DNA Microgels as a Platform for Cell-Free Protein Expression and Display.

Protein expression and selection is an essential process in the modification of biological products. Expressed proteins are selected based on desired traits (phenotypes) from diverse gene libraries (genotypes), whose size may be limited due to the difficulties inherent in diverse cell preparation. In addition, not all genes can be expressed in cells, and linking genotype with phenotype further presents a great challenge in protein engineering. We present a DNA gel-based platform that demonstrates the versatility of two DNA microgel formats to address fundamental challenges of protein engineering, including high protein yield, isolation of gene sets, and protein display. We utilize microgels to show successful protein production and capture of a model protein, green fluorescent protein (GFP), which is further used to demonstrate a successful gene enrichment through fluorescence-activated cell sorting (FACS) of a mixed population of microgels containing the GFP gene. Through psoralen cross-linking of the hydrogels, we have synthesized DNA microgels capable of surviving denaturing conditions while still possessing the ability to produce protein. Lastly, we demonstrate a method of producing extremely high local gene concentrations of up to 32 000 gene repeats in hydrogels 1 to 2 µm in diameter. These DNA gels can serve as a novel cell-free platform for integrated protein expression and display, which can be applied toward more powerful, scalable protein engineering and cell-free synthetic biology with no physiological boundaries and limitations.

Engineering DNA-based functional materials.

While DNA is a genetic material, it is also an inherently polymeric material made from repeating units called nucleotides. Although DNA's biological functions have been studied for decades, the polymeric features of DNA have not been extensively exploited until recently. In this tutorial review, we focus on two aspects of using DNA as a polymeric material: (1) the engineering methods, and (2) the potential real-world applications. More specifically, various strategies for constructing DNA-based building blocks and materials are introduced based on DNA topologies, which include linear, branched/dendritic, and networked. Different applications in nanotechnology, medicine, and biotechnology are further reviewed.

Dynamic DNA material with emergent locomotion behavior powered by artificial metabolism.

Metabolism is a key process that makes life alive-the combination of anabolism and catabolism sustains life by a continuous flux of matter and energy. In other words, the materials comprising life are synthesized, assembled, dissipated, and decomposed autonomously in a controlled, hierarchical manner using biological processes. Although some biological approaches for creating dynamic materials have been reported, the construction of such materials by mimicking metabolism from scratch based on bioengineering has not yet been achieved. Various chemical approaches, especially dissipative assemblies, allow the construction of dynamic materials in a synthetic fashion, analogous to part of metabolism. Inspired by these approaches, here, we report a bottom-up construction of dynamic biomaterials powered by artificial metabolism, representing a combination of irreversible biosynthesis and dissipative assembly processes. An emergent locomotion behavior resembling a slime mold was programmed with this material by using an abstract design model similar to mechanical systems. Dynamic properties, such as autonomous pattern generation and continuous polarized regeneration, enabled locomotion along the designated tracks against a constant flow. Furthermore, an emergent racing behavior of two locomotive bodies was achieved by expanding the program. Other applications, including pathogen detection and hybrid nanomaterials, illustrated further potential use of this material. Dynamic biomaterials powered by artificial metabolism could provide a previously unexplored route to realize "artificial" biological systems with regenerating and self-sustaining characteristics.

Femtosecond laser ablation reveals antagonistic sensory and neuroendocrine signaling that underlie C. elegans behavior and development.

The specific roles of neuronal subcellular components in behavior and development remain largely unknown, even though advances in molecular biology and conventional whole-cell laser ablation have greatly accelerated the identification of contributors at the molecular and cellular levels. We systematically applied femtosecond laser ablation, which has submicrometer resolution in vivo, to dissect the cell bodies, dendrites, or axons of a sensory neuron (ASJ) in Caenorhabditis elegans to determine their roles in modulating locomotion and the developmental decisions for dauer, a facultative, stress-resistant life stage. Our results indicate that the cell body sends out axonally mediated and hormonal signals in order to mediate these functions. Furthermore, our results suggest that antagonistic sensory dendritic signals primarily drive and switch polarity between the decisions to enter and exit dauer. Thus, the improved resolution of femtosecond laser ablation reveals a rich complexity of neuronal signaling at the subcellular level, including multiple neurite and hormonally mediated pathways dependent on life stage.

Point-of-care nucleic acid detection using nanotechnology.

Recent developments in nanotechnology have led to significant advancements in point-of-care (POC) nucleic acid detection. The ability to sense DNA and RNA in a portable format leads to important applications for a range of settings, from on-site detection in the field to bedside diagnostics, in both developing and developed countries. We review recent innovations in three key process components for nucleic acid detection: sample preparation, target amplification, and read-out modalities. We discuss how the advancements realized by nanotechnology are making POC nucleic acid detection increasingly applicable for decentralized and accessible testing, in particular for the developing world.

Novel DNA materials and their applications.

The last two decades have witnessed the exponential development of DNA as a generic material instead of just a genetic material. The biological function, nanoscale geometry, biocompatibility, biodegradability, and molecular recognition capacity of DNA make it a promising candidate for the construction of novel functional nanomaterials. As a result, DNA has been recognized as one of the most appealing and versatile nanomaterial building blocks. Scientists have used DNA in this way to construct various amazing nanostructures, such as ordered lattices, origami, supramolecular assemblies, and even three-dimensional objects. In addition, DNA has been utilized as a guide and template to direct the assembly of other nanomaterials including nanowires, free-standing membranes, and crystals. Furthermore, DNA can also be used as structural components to construct bulk materials such as DNA hydrogels, demonstrating its ability to behave as a unique polymer. Overall, these novel DNA materials have found applications in various areas in the biomedical field in general, and nanomedicine in particular. In this review, we summarize the development of DNA assemblies, describe the innovative progress of multifunctional and bulk DNA materials, and highlight some real-world nanomedical applications of these DNA materials. We also show our insights throughout this article for the future direction of DNA materials.