Investigating and Modeling the Factors That Affect Genetic Circuit Performance.

Over the past 2 decades, synthetic biology has yielded ever more complex genetic circuits that are able to perform sophisticated functions in response to specific signals. Yet, genetic circuits are not immediately transferable to an outside-the-lab setting where their performance is highly compromised. We propose introducing a broader test step to the design-build-test-learn workflow to include factors that might contribute to unexpected genetic circuit performance. As a proof of concept, we have designed and evaluated a genetic circuit in various temperatures, inducer concentrations, nonsterilized soil exposure, and bacterial growth stages. We determined that the circuit's performance is dramatically altered when these factors differ from the optimal lab conditions. We observed significant changes in the time for signal detection as well as signal intensity when the genetic circuit was tested under nonoptimal lab conditions. As a learning effort, we then proceeded to generate model predictions in untested conditions, which is currently lacking in synthetic biology application design. Furthermore, broader test and learn steps uncovered a negative correlation between the time it takes for a gate to turn ON and the bacterial growth phases. As the synthetic biology discipline transitions from proof-of-concept genetic programs to appropriate and safe application implementations, more emphasis on test and learn steps (i.e., characterizing parts and circuits for a broad range of conditions) will provide missing insights on genetic circuit behavior outside the lab.

Engineered Riboswitch Nanocarriers as a Possible Disease-Modifying Treatment for Metabolic Disorders.

Both DNA- and RNA-based nanotechnologies are remarkably useful for the engineering of molecular devices in vitro and are applied in a vast collection of applications. Yet, the ability to integrate functional nucleic acid nanostructures in applications outside of the lab requires overcoming their inherent degradation sensitivity and subsequent loss of function. Viruses are minimalistic yet sophisticated supramolecular assemblies, capable of shielding their nucleic acid content in nuclease-rich environments. Inspired by this natural ability, we engineered RNA-virus-like particles (VLPs) nanocarriers (NCs). We showed that the VLPs can function as an exceptional protective shell against nuclease-mediated degradation. We then harnessed biological recognition elements and demonstrated how engineered riboswitch NCs can act as a possible disease-modifying treatment for genetic metabolic disorders. The functional riboswitch is capable of selectively and specifically binding metabolites and preventing their self-assembly process and its downstream effects. When applying the riboswitch nanocarriers to an in vivo yeast model of adenine accumulation and self-assembly, significant inhibition of the sensitivity to adenine feeding was observed. In addition, using an amyloid-specific dye, we proved the riboswitch nanocarriers' ability to reduce the level of intracellular amyloid-like metabolite cytotoxic structures. The potential of this RNA therapeutic technology does not apply only to metabolic disorders, as it can be easily fine-tuned to be applied to other conditions and diseases.

Genetically Encoding Ultrastable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria.

DNA nanotechnology is leading the field of in vitro molecular-scale device engineering, accumulating to a dazzling array of applications. However, while DNA nanostructures' function is robust under in vitro settings, their implementation in real-world conditions requires overcoming their rapid degradation and subsequent loss of function. Viruses are sophisticated supramolecular assemblies, able to protect their nucleic acid content in inhospitable biological environments. Inspired by this natural ability, we engineered in vitro and in vivo technologies, enabling the encapsulation and protection of functional DNA nanostructures inside MS2 bacteriophage virus-like particles (VLPs). We demonstrate the ssDNA-VLPs nanocomposites' (NCs) abilities to encapsulate single-stranded-DNA (ssDNA) in a variety of sizes (200-1500 nucleotides (nt)), sequences, and structures while retaining their functionality. Moreover, by exposing these NCs to hostile biological conditions, such as human blood serum, we exhibit that the VLPs serve as an excellent protective shell. These engineered NCs pose critical properties that are yet unattainable by current fabrication methods.

Nanoengineered Peptide-Based Antimicrobial Conductive Supramolecular Biomaterial for Cardiac Tissue Engineering.

Owing to their dynamic nature and ordered architecture, supramolecular materials strikingly resemble organic components of living systems. Although short-peptide self-assembled nanostructured hydrogels are regarded as intriguing supramolecular materials for biotechnology, their application is often limited due to their low stability and considerable challenge of combining other desirable properties. Herein, a di-Fmoc-based hydrogelator containing the cell-adhesive Arg-Gly-Asp (RGD) fragment that forms a mechanically stable, self-healing hydrogel is designed. Molecular dynamics simulation reveals the presence of RGD segments on the surface of the hydrogel fibers, highlighting their cell adherence capacity. Aiming to impart conductivity, the 3D network of the hydrogel is further nanoengineered by incorporating polyaniline (PAni). The composite hydrogels demonstrate semiconductivity, excellent antibacterial activity, and DNA binding capacity. Cardiac cells grown on the surface of the composite hydrogels form functional synchronized monolayers. Taken together, the combination of these attributes in a single hydrogel suggests it as an exceptional candidate for functional supramolecular biomaterial designed for electrogenic tissue engineering.

Unusual Two-Step Assembly of a Minimalistic Dipeptide-Based Functional Hypergelator.

Self-assembled peptide hydrogels represent the realization of peptide nanotechnology into biomedical products. There is a continuous quest to identify the simplest building blocks and optimize their critical gelation concentration (CGC). Herein, a minimalistic, de novo dipeptide, Fmoc-Lys(Fmoc)-Asp, as an hydrogelator with the lowest CGC ever reported, almost fourfold lower as compared to that of a large hexadecapeptide previously described, is reported. The dipeptide self-assembles through an unusual and unprecedented two-step process as elucidated by solid-state NMR and molecular dynamics simulation. The hydrogel is cytocompatible and supports 2D/3D cell growth. Conductive composite gels composed of Fmoc-Lys(Fmoc)-Asp and a conductive polymer exhibit excellent DNA binding. Fmoc-Lys(Fmoc)-Asp exhibits the lowest CGC and highest mechanical properties when compared to a library of dipeptide analogues, thus validating the uniqueness of the molecular design which confers useful properties for various potential applications.

Programmable On-Chip Artificial Cell Producing Post-Translationally Modified Ubiquitinated Protein.

In nature, intracellular microcompartments have evolved to allow the simultaneous execution of tightly regulated complex processes within a controlled environment. This architecture serves as the blueprint for the construction of a wide array of artificial cells. However, such systems are inadequate in their ability to confine and sequentially control multiple central dogma activities (transcription, translation, and post-translational modifications) resulting in a limited production of complex biomolecules. Here, an artificial cell-on-a-chip comprising hierarchical compartments allowing the processing and transport of products from transcription, translation, and post-translational modifications through connecting channels is designed and fabricated. This platform generates a tightly controlled system, yielding directly a purified modified protein, with the potential to produce proteoform of choice. Using this platform, the full ubiquitinated form of the Parkinson's disease-associated α-synuclein is generated starting from DNA, in a single device. By bringing together all central dogma activities in a single controllable platform, this approach will open up new possibilities for the synthesis of complex targets, will allow to decipher diverse molecular mechanisms in health and disease and to engineer protein-based materials and pharmaceutical agents.

Metal-Ion Modulated Structural Transformation of Amyloid-Like Dipeptide Supramolecular Self-Assembly.

The misfolding of proteins and peptides potentially leads to a conformation transition from an α-helix or random coil to ß-sheet-rich fibril structures, which are associated with various amyloid degenerative disorders. Inhibition of the ß-sheet aggregate formation and control of the structural transition could therefore attenuate the development of amyloid-associated diseases. However, the structural transitions of proteins and peptides are extraordinarily complex processes that are still not fully understood and thus challenging to manipulate. To simplify this complexity, herein, the effect of metal ions on the inhibition of amyloid-like ß-sheet dipeptide self-assembly is investigated. By changing the type and ratio of the metal ion/dipeptide mixture, structural transformation is achieved from a ß-sheet to a superhelix or random coil, as confirmed by experimental results and computational studies. Furthermore, the obtained supramolecular metallogel exhibits excellent in vitro DNA binding and diffusion capability due to the positive charge of the metal/dipeptide complex. This work may facilitate the understanding of the role of metal ions in inhibiting amyloid formation and broaden the future applications of supramolecular metallogels in three-dimensional (3D) DNA biochip, cell culture, and drug delivery.

Go with the Flow-Microfluidics Approaches for Amyloid Research.

The rapid development of cost-efficient microfluidic devices has received tremendous attention from scientists of diverse fields. The growing potential of utilizing microfluidic platforms has further advanced the ability to integrate existing technology into microfluidic devices. Thus, allowing scientists to approach questions in fundamental fields, such as amyloid research, using new and otherwise unachievable conditions. Amyloids are associated with neurodegeneration and are in the forefront of many research efforts worldwide. The newly emerged microfluidic technology can serve as a novel research tool providing a platform for developing new methods in this field. In this review, we summarize the recent progress in amyloid research using microfluidic approaches. These approaches are driven from various fields, including physical chemistry, electrochemistry, biochemistry, and cell biology. Moreover, the new insights into novel microfluidic approaches for amyloid research reviewed here can be easily modified for other research interests.