1,721,018 research outputs found
"Supramolecular Polymeric Chains and Dynamic Objects of Nanometric Size Obtained by the Self-Assembly of DNA Oligonucleotides".
No full textIt is possible to create rigid nanostructures by a careful design of the supramolecular assembly of oligodeoxynucleotides.[1] Using this type of approach, a number of monomeric or polymeric, static or dynamic, nano-objects have assembled in our laboratory. Parallelogram shaped nanostructures made of 4 “four-way junctions” have been assembled in order to make flexible or rigid, linear, branched or circular, supramolecular polymers with a high degree of control. The biochemical and structural characterization of these has been performed using gel electrophoresis and atomic force microscopy.
By proper chemical functionalization of the ODNs used for the assembly, it is possible to include non-DNA objects on the structures: this seems a clever strategy towards objects with functional elements located at a controlled distance on a nanostructure, with the possibility of also modulating their dynamics. We have demonstrated this approach by assembling a DNA-based switch that can turn fluorescence on and off.[2]
1. B. Samorì and G. Zuccheri, Angew. Chem. Int. Ed., 2005, 44, 1166-1181; M. Brucale, G. Zuccheri, B. Samorì, Trends in Biotechnology, 2006.
2. M. Brucale, G. Zuccheri, B. Samorì, Org. Biomol. Chem. 2005, 3, 575
DNA Nanotechnology Methods and Protocols
No full textGiorgio 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..
The liquid crystal - linear dichroism (L.C.-L.D.) of organic molecules by a modulation technique. Part.2. The phenylthio and thiophten chromophores studied by an 'L.D. substitution approach'
No full textSurface-bound DNA self-assembly or enzymatic reactions toward the PCR-free amplification of nucleic acids biosensor signals
No full textINTRODUCTION: Single-molecule sensitivity can be nowadays achieved when trying to characterize minute amounts of macromolecules n in a research laboratory setting. The sensitivity is significantly worse when working in the field and trying to assess the presence of a pollutant or a pathogen as quickly as possible. Point-of-care testing is the realm of biosensors and the trend of research is towards simplicity of use, low cost, reliability and, last but not least, sensitivity. In the detection of nucleic acids, the hybridization of an oligonucleotide probe with its poly-nucleotidic target is monitored by changes in physico-chemical properties of an interface, commonly induced by the use of a specific label. The probe, often bound to an oligonucleotide itself, can be a fluorescent dye, an enzyme, an electroactive moiety or other functional element. Such probes impart specificity and sensitivity to the assay, but make it drift far from simplicity of use (and low cost). METHODS AND RESULTS: Recently, label-free techniques are developing in which the presence of the analyte macromolecule itself can induce detectable physico-chemical changes. The struggle is to make such techniques sufficiently sensitive and specific. In our work, we are trying to implement DNA-based surface-bound amplification strategies that can serve to amplify the read-out signal of DNA hybridization in a label-free biosensor, such as those measuring the electrochemical properties of an electrode interface. The ‘hybridization chain reaction’ [1] is an isothermal enzyme-free strategy to trigger polymerization of oligonucleotides into a long double-stranded DNA. We have demonstrated that the ‘hybridization chain reaction’ can be also implemented (making use of available software tools [2]) in a surface-bound configuration, leading to the self-assembly of many copies of oligonucleotides on a target DNA bound on an oligonucleotide self-assembled monolayer. In another attempt, we have shown that terminal transferase (a template-free DNA polymerase) can be used to build a long polynucleotide out of the target DNA that is bound to its immobilized oligonucleotide probe. The Rolling Circle Amplification reaction is a very promising strategy to produce large amounts of DNA without the need of thermal cycling or of an accurate control of temperature. Preliminary evaluations on the Primer-Generation RCA [3] show its interest as an additional strategy to increase the amount of target DNA response from a biosensor surface. DISCUSSION & CONCLUSIONS: All these strategies will lead to the accumulation of nucleic acids at the solid-liquid interface when triggered by the probe-target recognition. This produces an amplification of the signal of a label-free biosensor. Even though the amplification factors of these implementations are still as low as 10, their further development is still possible, with the hope of avoiding or reducing the need of polymerase chain reaction in the detection of low concentrations of nucleic acids. REFERENCES: 1Dirks, R. M. and N. A. Pierce (2004). "Triggered amplification by hybridization chain reaction." Proc Natl Acad Sci U S A, 101(43): 15275-8. 2Goodman, P. (2005). "NANEV: a program employing evolutionary methods for the design of nucleic acid nanostructures" BioTechniques, 38(4): 548–550. 3Murakami, Sumaoka and Komiyama (2009) "Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification" Nucleic Acids Research, 2009, 37, e19. ACKNOWLEDGEMENTS: This work was supported by Framework Programme 6 Integrated Project DINAMICS
Turning Synthetic DNA Oligonucleotides into Designed Nanostructures: Supramolecular Polymeric Chains and Dynamic Objects by Self-assembly
No full textDNA 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.[1] As suggested by Ned Seeman, the recognized pioneer of this field,[2] 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.[3] This tool is expected to expand further the possibilities of assemblying and controlling nanostructures made of DNA.
[1] M. Brucale, G. Zuccheri, B. Samorì, Trends in Biotechnology, May 2006.
[2] N. C. Seeman, Nature 2003, 421, 427.
[3] M. Brucale, G. Zuccheri, B. Samorì, Org Biomol Chem 2005, 3, 575
Deposition of Supercoiled DNA on Mica for Scanning Force Microscopy Imaging
Full text linkThe 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
Analysis of the conformational space of murine prion protein: an amyloidogenic protein involved in neurodegenerative disorders.
No full text"The interaction of DNA with surfaces and such other trifles".
No full textDNA is the molecule that can bear the highest possible informational content. The different codes embedded in DNA can rule its molecular behaviour, from the assembly of different single chains into double-, triple- quadruple-stranded molecules or complex nanostructures, to the interaction of a DNA molecule with other molecules and with surfaces [1]. The knowledge and the control of the interaction of DNA molecules with other molecules and surfaces is key to the mastering of the creation and control of a number of DNA-bases nanostructures and of their formation or adsorption at surfaces [2], and also to the creation of functional elements based on DNA [3].
Many methods are nowadays available to drive the adsorption of DNA on surfaces. Molecules ranging from short oligodeoxynucleotides to large genomic DNA’s can be controllably adsorbed on a variety of surfaces, either covalently or non-covalently. The atomic force microscope (AFM) has proved a valuable tool to study the interaction of DNA with surfaces and its modes of adsorption [4]. When the surface adsorption is under control, the AFM can also be used to gather a large quantity of interesting data on the adsorbed nucleic acids. Information such as the persistence length of single- and double-stranded DNA [5], or the sequence-dependent microscopic curvature and flexibility of double-stranded DNA [6] have been measured directly on the AFM micrographs, with distinct advantages with respect to other available techniques.
The refinement of the AFM techniques, for what concerns the data processing, the experimental procedures and the instruments, have lead to novel measurements in the last years. It has been possible to more intimately study the mode of adsorption of DNA on surfaces such as mica. For example, thanks to experiments conducted with the novel single-molecule force spectroscopy technique, it has been possible for us to study the forces necessary to unbind DNA molecules from surfaces [7]. Furthermore, it has been possible to study the dynamic behaviour of single molecules in quasi-physiologic environment, one at a time, and still gather quantitative thermodynamic data out of this incredibly small specimen [8].
Thanks to the quantitative evaluation of the conformation of adsorbed DNA molecules it has been possible for us to determine that there are orientational preferences for the adsorption of double-stranded molecules on flat surfaces so that, when equilibrated, molecules seem to expose to the surface their T-rich faces (thus exposing A-rich faces to the solvent) [9]. Such preference is especially measured for intrinsically curved DNA molecules and it has lead us to the conclusion that the same DNA sequence elements that lead to DNA curvature can be used to compile a code to direct the orientation of DNA adsorption at surfaces. Such code, which could have had practical importance in pre-biotic stages of life development, is an additional informational code embedded in the already rich DNA molecule.
[1] Samorì, B. and G. Zuccheri, DNA Codes for Nanoscience. Angew Chem Int Ed Engl, 2005. 44(8): p. 1166-1181; Zuccheri, G., M. Brucale, and B. Samorì, The Tube or the Helix? This is the Question: Towards the Fully Controlled DNA-Directed Assembly of Carbon Nanotubes. Small, 2005. 1(6): p. 590-592; Seeman, N.C., DNA in a material world. Nature, 2003. 421(6921): p. 427-31.
[2] Brucale, M., G. Zuccheri, and B. Samori, Mastering the Complexity of DNA Nanostructures. 2006. in press.
[3] Brucale, M., G. Zuccheri, and B. Samorì, The dynamic properties of an intramolecular transition from DNA duplex to cytosine-thymine motif triplex. Org Biomol Chem, 2005. 3(4): p. 575-7; Niemeyer, C.M. and M. Adler, Nanomechanical devices based on DNA. Angew. Chem. Int. Ed. Engl., 2002. 41(20): p. 3779-3783; Seeman, N.C., From genes to machines: DNA nanomechanical devices. Trends Biochem Sci, 2005. 30(3): p. 119-25.
[4] Rivetti, C., M. Guthold, and C. Bustamante, Scanning force micros..
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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