Publications

Hierarchically porous materials have broad applications in biotechnology and medicine, yet current fabrication methods often lack scalability and biocompatibility. Here, we present a peptide-coordination self-assembly approach to prepare hierarchically porous microspheres composed of naturally occurring carnosine dipeptide and coordinated Zn(II) ions. Metal coordination led to microsphere formation featuring interconnected channels with a hierarchically porous structure. Characterization with scanning electron and transmission electron microscopy, as well as with extended X-ray absorption fine structure, confirmed its nanofibrous architecture and local Zn(II) coordination environment. Liquid cell transmission electron microscopy, in turn, provided real-time insight into the assembly process, revealing a stepwise process from nanoclusters to nanofibers and ultimately to porous microspheres. The carnosine-Zn(II) microspheres exhibit intrinsic blue fluorescence and high porosity, containing both micropores and mesopores, which facilitate efficient mass transport and biomolecule immobilization. We leverage these properties to generate reusable, cell-free synthesis nanoreactors, to enhance DNA transcription and translation and protect against nuclease degradation.

RNA-based therapeutics have revolutionized precision medicine due to their unprecedented potency, specificity, and adaptability. However, the inherent limited stability of RNA, including mRNA used in vaccines, is a major obstacle to the full realization of their potential. This instability, coupled with the centralized nature of vaccine production, currently limits the generation of RNA therapeutics at the point of care, which will otherwise fully harness the potential of these agents. Here, a microfluidic platform is presented for on-demand, personalized synthesis of modified mRNA stabilized by lipid nanoparticles. The design includes trapped biotinylated DNA, tagged T7 RNA polymerase, and a Tesla mixer, allowing the on-chip synthesis, purification, and encapsulation of mRNA in uniform lipid nanoparticles (LNPs), all conducted seamlessly on the same microfluidic device. This on-chip microfluidic synthesis approach is found to match standardized mRNA production yields, yet surpasses typical purification methods. Furthermore, as a proof-of-concept, the versatility and efficacy of the platform are demonstrated by generating diverse RNA sequences and structures, exhibiting functionality in human cell lines and mouse models. Moreover, an active SARS-CoV-2 vaccine is successfully engineered, highlighting the platform's potential for personalized vaccination strategies and offering a promising avenue for high throughput, decentralized vaccine delivery, reduced cold chain dependence, and even advancing current personalized medicine approaches through custom RNA therapeutics.

R2 elements, a class of non-long terminal repeat (non-LTR) retrotransposons, have the potential to be harnessed for transgene insertion. However, efforts to achieve this are limited by our understanding of the retrotransposon mechanisms. Here, we structurally and biochemically characterize R2 from Taeniopygia guttata (R2Tg). We show that R2Tg cleaves both strands of its ribosomal DNA target and binds a pseudoknotted RNA element within the R2 3 UTR to initiate target-primed reverse transcription. Guided by these insights, we engineer and characterize an all-RNA system for transgene insertion. We substantially reduce the systems size and insertion scars by eliminating unnecessary R2 sequences on the donor. We further improve the integration efficiency by chemically modifying the 5 end of the donor RNA and optimizing delivery, creating a compact system that achieves over 80% integration efficiency in several human cell lines. This work expands the genome engineering toolbox and provides mechanistic insights that will facilitate future development of R2-mediated gene insertion tools.

Despite ongoing efforts to study CRISPR systems, the evolutionary origins giving rise to reprogrammable RNA-guided mechanisms remain poorly understood. Here, we describe an integrated sequence/structure evolutionary tracing approach to identify the ancestors of the RNA-targeting CRISPR-Cas13 system. We find that Cas13 likely evolved from AbiF, which is encoded by an abortive infection-linked gene that is stably associated with a conserved non-coding RNA (ncRNA). We further characterize a miniature Cas13, classified here as Cas13e, which serves as an evolutionary intermediate between AbiF and other known Cas13s. Despite this relationship, we show that their functions substantially differ. Whereas Cas13e is an RNA-guided RNA-targeting system, AbiF is a toxin-antitoxin (TA) system with an RNA antitoxin. We solve the structure of AbiF using cryoelectron microscopy (cryo-EM), revealing basic structural alterations that set Cas13s apart from AbiF. Finally, we map the key structural changes that enabled a non-guided TA system to evolve into an RNA-guided CRISPR system.

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 circuits 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.

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.

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.

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 ArgGlyAsp (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.

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.

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.

The misfolding of proteins and peptides potentially leads to a conformation transition from an-helix or random coil to-sheet-rich fibril structures, which are associated with various amyloid degenerative disorders. Inhibition of the-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-sheet dipeptide self-assembly is investigated. By changing the type and ratio of the metal ion/dipeptide mixture, structural transformation is achieved from a-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.

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.

Deviations from the normal nucleoplasmic protein O-GlcNAcylation, as well as from normal protein sialylation and N-glycosylation in the secretory pathway, have been reported in Alzheimers disease (AD). However, the interplay between the cytoplasmic protein O-GlcNAcylation and the secretory N-/O-glycosylation in AD has not been described. We present a comprehensive analysis of the N-, O-, and O-GlcNAcglycomes in AD-affected brain regions as well as in AD patient serum. We detected marked differences in levels of glycan involved in both protein O-GlcNAcylation and N-/O-glycosylation between patients and healthy individuals and revealed brain regionspecific glycosylation-related pathology in patients. These alterations are not general for other neurodegenerative conditions, such as frontotemporal dementia and corticobasal degeneration. The alterations in the AD glycome in the serum could potentially lead to novel glyco-based biomarkers for AD progression. Strikingly, negative interrelationship was found between the pathways of protein O-GlcNAcylation and N-/O-glycosylation, suggesting a novel intracellular cross-talk.