1,721,018 research outputs found
Scanning Force Microscopy Studies on the Structure and Dynamics of Single DNA Molecules
No full textIn the last years, the Scanning Force Microscope imposed as a new and useful tool for the
analysis of the structure of nucleic acids. The applications of this class of techniques goes
from high resolution imaging of the double-helix, to the study of DNA-protein complexes, to
the analysis of the dynamics of fully hydrated nucleic acids. In this chapter, we present an
overview of the techniques and protocols available for the study of nucleic acids with the
scanning force microscope. We devote particular attention to the study of the dynamics, the
curvature and the flexibility of double-stranded DNA
DNA self-assembly for signal enhancement in nucleic acids biosensors
No full textSome of the possible perspective advantages of the uptake of nucleic acids biosensor technology are already within reach, still, the sometimes limited sensitivity can seriously inhibit the application of biosensor-based methods when these could be useful towards detection of nucleic acids variants present only at a very low concentration.
We have adapted and succeeded at using the hybridization chain reaction (HCR)[1] towards enhancing the signal due to the specific recognition and binding of soluble nucleic acids to a surface-bound probe. The enhancement strategy consists in a triggered supramolecular polymerization of DNA sequences or nanostructures at the location of specific nucleic acids recognition. We have showed that the method can be used towards the detection of an arbitrary DNA target through proper design of the sequences of the components[2]. Preliminary experimental evidence shows a significant enhancement of the signal, which could prove useful in some applications. We also proved that HCR can have single-nucleotide sensitivity for the detection and signal enhancement.
We have recently worked at the extension of HCR towards the detection of circulating miRNA targets, biomarkers of considerable interest for diagnostics. We have showed that HCR reagents can be designed to distinguish between closely-related miRNA targets, as it would be needed in diagnostics. Modifications of the HCR design can yield hyperbranched or target-recycling assembly and thus significantly increase the detection signal. We have preliminary results on a branched HCR design that could be compatible with surface-bound biosensors implementation (electrochemical, fluorescence, luminescence, SPR).
We showed sensitivity to single-nucleotide mismatch and proved that the sensing strategy could be applicable to the detection of nucleic acids from pathogens or of circulating miRNA
Self-assembled functional DNA Nanostructures as intracellular biosensors in single live human cells
Full text linkIn the last decade, in vivo studies have revealed that even subtle differences in size, concentration of components, or cell-cycle stage, make genetically-identical cells in a population respond differently to the same stimulus. In order to characterize such complexity of behavior and shed more light on the functioning and communication amongst cells, researchers are developing strategies to study single live cells in a population.
Self-assembled DNA nanostructures can be introduced in live cells, even without the aid of transfecting agents or by altering the membrane permeability otherwise. Recently, we have worked on the methods to design and prepare DNA-based fluorescent tetrahedral nanostructures, to deliver them to live cells and characterize such cells with fluorescence microscopy [3]. We have designed two types of sensitive DNA nanostructures (nanobiosensors). These are variation on the DNA tetrahedron motif introduced by the Turberfield group. Sensing of the desired target leads to a significant conformational change in the nanostructure that can be visualized (in solution or in the cells) via the spectroscopic properties of a FRET pair.
For example, by exploiting and tuning the properties of an intramolecular CT-motif triple helix, we designed and tested a nanostructure that can undergo a conformational transition driven by a change in pH, in a physiologically-relevant range. We showed the functioning of such functional nanostructures and their internalization in live cultured cells
Time-lapse imaging of conformational changes in supercoiled DNA by scanning force microscopy
No full textMost of the scanning force microscopy (SFM) images of supercoiled DNA on untreated mica thus far reported have not shown tight plectonemic structure seen by electron microscopy, but instead less coiled molecules and sometimes a partly "condensed" state with intimate chain-chain interactions. By observing time-lapse images of conformational changes of DNA induced by decreasing ionic strength of imaging buffer in solution SFM, we could show that the process of water rinsing, an indispensable step for preparation of dried samples, may be responsible for some of the conformational anomalies in the images previously reported. We have studied several protocols to observe supercoiled DNA molecules by SFM and discuss the merits and the demerits. Images obtained following uranyl acetate treatment may be ideal for the detection of DNA damage, as the supercoiled and nicked forms are easily distinguishable. © 2001 Elsevier Science
Scanning force microscopy study on a single-stranded DNA: The genome of parvovirus B19
No full textThe genome of parvovirus B19 is a 5600-base-long single-stranded DNA molecule with peculiar sequence symmetries. Both complementary forms of this single-stranded DNA are contained in distinct virions and they hybridize intermolecularly to double-stranded DNA if extracted from the capsids with traditional methods, thus losing some of their native structural features. A scanning force microscopy analysis of these double-stranded DNA molecules after thermal denaturation and renaturation gave us the chance to study the possible states that this DNA can assume in both its single-stranded and double-stranded forms. A novel but still poorly reproducible in situ lysis experiment that we have conducted on single virions with the scanning force microscope made it possible to image the totally unpaired state that the single-stranded DNA molecule most likely assumes inside the viral particle. Structural considerations on single molecules offer the opportunity for the formulation of plausible hypotheses on the interaction between the DNA and the viral structural proteins that could prove important for the DNA packaging in the capsid and, possibly, the viral infection mechanisms. © WILEY-VCH-Verlag GmbH, D-69451 Weinheim, 2001
DNA Supercoiling Imaged in Three Dimensions by Scanning Force Microscopy
No full textUntil now only planar projections of the molecular shape of supercoiled circular DNA molecules could be imaged with scanning force microscopy (SFM). In the three‐dimensional image of a supercoiled pBR322 DNA molecule in this report, the chirality and writhing number can be “read” directly. Local changes in the supercoiling in individual DNA molecules can be monitored, which should be important for our understanding of biological processes such as DNA transcription. Copyright © 1993 by VCH Verlagsgesellschaft mbH, German
Cromolyn-crosslinked chitosan nanoparticles for the treatment of allergic rhinitis
No full textThe aim of this work was to prepare new mucoadhesive nasal decongestant nanoparticles obtained by direct crosslinking between the cationic polymer chitosan and the anionic drug cromolyn. Different chitosan/cromolyn molar ratios were used in order to obtain nanoparticles of suitable size, encapsulation efficiency/drug loading and mucoadhesion. Moreover, the ability of the nanoparticles to deliver cromolyn into and through the nasal mucosa was evaluated. The obtained positively charged nanoparticles, sized 180-400 nm, showed interesting properties in terms of yield, mucoadhesion, encapsulation efficiency and drug loading. Release and permeation/penetration data indicated the ability of the nanoparticles to retain a high amount of cromolyn inside the mucosa, which is rich in mast cells. These findings suggest developing decongestant nanoparticles for potential treatment of allergic rhinitis
Polynucleotide: Adenosine glycosidase is the sole activity of ribosome-inactivating proteins on DNA
Full text linkPolynucleotide: adenosine glycosidases (PNAG) are a class of plant and bacterial enzymes commonly known as ribosome-inactivating proteins (RIP). They are presently classified as rRNA N-glycosidases in the enzyme nomenclature [EC 3.2.2.22]. Several activities on nucleic acids, other than depurination, have been attributed to PNAG: in particular modifications induced in circular plasmids, including linearisation and topological changes, and cleavage of guanidinic residues. Here we describe a chromatographic procedure to obtain nuclease-free PNAG by dye-chromatography onto Procion Red derivatized Sepharose®. Highly purified enzymes depurinate extensively pBR322 circular, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycolyase activities, do not cause topological changes, and adenine is the only base released from DNA and rRNA, even at very high enzyme concentrations. A scanning force microscopy (SFM) study of pBR322 treated with saporin-S6 confirmed that (i) this PNAG binds extensively to the plasmid, (ii) the distribution of the bound saporin-S6 molecules along the DNA chain is markedly variable, (iii) plasmids already digested with saporin-S6 do not appear fragmented or topologically modified. The observations here described demonstrate that polynucleotide:adenosine glycosidase is the sole enzymatic activity of the four ribosome-inactivating proteins gelonin, momordin I, pokeweed antiviral protein from seeds and saporin-S6. These proteins belong to different families, suggesting that the findings here described may be generalized to all PNAG
Visualization and analysis of chromatin by scanning force microscopy
No full textThe use of the scanning force microscope (SFM) to visualize and analyze chromatin fiber structures is presented. Protocols to prepare chromatin fibers for SFM imaging of fibers in air and in buffer are first discussed. Next, the conditions for acquiring high-quality SFM images such as optimal instrumental parameters, appropriate deposition substrates, and adequate procedures of sample deposition are described. It is shown that analysis and quantitation of the SFM images support an irregular, three-dimensional arrangement of nucleosomes in the native chromatin fiber. This structure is lost in linker histone-depleted fibers, which show, instead, a beads-on-a-string structure. Molecular modeling of the chromatin fiber structures and computer simulation of the SFM imaging process indicate that the natural variability of the linker length may be the major determinant of the structural irregularity of the native chromatin fiber. Removal of linker histones (H1/H5) may change the amount of DNA wrapped around the histone octamer, which in turn may induce the transition from a three-dimensional irregular helix to an extended beads-on-a-string structure. Studies of trinucleosomes indicate that both the average successive nucleosome center-to-center distance and the average angle between two successive linkers increase upon the removal of linker histone
Erratum: Mapping the intrinsic curvature and flexibility along the DNA chain (Proceedings of the National Academy of Sciences of the United States of America (2001) 98:6 (3074-3079))
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