Rapid Diagnosis of Coronavirus by RNA-Directed RNA Transcription Using an Engineered RNA-based Platform.

A coronavirus disease (COVID-19) outbreak associated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading widely through person-to-person transmission. Various detection approaches have been developed involving quantitative polymerase chain reaction (qPCR) methods, CRISPR-based systems, and direct targeting of specific coronavirus proteins. However, there have only been a few reports on the detection of RNA-dependent RNA polymerase (RdRP), the primer-independent RNA-replicable protein produced by the RNA genes of coronavirus. Here, we introduce a novel diagnostic methodology for COVID-19 using the RNA-directed and de novo RNA replicable function of RdRP. We devised an RNA platform for RdRP-induced transcription (RPRIT) that includes an RNA template that can be directly transcribed by RdRP. By utilizing RPRIT, the presence of RdRP can be readily confirmed within 30 min using isothermal incubation without PCR. This RdRP detection method can provide a new route for rapid diagnosis of RNA virus-infected patients.

Engineered extracellular vesicles and their mimetics for clinical translation.

Cells secrete extracellular vesicles (EVs) to external environments to achieve cellular homeostasis and cell-to-cell communication. Their therapeutic potential has been constantly spotlighted since they mirror both cytoplasmic and membranous components of parental cells. Meanwhile, growing evidence suggests that EV engineering could further promote EVs with a maximized capacity. In this review, a range of engineering techniques as well as upscaling approaches to exploit EVs and their mimetics are introduced. By laying out the pros and cons of each technique from different perspectives, we sought to provide an overview potentially helpful for understanding the current state of the art EV engineering and a guideline for choosing a suitable technique for engineering EVs. Furthermore, we envision that the advances in each technique will give rise to the combinatorial engineering of EVs, taking us a step closer to a clinical translation of EV-based therapeutics.

Formation of RNA Beads from Various DNA Nanoring Structures.

By taking advantages of rolling circle transcription, one of the powerful methods to overcome the instability of RNA, we successfully synthesized RNA particles having high copy numbers of RNA strands. To examine dependence of RNA particle formation to template circular DNA strands, we synthesized RNA beads with 65-nt, 92-nt and 200-nt DNA nanoring structures as template circular DNA. Our conclusion is that characteristics of RNA beads could be controlled with various template circular DNA for RCT. It is feasible that our RNA beads could be used for RNAzyme-based metal sensors such as aqueous lead ion sensing. In this case, we expect that multi-metal ion detection would be possible by using 200-nt circular DNA bearing multiple desired functions as a template for RNA bead formation. Furthermore, certain features of RNA beads such as sensitivity to nuclease digestion and maximum loading amount of drugs when used as a carrier are expected to be further adjusted by choosing appropriate porosity and size.

Enzymatic Polymerization on DNA Modified Gold Nanowire for Label-Free Detection of Pathogen DNA.

This paper presents a label-free biosensor for the detection of single-stranded pathogen DNA through the target-enhanced gelation between gold nanowires (AuNW) and the primer DNAs branched on AuNW. The target DNA enables circularization of the linear DNA template, and the primer DNA is elongated continuously via rolling circle amplification. As a result, in the presence of the target DNA, a macroscopic hydrogel was fabricated by the entanglement of the elongated DNA with AuNWs as a scaffold fiber for effective gelation. In contrast, very small separate particles were generated in the absence of the target DNA. This label-free biosensor might be a promising tool for the detection of pathogen DNAs without any devices for further analysis. Moreover, the biosensor based on the weaving of AuNW and DNAs suggests a novel direction for the applications of AuNWs in biological engineering.

Investigation of Förster resonance energy transfer (FRET) and competition of fluorescent dyes on DNA microparticles.

Fluorescent labeling is widely used to investigate the structural stability and changes to DNA nano- and microstructures. Despite this, the conventional method for observing DNA structures has several limitations in terms of cost-efficiency. This paper introduces a DNA spherical particle stained with DNA intercalating dyes (SYBR Green and SYTOX Orange) as tracers and reports the interaction between multiple dyes. The interference between the dyes was analyzed in terms of Förster resonance energy transfer (FRET) and competition. The changes in the fluorescence intensity by FRET were uniform, regardless of the sequence. The competition effect could occur when several dyes were added simultaneously. These properties are expected to help in the design of multicolor tracers in bioimaging and environmental applications.

Injectable Micro-Hydrogel for DNA Delivery: A Promising Therapeutic Platform.

Utilizing the immune system as a strategy for disease prevention and treatment is promising, especially with dendritic cells (DCs) playing a central role in adaptive immune responses. The unique properties of DCs drive interest in developing materials for cell-based therapy and immune modulation. Injectable systems require syringe-compatible scaffolds, while hydrogels, like alginate, known for their programmability and biocompatibility, offer a versatile platform for immune medicine enhancement through easy preparation and room-temperature cross-linking. In this study, we synthesized alginate balls loaded with DCs or cytosine-phosphorothioate-guanine deoxyribonucleotide (CpG DNA) microparticles, aiming for long-term immune cell culture with potential immune stimulation effects. Encapsulated DCs exhibited proliferation within the alginate balls for up to 7 days, and CpG MPs were uniformly dispersed, which can facilitate uptake by DCs. This was supported by the result that DCs effectively phagocytosed CpG microparticles in a 2D environment. After the uptake of CpG MPs, the alginate balls with CpG-MP-uptaken DCs were synthesized successfully. The injectable properties of the alginate balls were easily modulated by adjusting the syringe needle gauges. This innovative strategy holds substantial promise for advancing medical treatments, offering effective and comfortable solutions for controlled immune modulation.

Efficient circular RNA synthesis through Gap-DNA splint-mediated ligation.

The COVID-19 pandemic heightened interest in circular RNA (C-RNA) for RNA therapeutics, offering advantages over linear mRNAs. Circular mRNA facilitates uncapped molecule development, and C-RNAs ensure stability in RNA interference therapeutics. The synthesis method, RNA ligation, is employed in C-RNA-based therapeutics. Stable DNA-RNA hybrid constructs enable efficient RNA ligase-based circularization.

Label-free and liquid state SERS detection of multi-scaled bioanalytes via light-induced pinpoint colloidal assembly.

Surface-enhanced Raman scattering (SERS) has been extensively applied to detect complex analytes due to its ability to enhance the fingerprint signals of molecules around nanostructured metallic surfaces. Thus, it is essential to design SERS-active nanostructures with abundant electromagnetic hotspots in a probed volume according to the dimensions of the analytes, as the analytes must be located in their hotspots for maximum signal enhancement. Herein, we demonstrate a simple method for detecting robust SERS signals from multi-scaled bioanalytes, regardless of their dimensions in the liquid state, through a photothermally driven co-assembly with colloidal plasmonic nanoparticles as signal enhancers. Under resonant light illumination, plasmonic nanoparticles and analytes in the solution quickly assemble at the focused surface area by convective movements induced by the photothermal heating of the plasmonic nanoparticles without any surface modification. Such collective assemblies of plasmonic nanoparticles and analytes were optimized by varying the optical density and surface charge of the nanoparticles, the viscosity of the solvent, and the light illumination time to maximize the SERS signals. Using these light-induced co-assemblies, the intrinsic SERS signals of small biomolecules can be detected down to nanomolar concentrations based on their fingerprint spectra. Furthermore, large-sized biomarkers, such as viruses and exosomes, were successfully detected without labels, and the complexity of the collected spectra was statistically analyzed using t-distributed stochastic neighbor embedding combined with support vector machine (t-SNE + SVM). The proposed method is expected to provide a robust and convenient method to sensitively detect biologically and environmentally relevant analytes at multiple scales in liquid samples.

Multimodal Golden DNA Superstructures (GDSs) for Highly Efficient Photothermal Immunotherapy.

DNA-templated metallization has emerged as an efficient strategy for creating nanoscale-metal DNA hybrid structures with a desirable conformation and function. Despite the potential of DNA-metal hybrids, their use as combinatory therapeutic agents has rarely been examined. Herein, we present a simple approach for fabricating a multipurpose DNA superstructure that serves as an efficient photoimmunotherapy agent. Specifically, we adsorb and locally concentrate Au ions onto DNA superstructures through induced local reduction, resulting in the formation of Au nanoclusters. The mechanical and optical properties of these metallic nanoclusters can be rationally controlled by their conformations and metal ions. The resulting golden DNA superstructures (GDSs) exhibit significant photothermal effects that induce cancer cell apoptosis. When sequence-specific immunostimulatory effects of DNA are combined, GDSs provide a synergistic effect to eradicate cancer and inhibit metastasis, demonstrating potential as a combinatory therapeutic agent for tumor treatment. Altogether, the DNA superstructure-templated metal casting system offers promising materials for future biomedical applications.

Self-assembled RNA interference microsponges for efficient siRNA delivery.

The encapsulation and delivery of short interfering RNA (siRNA) has been realized using lipid nanoparticles, cationic complexes, inorganic nanoparticles, RNA nanoparticles and dendrimers. Still, the instability of RNA and the relatively ineffectual encapsulation process of siRNA remain critical issues towards the clinical translation of RNA as a therapeutic. Here we report the synthesis of a delivery vehicle that combines carrier and cargo: RNA interference (RNAi) polymers that self-assemble into nanoscale pleated sheets of hairpin RNA, which in turn form sponge-like microspheres. The RNAi-microsponges consist entirely of cleavable RNA strands, and are processed by the cell's RNA machinery to convert the stable hairpin RNA to siRNA only after cellular uptake, thus inherently providing protection for siRNA during delivery and transport to the cytoplasm. More than half a million copies of siRNA can be delivered to a cell with the uptake of a single RNAi-microsponge. The approach could lead to novel therapeutic routes for siRNA delivery.

Aptamer-based microspheres for highly sensitive protein detection using fluorescently-labeled DNA nanostructures.

Many highly sensitive protein detection techniques have been developed and have played an important role in the analysis of proteins. Herein, we report a novel technique that can detect proteins sensitively and effectively using aptamer-based DNA nanostructures. Thrombin was used as a target protein and aptamer was used to capture fluorescent dye-labeled DNA nanobarcodes or thrombin on a microsphere. The captured DNA nanobarcodes were replaced by a thrombin and aptamer interaction. The detection ability of this approach was confirmed by flow cytometry with different concentrations of thrombin. Our detection method has great potential for rapid and simple protein detection with a variety of aptamers.

Multiplexing enhancement for the detection of multiple pathogen DNA.

Various methods for the detection of pathogens have been researched and developed. However, most of detection methods are cost-ineffective and laborious. To minimize costs and labor, multi-detection of pathogen have been widely used. In this paper, we propose a novel multi-detection method, which can enhance multi-detection capability using fluorescent dye labeled DNA nanostructures that is named DNA nanobarcodes. By using three fluorescence colors, multi-detection capability is significantly increased because of the increased combination of three colors. Moreover, our approach uses a relatively simple DNA nanostructure to precisely control the fluorescence intensity ratio. Therefore, high multi-detection ability is achieved without constructing a complicated DNA nanostructure probe. Our novel detection method can overcome the obstacles of conventional methods and enhance multi-detection capability effectively. By using our new system, we were able to successfully detect nine different DNA pathogens simultaneously. Our system can easily increase the multiplexibility by using more fluosrescent colors on DNA nanobarcode.

A microfluidic device with 3-d hydrogel villi scaffold to simulate intestinal absorption.

The absorption of drugs via oral route is a subject of a great interest in drug development process. The current in vitro method for measuring the kinetics of drug absorption relies on 2-D monolayer culture of Caco-2 cells on a porous membrane, but physiologically unrealistic environment provided by this method often results in inaccurate drug absorption kinetics. Here we report a novel microfluidic system which better mimics the physiological environment of the human small intestine. Three dimensional geometries of villi of the small intestine were reproduced via novel hydrogel microfabrication technique, and the fluid flow in the apical and basolateral sides of intestinal tract was reproduced with a two-layer microfluidic device. A wide range of flow rates was achieved by using gravity-induced flow, potentially facilitating easier high-throughput implementation. The kinetics of diffusion process through the 3-D villi scaffold in the microfluidic device was measured and mathematically modeled. When combined with intestinal cell culture model, this novel 3-D microfluidic system can serve as an in vitro platform that better mimics the in vivo environment.

Self-assembly of a multimeric genomic hydrogel via multi-primed chain reaction of dual single-stranded circular plasmids for cell-free protein production.

Recent technical advances in cell-free protein synthesis (CFPS) offer several advantages over cell-based expression systems, including the application of cellular machinery, such as transcription and translation, in the test tube. Inspired by the advantages of CFPS, we have fabricated a multimeric genomic DNA hydrogel (mGD-gel) via rolling circle chain amplification (RCCA) using dual single-stranded circular plasmids with multiple primers. The mGD-gel exhibited significantly enhanced protein yield. In addition, mGD-gel can be reused at least five times, and the shape of the mGD-gel can be easily manipulated without losing the feasibility of protein expression. The mGD-gel platform based on the self-assembly of multimeric genomic DNA strands (mGD strands) has the potential to be used in CFPS systems for a variety of biotechnological applications.

Therapeutic Extracellular Vesicles from Tonsil-Derived Mesenchymal Stem Cells for the Treatment of Retinal Degenerative Disease.

BACKGROUND: Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic. METHODS: To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa. RESULTS: T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dose-dependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat. CONCLUSIONS: Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.

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.

Controlling in vivo stability and biodistribution in electrostatically assembled nanoparticles for systemic delivery.

This paper demonstrates the generation of systemically deliverable layer-by-layer (LbL) nanoparticles for cancer applications. LbL-based nanoparticles designed to navigate the body and deliver therapeutics in a programmable fashion are promising new and alternative systems for drug delivery, but there have been very few demonstrations of their systemic delivery in vivo due to a lack of knowledge in building LbL nanofilms that mimic traditional nanoparticle design to optimize delivery. The key to the successful application of these nanocarriers in vivo requires a systematic analysis of the influence of film architecture and adsorbed polyelectrolyte outer layer on their pharmacokinetics, which has thus far not been examined for this new approach to nanoparticle delivery. Herein, we have taken the first steps in stabilizing and controlling the systemic distribution of multilayer nanoparticles. Our findings highlight the unique character of LbL systems; the electrostatically assembled nanoparticles gain increased stability in vivo with larger numbers of deposited layers, and the final layer adsorbed generates a critical surface cascade, which dictates the surface chemistry and biological properties of the nanoparticle. This outer polyelectrolyte layer dramatically affects not only the degree of nonspecific particle uptake, but also the nanoparticle biodistribution. For hyaluronic acid (HA) outer layers, a long blood elimination half-life (∼9 h) and low accumulation (∼10-15% recovered fluorescence/g) in the liver were observed, illustrating that these systems can be designed to be highly appropriate for clinical translation.

RNA polymerization actuating nucleic acid membrane (RANAM)-based biosensing for universal RNA virus detection.

The coronavirus disease (COVID-19) pandemic has shown the importance of early disease diagnosis in preventing further infection and mortality. Despite major advances in the development of highly precise and rapid detection approaches, the time-consuming process of designing a virus-specific diagnostic kit has been a limiting factor in the early management of the pandemic. Here, we propose an RNA polymerase activity-sensing strategy utilizing an RNA polymerization actuating nucleic acid membrane (RANAM) partially metallized with gold for colorimetric RNA virus detection. Following RANAM-templated amplification of newly synthesized RNA, the presence of the RNA polymerase was determined by visualization of the inhibition of an oxidation/reduction (redox) reaction between 3,3',5,5'-tetramethylbenzidine (TMB) and blocked Au3+. As a proof of concept, a viral RNA-dependent RNA polymerase (RdRP), which is found in various RNA virus-infected cells, was chosen as a target molecule. With this novel RANAM biosensor, as little as 10 min of RdRP incubation could significantly reduce the colorimetric signal. Further development into an easy-to-use prototype kit in viral infection diagnosis detected RdRP present at levels even as low as 100 aM. Color formation based on the presence of RdRP could be simply and clearly confirmed through smartphone-assisted color imaging of the prototype kit. This study provides a non-PCR-based RNA virus detection including its variants using RdRP-mediated polymerization.

DNA Optoelectronics: Versatile Systems for On-Demand Functional Electrochemical Applications.

Herein, we propose innovative deoxyribonucleic acid (DNA)-based gels and their applications in diverse optoelectronics. We prepared the optoelectronic DNA-based gels (OpDNA Gel) through molecular complexation, that is, groove binding and ionic interactions of DNA and 1,1'-diheptyl-4,4'-bipyridinium (DHV). This process is feasible even with sequence-nonspecific DNA extracted from nature (e.g., salmon testes), resulting in the expansion of the application scope of DNA-based gels. OpDNA Gel possessed good mechanical characteristics (e.g., high compressibility, thermoplasticity, and outstanding viscoelastic properties) that have not been observed in typical DNA hydrogels. Moreover, the electrochromic (EC) characteristics of DHV were not lost when combined with OpDNA Gel. By taking advantage of the facile moldability, voltage-tunable EC behavior, and biocompatibility/biodegradability of OpDNA Gel, we successfully demonstrated its applicability in a variety of functional electrochemical systems, including on-demand information coding systems, user-customized EC displays, and microorganism monitoring systems. The OpDNA Gel is a promising platform for the application of DNA-based biomaterials in electrochemical optoelectronics.

Multimeric RNAs for efficient RNA-based therapeutics and vaccines.

There has been a growing interest in RNA therapeutics globally, and much progress has been made in this area, which has been further accelerated by the clinical applications of RNA-based vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Following these successful clinical trials, various technologies have been developed to improve the efficacy of RNA-based drugs. Multimerization of RNA therapeutics is one of the most attractive approaches to ensure high stability, high efficacy, and prolonged action of RNA-based drugs. In this review, we offer an overview of the representative approaches for generating repetitive functional RNAs by chemical conjugation, structural self-assembly, enzymatic elongation, and self-amplification. The therapeutic and vaccine applications of engineered multimeric RNAs in various diseases have also been summarized. By outlining the current status of multimeric RNAs, the potential of multimeric RNA as a promising treatment strategy is highlighted.