervoort J. D. and Patchett P. J. (2008) The Lu–Hf and Sm–Nd isotopic composition of CHUR: Constraints from unequilibrated chondrites and implications for the bulk composition of terrestrial planets. Earth Planet. Sci. Lett.273, 48–57]. To better understand the contrast between Sm–Nd and Lu–Hf systems, the REE and Hf distribution among mineral phases during metamorphism of Karoonda (CK) and Vigarano-type (CV) carbonaceous chondrites has been examined. Mineral modes were determined from elemental mapping on a set of five CK chondrites (from types 3–6) and one CV3 chondrite. Trace-element patterns are obtained for the first time in all the chondrite-forming minerals of a given class (CK chondrites) as well as one CV3 sample. This study reveals that REE are distributed among both phosphates and silicates. Only 30–50% of Sm and Nd are stored in phosphates (at least in chondrites types 3–5); as such, they are not mobilized during early stages of metamorphism. The remaining fraction of Sm and Nd is distributed among the same mineral phases; these elements are therefore not decoupled during metamorphism. Of the whole-rock total of Lu, the fraction held in phosphate decreases significantly as the degree of metamorphism increases (30% for types 3 and 4, less than 5% in type 6). In contrast to Lu, Hf is mainly hosted by silicates with little contribution from phosphates throughout the CK metamorphic sequence. A significant part of Sm and Nd are stored in phosphates in types 3–5, and these elements behave similarly during CK chondrite metamorphism. That explains the robustness of the Sm/Nd ratios in chondrites through metamorphism, and the slight discrepancies observed in the present-day isotopic Nd values in chondrites. On the contrary, Lu and Hf are borne by several different minerals and consequently they are redistributed during metamorphism–induced recrystallization. The Lu/Hf ratios are therefore significantly disturbed during chondrites metamorphism, leading to the high discrepancies observed in present-day Hf isotopic values in chondrites.

A reverse crystal Engineering approach

The enormous impact of crystal engineering in modern solid state chemistry takes advantage from the connection between a typical basic science field and the word engineering. Regrettably, the engineering aspect of organic or metal organic crystalline materials are limited, so far, to descriptive structural features, sometime entangled with topological aspects, but only rarely with true material design. This should include not only the fabrication and structural description at micro- and nano-scopic level of the solids, but also a proper reverse engineering, a fundamental discipline for engineers. Translated into scientific language, the reverse crystal engineering refers to a dedicated and accurate analysis of how the building blocks contribute to generate a given material property. This would enable a more appropriate design of new crystalline material. We propose here the application of reverse crystal engineering to optical properties of organic and metal organic framework structures, applying the distributed atomic polarizability approach that we have extensively investigated in the past few years[1,2].

From Broadstone to Zackenberg

Ecological networks are typically complex constructions of species and their interactions. During the last decade, the study of networks has moved from static to dynamic analyses, and has attained a deeper insight into their internal structure, heterogeneity, and temporal and spatial resolution. Here, we review, discuss and suggest research lines in the study of the spatio-temporal heterogeneity of networks and their hierarchical nature. We use case study data from two well-characterized model systems (the food web in Broadstone Stream in England and the pollination network at Zackenberg in Greenland), which are complemented with additional information from other studies. We focus upon eight topics: temporal dynamic space-for-time substitutions linkage constraints habitat borders network modularity individual-based networks invasions of networks and super networks that integrate different network types. Few studies have explicitly examined temporal change in networks, and we present examples that span from daily to decadal change: a common pattern that we see is a stable core surrounded by a group of dynamic, peripheral species, which, in pollinator networks enter the web via preferential linkage to the most generalist species. To some extent, temporal and spatial scales are interchangeable (i.e. networks exhibit ‘ergodicity’) and we explore how space-for-time substitutions can be used in the study of networks. Network structure is commonly constrained by phenological uncoupling (a temporal phenomenon), abundance, body size and population structure. Some potential links are never observed, that is they are ‘forbidden’ (fully constrained) or ‘missing’ (a sampling effect), and their absence can be just as ecologically significant as their presence. Spatial habitat borders can add heterogeneity to network structure, but their importance has rarely been studied: we explore how habitat generalization can be related to other resource dimensions. Many networks are hierarchically structured, with modules forming the basic building blocks, which can result in self-similarity. Scaling down from networks of species reveals another, finer-grained level of individual-based organization, the ecological consequences of which have yet to be fully explored. The few studies of individual-based ecological networks that are available suggest the potential for large intraspecific variance and, in the case of food webs, strong size-structuring. However, such data are still scarce and more studies are required to link individual-level and species-level networks. Invasions by alien species can be tracked by following the topological ‘career’ of the invader as it establishes itself within a network, with potentially important implications for conservation biology. Finally, by scaling up to a higher level of organization, it is possible to combine different network types (e.g. food webs and mutualistic networks) to form super networks, and this new approach has yet to be integrated into mainstream ecological research. We conclude by listing a set of research topics that we see as emerging candidates for ecological network studies in the near future.

Molecular beacons with a homo-DNA stem: improving target selectivity

Molecular beacons (MBs) are stem-loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivity

Fluorinated olefinic peptide nucleic acid: synthesis and pairing properties with complementary DNA

The fluorinated olefinic peptide nucleic acid (F-OPA) system was designed as a peptide nucleic acid (PNA) analogue in which the base carrying amide moiety was replaced by an isostructural and isoelectrostatic fluorinated C-C double bond, locking the nucleobases in one of the two possible rotameric forms. By comparison of the base-pairing properties of this analogue with its nonfluorinated analogue OPA and PNA, we aimed at a closer understanding of the role of this amide function in complementary DNA recognition. Here we present the synthesis of the F-OPA monomer building blocks containing the nucleobases A, T, and G according to the MMTr/Acyl protecting group scheme. Key steps are a selective desymmetrization of the double bond in the monomer precursor via lactonization as well as a highly regioselective Mitsunobu reaction for the introduction of the bases. PNA decamers containing single F-OPA mutations and fully modified F-OPA decamers and pentadecamers containing the bases A and T were synthesized by solid-phase peptide chemistry, and their hybridization properties with complementary parallel and antiparallel DNA were assessed by UV melting curves and CD spectroscopic methods. The stability of the duplexes formed by the decamers containing single (Z)-F-OPA modifications with parallel and antiparallel DNA was found to be strongly dependent on their position in the sequence with T(m) values ranging from +2.4 to -8.1 degrees C/modification as compared to PNA. Fully modified F-OPA decamers and pentadecamers were found to form parallel duplexes with complementary DNA with reduced stability compared to PNA or OPA. An asymmetric F-OPA pentadecamer was found to form a stable self-complex (T(m) approximately 65 degrees C) of unknown structure. The generally reduced affinity to DNA may therefore be due to an increased propensity for self-aggregation

Influence of perylenediimide–pyrene supramolecular interactions on the stability of DNA-based hybrids: Importance of electrostatic complementarity

Aromatic pi–pi stacking interactions are ubiquitous in nature, medicinal chemistry and materials sciences. They play a crucial role in the stacking of nucleobases, thus stabilising the DNA double helix. The following paper describes a series of chimeric DNA–polycyclic aromatic hydrocarbon (PAH) hybrids. The PAH building blocks are electron-rich pyrene and electron-poor perylenediimide (PDI), and were incorporated into complementary DNA strands. The hybrids contain different numbers of pyrene–PDI interactions that were found to directly influence duplex stability. As the pyrene–PDI ratio approaches 1:1, the stability of the duplexes increases with an average value of 7.5 °C per pyrene–PDI supramolecular interaction indicating the importance of electrostatic complementarity for aromatic pi–pi stacking interactions.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.[1] It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photolabile building blocks via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photo-labile moieties that show nanomolar binding affinities for the orthosteric binding site. Further on we developed a stable 5-HT3R overexpressing cell line and a purification method to yield the receptor in a high purity. Currently we are investigating crosslinking experiments and subsequent MS – analysis.

Assembling Multiporphyrin Stacks Inside the DNA Double Helix

Double stranded DNA hybrids containing up to four consecutive, face-to-face stacked porphyrins are described. Non-nucleosidic, 5,15-bisphenyl-substituted porphyrin building blocks were incorporated into complementary oligonucleotide strands. Upon hybridization multiple porphyrins are well accommodated inside the DNA scaffold without disturbing the overall B-DNA structure. The formation of double strands containing up to four free base porphyrins is enabled without compromising duplex stability. UV/vis, fluorescence, and CD spectroscopy demonstrate the formation of porphyrins H-aggregates inside the DNA double helix and provide evidence for the existence of strong excitonic coupling between interstrand stacked porphyrins. H-aggregation results in considerable fluorescence quenching. Most intense CD effects are observed in stacks containing four porphyrins. The findings demonstrate the value of DNA for the controlled formation of molecularly defined porphyrin aggregates.