Luvena L. Ong

Three-Dimensional Structures Self-Assembled from DNA Bricks

Building with DNA One route for assembling three-dimensional (3D) DNA nanostructures is to start with a long natural DNA single strand and attach short strands, or “staples,” that cause the entire “origami” structure to fold into a desired shape. Ke et al. (p. 1177, see the cover; see the Perspective by Gothelf) present an alternative approach to 3D assembly that builds upon modular assembly of 2D DNA tiles. One hundred and two distinct shapes were created from four-domain, 32-nucleotide single-stranded DNAs that assembled like childrens blocks; each block could bind to four neighboring blocks through specific pairing interactions. Computer design and stepwise assembly allowed assembly of hollow shapes with a variety of internal cavities. Stepwise assembly of 32-nucleotide DNA “bricks” can create a wide variety of nanoscale objects. 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.

Cooperative Nanoparticles for Tumor Detection and Photothermally Triggered Drug Delivery

The ability of one structural type to perform multiple medical diagnostic or therapeutic functions is often cited as an advantageous characteristic of nanomaterials that cannot be achieved with organic small molecules.[1–3] Although there are now many examples of nanosystems that integrate multiple functions into a single structure, the designs can reduce the efficacy of the individual functions due to space and surface-chemistry limitations in the tiny platforms. For example, magnetic nanoparticles and drug molecules can be co-encapsulated in liposomes to simultaneously perform multiple functions, such as magnetic resonance imaging, magnetic drug delivery and hyperthermia,[4] but the loading capacity and the stability are typically compromised relative to a single-component liposome. There has been some effort to develop intrinsically multifunctional nanomaterials such as magnetic nanocapsules and luminescent porous silicon nanoparticles to overcome such problems,[5,6] although these more complicated structures may lose versatility in terms of the types of payloads they can carry. Access to the payloads can also be limited, reducing the ability to control their release. Separating functions into two or more nanoparticle formulations is one means to simplify the problem. If two separate nanomaterials can be engineered to synergistically cooperate in their diagnostic or therapeutic functions, it is possible that the overall dosage can be reduced, minimizing side effects and providing a safer transition to the clinic.

Humanized mice with ectopic artificial liver tissues

“Humanized” mice offer a window into aspects of human physiology that are otherwise inaccessible. The best available methods for liver humanization rely on cell transplantation into immunodeficient mice with liver injury but these methods have not gained widespread use due to the duration and variability of hepatocyte repopulation. In light of the significant progress that has been achieved in clinical cell transplantation through tissue engineering, we sought to develop a humanized mouse model based on the facile and ectopic implantation of a tissue-engineered human liver. These human ectopic artificial livers (HEALs) stabilize the function of cryopreserved primary human hepatocytes through juxtacrine and paracrine signals in polymeric scaffolds. In contrast to current methods, HEALs can be efficiently established in immunocompetent mice with normal liver function. Mice transplanted with HEALs exhibit humanized liver functions persistent for weeks, including synthesis of human proteins, human drug metabolism, drug–drug interaction, and drug-induced liver injury. Here, mice with HEALs are used to predict the disproportionate metabolism and toxicity of “major” human metabolites using multiple routes of administration and monitoring. These advances may enable manufacturing of reproducible in vivo models for diverse drug development and research applications.

DNA brick crystals with prescribed depths.

We describe a general framework for constructing two-dimensional crystals with prescribed depth and sophisticated three-dimensional features. These crystals may serve as scaffolds for the precise spatial arrangements of functional materials for diverse applications. The crystals are self-assembled from single-stranded DNA components called DNA bricks. We demonstrate the experimental construction of DNA brick crystals that can grow to micron-size in the lateral dimensions with precisely controlled depth up to 80 nanometers. They can be designed to display user-specified sophisticated three-dimensional nanoscale features, such as continuous or discontinuous cavities and channels, and to pack DNA helices at parallel and perpendicular angles relative to the plane of the crystals.

Programmable self-assembly of three-dimensional nanostructures from 10,000 unique components

Nucleic acids (DNA and RNA) are widely used to construct nanometre-scale structures with ever increasing complexity, with possible application in fields such as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early kilodalton-scale examples containing typically tens of unique DNA strands. The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a desired structure, has provided access to megadalton-scale nanostructures that contain hundreds of unique DNA strands. Even larger DNA origami structures are possible, but manufacturing and manipulating an increasingly long scaffold strand remains a challenge. An alternative and more readily scalable approach involves the assembly of DNA bricks, which each consist of four short binding domains arranged so that the bricks can interlock. This approach does not require a scaffold; instead, the short DNA brick strands self-assemble according to specific inter-brick interactions. First-generation bricks used to create three-dimensional structures are 32 nucleotides long, consisting of four eight-nucleotide binding domains. Protocols have been designed to direct the assembly of hundreds of distinct bricks into well formed structures, but attempts to create larger structures have encountered practical challenges and had limited success. Here we show that DNA bricks with longer, 13-nucleotide binding domains make it possible to self-assemble 0.1–1-gigadalton, three-dimensional nanostructures from tens of thousands of unique components, including a 0.5-gigadalton cuboid containing about 30,000 unique bricks and a 1-gigadalton rotationally symmetric tetramer. We also assembled a cuboid that contains around 10,000 bricks and about 20,000 uniquely addressable, 13-base-pair ‘voxels’ that serves as a molecular canvas for three-dimensional sculpting. Complex, user-prescribed, three-dimensional cavities can be produced within this molecular canvas, enabling the creation of shapes such as letters, a helicoid and a teddy bear. We anticipate that with further optimization of structure design, strand synthesis and assembly procedure even larger structures could be accessible, which could be useful for applications such as positioning functional components.

Complex Reconfiguration of DNA Nanostructures

Nucleic acids have been used to create diverse synthetic structural and dynamic systems. Toehold-mediated strand displacement has enabled the construction of sophisticated circuits, motors, and molecular computers. Yet it remains challenging to demonstrate complex structural reconfiguration in which a structure changes from a starting shape to another arbitrarily prescribed shape. To address this challenge, we have developed a general structural-reconfiguration method that utilizes the modularly interconnected architecture of single-stranded DNA tile and brick structures. The removal of one component strand reveals a newly exposed toehold on a neighboring strand, thus enabling us to remove regions of connected component strands without the need to modify the strands with predesigned external toeholds. By using this method, we reconfigured a two-dimensional rectangular DNA canvas into diverse prescribed shapes. We also used this method to reconfigure a three-dimensional DNA cuboid.

A Compact DNA Cube with Side Length 10 nm

A small and compact DNA cube with zeptoliter volume is constructed by means of a generalized DNA brick concept using short synthetic oligonucleotides with varying lengths. By mimicking design principles from the DNA origami technique, the DNA cube offers higher stability and assembly yields compared to other approaches. Its potential application as nanoscale fluorescent probe is demonstrated using super-resolution imaging.

Barcode extension for analysis and reconstruction of structures

Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures.

Sub–100-nm metafluorophores with digitally tunable optical properties self-assembled from DNA

Programmable DNA-based metafluorophores allow simultaneous tagging and imaging with more than 100 virtual colors. Fluorescence microscopy allows specific target detection down to the level of single molecules and has become an enabling tool in biological research. To transduce the biological information to an imageable signal, we have developed a variety of fluorescent probes, such as organic dyes or fluorescent proteins with different colors. Despite their success, a limitation on constructing small fluorescent probes is the lack of a general framework to achieve precise and programmable control of critical optical properties, such as color and brightness. To address this challenge, we introduce metafluorophores, which are constructed as DNA nanostructure–based fluorescent probes with digitally tunable optical properties. Each metafluorophore is composed of multiple organic fluorophores, organized in a spatially controlled fashion in a compact sub–100-nm architecture using a DNA nanostructure scaffold. Using DNA origami with a size of 90 × 60 nm2, substantially smaller than the optical diffraction limit, we constructed small fluorescent probes with digitally tunable brightness, color, and photostability and demonstrated a palette of 124 virtual colors. Using these probes as fluorescent barcodes, we implemented an assay for multiplexed quantification of nucleic acids. Additionally, we demonstrated the triggered in situ self-assembly of fluorescent DNA nanostructures with prescribed brightness upon initial hybridization to a nucleic acid target.

Hierarchical Assembly of DNA Nanostructures Based on Four-Way Toehold-Mediated Strand Displacement

Because of its attractive cost and yield, hierarchical assembly, in which constituent structures of lower hierarchy share a majority of components, is an appealing approach to scale up DNA self-assembly. A few strategies have already been investigated to combine preformed DNA nanostructures. In this study, we present a new hierarchical assembly method based on four-way toehold-mediated strand displacement to facilitate the combination of preformed DNA structural units. Employing such a method, we have constructed a series of higher-order structures composed of 5, 7, 9, 11, 13, and 15 preformed units respectively.