Noves possibilitats estructurals per a la condensació dels cromosomes

La cromatina és el material amb què es formen els cromosomes. Generalment s'accepta que el DNA s'empaqueta en els cromosomes durant la mitosi seguint una successió de plegaments de fibres de cromatina. Segons un treball realitzat per investigadors de la UAB, la cromatina, a més de fibres, forma grànuls i plaques. Per tant, poden existir formes prèviament desconegudes d'empaquetament del DNA en els cromosomes.La cromatina es el material con el que se forman los cromosomas. Generalmente se acepta que el DNA se empaqueta en los cromosomas durante la mitosis siguiendo una sucesión de pliegues de fibras de cromatina. Según un trabajo realizado por investigadores de la UAB, la cromatina, además de fibras, forma gránulos y placas. Por tanto, pueden existir formas previamente desconocidas de empaquetamiento del DNA en los cromosomas.Chromosomes are composed of a material named chromatin. Generally it is accepted that DNA is packaged in chromosomes during mitosis following a succession of chromatin fiber folds. According to a study carried out by UAB researchers, in addition to fibers, chromatin forms granules and plates. Therefore, there could be previously unknown ways of DNA packaging in chromosomes

Nanotribology Results Show that DNA Forms a Mechanically Resistant 2D Network in Metaphase Chromatin Plates

AbstractIn a previous study, we found that metaphase chromosomes are formed by thin plates, and here we have applied atomic force microscopy (AFM) and friction force measurements at the nanoscale (nanotribology) to analyze the properties of these planar structures in aqueous media at room temperature. Our results show that high concentrations of NaCl and EDTA and extensive digestion with protease and nuclease enzymes cause plate denaturation. Nanotribology studies show that native plates under structuring conditions (5 mM Mg2+) have a relatively high friction coefficient (μ ≈ 0.3), which is markedly reduced when high concentrations of NaCl or EDTA are added (μ ≈ 0.1). This lubricant effect can be interpreted considering the electrostatic repulsion between DNA phosphate groups and the AFM tip. Protease digestion increases the friction coefficient (μ ≈ 0.5), but the highest friction is observed when DNA is cleaved by micrococcal nuclease (μ ≈ 0.9), indicating that DNA is the main structural element of plates. Whereas nuclease-digested plates are irreversibly damaged after the friction measurement, native plates can absorb kinetic energy from the AFM tip without suffering any damage. These results suggest that plates are formed by a flexible and mechanically resistant two-dimensional network which allows the safe storage of DNA during mitosis

Nou model estructural en capes primes per al cromosoma metafàsic

Experiments realitzats fent servir molt diverses tècniques de microscòpia han permès als investigadors del Laboratori de Cromatina de la UAB descobrir que, durant la divisió cel·lular, els cromosomes tenen el DNA empaquetat dins d'unes estructures planes que estan formades de moltes capes molt primes. Aquestes estructures planes estan apilades, omplen tot el volum dels cromosomes i probablement estan orientades perpendicularment respecte a l'eix central de les cromàtides.Experimentos realizados utilizando muy diversas técnicas demicroscopía han permitido a los investigadores del Laboratorio de Cromatina de la UAB descubrir que, durante la división celular, los cromosomas tienen el ADN empaquetado dentro de unas estructuras planas que están formadas de muchas capas muy delgadas. Estas estructuras planas están apiladas, llenan todo el volumen de los cromosomas y probablemente están orientadas perpendicularmente respecto al eje central de las cromátidasExperiments performed using several different microscopy techniques have allowed researchers at the UAB Chromatin Laboratory to discover that, during cell division, chromosome DNA is packaged with in planar structures formed by many extremely thin layers. These planar structuresare stacked, occupy the entire volume of the chromosomes, and are probably oriented perpendicular to the central chromatid axis. The planar geometry of these structures is very well defined, but the nucleosomes inside the successive layers are irregularly oriented

DNA-origami-aided lithography for sub-10 nanometer pattern printing

We report the first DNA-based origami technique that can print addressable patterns on surfaces with sub-10 nm resolution. Specifically, we have used a two-dimensional DNA origami as a template (DNA origami stamp) to transfer DNA with pre-programmed patterns (DNA ink) on gold surfaces. The DNA ink is composed of thiol-modified staple strands incorporated at specific positions of the DNA origami stamp to create patterns upon thiol-gold bond formation on the surface (DNA ink). The DNA pattern formed is composed of unique oligonucleotide sequences, each of which is individually addressable. As a proof-of-concept, we created a linear pattern of oligonucleotide-modified gold nanoparticles complementary to the DNA ink pattern. We have developed an in silico model to identify key elements in the formation of our DNA origami-driven lithography and nanoparticle patterning as well as simulate more complex nanoparticle patterns on surfaces

Estructura y propiedades de las placas de cromatina de los cromosomas metafásicos: Estudio mediante técnicas de microscopía TEM, AFM y Espectroscopia de Fuerza Atómica

En nuestro laboratorio el estudio de cromosomas en presencia de una elevada concentración de iones divalentes, permitió describir por primera vez las placas de cromatina como unidad estructural básica del cromosoma metafásico compacto. En el periodo experimental de esta tesis (5 años), se han investigado extensamente estas estructuras. El estudio exhaustivo mediante TEM de placas obtenidas utilizando diferentes métodos de preparación y los diferentes controles realizados, indican que las placas no son un artefacto preparativo de esta técnica. La microscopía de fuerza atómica (AFM) ha permitido visualizar e investigar las propiedades mecánicas de las placas en solución acuosa. Las placas son finas (∼6.5 nm; determinado mediante TEM y AFM) pero resistentes a la penetración por la punta de AFM: Su módulo de Young es de ∼0.2 GPa y el estrés requerido para penetrar la superficie es de ∼0.3 GPa (Mg2+ 5-20 mM). El estudio de la desnaturalización en tiempo real (mediante AFM) y las medidas de fricción a nanoescala (Nanontribología) han permitido estudiar las propiedades estructurales de las placas en medio acuoso a temperatura ambiente. Los resultados indican que las elevadas concentraciones de NaCl y EDTA, y la digestión extensa con proteasas y nucleasa micrococal causan la desnaturalización de las placas. Los resultados nanotribológicos indican que las placas en condiciones estructurantes (Mg2+ 5 mM) tienen un coeficiente de fricción relativamente elevado (μ∼0.3), el cual es marcadamente reducido cuando se añaden elevadas concentraciones de NaCl o EDTA (μ∼0.1). La digestión con protasas incrementa el coeficiente de fricción (μ∼0.5), pero la fricción más elevada se observa cuando el DNA se rompe por acción de la nucleasa micrococal (μ∼0.9), indicando que el DNA es el componente estructural principal de las placas. Estos resultados indican que los cromosomas nativos están formados por el apilamiento de placas, que están compuestas por una red bidimensional flexible y mecánicamente resistente de DNA y proteínas.In our laboratory the study of the chromosomes in the presence of high concentrations of divalent ions, has led us to report the chromatin plate-like structure for the first time as a fundamental element of the metaphase chromosome. In the experimental part of this Ph.D. Thesis (5 years), it has been analysed extensively this plate-like structures. The exhaustive TEM study of this planar structures obtained with different preparation procedures and the different performed controls, suggest that the plates are not an artifact of the sample preparation of this technique. The atomic force microscopy (AFM) has led us to image and investigate the mechanical properties of the plates in aqueous solution. The plates are thin (∼6.5 nm; determined by TEM and AFM) but compact and resistant to the penetration by the AFM tip: Their Young's modulus is ∼0.2 GPa and the stress required for surface penetration is ∼0.3 GPa (5-20 mM Mg2+. It has been applied a real time denaturation study (with AFM) and friction force measurements at the nanoscale (Nanotribology) to analyse the structural properties of the plates in aqueous solution at room temperature. The results show that at high concentrations of NaCl and EDTA, and extensive digestion with protease and nuclease enzymes cause plate denaturation. Nanotribology studies show that native plates under structuring conditions (5 mM Mg2+ have a relatively high friction coefficient (μ∼0.3), which is markedly reduced when high concentrations of NaCl or EDTA are added (μ∼0.1). Protease digestion increases the friction coefficient (μ∼0.5), but the highest friction is observed when DNA is cleaved by micrococcal nuclease (μ∼0.9), indicating that DNA is the main structural element of plates. This results suggest that the native chromosomes are formed by staked plates, that are composed by a flexible and mechanically resistant two-dimensional network of DNA and proteins

Interstrand interactions on DNA duplexes modified by TTF units at the 3′ or 5′-ends

Short DNA duplexes carrying TTF units at the same termini exhibit a high increase in melting temperature. When both TTF units were on opposite termini, salt-dependent aggregation is observed, yielding well defined spherical DNA supramolecular structures.This research was supported by the European Commission under Grant No. FP7-FUNMOL 213382, by the Spanish Ministry of Education (CTQ2010-20541), the Generalitat de Catalunya (2009/SGR/208), the Czech Science Foundation (P207/10/2214) and the Institute of Organic Chemistry and Biochemistry AS CR (this work is part of the Research Project Z4 055 0506). Dr. Radek Pohl is acknowledged for the NMR spectra measurement as well as the electron cryo-microscopy unit from CCiT of University of Barcelona for the use of the EM facility.Peer reviewe

DNA-Origami-Aided Lithography for Sub-10 Nanometer Pattern Printing

We report the first DNA-based origami technique that can print addressable patterns on surfaces with sub-10 nm resolution. Specifically, we have used a two-dimensional DNA origami as a template (DNA origami stamp) to transfer DNA with pre-programmed patterns (DNA ink) on gold surfaces. The DNA ink is composed of thiol-modified staple strands incorporated at specific positions of the DNA origami stamp to create patterns upon thiol-gold bond formation on the surface (DNA ink). The DNA pattern formed is composed of unique oligonucleotide sequences, each of which is individually addressable. As a proof-of-concept, we created a linear pattern of oligonucleotide-modified gold nanoparticles complementary to the DNA ink pattern. We have developed an in silico model to identify key elements in the formation of our DNA origami-driven lithography and nanoparticle patterning as well as simulate more complex nanoparticle patterns on surfaces

Biomedical applications of DNA-nanostructures

Nucleic acids are very important biomolecules in charge of the transmission of the genetic inheritance. In order to perform their biological functions, they have unique molecular recognition properties and they are chemically and physically stable. The self-assembly properties of nucleic acids have attracted a large interest in the scientific community for their use on nanosciences [1]. This is also a consequence of the existence of a robust method for the preparation of nucleic acid derivatives that allows the production of these compounds carrying a large variety of functional groups, molecules of interest and materials. In the present communication we will describe our recent work on the use of nucleic acids derivatives in the organization of molecules and materials on surfaces as well as for drug delivery purposes. These include the development of modified DNA origami [2] for the controlled deposition of nanoparticles [3] and for the study of interactions between proteins and aptamers with potential applications in the field of DNA repair [4]. The use of DNA nanostructures for drug delivery will be also described [5]. In this work, we exploited the ability of DNA nanostructures including DNA tetrahedron (Td) and rectangle DNA origami, to incorporate Floxuridine (FdUn) oligomers. Cholesterol moieties were synthetically attached to Td and DNA origami staples to improve cellular uptake. DNA nanoscaffolds functionalized with Floxuridine exhibited an enhanced cytotoxicity and higher ability to trigger apoptosis in colorectal cancer cells relative to conventional 5-fluorouracil (FU) and Floxuridine (FdU) drugs, especially having cholesterol as internalization helper. The cholesterol content mostly correlates with the increment of the FdUn nanoscaffolds cytotoxicity. DNA nanostructures bearing Floxuridine revealed to be able to circumvent 5-FU low sensitivity of drug-resistant colorectal cancer cells. Both DNA nanostructures attained comparable cytotoxic effect yet Td displays higher antiproliferative action. The present work suggests that self-assembled DNA nanoparticles are privileged carriers for delivering fluoropyrimidines, opening new avenues to the development of promising therapeutics for cancer treatment

Preparation and AFM-Characterization of Self-Assembled Monolayers Functionalized with a Thrombin Binding Aptamer

Self-assembled monolayers on silicon oxide surfaces functionalized with a unimolecular DNA G-quadruplex derived from the thrombin binding aptamer have been prepared. The formation of the G-quadruplex in the presence of potassium ions is observed by atomic force microscopy (AFM). The results presented here are interesting for the fabrication of nanostructured systems for sensing applications.This work was supported by the European Communities (NANO-3D, NMP-CT2005-014006 and FUNMOL, FP7-FUNMOL 213382) by the Spanish Ministry of Education (grants CTQ2010-20541-C03-01), and the Generalitat de Catalunya (2009/SGR/208).Peer reviewe

Estructura y propiedades de las placas de cromatina de los cromosomas metafásicos : estudio mediante técnicas de microscopía TEM, AFM y espectroscopia de fuerza atómica/

Descripció del recurs: el 27 de juny de 2011En nuestro laboratorio el estudio de cromosomas en presencia de una elevada concentración de iones divalentes, permitió describir por primera vez las placas de cromatina como unidad estructural básica del cromosoma metafásico compacto. En el periodo experimental de esta tesis (5 años), se han investigado extensamente estas estructuras. El estudio exhaustivo mediante TEM de placas obtenidas utilizando diferentes métodos de preparación y los diferentes controles realizados, indican que las placas no son un artefacto preparativo de esta técnica. La microscopía de fuerza atómica (AFM) ha permitido visualizar e investigar las propiedades mecánicas de las placas en solución acuosa. Las placas son finas (∼6.5 nm; determinado mediante TEM y AFM) pero resistentes a la penetración por la punta de AFM: Su módulo de Young es de ∼0.2 GPa y el estrés requerido para penetrar la superficie es de ∼0.3 GPa (Mg2+ 5-20 mM). El estudio de la desnaturalización en tiempo real (mediante AFM) y las medidas de fricción a nanoescala (Nanontribología) han permitido estudiar las propiedades estructurales de las placas en medio acuoso a temperatura ambiente. Los resultados indican que las elevadas concentraciones de NaCl y EDTA, y la digestión extensa con proteasas y nucleasa micrococal causan la desnaturalización de las placas. Los resultados nanotribológicos indican que las placas en condiciones estructurantes (Mg2+ 5 mM) tienen un coeficiente de fricción relativamente elevado (μ∼0.3), el cual es marcadamente reducido cuando se añaden elevadas concentraciones de NaCl o EDTA (μ∼0.1). La digestión con protasas incrementa el coeficiente de fricción (μ∼0.5), pero la fricción más elevada se observa cuando el DNA se rompe por acción de la nucleasa micrococal (μ∼0.9), indicando que el DNA es el componente estructural principal de las placas. Estos resultados indican que los cromosomas nativos están formados por el apilamiento de placas, que están compuestas por una red bidimensional flexible y mecánicamente resistente de DNA y proteínas.In our laboratory the study of the chromosomes in the presence of high concentrations of divalent ions, has led us to report the chromatin plate-like structure for the first time as a fundamental element of the metaphase chromosome. In the experimental part of this Ph.D. Thesis (5 years), it has been analysed extensively this plate-like structures. The exhaustive TEM study of this planar structures obtained with different preparation procedures and the different performed controls, suggest that the plates are not an artifact of the sample preparation of this technique. The atomic force microscopy (AFM) has led us to image and investigate the mechanical properties of the plates in aqueous solution. The plates are thin (∼6.5 nm; determined by TEM and AFM) but compact and resistant to the penetration by the AFM tip: Their Young's modulus is ∼0.2 GPa and the stress required for surface penetration is ∼0.3 GPa (5-20 mM Mg2+. It has been applied a real time denaturation study (with AFM) and friction force measurements at the nanoscale (Nanotribology) to analyse the structural properties of the plates in aqueous solution at room temperature. The results show that at high concentrations of NaCl and EDTA, and extensive digestion with protease and nuclease enzymes cause plate denaturation. Nanotribology studies show that native plates under structuring conditions (5 mM Mg2+ have a relatively high friction coefficient (μ∼0.3), which is markedly reduced when high concentrations of NaCl or EDTA are added (μ∼0.1). Protease digestion increases the friction coefficient (μ∼0.5), but the highest friction is observed when DNA is cleaved by micrococcal nuclease (μ∼0.9), indicating that DNA is the main structural element of plates. This results suggest that the native chromosomes are formed by staked plates, that are composed by a flexible and mechanically resistant two-dimensional network of DNA and proteins