Nanobiotechnology

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Nanobiotechnology

Nanobiotechnology, bionanotechnology, and nanobiology are terms that refer to the intersection of nanotechnology and biology. Given that the subject is one that has only emerged very recently, bionanotechnology and nanobiotechnology serve as blanket terms for various related technologies.

This discipline helps to indicate the merger of biological research with various fields of nanotechnology. Concepts that are enhanced through nanobiology include: nanodevices (such as biological machines), nanoparticles, and nanoscale phenomena that occurs within the discipline of nanotechnology. This technical approach to biology allows scientists to imagine and create systems that can be used for biological research. Biologically inspired nanotechnology uses biological systems as the inspirations for technologies not yet created. However, as with nanotechnology and biotechnology, bionanotechnology does have many potential ethical issues associated with it.

A ribosome is a biological machine. Protein domain dynamics can only be seen by neutron spin echo spectroscopy.

The most important objectives that are frequently found in nanobiology involve applying nanotools to relevant medical/biological problems and refining these applications. Developing new tools, such as peptoid nanosheets, for medical and biological purposes is another primary objective in nanotechnology. New nanotools are often made by refining the applications of the nanotools that are already being used. The imaging of native biomolecules, biological membranes, and tissues is also a major topic for nanobiology researchers. Other topics concerning nanobiology include the use of cantilever array sensors and the application of nanophotonics for manipulating molecular processes in living cells.

Recently, the use of microorganisms to synthesize functional nanoparticles has been of great interest. Microorganisms can change the oxidation state of metals. These microbial processes have opened up new opportunities for us to explore novel applications, for example, the biosynthesis of metal nanomaterials. In contrast to chemical and physical methods, microbial processes for synthesizing nanomaterials can be achieved in aqueous phase under gentle and environmentally benign conditions. This approach has become an attractive focus in current green bionanotechnology research towards sustainable development.

Terminology

The terms are often used interchangeably. When a distinction is intended, though, it is based on whether the focus is on applying biological ideas or on studying biology with nanotechnology. Bionanotechnology generally refers to the study of how the goals of nanotechnology can be guided by studying how biological "machines" work and adapting these biological motifs into improving existing nanotechnologies or creating new ones. Nanobiotechnology, on the other hand, refers to the ways that nanotechnology is used to create devices to study biological systems.

In other words, nanobiotechnology is essentially miniaturized biotechnology, whereas bionanotechnology is a specific application of nanotechnology. For example, DNA nanotechnology or cellular engineering would be classified as bionanotechnology because they involve working with biomolecules on the nanoscale. Conversely, many new medical technologies involving nanoparticles as delivery systems or as sensors would be examples of nanobiotechnology since they involve using nanotechnology to advance the goals of biology.

The definitions enumerated above will be utilized whenever a distinction between nanobio and bionano is made in this article. However, given the overlapping usage of the terms in modern parlance, individual technologies may need to be evaluated to determine which term is more fitting. As such, they are best discussed in parallel.

Concepts

Kinesin walking on a microtubule. It is a molecular biological machine that uses protein domain dynamics on nanoscales

Most of the scientific concepts in bionanotechnology are derived from other fields. Biochemical principles that are used to understand the material properties of biological systems are central in bionanotechnology because those same principles are to be used to create new technologies. Material properties and applications studied in bionanoscience include mechanical properties (e.g. deformation, adhesion, failure), electrical/electronic (e.g. electromechanical stimulation, capacitors, energy storage/batteries), optical (e.g. absorption, luminescence, photochemistry), thermal (e.g. thermomutability, thermal management), biological (e.g. how cells interact with nanomaterials, molecular flaws/defects, biosensing, biological mechanisms such as mechanosensation), nanoscience of disease (e.g. genetic disease, cancer, organ/tissue failure), as well as biological computing (e.g. DNA computing) and agriculture (target delivery of pesticides, hormones and fertilizers. The impact of bionanoscience, achieved through structural and mechanistic analyses of biological processes at nanoscale, is their translation into synthetic and technological applications through nanotechnology.

Nanobiotechnology takes most of its fundamentals from nanotechnology. Most of the devices designed for nano-biotechnological use are directly based on other existing nanotechnologies. Nanobiotechnology is often used to describe the overlapping multidisciplinary activities associated with biosensors, particularly where photonics, chemistry, biology, biophysics, nanomedicine, and engineering converge. Measurement in biology using wave guide techniques, such as dual-polarization interferometry, is another example.

Applications

Applications of bionanotechnology are extremely widespread. Insofar as the distinction holds, nanobiotechnology is much more commonplace in that it simply provides more tools for the study of biology. Bionanotechnology, on the other hand, promises to recreate biological mechanisms and pathways in a form that is useful in other ways.

Nanomedicine

Nanomedicine is a field of medical science whose applications are increasing.

Nanobots

The field includes nanorobots and biological machines, which constitute a very useful tool to develop this area of knowledge. In the past years, researchers have made many improvements in the different devices and systems required to develop functional nanorobots – such as motion and magnetic guidance. This supposes a new way of treating and dealing with diseases such as cancer; thanks to nanorobots, side effects of chemotherapy could get controlled, reduced and even eliminated, so some years from now, cancer patients could be offered an alternative to treat such diseases instead of chemotherapy, which causes secondary effects such as hair loss, fatigue or nausea killing not only cancerous cells but also the healthy ones. Nanobots could be used for various therapies, surgery, diagnosis, and medical imaging – such as via targeted drug-delivery to the brain (similar to nanoparticles) and other sites. Programmability for combinations of features such as "tissue penetration, site-targeting, stimuli responsiveness, and cargo-loading" makes such nanobots promising candidates for "precision medicine".

At a clinical level, cancer treatment with nanomedicine would consist of the supply of nanorobots to the patient through an injection that will search for cancerous cells while leaving the healthy ones untouched. Patients that are treated through nanomedicine would thereby not notice the presence of these nanomachines inside them; the only thing that would be noticeable is the progressive improvement of their health. Nanobiotechnology may be useful for medicine formulation.

"Precision antibiotics" has been proposed to make use of bacteriocin-mechanisms for targeted antibiotics.

Nanoparticles

Nanoparticles are already widely used in medicine. Its applications overlap with those of nanobots and in some cases it may be difficult to distinguish between them. They can be used to for diagnosis and targeted drug delivery, encapsulating medicine. Some can be manipulated using magnetic fields and, for example, experimentally, remote-controlled hormone release has been achieved this way.

One example advanced application under development are "Trojan horse" designer-nanoparticles that makes blood cells eat away – from the inside out – portions of atherosclerotic plaque that cause heart attacks and are the current most common cause of death globally.

Artificial cells

Artificial cells such as synthetic red blood cells that have all or many of the natural cells' known broad natural properties and abilities could be used to load functional cargos such as hemoglobin, drugs, magnetic nanoparticles, and ATP biosensors which may enable additional non-native functionalities.

Other

Nanofibers that mimic the matrix around cells and contain molecules that were engineered to wiggle was shown to be a potential therapy for spinal cord injury in mice.

Technically, gene therapy can also be considered to be a form of nanobiotechnology or to move towards it. An example of an area of genome editing related developments that is more clearly nanobiotechnology than more conventional gene therapies, is synthetic fabrication of functional materials in tissues. Researcher made C. elegans worms synthesize, fabricate, and assemble bioelectronic materials in its brain cells. They enabled modulation of membrane properties in specific neuron populations and manipulation of behavior in the living animals which might be useful in the study and treatments for diseases such as multiple sclerosis in specific and demonstrates the viability of such synthetic in vivo fabrication. Moreover, such genetically modified neurons may enable connecting external components – such as prosthetic limbs – to nerves.

Nanosensors based on e.g. nanotubes, nanowires, cantilevers, or atomic force microscopy could be applied to diagnostic devices/sensors

Nanobiotechnology

Nanobiotechnology (sometimes referred to as nanobiology) in medicine may be best described as helping modern medicine progress from treating symptoms to generating cures and regenerating biological tissues.

Three American patients have received whole cultured bladders with the help of doctors who use nanobiology techniques in their practice. Also, it has been demonstrated in animal studies that a uterus can be grown outside the body and then placed in the body in order to produce a baby. Stem cell treatments have been used to fix diseases that are found in the human heart and are in clinical trials in the United States. There is also funding for research into allowing people to have new limbs without having to resort to prosthesis. Artificial proteins might also become available to manufacture without the need for harsh chemicals and expensive machines. It has even been surmised that by the year 2055, computers may be made out of biochemicals and organic salts.

In vivo biosensors

Another example of current nanobiotechnological research involves nanospheres coated with fluorescent polymers. Researchers are seeking to design polymers whose fluorescence is quenched when they encounter specific molecules. Different polymers would detect different metabolites. The polymer-coated spheres could become part of new biological assays, and the technology might someday lead to particles which could be introduced into the human body to track down metabolites associated with tumors and other health problems. Another example, from a different perspective, would be evaluation and therapy at the nanoscopic level, i.e. the treatment of nanobacteria (25-200 nm sized) as is done by NanoBiotech Pharma.

In vitro biosensors

"Nanoantennas" made out of DNA – a novel type of nano-scale optical antenna – can be attached to proteins and produce a signal via fluorescence when these perform their biological functions, in particular for their distinct conformational changes. This could be used for further nanobiotechnology such as various types of nanomachines, to develop new drugs, for bioresearch and for new avenues in biochemistry.

Energy

It may also be useful in sustainable energy: in 2022, researchers reported 3D-printed nano-"skyscraper" electrodes – albeit micro-scale, the pillars had nano-features of porosity due to printed metal nanoparticle inks – (nanotechnology) that house cyanobacteria for extracting substantially more sustainable bioenergy from their photosynthesis (biotechnology) than in earlier studies.

Nanobiology

While nanobiology is in its infancy, there are a lot of promising methods that may rely on nanobiology in the future. Biological systems are inherently nano in scale; nanoscience must merge with biology in order to deliver biomacromolecules and molecular machines that are similar to nature. Controlling and mimicking the devices and processes that are constructed from molecules is a tremendous challenge to face for the converging disciplines of nanobiotechnology. All living things, including humans, can be considered to be nanofoundries. Natural evolution has optimized the "natural" form of nanobiology over millions of years. In the 21st century, humans have developed the technology to artificially tap into nanobiology. This process is best described as "organic merging with synthetic". Colonies of live neurons can live together on a biochip device; according to research from Gunther Gross at the University of North Texas. Self-assembling nanotubes have the ability to be used as a structural system. They would be composed together with rhodopsins; which would facilitate the optical computing process and help with the storage of biological materials. DNA (as the software for all living things) can be used as a structural proteomic system – a logical component for molecular computing. Ned Seeman – a researcher at New York University – along with other researchers are currently researching concepts that are similar to each other.

Bionanotechnology

Distinction from nanobiotechnology

Broadly, bionanotechnology can be distinguished from nanobiotechnology in that it refers to nanotechnology that makes use of biological materials/components – it could in principle or does alternatively use abiotic components. It plays a smaller role in medicine (which is concerned with biological organisms). It makes use of natural or biomimetic systems or elements for unique nanoscale structures and various applications that may not be directionally associated with biology rather than mostly biological applications. In contrast, nanobiotechnology uses biotechnology miniaturized to nanometer size or incorporates nanomolecules into biological systems. In some future applications, both fields could be merged.

DNA

DNA nanotechnology is one important example of bionanotechnology. The utilization of the inherent properties of nucleic acids like DNA to create useful materials or devices – such as biosensors – is a promising area of modern research.

DNA digital data storage refers mostly to the use of synthesized but otherwise conventional strands of DNA to store digital data, which could be useful for e.g. high-density long-term data storage that isn't accessed and written to frequently as an alternative to 5D optical data storage or for use in combination with other nanobiotechnology.

Membrane materials

Another important area of research involves taking advantage of membrane properties to generate synthetic membranes. Proteins that self-assemble to generate functional materials could be used as a novel approach for the large-scale production of programmable nanomaterials. One example is the development of amyloids found in bacterial biofilms as engineered nanomaterials that can be programmed genetically to have different properties.

Lipid nanotechnology

Lipid nanotechnology is another major area of research in bionanotechnology, where physico-chemical properties of lipids such as their antifouling and self-assembly is exploited to build nanodevices with applications in medicine and engineering. Lipid nanotechnology approaches can also be used to develop next-generation emulsion methods to maximize both absorption of fat-soluble nutrients and the ability to incorporate them into popular beverages.

Computing

"Memristors" fabricated from protein nanowires of the bacterium Geobacter sulfurreducens which function at substantially lower voltages than previously described ones may allow the construction of artificial neurons which function at voltages of biological action potentials. The nanowires have a range of advantages over silicon nanowires and the memristors may be used to directly process biosensing signals, for neuromorphic computing (see also: wetware computer) and/or direct communication with biological neurons.

Other

Protein folding studies provide a third important avenue of research, but one that has been largely inhibited by our inability to predict protein folding with a sufficiently high degree of accuracy. Given the myriad uses that biological systems have for proteins, though, research into understanding protein folding is of high importance and could prove fruitful for bionanotechnology in the future.

Agriculture

In the agriculture industry, engineered nanoparticles have been serving as nano carriers, containing herbicides, chemicals, or genes, which target particular plant parts to release their content.

Previously nanocapsules containing herbicides have been reported to effectively penetrate through cuticles and tissues, allowing the slow and constant release of the active substances. Likewise, other literature describes that nano-encapsulated slow release of fertilizers has also become a trend to save fertilizer consumption and to minimize environmental pollution through precision farming. These are only a few examples from numerous research works which might open up exciting opportunities for nanobiotechnology application in agriculture. Also, application of this kind of engineered nanoparticles to plants should be considered the level of amicability before it is employed in agriculture practices. Based on a thorough literature survey, it was understood that there is only limited authentic information available to explain the biological consequence of engineered nanoparticles on treated plants. Certain reports underline the phytotoxicity of various origin of engineered nanoparticles to the plant caused by the subject of concentrations and sizes . At the same time, however, an equal number of studies were reported with a positive outcome of nanoparticles, which facilitate growth promoting nature to treat plant. In particular, compared to other nanoparticles, silver and gold nanoparticles based applications elicited beneficial results on various plant species with less and/or no toxicity. Silver nanoparticles (AgNPs) treated leaves of Asparagus showed the increased content of ascorbate and chlorophyll. Similarly, AgNPs-treated common bean and corn has increased shoot and root length, leaf surface area, chlorophyll, carbohydrate and protein contents reported earlier. The gold nanoparticle has been used to induce growth and seed yield in Brassica juncea.

Nanobiotechnology is used in tissue cultures. The administration of micronutrients at the level of individual atoms and molecules allows for the stimulation of various stages of development, initiation of cell division, and differentiation in the production of plant material, which must be qualitatively uniform and genetically homogeneous. The use of nanoparticles of zinc (ZnO NPs) and silver (Ag NPs) compounds gives very good results in the micropropagation of chrysanthemums using the method of single-node shoot fragments.

Tools

This field relies on a variety of research methods, including experimental tools (e.g. imaging, characterization via AFM/optical tweezers etc.), x-ray diffraction based tools, synthesis via self-assembly, characterization of self-assembly (using e.g. MP-SPR, DPI, recombinant DNA methods, etc.), theory (e.g. statistical mechanics, nanomechanics, etc.), as well as computational approaches (bottom-up multi-scale simulation, supercomputing).

Risk management

Further information: Technological fix § Concerns, and Differential technological development

As of 2009, the risks of nanobiotechnologies are poorly understood and in the U.S. there is no solid national consensus on what kind of regulatory policy principles should be followed. For example, nanobiotechnologies may have hard to control effects on the environment or ecosystems and human health. The metal-based nanoparticles used for biomedical prospectives are extremely enticing in various applications due to their distinctive physicochemical characteristics, allowing them to influence cellular processes at the biological level. The fact that metal-based nanoparticles have high surface-to-volume ratios makes them reactive or catalytic. Due to their small size, they are more likely to be able to penetrate biological barriers such as cell membranes and cause cellular dysfunction in living organisms. Indeed, the high toxicity of some transition metals can make it challenging to use mixed oxide NPs in biomedical uses. It triggers adverse effects on organisms, causing oxidative stress, stimulating the formation of ROS, mitochondrial perturbation, and the modulation of cellular functions, with fatal results in some cases.

Bonin notes that "Nanotechnology is not a specific determinate homogenous entity, but a collection of diverse capabilities and applications" and that nanobiotechnology research and development is – as one of many fields – affected by dual-use problems.

Development of the nervous system

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Development_of_the_nervous_system 

The development of the nervous system, or neural development (neurodevelopment), refers to the processes that generate, shape, and reshape the nervous system of animals, from the earliest stages of embryonic development to adulthood. The field of neural development draws on both neuroscience and developmental biology to describe and provide insight into the cellular and molecular mechanisms by which complex nervous systems develop, from nematodes and fruit flies to mammals.

Defects in neural development can lead to malformations such as holoprosencephaly, and a wide variety of neurological disorders including limb paresis and paralysis, balance and vision disorders, and seizures, and in humans other disorders such as Rett syndrome, Down syndrome and intellectual disability.

Vertebrate brain development

Diagram of the vertebrate nervous system

Further information: Development of the nervous system in humans

The vertebrate central nervous system (CNS) is derived from the ectoderm—the outermost germ layer of the embryo. A part of the dorsal ectoderm becomes specified to neural ectoderm – neuroectoderm that forms the neural plate along the dorsal side of the embryo. This is a part of the early patterning of the embryo (including the invertebrate embryo) that also establishes an anterior-posterior axis. The neural plate is the source of the majority of neurons and glial cells of the CNS. The neural groove forms along the long axis of the neural plate, and the neural plate folds to give rise to the neural tube. This process is known as neurulation. When the tube is closed at both ends it is filled with embryonic cerebrospinal fluid. As the embryo develops, the anterior part of the neural tube expands and forms three primary brain vesicles, which become the forebrain (prosencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon). These simple, early vesicles enlarge and further divide into the telencephalon (future cerebral cortex and basal ganglia), diencephalon (future thalamus and hypothalamus), mesencephalon (future colliculi), metencephalon (future pons and cerebellum), and myelencephalon (future medulla). The CSF-filled central chamber is continuous from the telencephalon to the central canal of the spinal cord, and constitutes the developing ventricular system of the CNS. Embryonic cerebrospinal fluid differs from that formed in later developmental stages, and from adult CSF; it influences the behavior of neural precursors. Because the neural tube gives rise to the brain and spinal cord any mutations at this stage in development can lead to fatal deformities like anencephaly or lifelong disabilities like spina bifida. During this time, the walls of the neural tube contain neural stem cells, which drive brain growth as they divide many times. Gradually some of the cells stop dividing and differentiate into neurons and glial cells, which are the main cellular components of the CNS. The newly generated neurons migrate to different parts of the developing brain to self-organize into different brain structures. Once the neurons have reached their regional positions, they extend axons and dendrites, which allow them to communicate with other neurons via synapses. Synaptic communication between neurons leads to the establishment of functional neural circuits that mediate sensory and motor processing, and underlie behavior.

Flowchart of human brain development

Induction

During early embryonic development of the vertebrate, the dorsal ectoderm becomes specified to give rise to the epidermis and the nervous system; a part of the dorsal ectoderm becomes specified to neural ectoderm to form the neural plate which gives rise to the nervous system. The conversion of undifferentiated ectoderm to neuroectoderm requires signals from the mesoderm. At the onset of gastrulation presumptive mesodermal cells move through the dorsal blastopore lip and form a layer of mesoderm in between the endoderm and the ectoderm. Mesodermal cells migrate along the dorsal midline to give rise to the notochord that develops into the vertebral column. Neuroectoderm overlying the notochord develops into the neural plate in response to a diffusible signal produced by the notochord. The remainder of the ectoderm gives rise to the epidermis. The ability of the mesoderm to convert the overlying ectoderm into neural tissue is called neural induction.

In the early embryo, the neural plate folds outwards to form the neural groove. Beginning in the future neck region, the neural folds of this groove close to create the neural tube. The formation of the neural tube from the ectoderm is called neurulation. The ventral part of the neural tube is called the basal plate; the dorsal part is called the alar plate. The hollow interior is called the neural canal, and the open ends of the neural tube, called the neuropores, close off.

A transplanted blastopore lip can convert ectoderm into neural tissue and is said to have an inductive effect. Neural inducers are molecules that can induce the expression of neural genes in ectoderm explants without inducing mesodermal genes as well. Neural induction is often studied in Xenopus embryos since they have a simple body plan and there are good markers to distinguish between neural and non-neural tissue. Examples of neural inducers are the molecules noggin and chordin.

When embryonic ectodermal cells are cultured at low density in the absence of mesodermal cells they undergo neural differentiation (express neural genes), suggesting that neural differentiation is the default fate of ectodermal cells. In explant cultures (which allow direct cell-cell interactions) the same cells differentiate into epidermis. This is due to the action of BMP4 (a TGF-β family protein) that induces ectodermal cultures to differentiate into epidermis. During neural induction, noggin and chordin are produced by the dorsal mesoderm (notochord) and diffuse into the overlying ectoderm to inhibit the activity of BMP4. This inhibition of BMP4 causes the cells to differentiate into neural cells. Inhibition of TGF-β and BMP (bone morphogenetic protein) signaling can efficiently induce neural tissue from pluripotent stem cells.

Regionalization

In a later stage of development the superior part of the neural tube flexes at the level of the future midbrain—the mesencephalon, at the mesencephalic flexure or cephalic flexure. Above the mesencephalon is the prosencephalon (future forebrain) and beneath it is the rhombencephalon (future hindbrain).

The alar plate of the prosencephalon expands to form the telencephalon which gives rise to the cerebral hemispheres, whilst its basal plate becomes the diencephalon. The optical vesicle (which eventually become the optic nerve, retina and iris) forms at the basal plate of the prosencephalon.^{[citation needed]}

Patterning

In chordates, dorsal ectoderm forms all neural tissue and the nervous system. Patterning occurs due to specific environmental conditions - different concentrations of signaling molecules.

Dorsoventral axis

The ventral half of the neural plate is controlled by the notochord, which acts as the 'organiser'. The dorsal half is controlled by the ectoderm plate, which flanks either side of the neural plate.

Ectoderm follows a default pathway to become neural tissue. Evidence for this comes from single, cultured cells of ectoderm, which go on to form neural tissue. This is postulated to be because of a lack of BMPs, which are blocked by the organiser. The organiser may produce molecules such as follistatin, noggin and chordin that inhibit BMPs.

The ventral neural tube is patterned by sonic hedgehog (Shh) from the notochord, which acts as the inducing tissue. Notochord-derived Shh signals to the floor plate, and induces Shh expression in the floor plate. Floor plate-derived Shh subsequently signals to other cells in the neural tube, and is essential for proper specification of ventral neuron progenitor domains. Loss of Shh from the notochord and/or floor plate prevents proper specification of these progenitor domains. Shh binds Patched1, relieving Patched-mediated inhibition of Smoothened, leading to activation of the Gli family of transcription factors (GLI1, GLI2, and GLI3).

In this context Shh acts as a morphogen - it induces cell differentiation dependent on its concentration. At low concentrations it forms ventral interneurons, at higher concentrations it induces motor neuron development, and at highest concentrations it induces floor plate differentiation. Failure of Shh-modulated differentiation causes holoprosencephaly.

The dorsal neural tube is patterned by BMPs from the epidermal ectoderm flanking the neural plate. These induce sensory interneurons by activating Sr/Thr kinases and altering SMAD transcription factor levels.

Rostrocaudal (Anteroposterior) axis

Signals that control anteroposterior neural development include FGF and retinoic acid, which act in the hindbrain and spinal cord. The hindbrain, for example, is patterned by Hox genes, which are expressed in overlapping domains along the anteroposterior axis under the control of retinoic acid. The 3′ (3 prime end) genes in the Hox cluster are induced by retinoic acid in the hindbrain, whereas the 5′ (5 prime end) Hox genes are not induced by retinoic acid and are expressed more posteriorly in the spinal cord. Hoxb-1 is expressed in rhombomere 4 and gives rise to the facial nerve. Without this Hoxb-1 expression, a nerve similar to the trigeminal nerve arises.

Neurogenesis

Neurogenesis is the process by which neurons are generated from neural stem cells and progenitor cells. Neurons are 'post-mitotic', meaning that they will never divide again for the lifetime of the organism.

Epigenetic modifications play a key role in regulating gene expression in differentiating neural stem cells and are critical for cell fate determination in the developing and adult mammalian brain. Epigenetic modifications include DNA cytosine methylation to form 5-methylcytosine and 5-methylcytosine demethylation. DNA cytosine methylation is catalyzed by DNA methyltransferases (DNMTs). Methylcytosine demethylation is catalyzed in several sequential steps by TET enzymes that carry out oxidative reactions (e.g. 5-methylcytosine to 5-hydroxymethylcytosine) and enzymes of the DNA base excision repair (BER) pathway.

Neuronal migration

Corticogenesis: younger neurons migrate past older ones using radial glia as a scaffolding. Cajal–Retzius cells (red) release reelin (orange).

Neuronal migration is the method by which neurons travel from their origin or birthplace to their final position in the brain. There are several ways they can do this, e.g. by radial migration or tangential migration. Sequences of radial migration (also known as glial guidance) and somal translocation have been captured by time-lapse microscopy.

Tangential migration of interneurons from ganglionic eminence

Radial

Neuronal precursor cells proliferate in the ventricular zone of the developing neocortex, where the principal neural stem cell is the radial glial cell. The first postmitotic cells must leave the stem cell niche and migrate outward to form the preplate, which is destined to become Cajal–Retzius cells and subplate neurons. These cells do so by somal translocation. Neurons migrating with this mode of locomotion are bipolar and attach the leading edge of the process to the pia. The soma is then transported to the pial surface by nucleokinesis, a process by which a microtubule "cage" around the nucleus elongates and contracts in association with the centrosome to guide the nucleus to its final destination.

Radial glial cells, whose fibers serve as a scaffolding for migrating cells and a means of radial communication mediated by calcium dynamic activity, act as the main excitatory neuronal stem cell of the cerebral cortex or translocate to the cortical plate and differentiate either into astrocytes or neurons. Somal translocation can occur at any time during development.

Subsequent waves of neurons split the preplate by migrating along radial glial fibres to form the cortical plate. Each wave of migrating cells travel past their predecessors forming layers in an inside-out manner, meaning that the youngest neurons are the closest to the surface. It is estimated that glial guided migration represents 90% of migrating neurons in human and about 75% in rodents.

Tangential

Most interneurons migrate tangentially through multiple modes of migration to reach their appropriate location in the cortex. An example of tangential migration is the movement of interneurons from the ganglionic eminence to the cerebral cortex. One example of ongoing tangential migration in a mature organism, observed in some animals, is the rostral migratory stream connecting subventricular zone and olfactory bulb.

Axophilic

Many neurons migrating along the anterior-posterior axis of the body use existing axon tracts to migrate along; this is called axophilic migration. An example of this mode of migration is in GnRH-expressing neurons, which make a long journey from their birthplace in the nose, through the forebrain, and into the hypothalamus. Many of the mechanisms of this migration have been worked out, starting with the extracellular guidance cues that trigger intracellular signaling. These intracellular signals, such as calcium signaling, lead to actin and microtubule cytoskeletal dynamics, which produce cellular forces that interact with the extracellular environment through cell adhesion proteins to cause the movement of these cells.

Multipolar

There is also a method of neuronal migration called multipolar migration. This is seen in multipolar cells, which in the human, are abundantly present in the cortical intermediate zone. They do not resemble the cells migrating by locomotion or somal translocation. Instead these multipolar cells express neuronal markers and extend multiple thin processes in various directions independently of the radial glial fibers.

Neurotrophic factors

The survival of neurons is regulated by survival factors, called trophic factors. The neurotrophic hypothesis was formulated by Victor Hamburger and Rita Levi Montalcini based on studies of the developing nervous system. Victor Hamburger discovered that implanting an extra limb in the developing chick led to an increase in the number of spinal motor neurons. Initially he thought that the extra limb was inducing proliferation of motor neurons, but he and his colleagues later showed that there was a great deal of motor neuron death during normal development, and the extra limb prevented this cell death. According to the neurotrophic hypothesis, growing axons compete for limiting amounts of target-derived trophic factors and axons that fail to receive sufficient trophic support die by apoptosis. It is now clear that factors produced by a number of sources contribute to neuronal survival.

• Nerve Growth Factor (NGF): Rita Levi Montalcini and Stanley Cohen purified the first trophic factor, Nerve Growth Factor (NGF), for which they received the Nobel Prize. There are three NGF-related trophic factors: BDNF, NT3, and NT4, which regulate survival of various neuronal populations. The Trk proteins act as receptors for NGF and related factors. Trk is a receptor tyrosine kinase. Trk dimerization and phosphorylation leads to activation of various intracellular signaling pathways including the MAP kinase, Akt, and PKC pathways.
• CNTF: Ciliary neurotrophic factor is another protein that acts as a survival factor for motor neurons. CNTF acts via a receptor complex that includes CNTFRα, GP130, and LIFRβ. Activation of the receptor leads to phosphorylation and recruitment of the JAK kinase, which in turn phosphorylates LIFRβ. LIFRβ acts as a docking site for the STAT transcription factors. JAK kinase phosphorylates STAT proteins, which dissociate from the receptor and translocate to the nucleus to regulate gene expression.
• GDNF: Glial derived neurotrophic factor is a member of the TGFb family of proteins, and is a potent trophic factor for striatal neurons. The functional receptor is a heterodimer, composed of type 1 and type 2 receptors. Activation of the type 1 receptor leads to phosphorylation of Smad proteins, which translocate to the nucleus to activate gene expression.

Synapse formation

Neuromuscular junction

Main article: Neuromuscular junction

Much of our understanding of synapse formation comes from studies at the neuromuscular junction. The transmitter at this synapse is acetylcholine. The acetylcholine receptor (AChR) is present at the surface of muscle cells before synapse formation. The arrival of the nerve induces clustering of the receptors at the synapse. McMahan and Sanes showed that the synaptogenic signal is concentrated at the basal lamina. They also showed that the synaptogenic signal is produced by the nerve, and they identified the factor as Agrin. Agrin induces clustering of AChRs on the muscle surface and synapse formation is disrupted in agrin knockout mice. Agrin transduces the signal via MuSK receptor to rapsyn. Fischbach and colleagues showed that receptor subunits are selectively transcribed from nuclei next to the synaptic site. This is mediated by neuregulins.

In the mature synapse each muscle fiber is innervated by one motor neuron. However, during development, many of the fibers are innervated by multiple axons. Lichtman and colleagues have studied the process of synapses elimination. This is an activity-dependent event. Partial blockage of the receptor leads to retraction of corresponding presynaptic terminals. Later they used a connectomic approach, i.e., tracing out all the connections between motor neurons and muscle fibers, to characterize developmental synapse elimination on the level of a full circuit. Analysis confirmed the massive rewiring, 10-fold decrease in the number of synapses, that takes place as axons prune their motor units but add more synaptic areas at the NMJs with which they remain in contact.

CNS synapses

Agrin appears not to be a central mediator of CNS synapse formation and there is active interest in identifying signals that mediate CNS synaptogenesis. Neurons in culture develop synapses that are similar to those that form in vivo, suggesting that synaptogenic signals can function properly in vitro. CNS synaptogenesis studies have focused mainly on glutamatergic synapses. Imaging experiments show that dendrites are highly dynamic during development and often initiate contact with axons. This is followed by recruitment of postsynaptic proteins to the site of contact. Stephen Smith and colleagues have shown that contact initiated by dendritic filopodia can develop into synapses.

Induction of synapse formation by glial factors: Barres and colleagues made the observation that factors in glial conditioned media induce synapse formation in retinal ganglion cell cultures. Synapse formation in the CNS is correlated with astrocyte differentiation suggesting that astrocytes might provide a synaptogenic factor. The identity of the astrocytic factors is not yet known.

Neuroligins and SynCAM as synaptogenic signals: Sudhof, Serafini, Scheiffele and colleagues have shown that neuroligins and SynCAM can act as factors that induce presynaptic differentiation. Neuroligins are concentrated at the postsynaptic site and act via neurexins concentrated in the presynaptic axons. SynCAM is a cell adhesion molecule that is present in both pre- and post-synaptic membranes.

Assembly of neural circuits

Further information: Activity-dependent plasticity

The processes of neuronal migration, differentiation and axon guidance are generally believed to be activity-independent mechanisms and rely on hard-wired genetic programs in the neurons themselves. Research findings however have implicated a role for activity-dependent mechanisms in mediating some aspects of these processes such as the rate of neuronal migration, aspects of neuronal differentiation and axon pathfinding.These three processes are directed by molecular cues that act as guidance forces for growing axons - Chemoattraction (through the use of Netrins), Chemorepulsion ( through the use of secreted Semaphorins), Contact attraction (Cadherins) and Contact repulsion (Semaphorins). Activity-dependent mechanisms influence neural circuit development and are crucial for laying out early connectivity maps and the continued refinement of synapses which occurs during development. There are two distinct types of neural activity we observe in developing circuits - early spontaneous activity and sensory-evoked activity. Spontaneous activity occurs early during neural circuit development even when sensory input is absent and is observed in many systems such as the developing visual system, auditory system, motor system, hippocampus, cerebellum and neocortex.

Experimental techniques such as direct electrophysiological recording, fluorescence imaging using calcium indicators and optogenetic techniques have shed light on the nature and function of these early bursts of activity. They have distinct spatial and temporal patterns during development and their ablation during development has been known to result in deficits in network refinement in the visual system. In the immature retina, waves of spontaneous action potentials arise from the retinal ganglion cells and sweep across the retinal surface in the first few postnatal weeks. These waves are mediated by neurotransmitter acetylcholine in the initial phase and later on by glutamate. They are thought to instruct the formation of two sensory maps- the retinotopic map and eye-specific segregation. Retinotopic map refinement occurs in downstream visual targets in the brain-the superior colliculus (SC) and dorsal lateral geniculate nucleus (LGN). Pharmacological disruption and mouse models lacking the β2 subunit of the nicotinic acetylcholine receptor has shown that the lack of spontaneous activity leads to marked defects in retinotopy and eye-specific segregation.

Recent studies confirm that microglia, the resident immune cell of the brain, establish direct contacts with the cell bodies of developing neurons, and through these connections, regulate neurogenesis, migration, integration and the formation of neuronal networks in an activity-dependent manner.

In the developing auditory system, developing cochlea generate bursts of activity which spreads across the inner hair cells and spiral ganglion neurons which relay auditory information to the brain. ATP release from supporting cells triggers action potentials in inner hair cells. In the auditory system, spontaneous activity is thought to be involved in tonotopic map formation by segregating cochlear neuron axons tuned to high and low frequencies. In the motor system, periodic bursts of spontaneous activity are driven by excitatory GABA and glutamate during the early stages and by acetylcholine and glutamate at later stages. In the developing zebrafish spinal cord, early spontaneous activity is required for the formation of increasingly synchronous alternating bursts between ipsilateral and contralateral regions of the spinal cord and for the integration of new cells into the circuit. Motor neurons innervating the same twitch muscle fibers are thought to maintain synchronous activity which allows both neurons to remain in contact with the muscle fiber in adulthood. In the cortex, early waves of activity have been observed in the cerebellum and cortical slices. Once sensory stimulus becomes available, final fine-tuning of sensory-coding maps and circuit refinement begins to rely more and more on sensory-evoked activity as demonstrated by classic experiments about the effects of sensory deprivation during critical periods.

Contemporary diffusion-weighted MRI techniques may also uncover the macroscopic process of axonal development. The connectome can be constructed from diffusion MRI data: the vertices of the graph correspond to anatomically labelled gray matter areas, and two such vertices, say u and v, are connected by an edge if the tractography phase of the data processing finds an axonal fiber that connects the two areas, corresponding to u and v.

Numerous braingraphs, computed from the Human Connectome Project can be downloaded from the http://braingraph.org site. The Consensus Connectome Dynamics (CCD) is a remarkable phenomenon that was discovered by continuously decreasing the minimum confidence-parameter at the graphical interface of the Budapest Reference Connectome Server. The Budapest Reference Connectome Server (http://connectome.pitgroup.org) depicts the cerebral connections of n=418 subjects with a frequency-parameter k: For any k=1,2,...,n one can view the graph of the edges that are present in at least k connectomes. If parameter k is decreased one-by-one from k=n through k=1 then more and more edges appear in the graph, since the inclusion condition is relaxed. The surprising observation is that the appearance of the edges is far from random: it resembles a growing, complex structure, like a tree or a shrub (visualized on the animation on the left).

It is hypothesized in  that the growing structure copies the axonal development of the human brain: the earliest developing connections (axonal fibers) are common at most of the subjects, and the subsequently developing connections have larger and larger variance, because their variances are accumulated in the process of axonal development.

Synapse elimination

Main article: Synaptic pruning

Synapse elimination is one of the most crucial part of refining the developing neural circuits during embryonic development. Initially, the nervous system creates an excess of neuron connections to ensure that all target cells are contacted. This mainly happens in the developing vertebrate nervous system. The reason why this happens is to make sure all the cells in the target population are innervated. Several motor neurons then compete for each neuromuscular junction, but only one survives until adulthood. Competition in vitro has been shown to involve a limited neurotrophic substance that is released, or that neural activity infers advantage to strong post-synaptic connections by giving resistance to a toxin also released upon nerve stimulation. In vivo, it is suggested that muscle fibres select the strongest neuron through a retrograde signal or that activity-dependent synapse elimination mechanisms determine the identity of the "winning" axon at a motor endplate. The winning axon is a representation of the 'pruning' process of the weaker and less active synapses to increase efficiency.

Mapping

Brain mapping can show how an animal's brain changes throughout its lifetime. As of 2021, scientists mapped and compared the whole brains (head ganglia) of eight C. elegans worms across their development on the neuronal level and the complete wiring of a single mammalian muscle from birth to adulthood.

Adult neurogenesis

Main article: Adult neurogenesis

Neurogenesis also occurs to generate functional neurons in adults. This occurs in specific parts of the adult brain such as the dentate gyrus of the hippocampus. Adult neurogenesis was first found in models of rats by Altman and Das, as it was only known to be present in embryonic development.

DNA nanotechnology

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/DNA_nanotechnology 

DNA nanotechnology involves forming artificial, designed nanostructures out of nucleic acids, such as this DNA tetrahedron. Each edge of the tetrahedron is a 20 base pair DNA double helix, and each vertex is a three-arm junction. The 4 DNA strands that form the 4 tetrahedral faces are color-coded.

DNA nanotechnology is the design and manufacture of artificial nucleic acid structures for technological uses. In this field, nucleic acids are used as non-biological engineering materials for nanotechnology rather than as the carriers of genetic information in living cells. Researchers in the field have created static structures such as two- and three-dimensional crystal lattices, nanotubes, polyhedra, and arbitrary shapes, and functional devices such as molecular machines and DNA computers. The field is beginning to be used as a tool to solve basic science problems in structural biology and biophysics, including applications in X-ray crystallography and nuclear magnetic resonance spectroscopy of proteins to determine structures. Potential applications in molecular scale electronics and nanomedicine are also being investigated.

The conceptual foundation for DNA nanotechnology was first laid out by Nadrian Seeman in the early 1980s, and the field began to attract widespread interest in the mid-2000s. This use of nucleic acids is enabled by their strict base pairing rules, which cause only portions of strands with complementary base sequences to bind together to form strong, rigid double helix structures. This allows for the rational design of base sequences that will selectively assemble to form complex target structures with precisely controlled nanoscale features. Several assembly methods are used to make these structures, including tile-based structures that assemble from smaller structures, folding structures using the DNA origami method, and dynamically reconfigurable structures using strand displacement methods. The field's name specifically references DNA, but the same principles have been used with other types of nucleic acids as well, leading to the occasional use of the alternative name nucleic acid nanotechnology.

History

The conceptual foundation for DNA nanotechnology was first laid out by Nadrian Seeman in the early 1980s. Seeman's original motivation was to create a three-dimensional DNA lattice for orienting other large molecules, which would simplify their crystallographic study by eliminating the difficult process of obtaining pure crystals. This idea had reportedly come to him in late 1980, after realizing the similarity between the woodcut Depth by M. C. Escher and an array of DNA six-arm junctions. Several natural branched DNA structures were known at the time, including the DNA replication fork and the mobile Holliday junction, but Seeman's insight was that immobile nucleic acid junctions could be created by properly designing the strand sequences to remove symmetry in the assembled molecule, and that these immobile junctions could in principle be combined into rigid crystalline lattices. The first theoretical paper proposing this scheme was published in 1982, and the first experimental demonstration of an immobile DNA junction was published the following year.

The woodcut Depth (pictured) by M. C. Escher reportedly inspired Nadrian Seeman to consider using three-dimensional lattices of DNA to orient hard-to-crystallize molecules. This led to the beginning of the field of DNA nanotechnology.

In 1991, Seeman's laboratory published a report on the synthesis of a cube made of DNA, the first synthetic three-dimensional nucleic acid nanostructure, for which he received the 1995 Feynman Prize in Nanotechnology. This was followed by a DNA truncated octahedron. It soon became clear that these structures, polygonal shapes with flexible junctions as their vertices, were not rigid enough to form extended three-dimensional lattices. Seeman developed the more rigid double-crossover (DX) structural motif, and in 1998, in collaboration with Erik Winfree, published the creation of two-dimensional lattices of DX tiles. These tile-based structures had the advantage that they provided the ability to implement DNA computing, which was demonstrated by Winfree and Paul Rothemund in their 2004 paper on the algorithmic self-assembly of a Sierpinski gasket structure, and for which they shared the 2006 Feynman Prize in Nanotechnology. Winfree's key insight was that the DX tiles could be used as Wang tiles, meaning that their assembly could perform computation. The synthesis of a three-dimensional lattice was finally published by Seeman in 2009, nearly thirty years after he had set out to achieve it.

New abilities continued to be discovered for designed DNA structures throughout the 2000s. The first DNA nanomachine—a motif that changes its structure in response to an input—was demonstrated in 1999 by Seeman. An improved system, which was the first nucleic acid device to make use of toehold-mediated strand displacement, was demonstrated by Bernard Yurke in 2000. The next advance was to translate this into mechanical motion, and in 2004 and 2005, several DNA walker systems were demonstrated by the groups of Seeman, Niles Pierce, Andrew Turberfield, and Chengde Mao. The idea of using DNA arrays to template the assembly of other molecules such as nanoparticles and proteins, first suggested by Bruche Robinson and Seeman in 1987, was demonstrated in 2002 by Seeman, Kiehl et al. and subsequently by many other groups.

In 2006, Rothemund first demonstrated the DNA origami method for easily and robustly forming folded DNA structures of arbitrary shape. Rothemund had conceived of this method as being conceptually intermediate between Seeman's DX lattices, which used many short strands, and William Shih's DNA octahedron, which consisted mostly of one very long strand. Rothemund's DNA origami contains a long strand which folding is assisted by several short strands. This method allowed forming much larger structures than formerly possible, and which are less technically demanding to design and synthesize. DNA origami was the cover story of Nature on March 15, 2006. Rothemund's research demonstrating two-dimensional DNA origami structures was followed by the demonstration of solid three-dimensional DNA origami by Douglas et al. in 2009, while the labs of Jørgen Kjems and Yan demonstrated hollow three-dimensional structures made out of two-dimensional faces.

DNA nanotechnology was initially met with some skepticism due to the unusual non-biological use of nucleic acids as materials for building structures and doing computation, and the preponderance of proof of principle experiments that extended the abilities of the field but were far from actual applications. Seeman's 1991 paper on the synthesis of the DNA cube was rejected by the journal Science after one reviewer praised its originality while another criticized it for its lack of biological relevance. By the early 2010s the field was considered to have increased its abilities to the point that applications for basic science research were beginning to be realized, and practical applications in medicine and other fields were beginning to be considered feasible. The field had grown from very few active laboratories in 2001 to at least 60 in 2010, which increased the talent pool and thus the number of scientific advances in the field during that decade.

Fundamental concepts

These four strands associate into a DNA four-arm junction because this structure maximizes the number of correct base pairs, with A matched to T and C matched to G. See this image for a more realistic model of the four-arm junction showing its tertiary structure.

This double-crossover (DX) supramolecular complex consists of five DNA single strands that form two double-helical domains, on the top and the bottom in this image. There are two crossover points where the strands cross from one domain into the other.

Properties of nucleic acids

Nanotechnology is often defined as the study of materials and devices with features on a scale below 100 nanometers. DNA nanotechnology, specifically, is an example of bottom-up molecular self-assembly, in which molecular components spontaneously organize into stable structures; the particular form of these structures is induced by the physical and chemical properties of the components selected by the designers. In DNA nanotechnology, the component materials are strands of nucleic acids such as DNA; these strands are often synthetic and are almost always used outside the context of a living cell. DNA is well-suited to nanoscale construction because the binding between two nucleic acid strands depends on simple base pairing rules which are well understood, and form the specific nanoscale structure of the nucleic acid double helix. These qualities make the assembly of nucleic acid structures easy to control through nucleic acid design. This property is absent in other materials used in nanotechnology, including proteins, for which protein design is very difficult, and nanoparticles, which lack the capability for specific assembly on their own.

The structure of a nucleic acid molecule consists of a sequence of nucleotides distinguished by which nucleobase they contain. In DNA, the four bases present are adenine (A), cytosine (C), guanine (G), and thymine (T). Nucleic acids have the property that two molecules will only bind to each other to form a double helix if the two sequences are complementary, meaning that they form matching sequences of base pairs, with A only binding to T, and C only to G. Because the formation of correctly matched base pairs is energetically favorable, nucleic acid strands are expected in most cases to bind to each other in the conformation that maximizes the number of correctly paired bases. The sequences of bases in a system of strands thus determine the pattern of binding and the overall structure in an easily controllable way. In DNA nanotechnology, the base sequences of strands are rationally designed by researchers so that the base pairing interactions cause the strands to assemble in the desired conformation. While DNA is the dominant material used, structures incorporating other nucleic acids such as RNA and peptide nucleic acid (PNA) have also been constructed.

Subfields

DNA nanotechnology is sometimes divided into two overlapping subfields: structural DNA nanotechnology and dynamic DNA nanotechnology. Structural DNA nanotechnology, sometimes abbreviated as SDN, focuses on synthesizing and characterizing nucleic acid complexes and materials that assemble into a static, equilibrium end state. On the other hand, dynamic DNA nanotechnology focuses on complexes with useful non-equilibrium behavior such as the ability to reconfigure based on a chemical or physical stimulus. Some complexes, such as nucleic acid nanomechanical devices, combine features of both the structural and dynamic subfields.

The complexes constructed in structural DNA nanotechnology use topologically branched nucleic acid structures containing junctions. (In contrast, most biological DNA exists as an unbranched double helix.) One of the simplest branched structures is a four-arm junction that consists of four individual DNA strands, portions of which are complementary in a specific pattern. Unlike in natural Holliday junctions, each arm in the artificial immobile four-arm junction has a different base sequence, causing the junction point to be fixed at a certain position. Multiple junctions can be combined in the same complex, such as in the widely used double-crossover (DX) structural motif, which contains two parallel double helical domains with individual strands crossing between the domains at two crossover points. Each crossover point is, topologically, a four-arm junction, but is constrained to one orientation, in contrast to the flexible single four-arm junction, providing a rigidity that makes the DX motif suitable as a structural building block for larger DNA complexes.

Dynamic DNA nanotechnology uses a mechanism called toehold-mediated strand displacement to allow the nucleic acid complexes to reconfigure in response to the addition of a new nucleic acid strand. In this reaction, the incoming strand binds to a single-stranded toehold region of a double-stranded complex, and then displaces one of the strands bound in the original complex through a branch migration process. The overall effect is that one of the strands in the complex is replaced with another one.^[23] In addition, reconfigurable structures and devices can be made using functional nucleic acids such as deoxyribozymes and ribozymes, which can perform chemical reactions, and aptamers, which can bind to specific proteins or small molecules.

Structural DNA nanotechnology

Structural DNA nanotechnology, sometimes abbreviated as SDN, focuses on synthesizing and characterizing nucleic acid complexes and materials where the assembly has a static, equilibrium endpoint. The nucleic acid double helix has a robust, defined three-dimensional geometry that makes it possible to simulate, predict and design the structures of more complicated nucleic acid complexes. Many such structures have been created, including two- and three-dimensional structures, and periodic, aperiodic, and discrete structures.

Extended lattices

The assembly of a DX array. Left, schematic diagram. Each bar represents a double-helical domain of DNA, with the shapes representing complementary sticky ends. The DX complex at top will combine with other DX complexes into the two-dimensional array shown at bottom. Right, an atomic force microscopy image of the assembled array. The individual DX tiles are clearly visible within the assembled structure. The field is 150 nm across.

Left, a model of a DNA tile used to make another two-dimensional periodic lattice. Right, an atomic force micrograph of the assembled lattice.

An example of an aperiodic two-dimensional lattice that assembles into a fractal pattern. Left, the Sierpinski gasket fractal. Right, DNA arrays that display a representation of the Sierpinski gasket on their surfaces

Small nucleic acid complexes can be equipped with sticky ends and combined into larger two-dimensional periodic lattices containing a specific tessellated pattern of the individual molecular tiles. The earliest example of this used double-crossover (DX) complexes as the basic tiles, each containing four sticky ends designed with sequences that caused the DX units to combine into periodic two-dimensional flat sheets that are essentially rigid two-dimensional crystals of DNA. Two-dimensional arrays have been made from other motifs as well, including the Holliday junction rhombus lattice, and various DX-based arrays making use of a double-cohesion scheme. The top two images at right show examples of tile-based periodic lattices.

Two-dimensional arrays can be made to exhibit aperiodic structures whose assembly implements a specific algorithm, exhibiting one form of DNA computing. The DX tiles can have their sticky end sequences chosen so that they act as Wang tiles, allowing them to perform computation. A DX array whose assembly encodes an XOR operation has been demonstrated; this allows the DNA array to implement a cellular automaton that generates a fractal known as the Sierpinski gasket. The third image at right shows this type of array. Another system has the function of a binary counter, displaying a representation of increasing binary numbers as it grows. These results show that computation can be incorporated into the assembly of DNA arrays.

DX arrays have been made to form hollow nanotubes 4–20 nm in diameter, essentially two-dimensional lattices which curve back upon themselves. These DNA nanotubes are somewhat similar in size and shape to carbon nanotubes, and while they lack the electrical conductance of carbon nanotubes, DNA nanotubes are more easily modified and connected to other structures. One of many schemes for constructing DNA nanotubes uses a lattice of curved DX tiles that curls around itself and closes into a tube. In an alternative method that allows the circumference to be specified in a simple, modular fashion using single-stranded tiles, the rigidity of the tube is an emergent property.

Forming three-dimensional lattices of DNA was the earliest goal of DNA nanotechnology, but this proved to be one of the most difficult to realize. Success using a motif based on the concept of tensegrity, a balance between tension and compression forces, was finally reported in 2009.

Discrete structures

Researchers have synthesized many three-dimensional DNA complexes that each have the connectivity of a polyhedron, such as a cube or octahedron, meaning that the DNA duplexes trace the edges of a polyhedron with a DNA junction at each vertex. The earliest demonstrations of DNA polyhedra were very work-intensive, requiring multiple ligations and solid-phase synthesis steps to create catenated polyhedra. Subsequent work yielded polyhedra whose synthesis was much easier. These include a DNA octahedron made from a long single strand designed to fold into the correct conformation, and a tetrahedron that can be produced from four DNA strands in one step, pictured at the top of this article.

Nanostructures of arbitrary, non-regular shapes are usually made using the DNA origami method. These structures consist of a long, natural virus strand as a "scaffold", which is made to fold into the desired shape by computationally designed short "staple" strands. This method has the advantages of being easy to design, as the base sequence is predetermined by the scaffold strand sequence, and not requiring high strand purity and accurate stoichiometry, as most other DNA nanotechnology methods do. DNA origami was first demonstrated for two-dimensional shapes, such as a smiley face, a coarse map of the Western Hemisphere, and the Mona Lisa painting. Solid three-dimensional structures can be made by using parallel DNA helices arranged in a honeycomb pattern, and structures with two-dimensional faces can be made to fold into a hollow overall three-dimensional shape, akin to a cardboard box. These can be programmed to open and reveal or release a molecular cargo in response to a stimulus, making them potentially useful as programmable molecular cages.

Templated assembly

Nucleic acid structures can be made to incorporate molecules other than nucleic acids, sometimes called heteroelements, including proteins, metallic nanoparticles, quantum dots, amines, and fullerenes. This allows the construction of materials and devices with a range of functionalities much greater than is possible with nucleic acids alone. The goal is to use the self-assembly of the nucleic acid structures to template the assembly of the nanoparticles hosted on them, controlling their position and in some cases orientation. Many of these schemes use a covalent attachment scheme, using oligonucleotides with amide or thiol functional groups as a chemical handle to bind the heteroelements. This covalent binding scheme has been used to arrange gold nanoparticles on a DX-based array, and to arrange streptavidin protein molecules into specific patterns on a DX array. A non-covalent hosting scheme using Dervan polyamides on a DX array was used to arrange streptavidin proteins in a specific pattern on a DX array. Carbon nanotubes have been hosted on DNA arrays in a pattern allowing the assembly to act as a molecular electronic device, a carbon nanotube field-effect transistor. In addition, there are nucleic acid metallization methods, in which the nucleic acid is replaced by a metal which assumes the general shape of the original nucleic acid structure, and schemes for using nucleic acid nanostructures as lithography masks, transferring their pattern into a solid surface.

Dynamic DNA nanotechnology

Dynamic DNA nanotechnology often makes use of toehold-mediated strand displacement reactions. In this example, the red strand binds to the single stranded toehold region on the green strand (region 1), and then in a branch migration process across region 2, the blue strand is displaced and freed from the complex. Reactions like these are used to dynamically reconfigure or assemble nucleic acid nanostructures. In addition, the red and blue strands can be used as signals in a molecular logic gate.

Dynamic DNA nanotechnology focuses on forming nucleic acid systems with designed dynamic functionalities related to their overall structures, such as computation and mechanical motion. There is some overlap between structural and dynamic DNA nanotechnology, as structures can be formed through annealing and then reconfigured dynamically, or can be made to form dynamically in the first place.

Nanomechanical devices

Main article: DNA machine

DNA complexes have been made that change their conformation upon some stimulus, making them one form of nanorobotics. These structures are initially formed in the same way as the static structures made in structural DNA nanotechnology, but are designed so that dynamic reconfiguration is possible after the initial assembly. The earliest such device made use of the transition between the B-DNA and Z-DNA forms to respond to a change in buffer conditions by undergoing a twisting motion. This reliance on buffer conditions caused all devices to change state at the same time. Subsequent systems could change states based upon the presence of control strands, allowing multiple devices to be independently operated in solution. Some examples of such systems are a "molecular tweezers" design that has an open and a closed state, a device that could switch from a paranemic-crossover (PX) conformation to a (JX2) conformation with two non-junction juxtapositions of the DNA backbone, undergoing rotational motion in the process, and a two-dimensional array that could dynamically expand and contract in response to control strands. Structures have also been made that dynamically open or close, potentially acting as a molecular cage to release or reveal a functional cargo upon opening. In another example, a DNA origami nanostructure was coupled to T7 RNA polymerase and could thus be operated as a chemical energy-driven motor that can be coupled to a passive follower, which it then drives.

DNA walkers are a class of nucleic acid nanomachines that exhibit directional motion along a linear track. A large number of schemes have been demonstrated. One strategy is to control the motion of the walker along the track using control strands that need to be manually added in sequence. It is also possible to control individual steps of a DNA walker by irradiation with light of different wavelengths. Another approach is to make use of restriction enzymes or deoxyribozymes to cleave the strands and cause the walker to move forward, which has the advantage of running autonomously. A later system could walk upon a two-dimensional surface rather than a linear track, and demonstrated the ability to selectively pick up and move molecular cargo. In 2018, a catenated DNA that uses rolling circle transcription by an attached T7 RNA polymerase was shown to walk along a DNA-path, guided by the generated RNA strand. Additionally, a linear walker has been demonstrated that performs DNA-templated synthesis as the walker advances along the track, allowing autonomous multistep chemical synthesis directed by the walker. The synthetic DNA walkers' function is similar to that of the proteins dynein and kinesin.

Strand displacement cascades

Cascades of strand displacement reactions can be used for either computational or structural purposes. An individual strand displacement reaction involves revealing a new sequence in response to the presence of some initiator strand. Many such reactions can be linked into a cascade where the newly revealed output sequence of one reaction can initiate another strand displacement reaction elsewhere. This in turn allows for the construction of chemical reaction networks with many components, exhibiting complex computational and information processing abilities. These cascades are made energetically favorable through the formation of new base pairs, and the entropy gain from disassembly reactions. Strand displacement cascades allow isothermal operation of the assembly or computational process, in contrast to traditional nucleic acid assembly's requirement for a thermal annealing step, where the temperature is raised and then slowly lowered to ensure proper formation of the desired structure. They can also support catalytic function of the initiator species, where less than one equivalent of the initiator can cause the reaction to go to completion.

Strand displacement complexes can be used to make molecular logic gates capable of complex computation. Unlike traditional electronic computers, which use electric current as inputs and outputs, molecular computers use the concentrations of specific chemical species as signals. In the case of nucleic acid strand displacement circuits, the signal is the presence of nucleic acid strands that are released or consumed by binding and unbinding events to other strands in displacement complexes. This approach has been used to make logic gates such as AND, OR, and NOT gates. More recently, a four-bit circuit was demonstrated that can compute the square root of the integers 0–15, using a system of gates containing 130 DNA strands.

Another use of strand displacement cascades is to make dynamically assembled structures. These use a hairpin structure for the reactants, so that when the input strand binds, the newly revealed sequence is on the same molecule rather than disassembling. This allows new opened hairpins to be added to a growing complex. This approach has been used to make simple structures such as three- and four-arm junctions and dendrimers.

Applications

DNA nanotechnology provides one of the few ways to form designed, complex structures with precise control over nanoscale features. The field is beginning to see application to solve basic science problems in structural biology and biophysics. The earliest such application envisaged for the field, and one still in development, is in crystallography, where molecules that are difficult to crystallize in isolation could be arranged within a three-dimensional nucleic acid lattice, allowing determination of their structure. Another application is the use of DNA origami rods to replace liquid crystals in residual dipolar coupling experiments in protein NMR spectroscopy; using DNA origami is advantageous because, unlike liquid crystals, they are tolerant of the detergents needed to suspend membrane proteins in solution. DNA walkers have been used as nanoscale assembly lines to move nanoparticles and direct chemical synthesis. Further, DNA origami structures have aided in the biophysical studies of enzyme function and protein folding.

DNA nanotechnology is moving toward potential real-world applications. The ability of nucleic acid arrays to arrange other molecules indicates its potential applications in molecular scale electronics. The assembly of a nucleic acid structure could be used to template the assembly of molecular electronic elements such as molecular wires, providing a method for nanometer-scale control of the placement and overall architecture of the device analogous to a molecular breadboard. DNA nanotechnology has been compared to the concept of programmable matter because of the coupling of computation to its material properties.

In a study conducted by a group of scientists from iNANO and CDNA centers in Aarhus University, researchers were able to construct a small multi-switchable 3D DNA Box Origami. The proposed nanoparticle was characterized by atomic force microscopy (AFM), transmission electron microscopy (TEM) and Förster resonance energy transfer (FRET). The constructed box was shown to have a unique reclosing mechanism, which enabled it to repeatedly open and close in response to a unique set of DNA or RNA keys. The authors proposed that this "DNA device can potentially be used for a broad range of applications such as controlling the function of single molecules, controlled drug delivery, and molecular computing."

There are potential applications for DNA nanotechnology in nanomedicine, making use of its ability to perform computation in a biocompatible format to make "smart drugs" for targeted drug delivery, as well as for diagnostic applications. One such system being investigated uses a hollow DNA box containing proteins that induce apoptosis, or cell death, that will only open when in proximity to a cancer cell. There has additionally been interest in expressing these artificial structures in engineered living bacterial cells, most likely using the transcribed RNA for the assembly, although it is unknown whether these complex structures are able to efficiently fold or assemble in the cell's cytoplasm. If successful, this could enable directed evolution of nucleic acid nanostructures. Scientists at Oxford University reported the self-assembly of four short strands of synthetic DNA into a cage which can enter cells and survive for at least 48 hours. The fluorescently labeled DNA tetrahedra were found to remain intact in the laboratory cultured human kidney cells despite the attack by cellular enzymes after two days. This experiment showed the potential of drug delivery inside the living cells using the DNA ‘cage’. A DNA tetrahedron was used to deliver RNA Interference (RNAi) in a mouse model, reported a team of researchers in MIT. Delivery of the interfering RNA for treatment has showed some success using polymer or lipid, but there are limits of safety and imprecise targeting, in addition to short shelf life in the blood stream. The DNA nanostructure created by the team consists of six strands of DNA to form a tetrahedron, with one strand of RNA affixed to each of the six edges. The tetrahedron is further equipped with targeting protein, three folate molecules, which lead the DNA nanoparticles to the abundant folate receptors found on some tumors. The result showed that the gene expression targeted by the RNAi, luciferase, dropped by more than half. This study shows promise in using DNA nanotechnology as an effective tool to deliver treatment using the emerging RNA Interference technology. The DNA tetrahedron was also used in an effort to overcome the phenomena multidrug resistance. Doxorubicin (DOX) was conjugated with the tetrahedron and was loaded into MCF-7 breast cancer cells that contained the P-glycoprotein drug efflux pump. The results of the experiment showed the DOX was not being pumped out and apoptosis of the cancer cells was achieved. The tetrahedron without DOX was loaded into cells to test its biocompatibility, and the structure showed no cytotoxicity itself. The DNA tetrahedron was also used as barcode for profiling the subcellular expression and distribution of proteins in cells for diagnostic purposes. The tetrahedral-nanostructured showed enhanced signal due to higher labeling efficiency and stability.

Applications for DNA nanotechnology in nanomedicine also focus on mimicking the structure and function of naturally occurring membrane proteins with designed DNA nanostructures. In 2012, Langecker et al. introduced a pore-shaped DNA origami structure that can self-insert into lipid membranes via hydrophobic cholesterol modifications and induce ionic currents across the membrane. This first demonstration of a synthetic DNA ion channel was followed by a variety of pore-inducing designs ranging from a single DNA duplex, to small tile-based structures, and large DNA origami transmembrane porins. Similar to naturally occurring protein ion channels, this ensemble of synthetic DNA-made counterparts thereby spans multiple orders of magnitude in conductance. The study of the membrane-inserting single DNA duplex showed that current must also flow on the DNA-lipid interface as no central channel lumen is present in the design that lets ions pass across the lipid bilayer. This indicated that the DNA-induced lipid pore has a toroidal shape, rather than cylindrical, as lipid headgroups reorient to face towards the membrane-inserted part of the DNA. Researchers from the University of Cambridge and the University of Illinois at Urbana-Champaign then demonstrated that such a DNA-induced toroidal pore can facilitate rapid lipid flip-flop between the lipid bilayer leaflets. Utilizing this effect, they designed a synthetic DNA-built enzyme that flips lipids in biological membranes orders of magnitudes faster than naturally occurring proteins called scramblases. This development highlights the potential of synthetic DNA nanostructures for personalized drugs and therapeutics.

Design

DNA nanostructures must be rationally designed so that individual nucleic acid strands will assemble into the desired structures. This process usually begins with specification of a desired target structure or function. Then, the overall secondary structure of the target complex is determined, specifying the arrangement of nucleic acid strands within the structure, and which portions of those strands should be bound to each other. The last step is the primary structure design, which is the specification of the actual base sequences of each nucleic acid strand.

Structural design

The first step in designing a nucleic acid nanostructure is to decide how a given structure should be represented by a specific arrangement of nucleic acid strands. This design step determines the secondary structure, or the positions of the base pairs that hold the individual strands together in the desired shape. Several approaches have been demonstrated:

• Tile-based structures. This approach breaks the target structure into smaller units with strong binding between the strands contained in each unit, and weaker interactions between the units. It is often used to make periodic lattices, but can also be used to implement algorithmic self-assembly, making them a platform for DNA computing. This was the dominant design strategy used from the mid-1990s until the mid-2000s, when the DNA origami methodology was developed.
• Folding structures. An alternative to the tile-based approach, folding approaches make the nanostructure from one long strand, which can either have a designed sequence that folds due to its interactions with itself, or it can be folded into the desired shape by using shorter, "staple" strands. This latter method is called DNA origami, which allows forming nanoscale two- and three-dimensional shapes (see Discrete structures above).
• Dynamic assembly. This approach directly controls the kinetics of DNA self-assembly, specifying all of the intermediate steps in the reaction mechanism in addition to the final product. This is done using starting materials which adopt a hairpin structure; these then assemble into the final conformation in a cascade reaction, in a specific order (see Strand displacement cascades below). This approach has the advantage of proceeding isothermally, at a constant temperature. This is in contrast to the thermodynamic approaches, which require a thermal annealing step where a temperature change is required to trigger the assembly and favor proper formation of the desired structure.

Sequence design

Main article: Nucleic acid design

After any of the above approaches are used to design the secondary structure of a target complex, an actual sequence of nucleotides that will form into the desired structure must be devised. Nucleic acid design is the process of assigning a specific nucleic acid base sequence to each of a structure's constituent strands so that they will associate into a desired conformation. Most methods have the goal of designing sequences so that the target structure has the lowest energy, and is thus the most thermodynamically favorable, while incorrectly assembled structures have higher energies and are thus disfavored. This is done either through simple, faster heuristic methods such as sequence symmetry minimization, or by using a full nearest-neighbor thermodynamic model, which is more accurate but slower and more computationally intensive. Geometric models are used to examine tertiary structure of the nanostructures and to ensure that the complexes are not overly strained.

Nucleic acid design has similar goals to protein design. In both, the sequence of monomers is designed to favor the desired target structure and to disfavor other structures. Nucleic acid design has the advantage of being much computationally easier than protein design, because the simple base pairing rules are sufficient to predict a structure's energetic favorability, and detailed information about the overall three-dimensional folding of the structure is not required. This allows the use of simple heuristic methods that yield experimentally robust designs. Nucleic acid structures are less versatile than proteins in their function because of proteins' increased ability to fold into complex structures, and the limited chemical diversity of the four nucleotides as compared to the twenty proteinogenic amino acids.

Materials and methods

Gel electrophoresis methods, such as this formation assay on a DX complex, are used to ascertain whether the desired structures are forming properly. Each vertical lane contains a series of bands, where each band is characteristic of a particular reaction intermediate.

The sequences of the DNA strands making up a target structure are designed computationally, using molecular modeling and thermodynamic modeling software. The nucleic acids themselves are then synthesized using standard oligonucleotide synthesis methods, usually automated in an oligonucleotide synthesizer, and strands of custom sequences are commercially available. Strands can be purified by denaturing gel electrophoresis if needed, and precise concentrations determined via any of several nucleic acid quantitation methods using ultraviolet absorbance spectroscopy.

The fully formed target structures can be verified using native gel electrophoresis, which gives size and shape information for the nucleic acid complexes. An electrophoretic mobility shift assay can assess whether a structure incorporates all desired strands. Fluorescent labeling and Förster resonance energy transfer (FRET) are sometimes used to characterize the structure of the complexes.

Nucleic acid structures can be directly imaged by atomic force microscopy, which is well suited to extended two-dimensional structures, but less useful for discrete three-dimensional structures because of the microscope tip's interaction with the fragile nucleic acid structure; transmission electron microscopy and cryo-electron microscopy are often used in this case. Extended three-dimensional lattices are analyzed by X-ray crystallography.