共查询到20条相似文献,搜索用时 15 毫秒
1.
Sam Kriegman Douglas Blackiston Michael Levin Josh Bongard 《Proceedings of the National Academy of Sciences of the United States of America》2021,118(49)
All living systems perpetuate themselves via growth in or on the body, followed by splitting, budding, or birth. We find that synthetic multicellular assemblies can also replicate kinematically by moving and compressing dissociated cells in their environment into functional self-copies. This form of perpetuation, previously unseen in any organism, arises spontaneously over days rather than evolving over millennia. We also show how artificial intelligence methods can design assemblies that postpone loss of replicative ability and perform useful work as a side effect of replication. This suggests other unique and useful phenotypes can be rapidly reached from wild-type organisms without selection or genetic engineering, thereby broadening our understanding of the conditions under which replication arises, phenotypic plasticity, and how useful replicative machines may be realized.Like the other necessary abilities life must possess to survive, replication has evolved into many diverse forms: fission, budding, fragmentation, spore formation, vegetative propagation, parthenogenesis, sexual reproduction, hermaphroditism, and viral propagation. These diverse processes however share a common property: all involve growth within or on the body of the organism. In contrast, a non–growth-based form of self-replication dominates at the subcellular level: molecular machines assemble material in their external environment into functional self-copies directly, or in concert with other machines. Such kinematic replication has never been observed at higher levels of biological organization, nor was it known whether multicellular systems were even capable of it.Despite this lack, organisms do possess deep reservoirs of adaptive potential at all levels of organization, allowing for manual or automated interventions that deflect development toward biological forms and functions different from wild type (1), including the growth and maintenance of organs independent of their host organism (2–4), or unlocking regenerative capacity (5–7). Design, if framed as morphological reconfiguration, can reposition biological tissues or redirect self-organizing processes to new stable forms without recourse to genomic editing or transgenes (8). Recent work has shown that individual, genetically unmodified prospective skin (9) and heart muscle (10) cells, when removed from their native embryonic microenvironments and reassembled, can organize into stable forms and behaviors not exhibited by the organism from which the cells were taken, at any point in its natural life cycle. We show here that if cells are similarly liberated, compressed, and placed among more dissociated cells that serve as feedstock, they can exhibit kinematic self-replication, a behavior not only absent from the donating organism but from every other known plant or animal. Furthermore, replication does not evolve in response to selection pressures, but arises spontaneously over 5 d given appropriate initial and environmental conditions. 相似文献
2.
INAUGURAL ARTICLE by a Recently Elected Academy Member:Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Yee-Hung M. Chan Bettina van Lengerich Steven G. Boxer 《Proceedings of the National Academy of Sciences of the United States of America》2009,106(4):979-984
Synthetic lipid–oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5′-coupled conjugate and the other the 3′-coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane–membrane spacing on fusion, a series of conjugates was constructed by adding 2–24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction. 相似文献
3.
Kwame Sefah Zunyi Yang Kevin M. Bradley Shuichi Hoshika Elizabeth Jiménez Liqin Zhang Guizhi Zhu Savita Shanker Fahong Yu Diane Turek Weihong Tan Steven A. Benner 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(4):1449-1454
Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson–Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS–SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS–SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS–SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS–SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.A quarter century ago, various laboratories independently sought to apply in vitro selection, commonly called “SELEX” (systematic evolution of ligands by exponential enrichment), to create DNA and RNA (collectively “xNA”) “aptamers”, xNA molecules that bind specifically to many different types of targets (1–3). SELEX follows a simple recipe that includes (i) making a library of nucleic acid molecules; (ii) placing the library in contact with the target to separate molecules in the library that bind from those that do not; (iii) amplifying “survivors” by PCR; and (iv) after a sufficient number of cycles of selection, recovering individual aptamers that are sequenced, resynthesized, and characterized as molecular entities having unambiguous molecular structures.SELEX has had substantial success, with xNA aptamers now known for many targets, including small molecules, carbohydrates, peptides, and various kinds of cancer cells (4–7). Nevertheless, considerable effort has been devoted to improving SELEX (8–10). Improvements were initially sought by adding functionalized side chains to one or more of the standard nucleotides used in the SELEX library (11–13). More recently, Perrin and coworkers have had notable success obtaining catalytic DNAzymes using heavily functionalized DNA libraries (14). Following a slightly different rationale, SomaLogic has obtained slow off-rate modified aptamers (SOMAmers) by appending hydrophobic side chains (e.g., benzyl, naphthyl, tryptamino, and isobutyl) to nucleobases (15). In one set of SOMAmers targeted against ∼800 different human proteins, affinities in the 0.1 pM–1 μM range are reported (16).However, another approach to increase the power of aptamers is to increase the number of independently replicating nucleotides in the xNA library. For example, Hirao and coworkers recently added a fifth nucleotide to aptamers against IFN-gamma and VEGF-165 (17). Adding nucleotides to xNA libraries offers the possibility of obtaining higher information density in aptamer sequences, a richer variety of folds, more control over folding interactions, and xNA libraries having sequences and functional diversity more like proteins.Another approach starts with the recognition that the two nucleobase pairs found in natural xNA [G:C and A:T(U)] do not exploit all possible hydrogen bonding patterns (Fig. 1). Therefore, rearranging hydrogen bond donor and acceptor groups on the nucleobases can increase the number of independently replicable nucleosides in xNA from 4 to 12 (forming six nucleobase pairs) (18). In this artificially expanded genetic information system (AEGIS), the 12 different nucleotide “letters” pair via six distinguishable hydrogen bonding patterns to give a system that can pair, be copied, and evolve like natural DNA, but with higher information density and more functional groups (19). This laid the grounds for the work reported here, an “AEGIS–SELEX.”Open in a separate windowFig. 1.(Left) Watson–Crick pairing follows two rules of complementarity: (i) size complementarity (large purines pair with small pyrimidines) and (ii) hydrogen bonding complementarity [hydrogen bond donors (blue) pair with hydrogen bond acceptors (red)]. Rearranging hydrogen bond donors and acceptors on the bases gives an artificially expanded genetic information system (AEGIS). An xNA biopolymer having functionalized AEGIS components may allow SELEX to yield protein-like aptamers better than the standard DNA and RNA biopolymers. The example of AEGIS–SELEX reported here adds the Z:P pair, shown at the bottom of the molecular structures. (Right) The images show the general idea behind AEGIS in cartoon form.We report here a unique example of AEGIS–SELEX. This AEGIS–SELEX exploits two additional nucleotides [2-amino-8-(1′-β-d-2-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)one, trivially called P, and 6-amino-5-nitro-3-(1′-β-d-2′-deoxyribofuranosyl)-2(1H)-pyridone, trivially called Z] (20–23). GACTZP AEGIS–SELEX is based on the following features of a molecular biology for “second generation” AEGIS that have been developed over the past few years in these laboratories:
- (i) Methods to synthesize libraries containing 1012–1014 different AEGIS oligonucleotides (20).
- (ii) Procedures to PCR amplify AEGIS nucleotides (22, 24).
- (iii) Procedures to sequence AEGIS DNA molecules that emerge after multiple rounds of selection (22).
4.
5.
Shigeki Sawada Chie Furihata Taijiro Matsushima 《Journal of cancer research and clinical oncology》1989,115(4):345-350
Summary A short-term in vivo method for assay of repair and replication of rat liver DNA has been developed, by which possible hepatocarcinogens could be identified in a few days. F344 rats were treated orally with two genotoxic hepatocarcinogens, dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2AAF), or a nongenotoxic hepatocarcinogen, carbon tetrachloride (CCl4). Then at suitable times after treatment, their hepatocytes were isolated by a twostep collagenase perfusion technique in situ and incubated with [3H]dThd with or without hydroxyurea, which inhibits DNA replication. Their nuclear DNA was then extracted and the incorporation of [3H]dThd into nuclear DNA was determined in a liquid scintillation counter. Unscheduled DNA synthesis (DNA repair), induced by DMN at doses of 2.5–10 mg/kg body weight and by 2AAF at doses of 12.5–50 mg/kg body weight, could be detected 2 h and 4 h after their administration as an increase of DNA synthesis of up to 5.8-fold and 6.0-fold, respectively, in the presence of hydroxyurea. Replicative DNA synthesis, induced by CCl4 at a dose of 200 mg/kg body weight, could be detected 48 h after its administration as a 23-fold increase of DNA synthesis in the absence of hydroxyurea and was inhibited approximately 97%–99% by hydroxyurea. Replicative DNA synthesis induced by 2AAF at a dose of 25 mg/kg body weight 16 h after its administration could be detected as a 6.8-fold increase of DNA synthesis in the absence of hydroxyurea. These results show that unscheduled and replicative DNA synthesis can be clearly distinguished by simultaneous measurements of the incorporation of [3H]dThd into nuclear DNA in the presence and absence of hydroxyurea.Abbreviations used 2AAF
2-acetylaminofluorene
- DMN
dimethylnitrosamine
- GTA
[ethylene-bis(oxyethylenenitrilo)]tetraacetic acid HBSS(-)
- Ca2+
free Hanks' balanced salt solution
- [3H]dThd
[CH3–3H]thymidine
- HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan and a grant from the Foundation for Promotion of Cancer Research of Japan 相似文献
6.
Feng Zhou Ruojie Sha Heng Ni Nadrian Seeman Paul Chaikin 《Proceedings of the National Academy of Sciences of the United States of America》2021,118(50)
Artificial self-replication and exponential growth holds the promise of gaining a better understanding of fundamental processes in nature but also of evolving new materials and devices with useful properties. A system of DNA origami dimers has been shown to exhibit exponential growth and selection. Here we introduce mutation and growth advantages to study the possibility of Darwinian-like evolution. We seed and grow one dimer species, AB, from A and B monomers that doubles in each cycle. A similar species from C and D monomers can replicate at a controlled growth rate of two or four per cycle but is unseeded. Introducing a small mutation rate so that AB parents infrequently template CD offspring we show experimentally that the CD species can take over the system in approximately six generations in an advantageous environment. This demonstration opens the door to the use of evolution in materials design.Nature has been very successful in making a wide variety of functional materials, devices, and organisms through natural selection. DNA origamis are convenient starting points for artificial materials evolution. Not only can they be designed to form almost arbitrary shapes (1–6), but also they can be joined programmably with each other to make objects on a larger scale (7–9). Further, they can precisely organize other materials such as nanoparticles (10, 11), nanotubes (12, 13), and enzymes (14). Previously we have demonstrated self-replication, exponential growth, and selection in DNA origami tiles. The basic replication scheme is to template the transfer of structure and information from one generation to the next (15–18). This involves specific recognition of elementary units, organization, and reversible and irreversible binding. However, the initial efforts were to make the system as error-free as possible, and in fact the experiments showed only copies of the seeded patterns. The recognition strands were sufficiently specific that they would only hybridize to their complementary strands ensuring correct templating. It has also been demonstrated that exponential growth with a well-controlled growth rate can be achieved in the DNA origami artificial self-replication system (19, 20). As a start on evolutionary pressure to evolve new and better materials and devices we now want to introduce mistakes, mutations.Errors, i.e., mutations, can occur in self-replication when we change the recognition strands. The original DNA origami species were self-complementary in the recognition strands sequence. Now we make them self-complementary and almost complementary to the mutated species by using strands that differ by one to five nucleotides between the two species. With the cross-over we should have a mixture of original and mutated species after many replication cycles, similar to the historical example of the peppered moth where the fittest species dominated but neither species went extinct (21).Here, we report the study of the mutation and evolution of an artificial self-replication system of DNA origami dimer rafts. This represents a first step toward using such mutations toward directed evolution of an artificial system and illustrates some of the basic principles of natural selection. We designed two self-replicating species AB and CD which share the same replication procedure, but with a controllable growth rate. Starting with monomers, A, B or C, D, there is negligible growth of dimers in the absence of AB or CD templating seeds; any dimers that are formed are sterile and do not replicate (19). If we were to introduce an AB seed with no mutation possible then AB would exponentially grow and there would be negligible CD. By introducing a small error (3 bases) in the AB recognition sticky strands (42 bases in total), there was a small chance of ∼3% for the system to mutate templating a CD which can replicate itself effectively, starting a new species. The mutation rate is small, but the mutated nanostructure shares the same replication ability as the original dimer. Although the original dimers are dominant at the beginning of self-replication, after many replication cycles we should have an equal mixture of mutated and original dimers if AB and CD have the same growth rates. In addition, we can create differences in the self-replication rates of AB and CD species and give growth advantage to the CD structures; then, after many replication cycles, the mutated species will take over the system. We can use the functionality of the different species to affect this takeover. Mutation and population domination by the fittest species would amount to natural selection in this artificial system. With an eye toward using this process for directed evolution and the fact that a high mutation rate leads to an Eigen catastrophe (22), or a species does not persist long enough to take advantage of its evolutionary advantage, we have kept the mutation rate low, although not yet as low as in living systems. In the present case a low mutation rate is particularly important in that the forward and reverse mutations are equally limiting the final ratio of the species with high and low growth advantage. 相似文献
7.
8.
Alexander E. Marras Lifeng Zhou Hai-Jun Su Carlos E. Castro 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(3):713-718
DNA origami enables the precise fabrication of nanoscale geometries. We demonstrate an approach to engineer complex and reversible motion of nanoscale DNA origami machine elements. We first design, fabricate, and characterize the mechanical behavior of flexible DNA origami rotational and linear joints that integrate stiff double-stranded DNA components and flexible single-stranded DNA components to constrain motion along a single degree of freedom and demonstrate the ability to tune the flexibility and range of motion. Multiple joints with simple 1D motion were then integrated into higher order mechanisms. One mechanism is a crank–slider that couples rotational and linear motion, and the other is a Bennett linkage that moves between a compacted bundle and an expanded frame configuration with a constrained 3D motion path. Finally, we demonstrate distributed actuation of the linkage using DNA input strands to achieve reversible conformational changes of the entire structure on ∼minute timescales. Our results demonstrate programmable motion of 2D and 3D DNA origami mechanisms constructed following a macroscopic machine design approach.The ability to control, manipulate, and organize matter at the nanoscale has demonstrated immense potential for advancements in industrial technology, medicine, and materials (1–3). Bottom-up self-assembly has become a particularly promising area for nanofabrication (4, 5); however, to date designing complex motion at the nanoscale remains a challenge (6–9). Amino acid polymers exhibit well-defined and complex dynamics in natural systems and have been assembled into designed structures including nanotubes, sheets, and networks (10–12), although the complexity of interactions that govern amino acid folding make designing complex geometries extremely challenging. DNA nanotechnology, on the other hand, has exploited well-understood assembly properties of DNA to create a variety of increasingly complex designed nanostructures (13–15).Scaffolded DNA origami, the process of folding a long single-stranded DNA (ssDNA) strand into a custom structure (16–18), has enabled the fabrication of nanoscale objects with unprecedented geometric complexity that have recently been implemented in applications such as containers for drug delivery (19, 20), nanopores for single-molecule sensing (21–23), and templates for nanoparticles (24, 25) or proteins (26–28). The majority of these and other applications of DNA origami have largely focused on static structures. Natural biomolecular machines, in contrast, have a rich diversity of functionalities that rely on complex but well-defined and reversible conformational changes. Currently, the scope of biomolecular nanotechnology is limited by an inability to achieve similar motion in designed nanosystems.DNA nanotechnology has enabled critical steps toward that goal starting with the work of Mao et al. (29), who developed a DNA nanostructure that took advantage of the B–Z transition of DNA to switch states. Since then, efforts to fabricate dynamic DNA systems have primarily focused on strand displacement approaches (30) mainly on systems comprising a few strands or arrays of strands undergoing ∼nm-scale motions (31–37) in some cases guided by DNA origami templates (38–40). More recently, strand displacement has been used to reconfigure DNA origami nanostructures, for example opening DNA containers (19, 41, 42), controlling molecular binding (43, 44), or reconfiguring structures (45). The largest triggerable structural change was achieved by Han et al. in a DNA origami Möbius strip (one-sided ribbon structure) that could be opened to approximately double in size (45). Constrained motion has been achieved in systems with rotational motion (19, 20, 32, 41, 44, 46, 47) in some cases to open lid-like components (19, 20, 41) or detect molecular binding (44, 48, 49). A few of these systems achieved reversible conformational changes (32, 41, 44, 46), although the motion path and flexibility were not studied. Constrained linear motion has remained largely unexplored. Linear displacements on the scale of a few nanometers have been demonstrated via conformational changes of DNA structure motifs (50–55), strand invasion to open DNA hairpins (36, 55, 56), or the reversible sliding motion of a DNA tile actuator (56); these cases also did not investigate the motion path or flexibility of motion.Building on these prior studies, this work implements concepts from macroscopic machine design to build modular parts with constrained motion. We demonstrate an ability to tune the flexibility and range of motion and then integrate these parts into prototype mechanisms with designed 2D and 3D motion. We further demonstrate reversible actuation of a mechanism with complex conformational changes on minute timescales. 相似文献
9.
Shimyn Slomovic Keith Pardee James J. Collins 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(47):14429-14435
There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms. 相似文献
10.
Zeevi V Tovkach A Tzfira T 《Proceedings of the National Academy of Sciences of the United States of America》2008,105(35):12785-12790
The ability to accurately digest and ligate DNA molecules of different origins is fundamental to modern recombinant DNA research. Only a handful of enzymes are capable of recognizing and cleaving novel and long DNA sequences, however. The slow evolution and engineering of new restriction enzymes calls for alternative strategies to design novel and unique restriction enzymes capable of binding and digesting specific long DNA sequences. Here we report on the use of zinc finger nucleases (ZFNs)—hybrid synthetic restriction enzymes that can be specifically designed to bind and cleave long DNA sequences—for the purpose of DNA recombination. We show that novel ZFNs can be designed for the digestion of specific sequences and can be expressed and used for cloning purposes. We also demonstrate the power of ZFNs in DNA cloning by custom-cloning a target DNA sequence and assembling dual-expression cassettes on a single target plasmid, a task that rarely can be achieved using type-II restriction enzymes. We demonstrate the flexibility of ZFN design and the ability to shuffle monomers of different ZFNs for the digestion of compatible recognition sites through ligation of compatible ends and their cleavage by heterodimer ZFNs. Of no less importance, we show that ZFNs can be designed to recognize and cleave existing DNA sequences for the custom-cloning of native target DNA molecules. 相似文献
11.
Rizal F. Hariadi Erik Winfree Bernard Yurke 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(45):E6086-E6095
Quantifying the mechanical forces produced by fluid flows within the ocean is critical to understanding the ocean’s environmental phenomena. Such forces may have been instrumental in the origin of life by driving a primitive form of self-replication through fragmentation. Among the intense sources of hydrodynamic shear encountered in the ocean are breaking waves and the bursting bubbles produced by such waves. On a microscopic scale, one expects the surface-tension–driven flows produced during bubble rupture to exhibit particularly high velocity gradients due to the small size scales and masses involved. However, little work has examined the strength of shear flow rates in commonly encountered ocean conditions. By using DNA nanotubes as a novel fluid flow sensor, we investigate the elongational rates generated in bursting films within aqueous bubble foams using both laboratory buffer and ocean water. To characterize the elongational rate distribution associated with a bursting bubble, we introduce the concept of a fragmentation volume and measure its form as a function of elongational flow rate. We find that substantial volumes experience surprisingly large flow rates: during the bursting of a bubble having an air volume of 10 mm3, elongational rates at least as large as s−1 are generated in a fragmentation volume of ~ 2 × 10?6
μL. The determination of the elongational strain rate distribution is essential for assessing how effectively fluid motion within bursting bubbles at the ocean surface can shear microscopic particles and microorganisms, and could have driven the self-replication of a protobiont.Functioning like a giant heat engine between the high-temperature heat bath of the sun and the low-temperature heat bath of outer space, the earth’s atmosphere generates wind and rain with intense fluid flows. The mechanical stresses produced by these hydrodynamic flows are among the environmental stresses that biological organisms must cope with. Organisms often exploit these stresses and fluid flows, most notably to aid reproduction. A well-known example at the macroscopic scale is the wind dispersal of spores, seeds, and pollen. Less well-known, fragmentation resulting from fluid-flow-induced stress is used by a number of marine organisms as a means of vegetative reproduction, such as macrophyte algae (1) and sponge and coral colony (2, 3) propagation by storm-induced fragmentation. Can analogous mechanical forces facilitate vegetative reproduction at the microscale? Current evidence is at best indirect: Filamentous cyanobacteria are known to fragment under environmental stress (4, 5), suggesting that prokaryotes may use fluid-flow-induced fragmentation as a means of clonal reproduction and dispersal as well.Several origin-of-life hypotheses invoke processes in which environmentally produced microscale mechanical forces drive self-replication through fragmentation. Oparin proposed that fragmentation of coacervates may have constituted a primitive form of self-replication allowing for Darwinian evolution by natural selection (6). More concretely, Cairns-Smith proposed that life arose from mineral crystals that replicated by fragmentation into new seed crystals, thereby propagating genetic information consisting of the patterns of defects within the mother crystal (7–9). Szostak’s group also proposed that division of protocells (having a lipid bilayer) can be driven by fluid flow (10–12). Inspired by Cairns-Smith’s proposal, Schulman, Yurke, and Winfree used DNA tile self-assembly to construct a self-replicating system in which fragmentation was induced by intense elongational flow at a constriction in a flow channel (13). This synthetic system is analogous to an in vitro system in which exponential growth of prions is driven via fragmentation by mechanical shearing of amyloid fibrils (14). Similarities between regeneration (self-healing) and asexual reproduction in modern organisms have led some to postulate fragmentation-and-regeneration as a primordial form of reproduction (15).To effectively shear microscopic objects such as bacteria or protobionts, fluid flows must exhibit high-velocity gradients over the length scale of the object. Such small-scale high-velocity-gradient flows occur naturally in breaking ocean waves that produce whitecaps (16). Within these waves, the highest velocity gradients are expected to occur in the films of bursting bubbles due to the rapid acceleration produced by surface tension forces acting on the small fluid mass. Moreover, bursting bubbles can generate mechanical stresses of sufficient intensity to be biologically relevant to organisms living in the sea surface microlayer (neuston) (17). As a technological example, cell death at the air–liquid interface during the sparging of bioreactors to enhance oxygen diffusion has been attributed to bubble bursting (18).The fluid flows most effective at shearing small free-floating objects are those that exhibit strain deformation. In such flows, a rod-shaped object will tend to align itself along the direction of maximum fluid extension. In this orientation the rod experiences the greatest tensile stress and is most susceptible to fragmentation. In a given fluid element, the rate of fluid extension––i.e., the rate at which two points in the fluid separate, divided by the distance between them––has a maximum that is referred to as the elongational rate and is here denoted by . A useful way to conceptualize the meaning of is to consider the case when it is constant. In this case, the time it takes for the fluid element to double its length is .Fig. 1 shows a mechanism by which rod-shaped objects within bursting fluid films can be fragmented. As the hole produced in the bubble film expands, its circumference increases (Fig. 1B). Due to this, fluid elements near the hole’s edge will experience elongation in the direction perpendicular to the velocity of the hole edge. As shown in Fig. 1 C and D, rod-shaped structures within the bubble film will align along the circumference of the expanding hole. If the tension generated along the length of the structure exceeds its tensile strength, the structure will fragment (Fig. 1E).Open in a separate windowFig. 1.DNA nanotubes fragmentation by bursting bubbles. Side (A) and top (B) views of a bubble filled with air bursting on a water surface. Color gradient loosely corresponds to the expected magnitude of the hydrodynamic forces. (B–E) As the hole travels outward driven by surface tension, the liquid film is accumulated into a growing toroidal rim (gray rings). The enlargement of the hole produces elongational flow, with rate , which is tangential to the perimeter where r is the hole radius and v is the outward velocity of the hole perimeter. The two blue arcs are the two volume elements of the bursting film. (C and D). As the hole expands, the fluid flow orients DNA nanotubes (black, red). (D and E) The elongational flow breaks sufficiently long DNA nanotubes (black) of length l into fragments of length l1 and l2 due to tension applied to the nanotube by the elongational fluid flow. Short nanotubes (red) are not fragmented due to insufficient build-up of tension.The elongational rates generated by this mechanism can be estimated using a model of film hole dynamics, for a film of uniform thickness. Initially considered by Dupré (19), Rayleigh (20), and Ranz (21), then corrected by Culick (22) and Taylor (23), the model treats the rupture as a circular hole that propagates outward with the film fluid accumulating in a toroid at the hole perimeter. From momentum balance, the hole propagates outward with a constant speed , where σ is the surface tension of the film, ρ is the fluid density, and δ is the film thickness. The elongational rate of the circumference is given by , where r is the hole radius. The volume of fluid subjected to elongational rates greater than , in this simple model, is given by , which is, surprisingly, independent of film thickness. Such volumes provide a natural way to characterize the ability of a bursting bubble to fragment objects suspended within the bubble film that will shear under given elongational rate . Importantly, can be defined in a model-independent way as the volume of fluid that experiences elongational rates greater than during the course of bubble bursting. We will refer to such volumes as fragmentation volumes.Although easiest to explain, the Culick and Taylor model does not describe the only type of elongational flow that can be generated within a bursting bubble; therefore the estimate for based on this model should be considered a loose lower bound for the true value. In fact, only half the surface-tension energy released is converted into the kinetic energy of the outward motion of the fluid (24). This suggests that the other half of the surface-tension energy must be dissipated within the film near the edge of the hole. For fluids with low viscosity, such as water, this implies that there are intense small-scale fluid flows near the edge of the hole (18) in addition to those illustrated in Fig. 1. Hydrodynamic instabilities, particularly with larger bubbles, can occur along the perimeter, resulting in fingering and the formation of droplets (25). Also, particularly for small bubbles, the expanding hole produces an inward propagating wave at the bottom surface of the bubble that forms a jet that may launch droplets (26, 27). High-flow gradients are expected in the region where these jets pinch to form droplets. A recent discussion of droplet production during bubble bursting in ocean-like (i.e., not soapy) water was given by Lhuissier and Villermaux (28).As discussed by Lhuissier and Villermaux, at the instant the bubble bursts it possesses a cap of uniform thickness that, at a well-defined edge, joins with the bulk fluid in a region where the thickness rapidly increases with distance from the center of the bubble (Fig. 1A). The critical thickness at which bubble films spontaneously burst depends on the bubble radius (28), increasing from 0.05 to 30 μm as the bubble radius increases from 1 to 20 mm. For an object such as a microorganism to be fully impacted by the mechanical stresses produced by bubble bursting, it would have to reside in the cap film or close to the cap boundary; that is, its thinnest dimension would have to be smaller than the film thickness. Nevertheless, mechanical stresses produced by bursting bubbles are among the stresses that microorganisms, particularly those that occupy the niche consisting of the neuston or sea surface microlayer (29–31), must cope with. We hypothesize that protobionts, small enough to be suspended within the bubble film, also occupied this niche and used these stresses to facilitate replication. However, little work seems to have been done to characterize the elongational rates produced during the bursting of a bubble that would facilitate assessing whether forces of sufficient magnitude are generated.In this study the fragmentation of DNA nanotubes is used to characterize both the magnitude of the elongational rates produced and the volume of fluid subjected to these elongational rates during bubble bursting. These nanotubes are constructed from short DNA oligomers referred to as single-stranded tiles, which each have four sequence domains by which a given oligomer binds with four neighboring oligomers via Watson–Crick base pairing. Thus, whereas each DNA oligomer is held together by covalent bonds, the entire tube assembly is held together by the supramolecular interactions that enable two complementary single-stranded oligomers to form duplex DNA. Base sequences of the oligomers are designed so that the axes of the duplex DNA are parallel to the long axis of the tube. The supramolecular Watson–Crick bonds between neighboring single-stranded tiles are much weaker than the covalent bonds of the phosphate backbone of a single-stranded tile (32). Under sudden tension along the axis of the duplex DNA (33), the tensile force at which the supramolecular bonding fails is referred to as the overstretching force fc, which has a value of about 65 pN (34, 35). For a tube in which there are n duplex strands in cross-section, the tensile force will be Tc = nfc. For the tubes used in the experiments reported here, n = 7 (SI Appendix, Fig. S1) and consequently the tubes fragment when subjected to tensile forces in excess of 455 pN. The tubes have a radius of 4 nm, a persistence length of 5 μm (36), and a length distribution that peaks at 5 μm at the start of the experiment (SI Appendix, Fig. S2; see Fig. 3 B and G).Open in a separate windowFig. 3.Nanotube length distributions for bubble bursting experiments in assay buffer or in ocean water. Fluorescence microscopy images and fragment length distributions of DNA nanotubes withdrawn from a sample with an initial volume of 100 μL after 0 mL (A and B), 60 mL (C and D), and 360 mL (E and F) of air had passed through the sample at a flow rate of 18 mL/min. The mean tube length ?l? for each distribution is given at the top of each histogram. Nanotube length distribution in bubble bursting experiment with ocean water after 0 mL (G), 60 mL (H), and 360 mL (I) of air.Some of these tubes will be trapped in the bubble film and will be subjected to elongational forces in the manner illustrated in Fig. 1. Although nanotubes will necessarily also be subject to compressive fluid flows, they are not fragile under compression. The junctions at which a given single-stranded tile connects with two neighboring tiles are flexible, allowing the tube to crumple and then straighten when the compressive forces are relieved. This collapse into a coil configuration followed by stretching has been studied for other stiff linear biopolymers and synthetic fibers in hydrodynamic flows near stagnation points (33, 37, 38). Generally, the tubes will crumple under the compressive flow and reorient and stretch along the axis of the elongational flow (Movie S1 and SI Appendix, section 10). A further complication is that Brownian motion will tend to counteract the alignment produced by the elongational flow. However, as will be shown, under the conditions in which our DNA nanotubes break, the Péclet number––which is the ratio of the active transport rate to the diffusive transport rate––is in excess of 1.4 × 104, indicating that diffusive misalignment of the nanotubes plays a negligible role in our experiments. Due to the viscous stresses exerted on the DNA nanotube as it reorients along the direction of maximum extension flow, the tension experienced by the tube will be greatest at the center of the tube and will be greater for longer tubes, scaling as , where l is the tube length and R the tube radius (34). If the tensile force is exceeded, the tube will break into two fragments of nearly equal length. If the elongational flow continues to intensify so that the two fragments experience a tension at their centers that exceeds the tensile force, each of these in turn will fragment into two shorter pieces of equal length. This cascading process will continue until the elongational rate reaches its maximum value.DNA nanotubes are well-suited to serve as probes of hydrodynamic flows within bubble films for three reasons. First, their fragmentation in elongational flows has already been extensively characterized (34). Second, they are highly soluble in water and do not exhibit a surfactant-like tendency to stick to the air–water interface, unlike many proteins. Third, it is straightforward to measure histograms of nanotube lengths using fluorescence microscopy. Here, from the evolution of the DNA nanotube fragment length during the course of bubbling, we were able to determine the fragmentation volumes for elongational rates over five orders of magnitude, although our experimental techniques were not able to distinguish where the DNA nanotubes were broken within the bubble. Our findings suggest that, via bubbles, ocean waves provide a source of strong mechanical forces at the micron-scale mechanical stresses that ocean surface-dwelling microbes must cope with, that may be involved in the natural breakdown of pollutants, and that would have been available for protobionts to use as a means of driving self-replication. 相似文献
12.
Silvia Biffi Roberto Cerbino Francesca Bomboi Elvezia Maria Paraboschi Rosanna Asselta Francesco Sciortino Tommaso Bellini 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(39):15633-15637
Colloidal particles with directional interactions are key in the realization of new colloidal materials with possibly unconventional phase behaviors. Here we exploit DNA self-assembly to produce bulk quantities of “DNA stars” with three or four sticky terminals, mimicking molecules with controlled limited valence. Solutions of such molecules exhibit a consolution curve with an upper critical point, whose temperature and concentration decrease with the valence. Upon approaching the critical point from high temperature, the intensity of the scattered light diverges with a power law, whereas the intensity time autocorrelation functions show a surprising two-step relaxation, somehow reminiscent of glassy materials. The slow relaxation time exhibits an Arrhenius behavior with no signs of criticality, demonstrating a unique scenario where the critical slowing down of the concentration fluctuations is subordinate to the large lifetime of the DNA bonds, with relevant analogies to critical dynamics in polymer solutions. The combination of equilibrium and dynamic behavior of DNA nanostars demonstrates the potential of DNA molecules in diversifying the pathways toward collective properties and self-assembled materials, beyond the range of phenomena accessible with ordinary molecular fluids.In recent years, a strong effort has been devoted to introduce a new generation of micro- and nanocolloids interacting via strongly anisotropic forces. Anisotropic interactions can simply arise from a nonspherical particle shape or from more sophisticated physical and/or chemical patterning of the particle surface (1–7). An alternative strategy to produce complex nanoparticles is to exploit the self-assembly of DNA oligomers. The rational design of the DNA sequences enables guiding the association of multiple DNA strands into a rich variety of nanosized objects, such as geometrical figures, hollow capsules, and nanomachines, as well as more complex meso- and macroscopic structures (8–13). The selectivity of DNA binding can also be exploited to control the mutual interactions between the structures (14, 15), whereas the spontaneous assembly of DNA sequences enables producing large ensembles of particles. These properties make DNA a powerful tool to explore fundamental phenomena of soft matter and statistical physics, as indicated by previous studies of liquid-crystalline ordering and phase separations in solutions of short DNA oligomers (16–18). Here we exploit DNA self-assembly to experimentally address the phase behavior of particles interacting with specific valence, strength, and selectivity.Colloidal particles with controlled valence are the next step toward the realization of new colloidal materials and phases dependent on the presence of a small number of bonds (1–7). Theoretical and numerical studies (19) predict that a solution of low-valence particles should exhibit phase coexistence—the colloidal analog of the vapor–liquid coexistence in simple liquids—but only at very small concentrations. The unstable region in the temperature–concentration plane is expected to significantly shrink, with critical temperature and critical concentration decreasing and approaching zero as the valence is reduced. Indirect support to theoretical predictions comes from recent experiments (20), which have interpreted the irreversible aging dynamics of a synthetic clay as an equilibrium gelation process (21) by invoking an effective (although unknown) limited valence of the clay particles. However, despite these promising findings, the absence of a strategy for realizing bulk quantities of particles with reversible interactions and with controlled valence (1) has until now hampered the experimental investigation of the systematic dependence of the coexistence curve on the valence.Here we focus on DNA molecules with valence f = 3 and 4, the latter potentially reproducing on a colloidal length scale the behavior of silica and of other network-forming molecules like water (22). Particles are shaped as nanostars having f arms with sticky tips. At variance with DNA structures aimed at the formation of 2D and 3D crystals, which required a big effort in carefully determining the optimal location of the interacting sequences, the liquid–vapor transition is expected to be rather insensitive to the position of the sticky spots (23), giving us the benefit of basing our study on the simplest structures granting controlled valence. DNA nanostars are obtained by dissolving in water equimolar quantities of f distinct 49-base-long oligonucleotides. Sequences are designed to self-assemble around , forming structures with f double-stranded arms of 20 bases each (Fig. 1 A and B). To enable angular flexibility between different arms, bases with no complementary partner were inserted between the arm-forming sequences. Each arm terminates with an equal six-nucleotide-long overhang of sequence CGATCG. This self-complementary sequence promotes nanostars association via Watson–Crick pairing of the overhangs of close-by structures. We used the same overhang sequence for and nanostars to provide identical interaction strength in structures of different f. Details about sequences, sample preparation, nanostars assembly, and energy evaluations are given in Materials and Methods and in SI Text. Because the binding between sticky overhangs is stronger than all other interparticle interactions (excluded volume, van der Waals, electrostatic), DNA nanostars provide an optimal model for highlighting the role of the valence. Similar DNA nanostars were studied by Luo and coworkers (24, 25) to investigate their gelation in the presence of enzymatic catalysis. We operate in the absence of any enzymes to benefit the reversibility of the DNA interaction and systematically investigate the equilibrium phase behavior.Open in a separate windowFig. 1.Phase behavior of DNA nanostars with valence and . (A) The and (B) nanostars are formed by the self-assembly of three and four oligomers, respectively. Arm tips terminate with one sticky overhang each. (C) Fluorescent emission from a capillary tube containing a sample of EtBr-marked nanostars photographed after the sample was centrifuged at two different T (as indicated by the green and magenta dots in E). At low-enough T (magenta-framed picture, Right), the system phase separates into DNA-rich and DNA-poor phases. (D) Above , DNA is single stranded. For , single strands hybridize, leading to the self-assembly of stable (blue frame, Left) and (orange frame, Right) nanostars. For , nanostars are independent. Below , interactions between sticky overhangs (see schematic at the bottom) promote the formation of clusters that grow progressively larger as T is lowered. (E) Experimentally determined consolution curve for nanostars with (blue dots) and (red dots). The nanostars have a markedly reduced coexistence region with respect to nanostars. The concentrations of the dense phases at low T correspond to nanostars molarities of 0.20 mM and 0.29 mM for and , respectively. As T is lowered from stable homogeneous conditions (green dot and C Left, green-framed picture) to a temperature within the consolution curve (magenta dot and C Right, magenta-framed picture) along the critical isochores (dashed gray arrows), the system phase separates into two coexisting phases whose concentration is indicated by the magenta tie line.We studied the phase diagram by characterizing the behavior of samples prepared at different DNA nanostars concentrations . We found a large T interval in which nanostars with desired valence were well formed but weakly interacting (Fig. 1D), coherently with the expectation that the binding between overhangs of different nanostars should start becoming relevant at . In this range of T, samples remained homogeneous with no detectable sign of a phase separation. Upon cooling enough, all investigated samples were found to phase separate into coexisting small droplets, providing evidence of a phase separation process between two phases differing into particle concentration. To properly evaluate the coexisting concentrations, each sample was centrifuged at a fixed T for several hours. For each we found a temperature such that samples centrifuged at developed a clear meniscus, whereas for no sign of a phase separation was detected (Fig. 1C). The measurement of DNA concentration via UV absorption in the two phases (SI Text) allowed us to determine the T dependence of the concentration of the coexisting phases for both and nanostars and to build the phase diagram reported in Fig. 1E.The range of where separation takes place is rather limited and decreases from to . The concentration of the dense phase is comparable to the concentration of regular networks in which DNA nanostars are fully bonded with f neighbors each. Indeed, simple geometrical considerations indicate that the DNA concentration of such networks is rather small and strongly depends on the nanostar valence. A diamond lattice formed by nanostars in which all paired arms were perfectly aligned would have a density of (see SI Text for more detail). Our findings thus indicate that the dense fluid phase has indeed a density comparable to the fully bonded network state. We also find that the critical temperature decreases with decreasing f, again in agreement with qualitative arguments that take into account the T dependence of the DNA binding free energy (SI Text). These considerations, together with the agreement of our results with theoretical predictions (19), indicate that the dependence of the coexistence region from the particles valence that we observe with the DNA nanostars is universal for limited valence systems.The experimentally determined consolution curve necessarily terminates, at high T, in a critical point marking the divergence of DNA concentration fluctuations. To characterize such critical behavior we investigated amplitude and dynamics of the pretransitional concentration fluctuations by preparing samples at and and lowering T to approach . Measurements were done via static and dynamic light-scattering experiments for six different angles covering the wave vector range . This experimental approach takes advantage of the large refractive index of DNA, enabling an effective detection of concentration fluctuations.Fig. 2A shows the intensity scattered by the solutions of nanostars along the critical isochore at various scattering angles, ranging from to . All data can be simultaneously fitted by a Lorentzian shapewhich expresses the dependence of the susceptibility on T and on the scattering wave vector q in the critical region. In Eq. 1, diverges as , whereas ξ is the correlation length, diverging as . and provide the reference values of the critical scattering intensity and of the thermal correlation length far from the critical point. accounts for the (small) noncritical component of the scattered intensity. The appropriate Ising exponents are and (26). The simultaneous best fit of the scattered intensity at all measured T and q values (lines in Fig. 2A) provides a robust estimate for the four fit parameters , , , and . Analogous analysis has been performed on solutions of nanostars (SI Text). The resulting values for the critical temperatures are and for and , respectively (vertical lines in Fig. 2B). The best fit yields and for and , respectively. These values are in the range of the hydrodynamic radius of the nanostar ( and 4.7 nm for and ) and reflect the different critical density of the two systems, smaller in the case of . The marked increase of the intensity scattered by both and nanostars upon lowering T, compared in Fig. 2B for a scattering angle equal to , our smallest accessible scattering angle, provides evidence of the growth of critical concentration fluctuations. To the best of our knowledge, no other example of critical behavior in DNA solutions was previously reported.Open in a separate windowFig. 2.Pretransitional behavior of DNA nanostars along the critical isochore. (A) Scattered intensity measured as a function of T at various scattering angles. The scattering angles and the corresponding scattering vectors explored in this experiments are 30° (q = 8.15 μm−1), 45° (q = 12.1 μm−1), 68° (q = 17.6 μm−1), 90° (q = 22.3 μm−1), 101° (q = 24.3 μm−1), and 152° (q = 30.6 μm−1). (B) T dependence of the scattered intensity measured at 30° (full symbols) for both and systems. Dotted lines in A and B are the best fit by the Lorentzian function in Eq. 1 (dotted lines). Dashed vertical lines indicate as determined by the best fit. Scattered intensities relative to the structures have been divided by a factor of 2 to make them overlap with data at high T. (C) Field correlation functions measured at in the system for various T (symbols). Data are fitted to a sum of two stretched exponentials (lines). (D) Scattering intensity associated to the fast and slow contributions of at scattering angle . The line is the fit to the total scattering intensity already reported in B.Though the divergence in Fig. 2 A and B follows expectations, the dynamic behavior is richer than anticipated. The theory of dynamic critical phenomena predicts an exponential decay of the correlation function and a single characteristic time diverging as power law upon approaching the critical point, the so-called “critical slowing down.” Instead, as the critical point is approached, the field correlation functions become characterized by a two-step relaxation process (Fig. 2C), with the clear insurgence of a plateau whose height (the so-called “nonergodicity factor” in glass physics) (27) increases on cooling. All correlation functions can be well fitted to the sum of two stretched exponentialswhere and ; and ; and and are the amplitudes (with ), characteristic times, and stretching exponents of the fast and slow components, respectively.The clear separation in two fast and slow components allows us to decouple the contribution of the two processes to the static scattering, by weighting the total intensity with the two amplitudes and . The result of such a procedure, shown in Fig. 2D, indicates that the critical growth of the scattered intensity is entirely associated with the slow decay.As T decreases, the two characteristic times and behave very differently (Fig. 3A). At high T, the dynamics is the one expected for independent, free-diffusing DNA nanostars, with a single exponential decay process having a characteristic time coherent to the time expected for the nanostar radius. As T is lowered, such relaxation develops continuously into the fast component, with the stretching exponent decreasing (Fig. 3B) and changing only very mildly, not unlike what is expected for the T dependence of free diffusion. Quite different is the behavior of , which slows down more than three orders of magnitude in a continuous fashion. Fig. 3C shows that behaves as an Arrhenius-activated process , where is the characteristic time found in the limit of high T. By fitting the slope of vs. , we determined , the enthalpic component of . We find and for and nanostars, respectively. These values correspond to ∼2.0 and 3.0 times the enthalpic component expected for the binding of the sticky overhangs (SI Text). The entropic component associated with slow relaxation is instead less immediately accessible, because to extract it, it is necessary to have an independent estimate of . A simple choice is to assume . Under this assumption, we obtain and for the and systems, respectively. These values correspond, respectively, to ∼2.0 and 3.0 times the entropic component expected for the binding of the sticky overhangs (SI Text), in line with that obtained for the enthalpic component. This analysis indicates that as critical fluctuations start to develop, ergodicity is achieved via the breaking of bonds between nanostars. Indeed, density fluctuations can be viewed as the buildup of networks of bonded nanostars. Local disruptions of bonds enables the readjustment of the network either through nanostars “evaporating” away from the network and reconnecting elsewhere or through the rearrangements of network portions made flexible by the opening of bonds. The kinetics of this process is intrinsically limited by the rate of unbinding events, easily spanning into the millisecond regime (28–30) (see SI Text for further discussion).Open in a separate windowFig. 3.Dynamic behavior of and DNA nanostars along the critical isochore. (A) T dependence of the slow and fast decay times. The black line shows the expected T dependence of the diffusive τ for independent nanostars. (B) T dependence of the stretching exponent for the fast and for the slow components. (C) plotted as function of 1/T and fitted by an Arrhenius law. (D) Comparison, in a narrow T interval close to , of the extrapolated Arrhenius T dependence observed for for the DNA nanostars with the correlation times expected on the basis of the critical slowing down (SI Text).Our results show that the characteristic time of the slow process, despite being associated with critical density fluctuations, has a temperature dependence with no sign of the power-law divergence expected for critical slowing down. We interpret the dominance of the activated dynamics over the conventional slowing down as a consequence of the long lifetime of the bonds between DNA structures, larger than the time necessary for free structures to diffuse over distances comparable to ξ. In this condition, the decay of density correlations hinges on the restructuring of the large clusters, and it is thus determined by the bond lifetime. As illustrated in Fig. 3D, the critical slowing down is in this system probably constrained in a narrow T interval around , where the collective diffusional time across ξ becomes larger than the time required for activated bond rupture. The simple estimates in Fig. 3D indicate that such an interval is too narrow to be experimentally accessed with our instrumentation.The breakdown of dynamic universality here observed has strong analogies with the behavior of polymeric solutions in the proximity of their critical demixing point. As in the present case of DNA nanostars, polymeric solutions also feature a double relaxation and follow an unconventional critical dynamics as the critical point is approached (31, 32). In both systems, the dynamics close to the critical point is modified by the presence of slow microscopic mechanical relaxations that overshadow the collective behavior. This unusual phenomenon is explained by dynamic coupling of critical concentration fluctuations with an additional slow viscoelastic mode intrinsic to polymer solutions. Such anomalous kinetics may extend in a large range of T, including temperatures close to , where the critical behavior is typically expected. As a result, the cross-over to the conventional critical slowing down is pushed to temperatures so close to to be practically undetectable. Similar coupling between a critical mode and a slow relaxational mode has been proposed as a universal feature distinctive to mixtures whose components have strong dynamic asymmetries, including polymer solutions, protein solutions, and colloidal dispersions (33). In the case of DNA nanostars, the noncritical dynamics result from a subtle combination of selectivity, interaction energy, and lifetime, which can all be very finely tuned using DNA strands with different sequence and length, offering powerful handles for the exploration of this class of dynamic phenomena.Our results demonstrate that DNA structures are unique particles for investigating the phase behavior of systems in which it is possible to tune binding selectivity (via the DNA sequence), strength of interaction (via the DNA length), and valence. Key in this application is the strong temperature dependence of the DNA duplex binding strength, which enables exploring a wide range of bond energies and bond lifetimes, as no other model system does. We foresee that a large variety of topics in statistical physics can be experimentally addressed through the use of DNA supermolecules, including reentrant behaviors induced by competitive interactions (34, 35), higher-order network–network critical points (36), and arrested states of matter (glasses and gels) (27). In the specific case of limited valence structures here discussed, our results set the basis to predict the thermal and kinetic stability of self-assembled DNA hydrogels (25), a finding with relevant implications in the design of commercial complex fluids with tailored properties. 相似文献
13.
Oliver G. Hayes Benjamin E. Partridge Chad A. Mirkin 《Proceedings of the National Academy of Sciences of the United States of America》2021,118(40)
The structural and functional diversity of materials in nature depends on the controlled assembly of discrete building blocks into complex architectures via specific, multistep, hierarchical assembly pathways. Achieving similar complexity in synthetic materials through hierarchical assembly is challenging due to difficulties with defining multiple recognition areas on synthetic building blocks and controlling the sequence through which those recognition sites direct assembly. Here, we show that we can exploit the chemical anisotropy of proteins and the programmability of DNA ligands to deliberately control the hierarchical assembly of protein–DNA materials. Through DNA sequence design, we introduce orthogonal DNA interactions with disparate interaction strengths (“strong” and “weak”) onto specific geometric regions of a model protein, stable protein 1 (Sp1). We show that the spatial encoding of DNA ligands leads to highly directional assembly via strong interactions and that, by design, the first stage of assembly increases the multivalency of weak DNA–DNA interactions that give rise to an emergent second stage of assembly. Furthermore, we demonstrate that judicious DNA design not only directs assembly along a given pathway but can also direct distinct structural outcomes from a single pathway. This combination of protein surface and DNA sequence design allows us to encode the structural and chemical information necessary into building blocks to program their multistep hierarchical assembly. Our findings represent a strategy for controlling the hierarchical assembly of proteins to realize a diverse set of protein–DNA materials by design.Hierarchical assembly is integral to the structural complexity and function of materials and systems that occur in nature. Muscle tissue (1), amyloid fibrils (2), and collagen networks (3) are all examples of highly organized supramolecular architectures that arise from bottom-up, multistep, regulated assembly processes. The well-controlled sequence of assembly steps along a given pathway and the specificity of interactions between components are critical to the observed structural complexity and diversity (4, 5). While nanoscale hierarchical assembly is prevalent and important in nature, and our ability to control the bottom-up assembly of synthetic nanoscale building blocks has been transformed over the past two decades (6–8), we are still limited in what can be programmed through hierarchical mechanisms (9, 10). This is due to difficulties in defining the number, type, and location of multiple interactions on synthetic building blocks, as well as limitations in controlling the interplay between orthogonal interactions to achieve a desired assembly pathway (11). The development of tools and strategies to program multistep assembly pathways of nanoscale building blocks would redefine how we control the bottom-up synthesis of materials and accelerate the discovery of novel structures with desirable properties and functions (12, 13). In this work, we address this gap by spatially encoding programmable interacting ligands (DNA) onto the surface of chemically addressable building blocks (proteins).Proteins are an important class of nanoscale building block because of their structural and functional roles in biology. As such, developing methods to synthetically engineer new materials from proteins is a common goal in the fields of synthetic biology, chemistry, and materials science, with diverse applications from catalysis (14) to immune evasion (15) and biological delivery (16). The chemical complexity of protein surfaces defines specific recognition between protein interfaces and is key to the hierarchical assembly processes observed in nature. However, their complex surfaces make it challenging to design protein building blocks that will transform into targeted materials by traversing an intended assembly pathway. While powerful de novo design strategies have been utilized to create proteins with predetermined interfaces and assembly outcomes (17, 18), this approach inherently deviates from the pool of naturally occurring protein building blocks that could be utilized for materials engineering. Other strategies have relied on introducing controlled molecular interactions to the surfaces of proteins ranging from metal coordination chemistries (19–21) to hydrophobic (22) and host–guest interactions (23, 24). Despite significant innovation in manipulating surface interactions through chemical modifications, less attention has been paid to designing protein building blocks that can undergo multistep assembly pathways mimicking those in nature (25–27), because it remains challenging to realize interactions that are simultaneously specific, orthogonal, and have tunable strengths. Indeed, methods to define interaction location and type on the surface of a building block, in conjunction with an understanding of how to control and regulate each interaction independently, are needed to successfully program hierarchical assembly pathways. Although a growing body of literature has examined assembly pathways in the context of protein crystal polymorphism (28, 29), the ability to design directional, multistep assembly processes remains elusive.In addition to programming the structures of protein assemblies using DNA origami templates with specific, directional interactions (16, 29–33), our group and others have shown that DNA ligands chemically tethered to the surfaces of proteins at specific locations can drive the assembly of proteins into one-dimensional (1D), two-dimensional (2D), and three-dimensional (3D) assemblies and crystals (34–45). Protein mutagenesis has been used to site-specifically encode multiple, orthogonal DNA interactions onto protein surfaces to program directional assembly (46). Furthermore, the programmable recognition properties of DNA surface ligands have been utilized to control the polymerization pathway of proteins (47). However, these examples all rely on a single assembly step to reach their target structure and do not teach us how to create more complex materials from multistep, hierarchical assembly of proteins, such as those observed in nature. Indeed, even when multistep DNA assembly was demonstrated for inorganic nanoparticles, the second assembly step could only be induced by chemical modification of the structure formed after an initial assembly step and the addition of more nanoparticle building blocks to the system (48).We hypothesized that, if we could define the specificity, strength, and spatial distribution of multiple specific DNA interactions on the surface of a protein, we would be able to synthesize protein building blocks that undergo spontaneous, programmed, multistep assembly processes. Here, by defining the chemical anisotropy of a protein’s surface via mutagenesis, we define DNA interactions spatially, that is, axially or equatorially with respect to the geometry of an anisotropic protein (Scheme 1A). Through careful DNA design, we modulate the relative interaction strengths of the axial and equatorial faces such that assembly via strong interactions in a single direction leads to an emergent, second interaction that can program assembly in an orthogonal direction (Scheme 1B). The emergence of this second interaction is a hallmark of hierarchical assembly observed in nature and is responsible for directing the assembly of proteins along specific, multistep pathways. This study focuses on articulating this concept for programming the assembly of nanoscale building blocks along specific, hierarchical pathways, rather than obtaining arbitrarily high registry in 2D and 3D protein materials.Open in a separate windowScheme 1.Design of Sp1m chemical surface and proposed hierarchical assembly schemes. (A) Native Sp1 (Left) presents multiple primary amines (lysines and N termini, blue) and no cysteines (red) on its surface. Three mutations were designed to remove two native lysines and introduce one cysteine per subunit. Due to the dodecameric structure of Sp1m, these mutations define the chemical anisotropy across the protein surface with amine residues only on the axial face and cysteines located only on the equatorial face. (B) Proposed assembly schemes for building blocks containing strong or weak surface interactions at their axial or equatorial positions. Strong interactions direct the first stage of assembly, leading to multivalency among weak interactions that direct the second stage of assembly. 相似文献
14.
Mario Sarbia Gabriele Molsberger Rainhart Willers Olaf Horstmann Christof Schröders Rainer Porschen Helmut E. Gabbert 《Journal of cancer research and clinical oncology》1996,122(3):186-188
DNA aneuploidy, as determined by flow cytometry, was detected in 36 out of 59 adenocarcinomas of the esophagogastric junction (61.0%). DNA aneuploidy was more frequent in tumors with infiltrative growth pattern and in high pT categories. No correlation was found with pN category, grading and Laurén's classification. In contrast to clinicopathological parameters, DNA ploidy had no impact on patient's survival in univariate survival analysis. 相似文献
15.
Ignacio Gispert James W. Hindley Colin P. Pilkington Hansa Shree Laura M. C. Barter Oscar Ces Yuval Elani 《Proceedings of the National Academy of Sciences of the United States of America》2022,119(42)
Intercellular communication is a hallmark of living systems. As such, engineering artificial cells that possess this behavior has been at the heart of activities in bottom-up synthetic biology. Communication between artificial and living cells has potential to confer novel capabilities to living organisms that could be exploited in biomedicine and biotechnology. However, most current approaches rely on the exchange of chemical signals that cannot be externally controlled. Here, we report two types of remote-controlled vesicle-based artificial organelles that translate physical inputs into chemical messages that lead to bacterial activation. Upon light or temperature stimulation, artificial cell membranes are activated, releasing signaling molecules that induce protein expression in Escherichia coli. This distributed approach differs from established methods for engineering stimuli-responsive bacteria. Here, artificial cells (as opposed to bacterial cells themselves) are the design unit. Having stimuli-responsive elements compartmentalized in artificial cells has potential applications in therapeutics, tissue engineering, and bioremediation. It will underpin the design of hybrid living/nonliving systems where temporal control over population interactions can be exerted.Artificial cells are engineered mimics of biological cells, constructed from the bottom up by bringing together defined molecular building blocks (1). They are designed to replicate the form, function, and behaviors of natural cells, and most often are based on enclosed compartments that contain biomolecular species responsible for imparting cell-like features (compartmentalization, sense/response, communication, etc.) (2, 3). In addition to being used as simplified cell models to decipher the rules of life through an “understanding by building” approach (4), a major motivation behind artificial cell research is their potential to act as devices that can be used in biomedical and biotechnological applications (5–7).There are several advantages associated with artificial cells compared with genetically engineered biological cells that are the preserve of top-down synthetic biology. This includes the ability to incorporate nonbiological functional components, reduced regulatory and biosafety considerations, and the removal of cell-burden limitations that arise when engineered cellular functions exist alongside native ones (8). However, perhaps the major attraction of artificial cells is that they only contain the minimal components required to perform their function and can thus be composed of only a small number of molecular species. It is therefore easier to engineer them to have user-defined features, so they are highly controllable and programmable. However, their reduced complexity means that they cannot currently match the metabolic, regulatory, and behavioral sophistication of their biological counterparts. Processes in biological cells may thus be more robust, with cells able to perform their functions even in the presence of perturbations or changes in their milieu (9), which is often the basis of their use in many applications (10).The programmability of artificial cells and the technological potential of biological cells have led to increased efforts at coupling the two together, and in the process, accumulating the advantages associated with both (11). This can be achieved by either forming hybrid living/artificial cells (12–14) or engineering communication routes between populations of living cells (bacteria or eukaryotes) and artificial ones (15–20). In so doing, not only could the extensive genetic modification of cells be bypassed (as the artificial cells are the engineered species), but also more complex functions could be reconstituted when both types of cells are incorporated into the same system.Integrating artificial and biological cells requires establishing communication pathways between them, for example, through quorum sensing using autoinducer molecules (21, 22). This approach was exploited to expand the sensory range of bacteria through the construction of artificial cells that operated as chemical translators that could sense a small molecule and release an inducer molecule that bacteria could respond to (23). Similarly, robust artificial cells were designed to act as sensor or reporter modules depending on the DNA program present in the artificial or bacterial cell (24). In recent years, engineered communication has been extended to artificial-eukaryotic cell networks (17, 25–27).Nevertheless, the current systems rely on chemical triggers, while the use of physical stimuli remains mostly unexplored. We intended to remedy this by engineering stimuli-responsive artificial cells which can communicate with bacteria.In so doing, stimuli-responsive artificial cells would constitute distributed “artificial organelles,” which are not encapsulated within living cells but endow bacteria with novel responsiveness to physical stimuli. This would also provide artificial/living hybrid cell systems with a control step to allow external activation by an end user on demand. Designing a library of stimuli-responsive platforms entails the next breakthrough in cellular bionics, since controlling cell behaviors using artificial cells as intermediaries would enable processes to take place in a temporally controlled manner, in a defined location, and in response to specific stimuli. There have been several examples of stimuli-responsive population communication operating between synthetic cell populations, including communication between cell-like compartments in synthetic tissues using a light-regulated DNA promoter (28), through divalent cation chelators (29), or using a photolabile DNA linker (30). In a recent exciting preprint, a light-activated DNA template was used to control communication between synthetic and bacterial cells using ultraviolet (UV) light (31). Nevertheless, to realize the incorporation of hybrid artificial/living cell systems in applications such as therapeutics, bioremediation, and biosensing, remote-controlled cell mimics able to communicate with a wide range of biological cells, using different stimuli, need to be further developed (32). Herein, we address this gap by designing a generalizable vesicle-based artificial cell platform capable of compartmentalizing content, sensing a physical cue, and releasing chemical messengers that can then activate a DNA program in Escherichia coli.To do this, we developed and exploited two types of synthetic cell compartments, one of which was light responsive and the other thermoresponsive. These house inducer molecules with them, which are ordinarily shielded from surrounding bacteria. Only upon encountering the relevant stimuli do these molecules get released and activate protein expression in the living cells. This allowed us to control protein expression in cells using artificial cells and intermediaries that translate the stimuli (light and heat) into a chemical signal that the cells respond to. 相似文献
16.
17.
18.
In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon. 总被引:30,自引:0,他引:30
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
E A Groisman B A Castilho M J Casadaban 《Proceedings of the National Academy of Sciences of the United States of America》1984,81(5):1480-1483
A mini-Mu bacteriophage containing a high copy number plasmid replicon was constructed to clone genes in vivo. A chloramphenicol resistance gene for independent selection and the lacZYA operon to form gene fusions were also incorporated into this phage. This mini-Mu element can transpose at a high frequency when derepressed, and it can be complemented by a helper Mu prophage for lytic growth. DNA sequences that are flanked by two copies of this mini-Mu can be packaged along with them. After infection, homologous recombination can occur between the mini-Mu sequences, resulting in the formation of plasmids carrying the transduced sequences. lac operon fusions can be formed with promoters and translation initiation sites on the cloned sequences in the resulting plasmids. The utility of this system was demonstrated by cloning genes from eight different Escherichia coli operons and by identifying lac fusions to the regulated araBAD operon among clones selected for the nearby leu operon. 相似文献
19.
Guolong Zhu Mark Hannel Ruojie Sha Feng Zhou Matan Yah Ben Zion Yin Zhang Kyle Bishop David Grier Nadrian Seeman Paul Chaikin 《Proceedings of the National Academy of Sciences of the United States of America》2021,118(21)
The programmability of DNA oligonucleotides has led to sophisticated DNA nanotechnology and considerable research on DNA nanomachines powered by DNA hybridization. Here, we investigate an extension of this technology to the micrometer-colloidal scale, in which observations and measurements can be made in real time/space using optical microscopy and holographic optical tweezers. We use semirigid DNA origami structures, hinges with mechanical advantage, self-assembled into a nine-hinge, accordion-like chemomechanical device, with one end anchored to a substrate and a colloidal bead attached to the other end. Pulling the bead converts the mechanical energy into chemical energy stored by unzipping the DNA that bridges the hinge. Releasing the bead returns this energy in rapid (>20 μm/s) motion of the bead. Force-extension curves yield energy storage/retrieval in these devices that is very high. We also demonstrate remote activation and sensing—pulling the bead enables binding at a distant site. This work opens the door to easily designed and constructed micromechanical devices that bridge the molecular and colloidal/cellular scales.Force, motion, and work are ubiquitously produced in living organisms by molecular motors fueled by ATP. Hydrolysis of ATP at ∼20 kBT/molecule is consumed by motors with <50% efficiency generating forces of 2 to 8 pN (1); DNA hybridization produces forces of 8 to 20 pN and supplies energies of 2 to 5 kBT per nucleotide (nt) pair (2). The comparable numbers suggest that DNA hybridization may prove a useful way to power microdevices or store energy microscopically, particularly since the fuel and mechanical device can be the same molecule. Previous DNA devices (3–5) are important demonstrations typically on the nanoscale but relatively slow (min/h). Here, we demonstrate that DNA hybridization can be leveraged through mechanical advantage to power rapid motion.Our basic mechanical device is an extended hinge (Fig. 1A), which can be closed or opened by zipping or unzipping complementary DNA strands via applied light, heat, or mechanical force. It consists of two semirigid DNA origami six-helix bundles (6HBs, 410 nm long) (6, 7) joined end to end by short semiflexible single DNA strands (ssDNA) (8). On each rod, there are complementary DNA sticky ends proximal (14 nm) to the hinge vertex, forming a “bridging DNA zipper.” The mechanical advantage, lever arm ratio, is 410/14 nm ∼30 for a single hinge. Attaching one arm to a substrate and extending the other arm to two 6HBs makes it a trimer (Fig. 1B). With a bead attached to its end, we have a device with a throw of 1.6 μm. We use ssDNA (two 2 nt thymine) at the hinge vertex to open it and a bridging zipper with 16 paired nt to close it. We can open and close the hinge by cycling temperature. Fig. 1 C and D shows the bead positions at 16 and 42 °C from optical microscopy. Global heating/cooling cycles take seconds/minutes (SI Appendix, Fig. S2). We also cycle trimer hinges using azobenzene-modified (9) sticky ends with 420/360 nm light to open/close the hinge (SI Appendix, Fig. S4).Open in a separate windowFig. 1.Devices, hinges, accordions, and thermal cycling. (A) The basic device is made of 6HB DNA origami rods linked with a short ssDNA connection at the hinge vertex and closed by a bridging DNA zipper attached 14 nm from the vertex. (B) A simple DNA heat engine—one 6HB is attached to the substrate, and two rigidly connected rods attach to a bead. The DNA zippers close at 16 °C and open at 42 °C. (C) Bead positions in two full cycles of 16 to 42 °C to 16 to 42 °C. (D) The particle’s probability distribution along the x-axis in the hot and cold states. Dashed black and solid gray lines are calculated from models with thermal motion and elastic restoring force (SI Appendix, Fig. S1).For large extensions, we combine 10 rods into an “accordion,” attached to the substrate at one end and to a 500-nm colloidal bead (10–15) at the other end (Fig. 2A and SI Appendix, Fig, S5). Force-extension measurements (16, 17) on the accordion were made by pulling on the tethered bead with optical tweezers until the bead escapes the optical trap (Fig. 2B and SI Appendix, Fig, S7 and Movie S1). Mechanical advantage amplifies 6 pN on the bead to ∼180 pN on DNA zippers. The total throw of the accordion is 7.4 μm. The equilibrium particle position distribution and Boltzmann statistics yield the initial restoring force and equilibrium position (SI Appendix, Fig. S8). Force calibration is detailed in SI Appendix, Fig. S6.Open in a separate windowFig. 2.Force-extension curves for the accordion construct. (A) An “accordion” of 10 6HBs, with nine DNA zippers and two 4T ssDNA at the vertices. The first 6HB is bound to the substrate with sticky “legs” and the last to a bead with sticky “hands.” (B) Each red point is an average over 40 measurements on each of five samples. Dashed red is calculated from the Boltzmann distribution, measured with no force. Solid blue is a control experiment with no bridging DNA zippers. Dashed blue is from the equilibrium distribution of the control. Solid black is the Langevin function for a 10-mer freely jointed chain. The green shaded area (408 kBT) is the work done and energy stored in extending the accordion (ΔG for unzipped sequences is 418 kBT). (C) Measured and calculated curves to full extension.In the force-extension curve (Fig. 2 B and C), bridging DNA unzips in the range 8 to 20 pN or 0.27 to 0.67 pN applied to the accordion ends. A control with no bridging DNA zippers (blue) is well fit as a freely jointed chain, Langevin function (black) (SI Appendix, Fig. S10). The force-extension curve agrees well with the rms displacement (SI Appendix, Fig. S9) from an elastic spring model at low force and an entropic freely jointed chain at large force. The integrated area (shaded green) between the accordion (red) and the control (blue), 408 kBT (±8.3%), is the measured work done and energy stored in the unzipped DNA. By comparison, the calculated ΔG for the particular sequences used is 418 kBT (±5%) (18): The mechanical work done on the extension has been converted to recoverable DNA hybridization chemical energy that can be used to move the tethered bead through the viscous medium.The bridging DNA zippers used in Fig. 2 have 30 nt pairs plus 4 nt spacers at midpoint and remain hybridized with at least 8 nt pairs upon complete accordion extension. Shorter zippers (21 nt pairs), which are completely unzipped at full extension, show hysteresis (Fig. 3). At large extension, the only reactive force is entropic; therefore, the completely unzipped states lie on the same curve as the control, the freely jointed chain that lacks any bridging DNA zippers. At shorter extension, DNA zippers are partially hybridized, exerting an enthalpic force, in addition to the entropic force. The hinges are at least partially closed, and the force is transmitted from one hinge to the next by the elastic bending of the 6HB rods; see SI Appendix, Fig. S3. The losses only arise upon completely unzipping and then renucleating the hybridization, similar to losses in forming secondary structures in RNA or proteins on folding (19, 20).Open in a separate windowFig. 3.Hysteresis with complete unzipping of short DNA zippers. (A) The bead is pulled to 3,800 nm with 6 pN. Held with these forces, the extension is decreased. The trapping force is then lowered until the accordion pulls the bead out from the trap. The force and extension are recorded (red points). Without changing the trapping force, the bead is then pulled from its equilibrium position to where it is pulled out from the trap. Force and extension are recorded (blue points). Each trapping force corresponds to two extensions. An example is the two points just inside the dashed gray ellipse. The two states correspond to zipped and unzipped bridging DNA zippers, as illustrated above and below the ellipse. (B) Snapshots of the experiment for one set of points with green as full extension, red the position where the bead escapes the trap, yellow the equilibrium position to which the bead retracts, and blue the position that the bead can be pulled to with the same (escape) force, ∼0.33 pN. (Scale bar, 1 µm.)To quantify the time scale for such DNA devices, we pull the bead to near full extension and release, using the accordion with zippers long enough that they are always partially bound, and exhibit no hysteresis (Fig. 2). We use beads of two sizes, 500 (blue) and 1,000 nm (red). Both show a fast pull back within a fraction of second (Fig. 4B and Movie S2), with a peak velocity of 50 and 25 μm/s, respectively (Fig. 4C). The time and speed depend on the load rather than the folding time (10 to 100 μs) of the DNA hairpin (21). A model including particle and accordion Stokesian drag near a wall agrees with our observations (SI Appendix, Fig. S11). The force is mostly entropic at large extension and mostly elastic at short distances. The extension range in which the DNA hybridization is the dominant driver is from 3,000 to 1,000 nm. The collapse in this region happens within 0.1 s and at speeds exceeding 25 μm/s. Using shorter DNA zippers (SI Appendix, Fig. S12), it takes ∼1 s for the devices to return to 1,000 nm, as the complementary single strands must first diffuse to make contact, initiating hybridization.Open in a separate windowFig. 4.Accordion folding speed. (A) Schematic of the experiment. The bead is trapped in the laser tweezers, pulled to maximum extension, and released. (B) The extension is measured from micrograph movies at 30 frames/s, averaged over 40 runs. Red/blue are for 1-μm/500-nm beads, respectively. (C) Speed versus time from the data in Fig. 4B.To show directly that the bridging DNA is indeed unzipped upon accordion extension for short DNA zippers, we prepared a set of probe colloids coated with 21-base ssDNA, complementary to one side of a particular DNA zipper. Probe colloids held close to the unstretched accordion do not bind. When the accordion bead is pulled to full extension, the probe particle attaches to the designated open hinge strand. In three designs, with the probes programmed to bind to zippers at 400, 1,200 (Fig. 5A), and 2,000 nm (Fig. 5B), we observe binding. The binding is confirmed by pulling the bead around a circle at maximum extension and observing that each probe bead follows concentrically at its appropriate radius (SI Appendix, Fig. S13). Thus, our DNA accordion also exhibits remote sensing, a simple primitive form of mechanical allostery (22, 23), a displacement applied at one end of our construct allows binding at a distant site (Movie S3).Open in a separate windowFig. 5.Allostery. Probe colloids (900 nm, green) coated with ssDNA (21 nt) complementary to one strand of a specific bridging DNA zipper do not bind to the unstretched accordion. However, when the accordion is stretched, unzipping the bridging DNA zippers, the colloids attach to their specific positions. As seen in the superposed micrographs, when the accordion bead (red) is pulled and rotated to four different angles, the probe follows the rotation at a smaller radius. Radial position of the probe bead, dashed green circle, is 1.2 μm in A and 2 μm in B; radial position of the accordion bead, dotted red circle, is ∼3.8 μm. (Scale bar, 1 μm.)There have been many previous experiments pulling on DNA in clever configurations: long unhybridized sequences that explore DNA as a prototype entropic polymer (24), hairpin structures (25) that quantify hybridization free energy calculations and explore kinetics, or beautifully designed DNA springs (26) that demonstrate efficient purely elastic behavior with no dehybridization upon stretching. The present study builds on these studies and extends them to quantify the speed and reversibility of storing and recovering, specifically the dehybridization energy stored on the mesoscopic (many micrometers) scale.We have demonstrated that the energy associated with DNA hybridization at the nanometer scale can be leveraged to produce controlled motion at the micrometer scale, with a 1.6-μm DNA construct activated by heat or by light. Furthermore, we have made an accordion, which demonstrates that DNA can be utilized at the ∼4-μm scale to store and reversibly recover energy on a time scale of fractions of a second, producing speeds up to 50 μm/s. Unlike biological motors, in which the machine and the chemical energy storage are separate, DNA constructs incorporate both functions in a single molecule (3). It is worth emphasizing that in our devices we make use of reversible hydrogen bonds in contrast to the lossy use of ATP hydrolysis in molecular motors. The present system, combining mechanical advantage and short DNA strands that hybridize without initiation and without folding entanglements, is easily extended to even larger distances by adding more hinges (27). It has the advantage that it avoids the entanglements and distortion concomitant with folding large DNA hairpins. This system suggests a paradigm for the design of devices on the nanometer to cellular scales. Combined with the sophistication developed over the past four decades in structural DNA nanotechnology (28–36), our work opens the door to programmable microdevices, artificial cilia, micromuscles, Escherichia coli–like swimmers with active flagella, and remote sensors. 相似文献
20.
Kracker S Imai K Gardès P Ochs HD Fischer A Durandy AH 《Proceedings of the National Academy of Sciences of the United States of America》2010,107(51):22225-22230
Ig class-switch recombination (CSR) is a region-specific process that exchanges the constant Ig heavy-chain region and thus modifies an antibody's effector function. DNA lesions in switch (S) regions are induced by activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase 2 (UNG2), subsequently processed to DNA breaks, and resolved by either the classical nonhomologous end-joining pathway or the alternative end-joining pathway (XRCC4/DNA ligase 4- and/or Ku70/Ku80-independent and prone to increased microhomology usage). We examined whether the induction of DNA lesions influences DNA end-joining during CSR by analyzing Sμ-Sα recombination junctions in various human Ig CSR defects of DNA lesion induction. We observed a progressive trend toward the usage of microhomology in Sμ-Sα recombination junctions from AID-heterozygous to AID-autosomal dominant to UNG2-deficient B lymphocytes. We thus hypothesize that impaired induction of DNA lesions in S regions during CSR leads to unusual end-processing of the DNA breaks, resulting in microhomology-mediated end-joining, which could be an indication for preferential processing by alternative end-joining rather than by classical nonhomologous end-joining. 相似文献