DNA Tile Assembly Research Articles

A biomimetic branching signal-passing tile assembly model with dynamic growth and disassembly.

Natural biological branching processes can form tree-like structures at all scales and, moreover, can perform various functions to achieve specific goals; these include receiving stimuli, performing two-way communication along their branches, and dynamically reforming (extending or retracting branches). They underlie many biological systems with considerable diversity, frequency, and geometric complexity; these include networks of neurons, organ tissue, mycorrhizal fungal networks, plant growth, foraging networks, etc. This paper presents a biomimetic DNA tile assembly model (Y-STAM) to implement dynamic branching processes. The Y-STAM is a relatively compact mathematical model providing a design space where complex, biomimetic branch-like growth and behaviour can emerge from the appropriate parametrization of the model. We also introduce a class of augmented models (Y-STAM+) that provide time- and space-dependent modulations of tile glue strengths, which enable further diverse behaviours that are not possible in the Y-STAM; these additional behaviours include refinement of network assemblies, obstacle avoidance, and programmable growth patterns. We perform and discuss extensive simulations of the Y-STAM and the Y-STAM+. We envision that these models could be applied at the mesoscale and the molecular scale to dynamically assemble branching DNA nanostructures and offer insights into complex biological self-assembly processes.

Diverse Chiral Nanotubes Assembled from Identical DNA Strands.

DNA nanotubes with controllable geometries hold a wide range of interdisciplinary applications. When preparing DNA nanotubes of varying widths or distinct chirality, existing methods require repeatedly designing and synthesizing specific DNA sequences, which can be costly and laborious. Here, we proposed an intercalator-assisted DNA tile assembly method which enables the production of DNA nanotubes of diverse widths and chirality using identical DNA strands. Through adjusting the concentration of intercalators during assembly, the twisting direction and extent of DNA tiles could be modulated, leading to the formation of DNA nanotubes featuring controllable widths and chirality. Moreover, through introducing additional intercalators and secondary annealing, right-handed nanotubes could be reconfigured into distinct left-handed nanotubes. We expect that this method could be universally applied to modulating the self-assembly pathways of various DNA tiles and other chiral materials, advancing the landscape of DNA tile assembly.

Seeded growth of adaptive tiles on DNA origami

Structural DNA nanotechnology has been applied to construct complex static and dynamic DNA structures. Seeded growth, intended to regulate the crucial nucleation step, has been used to control the assembly of DNA tiles. However, most of the seeded growth strategies were applied to fixed DNA tiles, whereas the seeded growth on dynamic DNA tiles remains unreported. Here, we propose a seeded growth strategy in which dynamic tiles adaptively change their shapes to match the architecture of a DNA origami seed. Furthermore, when more adaptive DNA tiles assemble on a reconfigurable DNA origami domino array (DODA) seed, the conformation of DODA seeds is reversibly affected by the spontaneous reconfiguration of adaptive tiles. The adaptive seeded growth provides a mechanism for the construction of complex DNA nanomachines and may offer a general and adaptable method for the advancement of responsive materials, with active, autonomous, and adaptive spatiotemporal control properties. • The shapes of adaptive tiles can change to match “red” or “blue” DNA origami seed • The adaptive tiles can transform to “blue” conformation when assembling on fixed seed • The adaptive tile can trigger the deformation of reconfigurable seed • A molecular “tug-of-war” occurs when adaptive tile assembling on reconfigurable seed Liu et al. develop a seeded growth strategy whereby adaptive DNA tiles can adaptively assemble on DNA origami seeds, with their shapes changing simultaneously. When the adaptive tiles assemble on reconfigurable seeds, the seeds change into the other conformation, with the transformation triggered by the growing tile lattice.

In situ hand-in-hand DNA tile assembly and CRISPR/Cas12a-based ultrasensitive detection for leukemia diagnosis

CRISPR/Cas technology has attracted increasing attention for use in accurate and sensitive nucleic acid detection, and has innovated the field of molecular diagnostics. Because it is programmability and shows high fidelity and effective signal amplification capabilities. Specifically, Cas12a has been evaluated through in-depth research because it triggers collateral trans-cleavage activity for the target. Here, we report a novel CRISPR/Cas12a fluorescent biosensor by in situ hand-in-hand DNA tile assembly for leukemia diagnosis, and it allows an active isothermal amplification for multi-channel collection of CRISPR/cas12a transducers for achieving three-stage signal amplification. When the target gene concentration was in a range from 1 fM to 50 nM, the fluorescence intensity exhibited an excellent linear relationship with the logarithm of the target gene concentration, and the detection limit reached 1 fM. Furthermore, good detection performance can still be maintained in actual sample analysis. The CRISPR/Cas biosensor for leukemia diagnosis is more advanced than the traditional CRISPR/Cas detection systems, which can be used in cancer gene analyses, kinship judgment, environmental monitoring, and so on.

In Situ Hand-in-Hand DNA Tile Assembly: A pH-Driven and Aptamer-Targeted DNA Nanostructure for TK1 mRNA Visualization and Synergetic Killing of Cancer Cells.

In situ stimuli-responsive molecular devices have gained much attention in biomedical areas due to their characteristics of increased image contrast and drug accumulation. Herein, we present a hand-in-hand in situ tile assembly for improved visualization of TK1 mRNA and killing of cancer cells. A pH-responsive and aptamer-functionalized tile motif (pH-Apt-TM) was first formed by four single-strand DNA, possessing pH-responsiveness and intracellular TK1 mRNA recognition capacity. When encountering target cells, the pH-Apt-TM could recognize target receptors on the cell surface through the aptamer domain. Meanwhile, the extracellular acidic pH gathered the pH-Apt-TM into a multifunctional hand-in-hand DNA tile assembly (HDTA) on the cells' surface. Compared to the pH-Apt-TM, studies revealed that the HDTA exhibited enhanced recognition, efficient cellular uptake, and improved visualization of TK1 mRNA, accompanied by gene silencing. Moreover, using Dox as a chemotherapeutic model, specific drug delivery and enhanced cell killing were achieved with target cells.

Coarse-Grained Simulations of DNA Reveal Angular Dependence of Sticky-End Binding.

Annealing between sticky ends of DNA is an intermediate step in ligation. It can also be utilized to program specific binding sites for DNA tile and origami assembly. This reaction is generally understood as a bimolecular reaction dictated by the local concentration of the sticky ends. Its dependence on the relative orientation between the sticky ends, however, is less understood. Here we report on the interactions between DNA sticky ends using the coarse-grained oxDNA model; specifically, we consider how the orientational alignment of the double-stranded DNA (dsDNA) segments affects the time required for the sticky ends to bind, τb. We specify the orientation of the dsDNA segments with three parameters: θ, which measures the angle between the helical axes, and ϕ1 and ϕ2, which measure rotations of each strand around the helical axis. We find that the binding time depends strongly on both θ and ϕ2: ∼20-fold change with θ and 10-fold change with ϕ2. The binding time is the fastest when the helical axes of duplexes are pointing toward each other and the sticky ends protrude from the farthest two points. Our result is relevant for predicting hybridization efficiency of sticky ends that are rotationally restricted.

Implementing logical inference based on DNA assembly

Algorithms and information processing, fundamental to biological system, are an essential aspect of many elementary physical phenomena, such as molecular self-assembly. Self-assembly system has been proved to be capable of performing many logic operations by the early work. A significant challenge related to the design of molecular information processing systems is to develop a programmable architecture that controls the states of individual molecular events. Here, a novel systematic implementation of logical inference is presented based on DNA tile assembly system. Exploiting the intrinsic programmable capability of molecular interactions, firstly a seed tile configuration is constructed to encode the input information of a logical inference problem, including all facts, all inference rules and their equivalent rules. Then, three tile assembly subsystems are discussed to fulfil the main logical deduction steps. We describe mechanisms for finding the successful solutions among the many parallel assemblies. A whole tile assembly system is established on the base of a seed configuration and three subsystems. This prototype is the first programming language to implement deduction operations based on two-dimensional DNA assembly. It is demonstrated that algorithmic DNA tile assembly system can be treated as an important way to implement logic inference, which will shed light on aspects of applications in the field of artificial intelligence in the future.

Light-Controlled, Toehold-Mediated Logic Circuit for Assembly of DNA Tiles.

Inspired by cytoskeletal structures that respond sensitively to environmental changes and chemical inputs, we report a strategy to trigger and finely control the assembly of stimulus-responsive DNA nanostructures with light under isothermal conditions. The strategy is achieved via integrating an upstream light-controlled, toehold-mediated DNA strand displacement circuit with a downstream DNA tile self-assembly process. By rationally designing an upstream DNA strand module, we further transform the upstream DNA strand displacement circuit to an "AND gate" circuit to control the assembly of DNA nanostructures. This example represents the demonstration of the spatial and temporal assembly of DNA nanostructures using a noninvasive chemical input. Such a light-controlled DNA logic circuit not only adds a new element to the tool box of DNA nanotechnology but also inspires us to assemble complex and responsive nanostructures.

Macromolecular Crowding and Confinement Effect on the Growth of DNA Nanotubes in Dextran and Hyaluronic Acid Media.

The dense medium modulates the molecular structure and bioreactions in living cells via both noncovalent interactions and macromolecular crowding and confinement effects. However, the interplay between the volume effect and noncovalent interactions remains unclear. In this work, we studied in detail on how electrostatic interactions influence the crowding and confinement effect by comparing the formation and elongation of DNA nanotubes in branched dextran and charged hyaluronic acid (HA) solution of a broad concentration range, with and without 150 mM NaCl. In all the studied cases, three concentration regimes are identified: a crowding regime, a double-effect regime, and a confinement regime. In the crowding and double-effect regimes, the addition of 150 mM NaCl enhances the assembly of DNA tiles by screening the electrostatic repulsion, and a higher dextran solution is required to confine the DNA assembly into nanotubes. However, the screening effect on the HA network is more than that on the DNA assembly, so DNA tubes formed in HA solution at much lower concentrations. In the confinement regime, the electrostatic interaction exhibits a negligible effect on the DNA assembly in both dextran medium and HA medium. Our study demonstrates that the volume effect and noncovalent interactions are system specific and concentration dependent. Their interplay governs the living processes in crowded cells.

Simulation of one dimensional staged DNA tile assembly by the signal-passing hierarchical TAM

The Tile Assembly Model, and its many variants, is one of the most fundamental algorithmic assembly formalism within DNA nanotechnology. Most of the research in this field is focused on the complexity of assembling different shapes and patterns. In many cases, the assembly process is intrinsically deterministic and the final product is unique, while the assembly process might evolve through several possible assembly strategies. In this study we consider the controlled assembly of one dimensional tile structures according to predefined assembly graphs. We provide algorithmic approaches for developing such controlled assembly protocols, using the signal-passing Tile Assembly Model, as well as probabilistic approaches for investigating the assembly of such tile-based one-dimensional structures. As a byproduct, we build a generalized TAS (tile assembly system) which generate specific non-local non-associative algebraic computations and we assamble n × n squares using only one tile, which is a better efficiency compared to the staged assembly model.

Terminating DNA Tile Assembly with Nanostructured Caps.

Precise control over the nucleation, growth, and termination of self-assembly processes is a fundamental tool for controlling product yield and assembly dynamics. Mechanisms for altering these processes programmatically could allow the use of simple components to self-assemble complex final products or to design processes allowing for dynamic assembly or reconfiguration. Here we use DNA tile self-assembly to develop general design principles for building complexes that can bind to a growing biomolecular assembly and terminate its growth by systematically characterizing how different DNA origami nanostructures interact with the growing ends of DNA tile nanotubes. We find that nanostructures that present binding interfaces for all of the binding sites on a growing facet can bind selectively to growing ends and stop growth when these interfaces are presented on either a rigid or floppy scaffold. In contrast, nucleation of nanotubes requires the presentation of binding sites in an arrangement that matches the shape of the structure's facet. As a result, it is possible to build nanostructures that can terminate the growth of existing nanotubes but cannot nucleate a new structure. The resulting design principles for constructing structures that direct nucleation and termination of the growth of one-dimensional nanostructures can also serve as a starting point for programmatically directing two- and three-dimensional crystallization processes using nanostructure design.

Construction of a Polyhedral DNA 12-Arm Junction for Self-Assembly of Wireframe DNA Lattices.

A variety of different tiles for the construction of DNA lattices have been developed since the structural DNA nanotechnology field was born. The majority of these are designed for the realization of close-packed structures, where DNA helices are arranged in parallel and tiles are connected through sticky ends. Assembly of such structures requires the use of cation-rich buffers to minimize repulsion between parallel helices, which poses limits to the application of DNA nanostructures. Wireframe structures, on the other hand, are less susceptible to salt concentration, but the assembly of wireframe lattices is limited by the availability of tiles and motifs. Herein, we report the construction of a polyhedral 12-arm junction for the self-assembly of wireframe DNA lattices. Our approach differs from traditional assembly of DNA tiles through hybridization of sticky ends. Instead, the assembly approach presented here uses small polyhedral shapes as connecting points and branch points of wires in a lattice structure. Using this design principle and characterization techniques, such as transmission electron microscopy, single-particle reconstruction, patterning of gold nanoparticles, dynamic light scattering, UV melting analyses, and small-angle X-ray scattering among others, we demonstrated formation of finite 12-way junction structures, as well as 1D and 2D short assemblies, demonstrating an alternative way of designing polyhedral structures and lattices.

Metrology of DNA arrays by super-resolution microscopy.

Recent results in the assembly of DNA into structures and arrays with nanoscale features and patterns have opened the possibility of using DNA for sub-10 nm lithographic patterning of semiconductor devices. Super-resolution microscopy is being actively developed for DNA-based imaging and is compatible with inline optical metrology techniques for high volume manufacturing. Here, we combine DNA tile assembly with state-dependent super-resolution microscopy to introduce crystal-PAINT as a novel approach for metrology of DNA arrays. Using this approach, we demonstrate optical imaging and characterization of DNA arrays revealing grain boundaries and the temperature dependence of array quality. For finite arrays, analysis of crystal-PAINT images provides further quantitative information of array properties. This metrology approach enables defect detection and classification and facilitates statistical analysis of self-assembled DNA nanostructures.

PH-Controlled Assembly of DNA Tiles.

We demonstrate a strategy to trigger and finely control the assembly of supramolecular DNA nanostructures with pH. Control is achieved via a rationally designed strand displacement circuit that responds to pH and activates a downstream DNA tile self-assembly process. We observe that the DNA structures form under neutral/basic conditions, while the self-assembly process is suppressed under acidic conditions. The strategy presented here demonstrates a modular approach toward building systems capable of processing biochemical inputs and finely controlling the assembly of DNA-based nanostructures under isothermal conditions. In particular, the presented architecture is relevant for the development of complex DNA devices able to sense and respond to molecular markers associated with abnormal metabolism.

Nondeterministic self-assembly with asymmetric interactions.

We investigate general properties of nondeterministic self-assembly with asymmetric interactions, using a computational model and DNA tile assembly experiments. By contrasting symmetric and asymmetric interactions we show that the latter can lead to self-limiting cluster growth. Furthermore, by adjusting the relative abundance of self-assembly particles in a two-particle mixture, we are able to tune the final sizes of these clusters. We show that this is a fundamental property of asymmetric interactions, which has potential applications in bioengineering, and provides insights into the study of diseases caused by protein aggregation.

From Ion-Channels to Porins: Engineering DNA-Based Synthetic Counterparts

DNA nanotechnology allows for the creation of membrane-spanning channels with customized shape and functionality. We present three novel designs with channel diameters spanning an order of magnitude from 0.8 nm to 8 nm. We utilize DNA tile assembly and scaffolded origami with two different scaffold lengths to create channels of variable size and architectural complexity. Bifunctional porphyrin- and cholesterol-tags serve as membrane anchors to facilitate insertion into the lipid membrane (J. R. Burns, K. Gopfrich et al., Angewandte Chemie, 2015). We compare the conductance of the channels and confirm the correspondence between engineered design and single-channel behaviour. Our channels span two orders of magnitude in conductance, comparable to protein pores encompassing small ion channels as well as large porins. Conductance states are dependent on transmembrane voltage (K. Gopfrich, A. Seifert, et al., ACS Nano, 2014). We demonstrate that self-assembly and membrane attachment of simple DNA channels can be achieved within a minute, making their creation scalable for applications in biology (K. Gopfrich et al., Nanoletters, 2015). Confocal imaging with ion-sensitive dyes serves as an independent proof of the cation-selective ion transport capabilities of our DNA channels. Our work showcases the versatility of artificial DNA-based pores inspired by the rich structural and functional diversity of natural membrane components.

Directed enzymatic activation of 1-D DNA tiles.

The tile assembly model is a Turing universal model of self-assembly where a set of square shaped tiles with programmable sticky sides undergo coordinated self-assembly to form arbitrary shapes, thereby computing arbitrary functions. Activatable tiles are a theoretical extension to the Tile assembly model that enhances its robustness by protecting the sticky sides of tiles until a tile is partially incorporated into a growing assembly. In this article, we experimentally demonstrate a simplified version of the Activatable tile assembly model. In particular, we demonstrate the simultaneous assembly of protected DNA tiles where a set of inert tiles are activated via a DNA polymerase to undergo linear assembly. We then demonstrate stepwise activated assembly where a set of inert tiles are activated sequentially one after another as a result of attachment to a growing 1-D assembly. We hope that these results will pave the way for more sophisticated demonstrations of activated assemblies.

Building DNA Nanostructures for Molecular Computation, Templated Assembly, and Biological Applications

CONSPECTUS: DNA is a critical biomolecule well-known for its roles in biology and genetics. Moreover, its double-helical structure and the Watson-Crick pairing of its bases make DNA structurally predictable. This predictability enables design and synthesis of artificial DNA nanostructures by suitable programming of the base sequences of DNA strands. Since the advent of the field of DNA nanotechnology in 1982, a variety of DNA nanostructures have been designed and used for numerous applications. In this Account, we discuss the progress made by our lab which has contributed toward the overall advancement of the field. Tile-based DNA nanostructures are an integral part of structural DNA nanotechnology. These structures are formed using several short, chemically synthesized DNA strands by programming their base sequences so that they self-assemble into desired constructs. Design and assembly of several DNA tiles will be discussed in this Account. Tiles include, for example, TX tiles with three parallel, coplanar duplexes, 4 × 4 cross-tiles with four arms, and weave-tiles with weave-like architecture. Another category of tiles we will present involve multiple parallel duplexes that assemble to form closed tubular structures. All of these tile types have been used to form micrometer-scale one- and two-dimensional arrays and lattices. Origami-based structures constitute another category where a long single-stranded DNA scaffold is folded into desired shapes by association with multiple short staple strands. This Account will describe the efforts by our lab in devising new strategies to improve the maximum size of origami structures. The various DNA nanostructures detailed here have been used in a wide variety of different applications. This Account will discuss the use of DNA tiles for logical computation, encoding information as molecular barcodes, and functionalization for patterning of other nanoscale organic and inorganic materials. Consequently, we have used DNA nanostructures for templating metallic nanowires as well as for programmed assembly of proteins and nanoparticles with controlled spacings. Among other applications, we have used DNA nanotechnology in biosensors that detect target DNA sequences and to affect cell surface receptor clustering for communicating with a cell signaling pathway. We used DNA weave-tiles to control the spacing between thrombin-binding aptamers which resulted in very high antithrombin and anticoagulant activity of the construct. We believe that the tremendous progress in DNA nanotechnology over the past three decades will open even more research avenues in the near future for applications in a wide variety of disciplines including electronics, photonics, biomedical engineering, biosensing, therapeutics, and nucleic-acid-based drug delivery.

Solving Vertex Cover Problem Using DNA Tile Assembly Model

DNA tile assembly models are a class of mathematically distributed and parallel biocomputing models in DNA tiles. In previous works, tile assembly models have been proved be Turing-universal; that is, the system can do what Turing machine can do. In this paper, we use tile systems to solve computational hard problem. Mathematically, we construct three tile subsystems, which can be combined together to solve vertex cover problem. As a result, each of the proposed tile subsystems consists ofΘ(1) types of tiles, and the assembly process is executed in a parallel way (like DNA’s biological function in cells); thus the systems can generate the solution of the problem in linear time with respect to the size of the graph.

Three-Dimensional Structures Self-Assembled from DNA Bricks

We describe a simple and robust method to construct complex three-dimensional (3D) structures by using short synthetic DNA strands that we call "DNA bricks." In one-step annealing reactions, bricks with hundreds of distinct sequences self-assemble into prescribed 3D shapes. Each 32-nucleotide brick is a modular component; it binds to four local neighbors and can be removed or added independently. Each 8-base pair interaction between bricks defines a voxel with dimensions of 2.5 by 2.5 by 2.7 nanometers, and a master brick collection defines a "molecular canvas" with dimensions of 10 by 10 by 10 voxels. By selecting subsets of bricks from this canvas, we constructed a panel of 102 distinct shapes exhibiting sophisticated surface features, as well as intricate interior cavities and tunnels.