236 research outputs found
DNA Computing: A Paradigm Shift from Silicon to Carbon
DNA computing, a fascinating frontier in the realm of biological computing, marks a paradigm shift from traditional silicon-based processing to the innovative realm of carbon-based computation. Rooted in the principles of molecular biology, DNA computing harnesses the inherent parallelism of biological systems, offering a revolutionary approach to data storage, processing, and solving complex problems
Single-molecule portrait of DNA and RNA double helices
This is a pre-copyedited, author-produced version of an article accepted for publication in Integrative Biology following peer review. The version of record Arias-Gonzalez, J. Ricardo. 2014. Single-Molecule Portrait of DNA and RNA Double Helices. Integr. Biol. 6 (10). Oxford University Press (OUP): 904 25. doi:10.1039/c4ib00163j is available online at: https://doi.org/10.1039/c4ib00163j[EN] The composition and geometry of the genetic information carriers were described as double-stranded right helices sixty years ago. The flexibility of their sugar¿phosphate backbones and the chemistry of their nucleotide subunits, which give rise to the RNA and DNA polymers, were soon reported to generate two main structural duplex states with biological relevance: the so-called A and B forms. Double-stranded (ds) RNA adopts the former whereas dsDNA is stable in the latter. The presence of flexural and torsional stresses in combination with environmental conditions in the cell or in the event of specific sequences in the genome can, however, stabilize other conformations. Single-molecule manipulation, besides affording the investigation of the elastic response of these polymers, can test the stability of their structural states and transition models. This approach is uniquely suited to understanding the basic features of protein binding molecules, the dynamics of molecular motors and to shedding more light on the biological relevance of the information blocks of life. Here, we provide a comprehensive single-molecule analysis of DNA and RNA double helices in the context of their structural polymorphism to set a rigorous interpretation of their material response both inside and outside the cell. From early knowledge of static structures to current dynamic investigations, we review their phase transitions and mechanochemical behaviour and harness this fundamental knowledge not only through biological sciences, but also for Nanotechnology and Nanomedicine.We are sincerely indebted to S. Hormeno, F. Moreno-Herrero, B. Ibarra, J. L. Carrascosa, J. M. Valpuesta, M. Fuentes-Perez and C. Carrasco for their work throughout the years. C. Flors and A. Villasante are acknowledged for critical revision. This work was supported by Fundacion IMDEA Nanociencia.Arias-Gonzalez, JR. (2014). Single-molecule portrait of DNA and RNA double helices. Integrative Biology. 6(10):904-925. https://doi.org/10.1039/c4ib00163jS904925610Ivanov, V. I., Minchenkova, L. E., Minyat, E. E., Frank-Kamenetskii, M. D., & Schyolkina, A. K. (1974). The B̄ to Ā transition of DNA in solution. Journal of Molecular Biology, 87(4), 817-833. doi:10.1016/0022-2836(74)90086-2FRANKLIN, R. E., & GOSLING, R. G. (1953). 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Interactions between the plant Golgi apparatus and the cytoskeleton
In animal cells, the relationship between the Golgi apparatus and cytoskeleton has been well characterised but not much is known in plants.
The functions of the Golgi apparatus are conserved amongst eukaryotes. It is one of the main stations in the secretory pathway and is involved in protein processing and sorting to different destinations. In plants, it is also involved in trafficking and positioning of cell wall components.
In tobacco epidermal cells, fluorescent labelling with Golgi marker proteins has shown that the Golgi apparatus is made of hundreds of individual units scattered in the cortical cytoplasm and moving on the actin cytoskeleton. The contribution of actin filaments to Golgi body motility in plant has been extensively described, but this actin-centric view has recently been challenged.
Emerging evidence suggests that microtubules may contribute to short distance movement and ‘fine tuning’ of Golgi body displacement. Moreover, proteomic studies linking the actin- cytoskeleton to microtubules have demonstrated that these two components of the cytoskeleton are closely related and a role of the microtubules in Golgi movement cannot be excluded.
In this thesis, automated tracking of Golgi bodies was used to understand and quantify the contribution of actin filaments and microtubules to the organelle dynamics. The tracking technique is also used to assess how the labelling of the cytoskeleton, with a novel fluorescent nanoprobe, affects the dynamics and stability of the actin filaments and the movement of Golgi bodies; FRAP analysis (fluorescent recovery after photo-bleaching) was also used to investigate the binding properties of the fluorescent nanoprobe to the actin filaments. The nanoprobe was compared with another cytoskeletal marker, Lifeact-GFP, to evaluate their suitability for studying the organelle’s motility in relation to the actin-cytoskeleton.
Micromanipulation of Golgi bodies with optical tweezers was used to test if there are physical links between the organelles and the cytoskeleton.
The widely accepted model is that organelles move on actin filaments and movement is powered by myosins. The hypothesis that actin filaments slide one of top of the other, and drag the organelles along, was tested using the FRAP technique.
Kinesin-13a is the only microtubule motor protein localized on Golgi bodies by immunochemical studies. Its localization was investigated in vivo to evaluate if it is involved in linking Golgi bodies to microtubules
Internalisation of biophotonic techniques : transfection, injection and thermometry
Single cell manipulation can offer great insights into the whole of an organism, the rapidly growing -omics fields are illustrating the heterogeneity that can be found within cell populations and where these subtle differences may be exploited, from fundamental knowledge to diagnostics and therapeutics. The cutting edge of this single cell work requires the application of interdisciplinary research to fully exploit the boundaries being pushed. Biophotonics is one such body of interdisciplinary research, employing light to manipulate biological samples. This work seeks to make use biophotonic techniques as analogues for conventional biological methods. High throughput raster scan photoporation is utilised for attempted transfection, multiple trap optical tweezers are used in an attempt to optically drive mechanical injection of cells and the thermal impact of these optical tweezers, which require high energy densities to confine particles, is tested, via the exploitation of the temperature sensitive emission of quantum dot nanoparticles
Dynamic detection of the bio-molecular interaction at the surface of plasmonic nanoarrays
Nanophysics and plasmonics have recently become fields of relevant interest in the
world of research and, in particular, in biosensing and biochemistry. Nanoparticles of noble metals
interact with incident light giving rise to the Localized Surface Plasmon Resonance (LSPR), a sharp
peak of the extinction spectra of the nanoparticles as a result of the collective oscillation at a
resonant frequency of the conduction electrons. The shape of the peak and its position strongly
depend on both nano system properties, as composition, size, shape, orientation, and on the local
dielectric environment. A change in the medium in which the nanoparticle is embedded is indeed
detected and transduced as a distortion and shift of the peak. This mechanism is at the basis of the
biosensing application of plasmonic structures, revealing binding events of molecules to the surface
or extremely small variation in concentration of substances in the proximity. For this reason, LSPR
plasmonic biosensors gained great popularity in a broad range of applications, in particular as
diagnostic devices able to quantitatively detect biomarker molecules. MicroRNA, among the others,
are biomolecules of prominent interest associated to thumoral or other kind of diseases. The aim of
this project is to realize and test a sensitive, specific and label-free plasmonic nanobiosensor able to
detect microRNA target molecules and to investigate the dynamics of the binding of the
biomolecules on the surface of the optical transducers. To accomplish this task, Au nanoprisms
arrays (NPA) are chosen as reference structure, with a LSPR wavelength around 800 nm and
nanofabricated via NanoSphere Lithography (NSL) and thermal evaporation deposition. All the
samples are morphologically characterized with AFM or SEM microscopy. Post-treating procedure
and functionalization protocols are employed to allow the binding of the analyte molecule to be
detected to the sensor, and all the functionalization signals are detected by linear optical
spectroscopy in the visible or near-infrared spectral range. Static measurements are performed to
control the peak shift of the sample after each functionalization step, and dynamic measurements
in a microfluidic setup allow to monitor the temporal evolution of the optical signal and to
reconstruct in real-time the hybridization kinetics at the surface of the plasmonic sensor. A
217nm/RIU bulk sensitivity and 50fMoles limit of detection is reached with the employed
structures, indicating that both the nanofabrication and functionalization strategy are successful in
the detection of analyte molecules down to low concentration limits. Of course, optimization is
desirable, to push even further the sensitivity and solve challenges as for example the aspecific
target binding on the sensor surface. Another purpose of the work is to extract interesting
information about the dynamics of the hybridization reaction that takes place when the analyte
microRNA is bound to the surface of the nanoarray. Hybridization kinetics is studied, determining
the time and affinity constants characterizing the reaction. The results obtained will prove the non-
ideal behaviour of the association, laying the basis for future and advanced outlook about the
building of a non-Langmuir association model able to analytically describe the bi-molecular binding
system
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