1,721,108 research outputs found
DNA-PAINT microscope data of a DNA nanostructure printer : Data for "A DNA molecular printer capable of programmable positioning and patterning in two dimensions"
No full textThis dataset consist of reconstructed DNA-PAINT images of DNA origami based molecular devices. This is the data from the paper "A DNA molecular printer capable of programmable positioning and patterning in two dimensions". The data is structures after the figure of the paper. It is reconstructed and can be opened using the DNA-PAINT software Picasso. The data is described by what DNA paint probe was used to image it, corresponding to multiple image channels. 'P1' is the DNA-PAINT docking handle used on the frame and the canvas, 'R1' is the DNA-PAINT docking handle used on the sleeve, and 'R3' is the DNA-PAINT docking handle used on the ink patterned on the canvas
DNA-based optical sensors for forces in cytoskeletal networks
Full text linkMechanical forces are relevant for many biological processes, from wound healing and tumor formation to cell migration and differentiation. Cytoskeletal actin is largely responsible for responding to forces and transmitting them in cells, while also maintaining cell shape and integrity. Here, we describe a FRET-based hybrid DNA-protein tension sensor that is designed to sample transient forces in actin networks by employing two actin-binding motifs with a fast off-rate attached to a central DNA hairpin loop. Such a sensor will be useful to monitor rapidly changing stresses in the cell cytoskeleton. We use fluorescence lifetime imaging to determine the FRET efficiency and thereby the conformational state of the sensor, which makes the measurement robust against intensity variations. We demonstrate the applicability of the sensor by confocal microscopy and by monitoring crosslinking activity in in vitro actin networks by bulk rheology
DNA motor-protein hybrids for molecular transport and self-organisation
Full text linkKinesin is a molecular motor which walks on microtubule tracks in the eukaryotic cytoskeleton. It transports cargo but is also involved in cytoskeletal organisation. This thesis demonstrates fusing kinesin and DNA to construct a molecular transport system using self-organised tracks and to study the mechanics of the minimal motor unit of kinesin. The programmability of DNA allows for the formation of nanostructures with controllable interactions. Kinesin is conjugated to various DNA nanostructures to accomplish different tasks. Instructions encoded into DNA sequences are used to direct the assembly of a polar array of microtubules, to control the loading, active concentration and unloading of cargo on this track network and to trigger the disassembly of the network. Fluorescence microscopy was used to observe these microtubule arrays and the movement of cargo. It was found that the DNA signals used to control the unloading of cargo and the disassembly of the network had to be actively transported, rather than relying on diffusion, for effective delivery of the signal. This work lead to a first author publication, Wollman et al. (2013). DNA was also used to study kinesin by linking defined numbers of minimal functional motor units, single kinesin heads, into teams of 4-12 heads and observing their movement along microtubules via fluorescent labelling. A minimum of 5 heads were required for sustained movement, in agreement with the predictions of Hancock and Howard (1998). The velocity of teams increased with more heads, up to 8, and then a decrease was observed in teams with more heads
Sequence-specific synthesis of macromolecules using DNA-templated chemistry
No full textUsing a strand exchange mechanism we have prepared, by DNA templated chemistry, two 10-mers with defined and tunable monomer sequences. An optimized reaction protocol achieves 85% coupling yield per step, demonstrating that DNA-templated chemistry is a powerful tool for the synthesis of macromolecules with full sequence control
A logical theranostic CRISPR/Cas system and novel carriers for its delivery
Full text linkNucleic acid-based therapies hold the promise to treat many diseases in a way that was not
previously possible: at the genetic level. Unfortunately, these therapies are limited by a
lack of efficient delivery systems. It is challenging to synthesise new delivery systems that
can effciently reach specific tissues. This is because new delivery systems need to overcome
many sophisticated biological barriers, from avoiding the immune system to escaping the
endosomal pathway. Better delivery systems would make nucleic acid therapies a reality.
I tackle this fundamental issue by developing two novel nucleic acid delivery systems: the
Polymeric Spherical Nucleic Acids (PSNAs) and the Cell-Derived Vesicles (CDVs). PSNAs
are PMPC25-PDPA75 polymersomes decorated with intra- and extravesicular coronas of DNA
oligos, which have a great potential as siRNA delivery systems. PSNAs have been designed
to overcome the endosomal barrier, through an osmotic shock caused by their pH-dependent
disassembly. Because of these characteristics, PSNAs are able to deliver therapeutic siRNA
to an Amyotrophic Lateral Sclerosis (ALS) in vitro model. CDVs are bio-inspired delivery
systems produced from purified cellular plasma membrane, which confer on them a complex
biological identity through their natural lipids and membrane proteins. These vesicles are
readily engineered, as plasma membrane from different cell types can be isolated, or cells
can be transfected/transduced to express heterologous proteins of interest. This versatility
is enhanced by their ability to encapsulate small nucleic acids, such as DNA oligos or
CRISPR/Cas9 sgRNA, and functionally deliver them. CDVs are promising delivery systems
that may be applied to different diseases.
While delivery is a paramount issue, there is always unspecific in vivo accumulation,
as seen by the typical retention of nanoparticles in the liver. For the CRISPR/Cas9 gene
editing system, this is dangerous. Unspecific delivery of CRISPR/Cas9 components can
result in off-target mutations. To address this problem, I have developed a theranostic
CRISPR/Cas system that can perform logic computations with endogenous cell signals, in
order to determine if it is in the right environment
Multistep DNA-templated reactions for the synthesis of functional sequence controlled oligomers
No full textA strand displacement mechanism was designed to permit DNA-templated synthesis of functional oligomers of arbitrary length (see scheme). Key features of the mechanism are that successive coupling reactions take place in near-identical environments and that purification is only necessary in the last synthesis step
Structures and mechanisms for synthetic DNA motors
Full text linkDNA provides an ideal substrate for nanoscale construction and programmable dynamic mechanisms. DNA mechanisms can be used to produce DNA motors which do mechanical work, e.g. transportation of a substrate along a track.
I explore a method for control of a DNA mechanism ubiquitous in DNA motor designs, toehold-mediated strand displacement, by which one strand in a duplex can be swapped for another. My method uses a mismatch between a pair of nucleotides in the duplex, which is repaired by displacement. I find that displacement rate can be fine-tuned by adjusting the position of the mismatch in the duplex, enabling the design of complex kinetic behaviours.
A bipedal motor [1, 2] is designed to walk along a single-stranded DNA track. Previously the motor has only taken a single step, due to a lack of designs to extend the single-stranded track. I present a novel design for track held under tension using a 3D DNA origami tightrope, and verify its assembly. The bipedal motor design is adapted and a method to specifically place motors on tightropes is demonstrated. Motor operation is investigated on truncated tracks and tightrope tracks by electrophoresis and spectrofluorometry. The motor does not accumulate appreciably at the track end; this is tentatively attributed to rearrangement of the motor between track sites without interaction with fuel.
Tightrope origami can hold single-stranded DNA under pN tension. I use tightropes to study hybridization kinetics under tension and find dramatic, non-monotonic changes in hybridization rate constants and dissociation constants with tension in the range ∼0-15 pN. Extended tracks for a 'burnt-bridges' motor which destroys its track as it moves [3] are created on the inside of DNA nanotubes, which can be polymerised to create tracks up to a few mm in length, and on tiles which I attempt to join in a specific order. Crossing of the motor between tubes is verified, and microscopy experiments provide some evidence that track is being cleaved by the motor, a requirement for movement along the track. Tile based tracks are imaged by super-resolution DNA PAINT [4], providing proof-of-principle for track observation to infer motor movement.</p
Artificial programmable polymerases
Full text linkThis thesis takes inspiration from the ribosome, an ancient nanomachine conserved throughout life for its fundamental role translating genes into proteins. In the last decade primitive replicas of the RNA ribosome have been designed and constructed using DNA as a building material, which I call ‘artificial programmable polymerases’. By autonomously synthesising small molecules and aperiodic oligomers, these nanomachines have potential applications spanning directed evolution, smart materials, in situ drug synthesis and synthetic biology. However, numerous challenges must be resolved to make polymers of useful lengths. In this thesis I have focused on discrete design challenges relating to the construction of artificial programmable polymerases made of DNA.
DNA sequences can be designed to self-assemble into an array of 2D and 3D structures of nanoscale dimensions. By modifying DNA strands with reactive moieties attached via synthetic linkers, DNA nanostructures can colocalise reactants. In this way, a specific order of chemical reactions can be programmed by the changing the spatial proximity of reactants in a DNA nanostructure. In turn, a specific sequence of reactions during polymer synthesis may be determined by a DNA ‘program’. The challenges associated with autonomous programmable polymerisation are addressed in different chapters characterising new architectures for DNA templated chemical reactions.
Chapter 1 articulates a vision for the field, introducing programmable polymerases, their role in natural systems and various approaches to build artificial mimics. This review reveals the challenges addressed in the following chapters. Chapter 2 and Chapter 3 focus on the use of enzymes to coordinate chemomechanical cycles of polymer synthesis and movement by a DNA nanomachine. Chapter 4 proposes DNA architectures where monomers react within an identical reaction environment during each step of polymer synthesis. Chapter 5 explores the protection of reactants from degradation to increase the yield of long polymers. These advances will be useful in general for dynamic DNA nanotechnology and specifically in the construction of artificial programmable polymerases
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