Artificial biochemical circuits are likely to play as large a role in biological engineering as electrical circuits have played in the engineering of electromechanical devices. Toward that end, nucleic acids provide a designable substrate for the regulation of biochemical reactions. However, it has been difficult to incorporate signal amplification components. We introduce a design strategy that allows a specified input oligonucleotide to catalyze the release of a specified output oligonucleotide, which in turn can serve as a catalyst for other reactions. This reaction, which is driven forward by the configurational entropy of the released molecule, provides an amplifying circuit element that is simple, fast, modular, composable, and robust. We have constructed and characterized several circuits that amplify nucleic acid signals, including a feedforward cascade with quadratic kinetics and a positive feedback circuit with exponential growth kinetics.
Practical components for three-dimensional molecular nanofabrication must be simple to produce, stereopure, rigid, and adaptable. We report a family of DNA tetrahedra, less than 10 nanometers on a side, that can self-assemble in seconds with near-quantitative yield of one diastereomer. They can be connected by programmable DNA linkers. Their triangulated architecture confers structural stability; by compressing a DNA tetrahedron with an atomic force microscope, we have measured the axial compressibility of DNA and observed the buckling of the double helix under high loads.
We are learning to build synthetic molecular machinery from DNA. This research is inspired by biological systems in which individual molecules act, singly and in concert, as specialized machines: our ambition is to create new technologies to perform tasks that are currently beyond our reach. DNA nanomachines are made by self-assembly, using techniques that rely on the sequence-specific interactions that bind complementary oligonucleotides together in a double helix. They can be activated by interactions with specific signalling molecules or by changes in their environment. Devices that change state in response to an external trigger might be used for molecular sensing, intelligent drug delivery or programmable chemical synthesis. Biological molecular motors that carry cargoes within cells have inspired the construction of rudimentary DNA walkers that run along self-assembled tracks. It has even proved possible to create DNA motors that move autonomously, obtaining energy by catalysing the reaction of DNA or RNA fuels.
DNA cages are nanometer-scale polyhedral structures formed by self-assembly from synthetic DNA oligonucleotides. Potential applications include in vivo imaging and the targeted delivery of macromolecules into living cells. We report an investigation of the ability of a model cage, a DNA tetrahedron, to enter live cultured mammalian cells. Cultured human embryonic kidney cells were treated with a range of fluorescently labeled DNA tetrahedra and subsequently examined using confocal microscopy and flow cytometry. Substantial uptake of tetrahedra into cells was observed both when the cells were treated with tetrahedra alone and when the cells were treated with a mixture of tetrahedra and a transfection reagent. Analysis of the subcellular localization of transfected tetrahedra using confocal microscopy and organelle staining indicates that the cages are located in the cytoplasm. FRET experiments indicate that the DNA cages remain substantially intact within the cells for at least 48 h after transfection. This is a first step toward the use of engineered DNA nanostructures to deliver and control the activity of cargoes within cells.
Controlled motion at the nanoscale can be achieved by using Watson-Crick base-pairing to direct the assembly and operation of a molecular transport system consisting of a track, a motor 1-12 and fuel [13][14][15] , all made from DNA. Here, we assemble a 100-nm-long DNA track on a two-dimensional scaffold 16 , and show that a DNA motor loaded at one end of the track moves autonomously and at a constant average speed along the full length of the track, a journey comprising 16 consecutive steps for the motor. Real-time atomic force microscopy allows direct observation of individual steps of a single motor, revealing mechanistic details of its operation. This precisely controlled, long-range transport could lead to the development of systems that could be programmed and routed by instructions encoded in the nucleotide sequences of the track and motor. Such systems might be used to create molecular assembly lines modelled on the ribosome.An effective linear molecular motor must traverse its track without dissociating [1][2][3][4][5][6][7]10,12 and run unidirectionally without external intervention [4][5][6][7][8][9][10][11][12] . Directionality may be imposed by the sequential addition of DNA instructions 1-3 or, for autonomous motors, by modifying the track sites that have been visited 5,6,12 , by coupling motion to a unidirectional reaction cycle 4,9,12 or by coordinating the conformation changes of different parts of the motor 11,12 . DNA motors that satisfy all these criteria have typically been demonstrated on tracks that allow only 1-3 steps, although a stochastic DNA 'spider' with many legs has been shown to move longer distances by biased diffusion 17 along a 100 nm track 18 .We have investigated the motion of a simple directional and processive motor fuelled by DNA hydrolysis 6 along an extended track consisting of a one-dimensional array of single-stranded attachment sites (stators), separated by 6 nm. An extended track of 15 identical stators, flanked with special start and stop stators 6 , was assembled on a rectangular DNA origami tile measuring 100 nm × 70 nm (ref. 16; Fig. 1, Supplementary Figs S1, S2). The tile comprises a 7,249-nucleotide (nt) single-stranded DNA template (genome of bacteriophage M13) hybridized to short synthetic staple strands such that the final tile consists of a raft of 24 parallel double helices tethered by the crossover of staples. Two tile designs were used. The helices of tile type A are crosslinked at 16 bp intervals, creating slight underwinding (10.7 bp per turn), which is expected to lead to a global right-handed twisting of the tile 19 . Tile type B is designed to reduce this twist: the average distance between crossovers is 15.6 bp (giving 10.4 bp per turn). The centre and ends of each staple are positioned on opposite surfaces of the tile. Selected staples were extended to include either the 22-nt stator sequence at the 5 ′ end or a hairpin at the centre 16 (Fig. 1a). Stators hybridized to complementary motor strands are visible in atomic force microscope (AFM) images...
Synthetic molecular motors can be fuelled by the hydrolysis or hybridization of DNA. Such motors can move autonomously and programmably, and long-range transport has been observed on linear tracks. It has also been shown that DNA systems can compute. Here, we report a synthetic DNA-based system that integrates long-range transport and information processing. We show that the path of a motor through a network of tracks containing four possible routes can be programmed using instructions that are added externally or carried by the motor itself. When external control is used we find that 87% of the motors follow the correct path, and when internal control is used 71% of the motors follow the correct path. Programmable motion will allow the development of computing networks, molecular systems that can sort and process cargoes according to instructions that they carry, and assembly lines that can be reconfigured dynamically in response to changing demands.
There's no turning back for an autonomous DNA walker that moves along a self‐assembled track, driven by the hydrolysis of ATP. The track contains three anchorages (A, B, C) at which the walker (★), a six‐nucleotide DNA fragment, can be bound (see figure). The motion of the walker is unidirectional. At each step it is ligated to the next anchorage, then cut from the previous one by a restriction endonuclease.
DNA nanotechnology makes use of the exquisite self-recognition of DNA in order to build on a molecular scale. Although static structures may find applications in structural biology and computer science, many applications in nanomedicine and nanorobotics require the additional capacity for controlled three-dimensional movement. DNA architectures can span three dimensions and DNA devices are capable of movement, but active control of well-defined three-dimensional structures has not been achieved. We demonstrate the operation of reconfigurable DNA tetrahedra whose shapes change precisely and reversibly in response to specific molecular signals. Shape changes are confirmed by gel electrophoresis and by bulk and single-molecule Förster resonance energy transfer measurements. DNA tetrahedra are natural building blocks for three-dimensional construction; they may be synthesized rapidly with high yield of a single stereoisomer, and their triangulated architecture conveys structural stability. The introduction of shape-changing structural modules opens new avenues for the manipulation of matter on the nanometre scale.
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