Expanding DNA Origami Design Freedom with De Novo Synthesized Scaffolds.

The construction of DNA origami nanostructures is heavily dependent on the folding of the scaffold strand, which is typically a single-stranded DNA genome extracted from a bacteriophage (M13). Custom scaffolds can be prepared in a number of methods, but they are not widely accessible to a broad user base in the DNA nanotechnology community. Here, we explored new design and construction possibilities with custom scaffolds prepared in our cost- and time-efficient production pipeline. According to the pipeline, we de novo produced a variety of scaffolds of specified local and global sequence characteristics and consequent origami constructs of modular arrangement in morphologies and functionalities. Taking advantage of this strategy of template-free scaffold production, we also designed and produced three-letter-coded scaffolds that can fold into designated morphologies rapidly at room temperature. The expanded design and construction freedom immediately brings in many new research opportunities and invites many more on the horizon.

Reconfiguration of DNA nanostructures induced by enzymatic ligation treatment.

Enzymatic ligation is a popular method in DNA nanotechnology for structural enforcement. When employed as stability switch for chosen components, ligation can be applied to induce DNA nanostructure reconfiguration. In this study, we investigate the reinforcement effect of ligation on addressable DNA nanostructures assembled entirely from short synthetic strands as the basis of structural reconfiguration. A careful calibration of ligation efficiency is performed on structures with programmable nicks. Systematic investigation using comparative agarose gel electrophoresis enables quantitative assessment of enhanced survivability with ligation treatment on a number of unique structures. The solid ligation performance sets up the foundation for the ligation-based structural reconfiguration. With the capability of switching base pairing status between permanent and transient (ON and OFF) by a simple round of enzymatic treatment, ligation induced reconfiguration can be engineered for DNA nanostructures accordingly.

Mesojunction-Based Design Paradigm of Structural DNA Nanotechnology.

Mesojunctions were introduced as a basic type of crossover configuration in the early development of structural DNA nanotechnology. However, the investigations of self-assembly from multiple mesojunction complexes have been overlooked in comparison to their counterparts based on regular junctions. In this work, we designed standardized component strands for the construction of complex mesojunction lattices. Three typical mesojunction configurations with three and four arms were showcased in the self-assembly of 1-, 2-, and 3-dimensional lattices constructed from both a scaffold-free tiling approach and a scaffolded origami approach.

Self-assembly of fully addressable DNA nanostructures from double crossover tiles.

DNA origami and single-stranded tile (SST) are two proven approaches to self-assemble finite-size complex DNA nanostructures. The construction elements appeared in structures from these two methods can also be found in multi-stranded DNA tiles such as double crossover tiles. Here we report the design and observation of four types of finite-size lattices with four different double crossover tiles, respectively, which, we believe, in terms of both complexity and robustness, will be rival to DNA origami and SST structures.

Reentrant DNA shells tune polyphosphate condensate size.

The inorganic biopolymer polyphosphate (polyP) occurs in all domains of life and affects myriad cellular processes. A longstanding observation is polyP's frequent proximity to chromatin, and, in many bacteria, its occurrence as magnesium (Mg2+)-enriched condensates embedded in the nucleoid region, particularly in response to stress. The physical basis of the interaction between polyP, DNA and Mg2+, and the resulting effects on the organization of the nucleoid and polyP condensates, remain poorly understood. Here, using a minimal system of polyP, Mg2+, and DNA, we find that DNA can form shells around polyP-Mg2+ condensates. These shells show reentrant behavior, that is, they form within a window of Mg2+ concentrations, representing a tunable architecture with potential relevance in other multicomponent condensates. This surface association tunes condensate size and DNA morphology in a manner dependent on DNA length and concentration, even at DNA concentrations orders of magnitude lower than found in the cell. Our work also highlights the remarkable capacity of two primordial inorganic species to organize DNA.

Monochromatic Fluorescent Barcodes Hierarchically Assembled from Modular DNA Origami Nanorods.

With the rapid advancement of fluorescence microscopy, there is a growing interest in the multiplexed detection and identification of various bioanalytes (e.g., nucleic acids and proteins) for efficient sample processing and analysis. We introduce in this work a simple and robust method to provide combinations for micrometer-scale fluorescent DNA barcodes of hierarchically assembled DNA origami superstructures for multiplexed molecular probing. In addition to optically resolvable dots, we placed fluorescent loci on adjacent origami within the diffraction limit of each other, rendering them as unresolvable bars of measurable lengths. We created a basic set of barcodes and trained a machine learning algorithm to process and identify individual barcodes from raw images with high accuracy. Moreover, we demonstrated that the number of combinations can be increased exponentially by generating longer barcodes, by controlling the number of incorporated fluorophores to create multiple levels of fluorescence intensity, and by employing super-resolution imaging. To showcase the readiness of the barcodes for applications, we used our barcodes to capture and identify target nucleic acid sequences and for simultaneous multiplexed characterization of binding kinetics of several orthogonal complementary nucleic acids.

Addressable DNA nanotubes with repetitive components.

Extended DNA nanostructures have already been constructed in a repetitive arrangement from millions of building blocks, many more than currently feasible with even the gold standard of addressable self-assembled structures. In order to construct addressable DNA nanostructures with more building blocks, it is desirable to arrange the addressable components repetitively. Accordingly, the overall size of the structure can be multiplied by the level of repetition in the addressable strands. In this study, we present a nanotube system that combines two seemingly conflicting features: addressability and repetitiveness. Based on an understanding of the tubulation resulting from the intrinsic curvature of the components, we produce DNA nanotubes with addressability available along the axial direction of the self-assembled tubes, which are also programmably repetitive along the lateral direction.

Complex wireframe DNA nanostructures from simple building blocks.

DNA nanostructures with increasing complexity have showcased the power of programmable self-assembly from DNA strands. At the nascent stage of the field, a variety of small branched objects consisting of a few DNA strands were created. Since then, a quantum leap of complexity has been achieved by a scaffolded 'origami' approach and a scaffold-free approach using single-stranded tiles/bricks-creating fully addressable two-dimensional and three-dimensional DNA nanostructures designed on densely packed lattices. Recently, wireframe architectures have been applied to the DNA origami method to construct complex structures. Here, revisiting the original wireframe framework entirely made of short synthetic strands, we demonstrate a design paradigm that circumvents the sophisticated routing and size limitations intrinsic to the scaffold strand in DNA origami. Under this highly versatile self-assembly framework, we produce a myriad of wireframe structures, including 2D arrays, tubes, polyhedra, and multi-layer 3D arrays.