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Several findings propose the altered tau protein network as an important target for Alzheimer's disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3? and tau aggregation process. On the basis of this consideration and on our interest in multitarget paradigms in AD, we report on the discovery of 2,4-thiazolidinedione derivatives endowed with such a profile. 28 and 30 displayed micromolar IC50 values toward GSK-3?, together with the capacity of inhibiting AcPHF6 aggregation of 60% and 80% at 10 ?M, respectively. In addition, they showed PAMPA-BBB permeability, together with a suitable cellular safety profile. 30 also displayed inhibition of both K18 and full-length tau aggregations. Finally, both compounds were able to improve cell viability in an okadaic acid-induced neurodegeneration cell model. To the best of our knowledge, 28 and 30 are the first balanced, nontoxic, dual-acting compounds hitting tau cascade at two different hubs.

Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population. (C) 2005 Wiley-Liss, Inc.

The microbiological safety of drinking-water is a powerful environmental determinant of health. Assurance of drinking-water quality has been a pillar of primary prevention for more than 150 years and continues to be the foundation for the prevention and control of waterborne diseases. Roughly 884 million people — 1 of every 8 in the world — still lack access to safe drinking water, according to the World Health Organization and UNICEF. Meanwhile, water use has increased by more than twice the rate of the world population growth during the past century. The population of the industrialized world trusts the quality of the drinking water that distribution systems provide, but it is now becoming a fact that the microbiological safety or drinking water can no longer be taken for granted. Waterborne diseases are one of the major world-wide threats to public health, despite significant advances in water and wastewater treatment technology (World Health Organization, 2003). Waterborne diseases are estimated to be responsible for 4 % of all deaths and 5.7 % of the total disease burden worldwide. The occurrences of natural waterborne disease outbreaks as result of a failure in the conventional water treatment barriers were documented. Outbreaks have occurred in developed countries such as the United States (Cryptosporidium, Milwaukee, 1993) and Canada (Escherichia coli O157:H7, Walkerton, Ontario, 2000) and also in the United Kingdom and Europe. In Europe, in 2007, 17 waterborne outbreaks were reported by 8 countries (EFSA, 2009), probably under-reporting the true number. These involved 10912 cases, with 232 hospitalisations. The main biological risks involved were Campylobacter, Norovirus, Giardia and Cryptosporidium. Erratic and extreme precipitation events can overwhelm water treatment facilities and lead to Cryptosporidium outbreaks due to oocysts infiltrating drinking-water reservoirs from springs and lakes and persisting in the water distribution system for a long time despite vigorous and repetitive flushing of the system. A study from England and Wales found that 20 % of waterborne outbreaks in the past century were associated with a sustained period of low rainfall, compared with 10 % associated with heavy rainfall. Droughts or extended dry spells can reduce the volume of river flow possibly increasing the concentration of effluent pathogens posing a problem for the clearance capacity of treatment plants. In Europe, flooding has rarely been associated with an increased risk of waterborne disease outbreaks, but a few exceptions exist in the UK, Finland, the Czech Republic, and Sweden. An outbreak of Cryptosporidium hominis in November 2010 in northern Sweden (in Östersund) is held responsible for about 12700 cases: in samples from 174 cases, Cryptosporidium was confirmed; the water supply tested positive for Cryptosporidium, both in raw and potable water, very likely due to sewage water being released to the lake serving as reservoir for drinking water. The recommendation to boil drinking water was lifted only in February 2011 after an upgrade of the water treatment plant (ECDC, 2011). Deliberate sabotage of large municipal water supplies is possible while difficult, especially due to the large amount of needed biological agents. Nevertheless, criminal acts perpetrated on smaller water supplies have been recorded and the complexity of water systems can offer many possible access points for deliberate contamination acts. Breakdown in water supply safety may lead to large scale contamination and potentially to detectable disease outbreaks. Other breakdowns and low-level, potentially repeated contaminations may lead to significant sporadic diseases, but it is unlikely for these to be associated with the drinking water source by public health surveillance. The consequences of the use of water of non-potable quality may be severe on food processing facilities and public health and they will depend on the use of the water and the subsequent processing of potentially contaminated materials. Water of a quality that may be tolerated occasionally in drinking water supply may be unacceptable for some uses in the food industry. Inefficient management of the water quality may result in a significant financial impact on food production, for example, through product recalls. Currently, there is no single method to collect, process, and analyze a water sample for all pathogenic microorganisms of interest. In fact, water is currently monitored through infrequent batch measurements procedures, which unfortunately, can miss transient, but problematic water safety events. Some of the difficulties in developing a universal method include the physical differences between the major pathogen groups (viruses, bacteria, protozoa), efficiently concentrating large volume water samples to detect low target concentrations of certain pathogen groups, removing co-concentrated inhibitors from the sample, and standardizing a culture-independent endpoint detection method. Integrating the disparate technologies into a single, universal, simple method and detection system would represent a significant advance in public health and microbiological water quality analysis. Recent advances in sample collection, on-line sample processing and purification, and DNA microarray technologies may form the basis of a universal method to detect known and emerging waterborne pathogens. Thus, specific detection methods are still required in order to trace the origin of etiological agents, identify lapses in water treatment, and identify new quality control processes and procedures. By the time the results of microbiological assays on the water supply are available, thousands of people may have consumed the water and become sick. As also conveyed by the Technical Task Force of the WHO/UNICEF (Villié-Morgon, France 16–18 November 2010) new tools should be used for rapid assessments. The need of a fast response to some of the threats related to drinking water pushes towards the development and validation of molecular-based and biosensor-based methods which are expected to speed up significantly the analysis with respect to state-of-the-art cell culturing. Still, technological challenges must be overcome. Microtechnology and nanotechnology can come in help: the speed and performance of microelectronics, the versatility of microfluidics, the self-assembly of nanostructures for the making and functioning of biosensing surfaces. In this context, the DINAMICS EU FP6 Collaborative Research Project put together the efforts of university laboratories, research institutes and private companies to deliver technology for point-of-need automated networkable microbiological analysis systems. The end-result was the development of technology for a prototype of a fully automatic system that could collect a large volume of water from the supply (also directly from the tap), concentrate the nano- and micro-particulate comprising the pathogens, lyse the cells, extract the nucleic acids and expose them to the sensing surface of an electronic (multi-pathogen) biosensor. The software managing the entire analytical process could then send a message exploiting the mobile telephone network, in the case of positive detection of pathogens. This book reports on some of the results and technologies of DINAMICS, through a number of chapters written by the project participants. Sabine Müller and Jonathan Loeffler (Steinbeis Europa Zentrum) give an overview of the European regulations for drinking water. Christian Mittermayr (Lambda, GmbH) describes the intricacy of microbiological risk assessment, the mathematical modelling that can turn analytical results in the input for decision making. Miloslava Prokšová and collaborators (at the Slovak Water Research Institute) tell about the sound procedures for water sampling. Christoph Zeis (Provenion Engineering) describes an automatic system for concentrating pathogens out of large volumes of water. Hunor Santha and co-workers (University of Technology and Economy of Budapest) illustrate their cell-lysis device. Theo Veenstra (LioniX, BV) zooms in on microfluidics devices designed to do wonders such as DNA extraction, on-chip PCR, mixers and valves, hybridization chambers with electrodes. Daniele Gazzola and co-workers (University of Bologna) review the characteristics of electrochemical biosensors and report on the type of such biosensor developed within DINAMICS. Alessandra Vinelli and collaborators (University of Bologna) reviews some of the modern nucleic acids technologies that can be used to enhance the signal coming from the recognition of pathogenic nucleic acids. Nicolaus Asproulis … . To make the picture more complete, researchers who did not participate in DINAMICS were invited to contribute their views and their results. Many enriched this book. Sophie Courtois (Suez Environment) describes the process from water concentration to microarrays developed within the HealthyWater EU Project. Joseph Faulkinham (Virginia Polytechnic Institute and State University) tell about quantitating Micobacteria. Vicente Catalan and co-workers (LabAqua) tells about new methods for the detection of Legionella. Johan Nordgren and co-workers (Linköping University) tell about how to detect viruses in water. Richard Christen and co-workers (Université de Nice) tells about how to design PCR primers to detect waterborne bacteria. N. Priezjev … . T. Karakasidis … . Georgios Fagas … . Of course, the technology and knowledge described in this book alone is not enough to revolutionize the microbiological safety testing of drinking water. Still, the process has started and we would not be surprised if micro- and nanotechnology will soon lead the molecular detection of pathogens into a mature technology-driven field. In a similar way as chemical analysis takes full advantage of automation nowadays, our cities and homes might be protected in the future against waterborne infections. We trust we and the authors of this book gave our useful contribution.

Giorgio Vasari, a painter, architect, and art historian during the Italian Renaissance, is credited with coining the expression “andare a bottega,” (“attending the studio”) referring to the internship that the apprentice would complete in the master’s studio in order to learn what could be uniquely transmitted in person and in that particular environment and that could then lead to making a unique artist of the apprentice. Nowadays, this same concept holds true in science, and despite the many opportunities for communication and “virtual presence”, the real physical permanence in a lab is still the best way for a scientist to learn a technique or a protocol, or a way of thinking. A book of protocols, such as this, humbly proposes itself as the second-best option. Not quite the same as being in person in a lab and witnessing the experts’ execution of a protocol, it still holds many more details and hints than the usually brief methods section found in research papers. This book of protocols for DNA nanotechnology was composed with this concept in mind: prolonging the tradition of Methods in Molecular Biology, it tries to simplify researchers’ lives when they are putting in practice protocols whose results they have learnt in scientific journals. DNA is playing a quite important and dual role in nanotechnology. First, its properties can nowadays be studied with unprecedented detail, thanks to the new instrumental nano(bio)technologies and new insight is being gathered on the biological behavior and function of DNA thanks to new instrumentation, smart experimental design, and protocols. Second, the DNA molecule can be decontextualized and “simply” used as a copolymer with designed interaction rules. The Watson–Crick pairing code can be harnessed towards implementing the most complicated and elegant molecular self-assembly reported to date. After Ned Seeman’s contribution, elegantly complicated branched structures can be braided and joined towards building nano-objects of practically any desired form. DNA nanotechnology is somewhat like watching professional tennis players: everything seems so simple, but then you set foot on the court and realize how difficult it is to hit a nice shot. When you see the structural perfection of a self-assembling DNA nanoobject, such as a DNA origami, you marvel at how smart DNA is as a molecule and wonder how many different constructs you could design and realize. Among the others, this book tries to show the procedures to follow in order to repeat some of the methods that lead to such constructs, or to the mastering of the characterization techniques used to study them. Many details and procedures are the fruit of the blending of artistry, science, and patience, which are often unseen in a journal paper, but that could be what makes the difference between a winning shot and hitting the net. Many research groups share their expertise with the readers in this book. For the sake of conciseness, we here mention the group leaders, while it is truly from the daily work of a complete team that the details of a protocol can be worked out. The chapters of this book can be roughly divided into two parts: some deal with the methods of preparing the nanostructures, from the rationale of the operations to the techniques for their handling; some other chapters deal more directly with advanced instrumental techniques that can manipulate and characterize molecules and nanostructures. As part of the first group, Roberto Corradini introduces the reader to the methods and choices for taming helix chirality, Alexander Kotlyar, Wolfgang Fritzsche, Naoki Sugimoto, and James Vesenka share their different methods in growing, characterizing, and modifying nanowires based on G tetraplexes; Hao Yan and Friedrich Simmel teach all the basics for implementing the self-assembly of branched DNA nanostructures, and then characterizing the assembly. Hanadi Sleiman tells about hybrid...

Prion diseases are neurodegenerative disorders characterized by the presence of oligomers and amyloid fibrils. These are the result of protein aggregation processes of the cellular prion protein (PrPC) into amyloidal forms denoted as prions or PrPSc. We employed atomic force microscopy (AFM) for single molecule pulling (single molecule force spectroscopy, SMFS) experiments on the recombinant truncated murine prion protein (PrP) domain to characterize its conformations and potential initial oligomerization processes. Our AFM-SMFS results point to a complex scenario of structural heterogeneity of PrP at the monomeric and dimer level, like other amyloid proteins involved in similar pathologies. By applying this technique, we revealed that the PrP C-terminal domain unfolds in a two-state process. We used two dimeric constructs with different PrP reciprocal orientations: one construct with two sequential PrP in the N- to C-terminal orientation (N-C dimer) and a second one in the C- to C-terminal orientation (C-C dimer). The analysis revealed that the different behavior in terms of unfolding force, whereby the dimer placed C-C dimer unfolds at a higher force compared to the N-C orientation. We propose that the C-C dimer orientation may represent a building block of amyloid fibril formation.

Photodynamic therapy (PDT) represents a promising therapeutic modality for cancer. Here we used an orthogonal nanoarchitectonics approach (genetic/chemical) to engineer M13 bacteriophages as targeted vectors for efficient photodynamic killing of cancer cells. M13 was genetically refactored to display on the phage tip a peptide (SYPIPDT) able to bind the epidermal growth factor receptor (EGFR). The refactored M13(EGFR) phages demonstrated EGFR-targeted tropism and were internalized by A431 cancer cells, that overexpress EGFR. Using an orthogonal approach to the genetic display, M13(EGFR) phages were then chemically modified, conjugating hundreds of Rose Bengal (RB) photosensitizing molecules on the capsid surface, without affecting the selective recognition of the SYPIPDT peptides. Upon internalization, the M13(EGFR)-RB derivatives generated intracellularly reactive oxygen species, activated by an ultralow intensity white light irradiation. The killing activity of cancer cells is observed at picomolar concentrations of the M13(EGFR) phage.

The dysregulation of the concentration of individual circulating microRNAs or small sets of them has been recognized as a marker of disease. For example, an increase of the concentration of circulating miR-17 has been linked to lung cancer and metastatic breast cancer, while its decrease has been found in multiple sclerosis and gastric cancer. Consequently, techniques for the fast, specific and simple quantitation of microRNAs are becoming crucial enablers of early diagnosis and therapeutic follow-up. DNA based biosensors can serve this purpose, overcoming some of the drawbacks of conventional lab-based techniques. Herein, we report a cost-effective, simple and robust biosensor based on localized surface plasmon resonance and hybridization chain reaction. Immobilized gold nanoparticles are used for the detection of miR-17. Specificity of the detection was achieved by the use of hairpin surface-tethered probes and the hybridization chain reaction was used to amplify the detection signal and thus extend the dynamic range of the quantitation. Less than 1 h is needed for the entire procedure that achieved a limit of detection of about 1 pM or 50 amol/measurement, well within the reported useful range for diagnostic applications. We suggest that this technology could be a promising substitute of traditional lab-based techniques for the detection and quantification of miRNAs after these are extracted from diagnostic specimens and their analysis is thus made possible., Highlights • Over- or underexpression of microRNAs can serve as biomarkers for diseases, such as cancer. • A miRNA biosensor based on localized plasmon resonance and enzyme-free amplification is here reported. • The biosensor can quantitate specific microRNA in 1 h and can be multiplexed. • The limit of detection and specificity of the biosensor is within a diagnostically-useful range.

Whey-derived alpha-casozepine bioactive peptide (YLGYLEQLLR) was associated with previously optimized guar-gum film-PLGA nanoparticles, aiming to increase both stability across gastrointestinal tract and permeability across absorptive epithelia. Oral films associated with nanoparticles (FNp) enhance buccal absorption along with protection of carried bioactive molecules that are swallowed, with inherent increase of bioavailability. None of developed formulations induced significant loss of cell viability. Permeability across both buccal and intestinal cell barriers was enhanced when alpha-casozepine was carried by FNp system, when compared with film and nanoparticles alone, in a simulated gastrointestinal tract environment. Moreover, differences in permeability profile across buccal and intestinal epithelia were in accordance with the slower erosion of PLGA nanoparticles in a media of neutral pH, resembling oral cavity conditions, and a faster erosion in acidic conditions, as occurs in stomach, as observed by a continuous analysis of nanoparticle morphology over 980 min by atomic force microscopy. Additionally, apparent permeability of alpha-casozepine across TR146 human buccal carcinoma cells and Caco-2/HT29-MTX co-culture, carried by FNp was indeed superior when compared with peptide loaded in PLGA nanoparticles and in films alone or with free peptide control solution. Both FNp and PLGA nanoparticles alone enhanced the permeability of relaxing peptide compared with guar-gum films alone. An increased tongue adhesion when PLGA nanoparticles were added to the guar-gum films was also observed. Developed formulations improved both buccal an intestinal absorption of carried bioactive molecules without compromising cell viability.

Contributions in the dossier La scortesia linguistica. Teoria, didattica e traduzione bring together different approaches to impoliteness, a pervasive phenomenon in interaction that has been widely investigated in language studies over the last two decades and has also more recently been discussed within applied linguistics. Rosa Pugliese and Greta Zanoni present an online learning unit of the LIRA repository (Lingua e cultura Italiana in Rete per l’Apprendimento). After presenting the teacher training and action-research project “Oggi facciamo pragmatica”, Stefania Ferrari describes a set of activities focusing on linguistic (im)politeness conducted with primary school fourth graders. From a linguistic perspective, Delia Chiaro examines how political discourse has become contaminated with elements of disgust and discusses why this might be the case. Wladyslaw Chlopicki outlines the major conventional expletives (vulgarisms) in contemporary spoken Polish. Geraldine Horan discusses the interlingual and cross-cultural significance of nazi insults in English and German. Adopting a translation perspective, Flavia Cavaliere and Serena Zuccheri focus on swearing in the English and Chinese translated versions of the movie and TV series Gomorra. Finally, Licia Reggiani analyses narratological and translating functions of impoliteness in two French contemporary fictions.

New low molar mass (M-n similar to 20 kg mol(-1)) polystyrene-b-poly(methyl methacrylate-co-perfluorohexylethyl acrylate) (PS-b-P(MMA-co-FA)) block copolymers were synthesized all consisting of similar to 70 mol% PS block and similar to 30 mol % P(MMA-co-FA) block. The amount of FA counits in the latter block was increased (0.5, 1, 3 and 4 mol%) in order to enhance the incompatibility between the two blocks and to form different self-assembled nanostructures. The block copolymers were thermally annealed by Rapid Thermal Processing (RTP) over wide ranges of temperatures and times. Complementary SEM and AFM analyses evidenced that the introduction of FA counits above a critical content (similar to 2 mol%) drove a self-assembly process through mixed morphologies. These were formed at a distance from the polymer-air interface and consisted of perpendicular cylinders of P(MMA-co-FA) in a PS matrix laying at the substrate- polymer interface, and surmounted PS stripes and PS dots. Modification by low amounts of FA produced block copolymer samples that self-assembled in new, mixed nanostructures, even though they possessed much lower molar masses than those of analogous well established PS-b-PMMA block copolymers.

An increasing number of inter- and intramolecular interactions can nowadays be probed using single-molecule manipulation techniques. Protein unfolding and refolding is the most representative--though complex--of these interactions. Herein, we review the main modes of performing a force unfolding experiment: the velocity clamp and the new force clamp mode. We also compare some of the physical aspects behind the two most frequently used single-molecule manipulation instrumentations: optical tweezers and atomic force microscopes.

The aim of this study was to develope hydrophilic nanoparticles able to encapsulate and deliver lipophilic curcumin to intestinal epithelial (I-407) and colorectal cancer (HT-29) cells.

Nanosized PTFE-based core-shell particles can be prepared by emulsifier-free seed emulsion polymerization technique starting from spherical or rod-like PTFE seeds of different size. The shell can be constituted by the relatively high Tg polystyrene and polymethylmethacrylate as well as by low Tg polyacrylic copolymers. Peculiar thermal behavior of the PTFE component is observed due to the high degree of PTFE compartmentalization. A very precise control over the particle size can be exerted by properly adjusting the ratio between the monomers and the PTFE seed. Samples with uniformity ratios suited to build 2D and 3D colloidal crystals are easily prepared. 2D colloidal crystal of spheres leads to very small 2D nanostructuration, useful for the preparation of masks with a combination of nanosphere lithography and reactive ion etching. 3D colloidal crystals were also obtained featuring excellent opal quality.

An Open Source/Real-Time AFM Architecture to Perform Customizable Force Spectroscopy Experiments

Several authors demonstrated that an oligonucleotide based pH-sensitive construct can act as a switch between an open and a closed state by changing the pH. To validate this process, specially designed fluorescence dye-quencher substituted oligonucleotide constructs were developed to probe the switching between these two states. This paper reports on bulk and single molecule fluorescence investigations of a duplex-triplex pH sensitive oligonucleotide switch. On the bulk level, only a partial quenching of the fluorescence is observed, similarly to what is observed for other published switches and is supposed to be due to intermolecular interactions between oligonucleotide strands. On the single molecule level, each DNA-based nanometric construct shows a complete switching. These observations suggest the tendency of the DNA construct to associate at high concentration.

Binary homopolymer blends of two hydroxyl-terminated polystyrene (PS-OH) and polymethylmethacrylate (PMMA-OH) homopolymers (Mn similar to 16000 g mol(-1)) were grafted on SiO2 substrates by high-temperature (T > 150 degrees C), short-time (t < 600 s) thermal treatments. The resulting brush layer was tested to screen preferential interactions of the SiO2 substrate with the different symmetric and asymmetric PS-b-PMMA block copolymers deposited on top of the grafted molecules. By properly adjusting the blend composition and the processing parameters, an efficient surface neutralization path was identified, enabling the formation, in the block copolymer film, of homogeneous textures of lamellae or cylinders perpendicularly oriented with respect to the substrate. A critical interplay between the phase segregation of the homopolymer blends and their grafting process on the SiO2 was observed. In fact, the polar SiO2 is preferential for the PMMA-rich phase that forms a homogeneous layer on the substrate, while the PS-rich phase is located at the polymer-air interface. During the thermal treatment, phase segregation and grafting proceed simultaneously. Complete wetting of the PS rich phase on the PMMA rich phase leads to the formation of a PS/PMMA bilayer. In this case, the progressive diffusion of PS chains toward the polymer-SiO2 interface during the thermal treatment allows tuning of the brush layer composition.

A multitude of DNA-based nanostructures of different size and dimensionality have appeared in the literature in the last few years. The programmed self-assembly of oligodeoxynucleotides can be employed to build DNA nanostructures by design in a very successful approach towards bottom-up nanofabrication. This review paper will focus on the developments in this area of research and on some of the properties of DNA that elicited the birth of this new area of research.

Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population.

Opportunely designed synthetic DNA oligonucleotides can spontaneously self-assemble into nanosized structures, and can then assume different conformations in response to a variety of external stimuli. Since objects that can modify their shape in response to a stimulus are in principle capable of functional utility, it has been proposed that DNA molecular constructs can be employed as devices. We describe the design, synthesis and characterization of a DNA structure2 capable of repeatedly and rapidly varying its conformation due to the pH-dependent formation and breakdown of a CT-motif DNA triple helix.3 The repeated cycling of the pH between opportune values causes the adduct to intermittently assume a folded or open state, as experimentally confirmed by means of UV, CD and fluorescence spectroscopy, as well as an electrophoretic mobility shift assay. By means of silane chemistry, the nanomotor was then covalently anchored on glass surfaces, and included in various polymer matrices, allowing for single-molecule fluorescence characterizations of its static and dynamic behaviour. This also confirmed that the smooth cycling of the structure is possible in conditions that could hamper the performance of other DNA nanomotors, i.e. in sterically hindered contexts such as at surfaces, and suggesting that it might also work in other systems where diffusion is dominant and the mobility of the species involved in the cycling is critical, e.g. in nano-sized pores. We also suggest potential applications of the structure as a nanodevice, and report preliminary experiments regarding their implementation.

Opportunely designed synthetic DNA molecules can spontaneously self-assemble into stable nanosized structures of desired shape, and can then assume a large variety of conformations in response to external stimuli [1]. By controlling both the structure and its conformations, its thus possible to obtain functional dynamic nano-structures capable of performing work in the nanoscale. We describe the computational and experimental characterization of a previously reported [2] nanomotor based on the controlled formation and breakdown of an intramolecular DNA triple-helix, and suggest its potential applications. The DNA molecular motor object of this report is driven by a fast and clean mechanism, based on the pH-dependent formation and breakdown of a CT-motif DNA triplehelix [3]. The repeated cycling of the pH between opportune values causes the adduct to intermittently assume a folded or open state, as experimentally confirmed by means of UV, CD and fluorescence spectroscopy, as well as an electrophoretic mobility shift assay. By means of silane chemistry, the nanomotor was then covalently anchored on glass surfaces, allowing for single-molecule fluorescence characterizations of its static and dynamic behaviour. This also confirmed that the smooth functioning of the nanomotor is possible in conditions that could hamper the performance of other DNA nanomotors, i.e. in sterically hindered contexts such as at surfaces, and suggesting that it might also work in other systems where diffusion is dominant and the mobility of the species involved in the cycling is critical, e.g. in nano-sized pores. A computational model of the nanomotor was created, that permitted the individuation of the intermediates occurring during the motors opening and closing events. [1] a) C. M. Niemeyer, M. Adler, Angew. Chem, Int. Ed. 2002, 41, 3779; b) B. Samorì, G. Zuccheri, Angew. Chem, Int. Ed. 2005, 44, 1166. [2] M. Brucale, G. Zuccheri, B. Samorì, Org. Biomol. Chem. 2005, 3, 575. [3] G. Felsenfeld, D. R. Davies, A. Rich, J. Am. Chem. Soc. 1957, 79, 2023.

Nowadays several methods used for the purification of recombinant proteins are based on the formation of a coordination bond between the nickel(II)-nitrilotriacetate (NTA) group present on a chromatographic matrix and a stretch of six consecutive histidine residues (6XHis-tag) appended to the primary sequence of the protein [1]. Measuring the force anchoring the His-tagged proteins on the Ni2+-NTA functionalized matrices at the single molecule level can provide an insight in the chemical details of the bond, which could then be used to design possible improvement strategies. This approach has been already explored by different groups which measured the force of the Ni2+-NTA-(His)6 bond obtaining highly variable results [2,3,4]. Probably, these differences derived from the different experimental setups used. We faced such a problem by using an internal force gauge constituted by a dsDNA linker tethering the nitrilotriacetate (NTA) group to the surface. The mechanical properties of dsDNA have been in fact thoroughly investigated by different groups at the single molecule level [5,6]. We constructed a DNA molecule presenting a Ni2+-NTA group at one end and a thiol group, for the binding onto a gold surface, at the other end. Force-distance curves between the AFM tip, previously functionalised with a CG6H6 peptide, and the DNA bound to the surface were collected. The formation of the desired coordination bond leads to a force curve with 3 phases: (1) entropic stretching of the DNA linker; (2) overstretching transition of the linker, generating a plateau whose length is equal to 70% of that of the employed DNA; (3) detachment of the probe, which corresponds to the breaking of the coordination bond. Preliminary results show the overstretching plateau at 50 pN, a value close to the earlier reported ones, followed by the two possible rupture force profiles: either a single entropic stretching represented by classical Worm Like Chain (WLC) or a more complex profile which seems to be the combination of two WLC. This means that there are two possible scenarios in our experiments. In the first case, after the overstretch, the DNA linker (in the S-DNA form) is extended till the force reaches the value of the rupture of the Ni2+-NTA-(His)6 bond; in the second case the stretching of the S-DNA is followed by a melting process leading to the partial separation of the DNA strands. A description of the experiments and the values of the forces measured for the Ni2+-NTA-(His)6 bonds, which seems in agreement with the one measured by the Hinterdorfer group [3], will be presented. [1] Hochuli E et al. (1987) J. Chromatogr., 411: 177. [2] Conti M et al. (2000) Angew. Chem. Int. Ed., 39: 215. [3] Kienberger F. et al (2000) Single Mol., 1: 59. [4] Schmitt L. et al. (2000) Biophys J., 78: 3275. [5] Smith, S. B. et al. (1996) Science. 271: 795. [6] Rief, M. et al. (1999) Nature Struct. Biol. 6: 346.

DNA is the molecule that encodes the hereditary information in living organisms. In the last years, the specific recognition abilities and the possibility to encode information that are intrinsic in the DNA molecule have been used to assemble nanoscale structures by design. As suggested by Ned Seeman, the recognized pioneer of this field,[1] the Holliday junction is the fundamental structural element around which a great variety of structures can be designed and implemented: this is a branching point where 4 chains of double-stranded DNA meet. By organizing a number of junctions in a proper fashion, it is possible to create rigid structures that overtake the intrinsic flexibility and stochasticity of polymers to create DNA nanoscale objects with the desired size and shape. Using this type of approach a number of monomeric or polymeric nano-objects can be assembled, with the added possibility of introducing tunable elements, that can change their geometry on an external signal, opening the way towards the construction of nanostructures with controllable dynamics. Using synthetic oligodeoxynucleotides (ODN), in our laboratory we have assembled parallelogram shaped nanostructures made of 4 blocked Holliday junctions that have “sticky ends” on their side. Programmed assembly of these ends brings to the construction of polymers that can be either flexible or rigid (100 nm of persistence length or more). Proper mixing and assembly of different monomer structures can yield different topologies: we can obtain linear, branched or circular nanostructures up to several hundred nanometers in size. The biochemical and structural characterization of these has been performed using gel eletrophoresis and atomic force microscopy. By proper functionalization of the ODNs used for the assembly, it is possible to include non-DNA objects on the structures: this seems a clever strategy to assemble (a desired number of) objects at a controlled distance on a nanostructure, with the possibility of also modulating their dynamics. As an example of this paradigm, we have assembled interacting fluorophores on a rigid parallelogram made of DNA, and we have measured a significant FRET. This does not take place if the fluorophores are, instead, free in solution, or even if they are assembled on incomplete (more flexible) parallelogram structures. Furthermore, by assemblying oligonucleotides in a pH-controlled triple helix, we have recently introduced a novel structural motif to the toolbox for DNA-based molecules constructions.[2] This tool is expected to expand further the possibilities of assemblying and controlling nanostructures made of DNA. [1] N. C. Seeman, Nature 2003, 421, 427. [2] M. Brucale, G. Zuccheri, B. Samorì, Org Biomol Chem 2005, 3, 575.