Deposition of Supercoiled DNA on Mica for Scanning Force Microscopy Imaging

The deposition of DNA molecules on mica is driven and controlled by the ionic densities around DNA and close to the surface of the substrate. Dramatic improvements in the efficiency and reproducibility of DNA depositions were due to the introduction of divalent cations in the deposition solutions. The ionic distributions on DNA and on mica determine the mobility of adsorbed DNA molecules, thus letting them assume thermodynamically equilibrated conformations, or alternatively trapping them in non-equilibrated conformations upon adsorption. With these prerequisites, mica does not seem like an inert substrate for DNA deposition for microscopy, and its properties greatly affect the efficiency of DNA deposition and the appearance of the molecules on the substrate. In our laboratory, we have some preliminary evidence that mica could also participate in DNA damage, most likely through its heavy metal impurities

Automated DNA Fragments Recognition and Sizing through AFM Image Processing

This paper presents an automated algorithm to determine DNA fragment size from atomic force microscope images and to extract the molecular profiles. The sizing of DNA fragments is a widely used procedure for investigating the physical properties of individual or protein-bound DNA molecules. Several atomic force microscope (AFM) real and computer-generated images were tested for different pixel and fragment sizes and for different background noises. The automated approach minimizes processing time with respect to manual and semi-automated DNA sizing. Moreover, the DNA molecule profile recognition can be used to perform further structural analysis. For computer-generated images, the root mean square error incurred by the automated algorithm in the length estimation is 0.6% for a 7.8 nm image pixel size and 0.34% for a 3.9 nm image pixel size. For AFM real images we obtain a distribution of lengths with a standard deviation of 2.3% of mean and a measured average length very close to the real one, with an error around 0.33%

Evidence of Orientation-Dependent Early States of Prion Protein Misfolded Structures from Single Molecule Force Spectroscopy

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

Preparation and Properties of PTFE-PMMA Core-Shell Nanoparticles and Nanocomposites

he preparation of polytetrafluoroethylene-poly(methyl methacrylate) (PTFE-PMMA) core-shell particles was described, featuring controlled size and narrow size distribution over a wide compositional range, through a seeded emulsion polymerization starting from a PTFE seed of 26 nanometers. Over the entire MMA/PTFE range, the particle size increases as the MMA/PTFE ratio increases. A very precise control over the particle size can be exerted by properly adjusting the ratio between the monomer and the PTFE seed. Particles in the 80240 nm range can be prepared with uniformity indexes suited to build 2D and 3D colloidal crystals. These core-shell particles were employed to prepare nanocomposites with different compositions, through an annealing procedure at a temperature higher than the glass transition temperature of the shell forming polymer. A perfect dispersion of the PTFE particles within the PMMA matrix was obtained and optically transparent nanocomposites were prepared containing a very high PTFE amount

Microstructured soft devices for the growth and analysis of populations of homogenous multicellular tumor spheroids

: Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments

Statistical Mechanics of Elastica on Plane as a Model of Supercoiled DNA-Origin of the MKdV hierarchy-

In this article, I have investigated statistical mechanics of a non-stretched elastica in two dimensional space using path integral method. In the calculation, the MKdV hierarchy naturally appeared as the equations including the temperature fluctuation.I have classified the moduli of the closed elastica in heat bath and summed the Boltzmann weight with the thermalfluctuation over the moduli. Due to the bilinearity of the energy functional,I have obtained its exact partition function.By investigation of the system,I conjectured that an expectation value at a critical point of this system obeys the Painlev\'e equation of the first kind and its related equations extended by the KdV hierarchy.Furthermore I also commented onthe relation between the MKdV hierarchy and BRS transformationin this system.Comment: AMS-Tex Us

Mechanics of the IL2RA Gene Activation Revealed by Modeling and Atomic Force Microscopy

Transcription implies recruitment of RNA polymerase II and transcription factors (TFs) by DNA melting near transcription start site (TSS). Combining atomic force microscopy and computer modeling, we investigate the structural and dynamical properties of the IL2RA promoter and identify an intrinsically negative supercoil in the PRRII region (containing Elf-1 and HMGA1 binding sites), located upstream of a curved DNA region encompassing TSS. Conformational changes, evidenced by time-lapse studies, result in the progressive positioning of curvature apex towards the TSS, likely facilitating local DNA melting. In vitro assays confirm specific binding of the General Transcription Factors (GTFs) TBP and TFIIB over TATA-TSS position, where an inhibitory nucleosome prevented preinitiation complex (PIC) formation and uncontrolled DNA melting. These findings represent a substantial advance showing, first, that the structural properties of the IL2RA promoter are encoded in the DNA sequence and second, that during the initiation process DNA conformation is dynamic and not static

preface

Giorgio Vasari, a painter, architect, and art historian during the Italian Renaissance, is credited with coining the expression \u201candare a bottega,\u201d (\u201cattending the studio\u201d) referring to the internship that the apprentice would complete in the master\u2019s 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 \u201cvirtual presence\u201d, 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\u2019 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\u2019 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 \u201csimply\u201d used as a copolymer with designed interaction rules. The Watson\u2013Crick pairing code can be harnessed towards implementing the most complicated and elegant molecular self-assembly reported to date. After Ned Seeman\u2019s 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..