ABSTRACT
We report a thorough investigation of the role of single-stranded thymidine (ssT) linkers in the stability and flexibility of minimal, multistranded DNA nanostructures. We systematically explore the impact of varying the number of ssTs in three-way junction motifs (3WJs) on their formation and properties. Through various UV melting experiments and molecular dynamics simulations, we demonstrate that while the number of ssTs minimally affects thermodynamic stability, the increasing ssT regions significantly enhance the structural flexibility of 3WJs. Utilizing this knowledge, we design triangular DNA nanoparticles with varying ssTs, all showing exceptional assembly efficiency except for the 0T triangle. All triangles demonstrate enhanced stability in blood serum and are nonimmunostimulatory and nontoxic in mammalian cell lines. The 4T 3WJ is chosen as the building block for constructing other polygons due to its enhanced flexibility and favorable physicochemical characteristics, making it a versatile choice for creating cost-effective, stable, and functional DNA nanostructures that can be stored in the dehydrated forms while retaining their structures. Our study provides valuable insights into the design and application of nucleic acid nanostructures, emphasizing the importance of understanding stability and flexibility in the realm of nucleic acid nanotechnology. Our findings suggest the intricate connection between these ssTs and the structural adaptability of DNA 3WJs, paving the way for more precise design and engineering of nucleic acid nanosystems suitable for broad biomedical applications.
Subject(s)
Nanoparticles , Nanostructures , Nucleic Acids , Animals , Nucleic Acid Conformation , Nanostructures/chemistry , Nanotechnology , DNA/chemistry , Nanoparticles/chemistry , MammalsABSTRACT
Recalcitrant staphylococcal osteomyelitis may be due, in part, to the ability of Staphylococcus aureus to invade bone cells. However, osteoclasts and osteoblasts are now recognized to shape host responses to bacterial infection and we have recently described their ability to produce IFN-ß following S. aureus infection and limit intracellular bacterial survival/propagation. Here, we have investigated the ability of novel, rationally designed, nucleic acid nanoparticles (NANPs) to induce the production of immune mediators, including IFN-ß, following introduction into bone cells. We demonstrate the successful delivery of representative NANPs into osteoblasts and osteoclasts via endosomal trafficking when complexed with lipid-based carriers. Their delivery was found to differentially induce immune responses according to their composition and architecture via discrete cytosolic pattern recognition receptors. Finally, the utility of this nanoparticle technology was supported by the demonstration that immunostimulatory NANPs augment IFN-ß production by S. aureus infected bone cells and reduce intracellular bacterial burden.
Subject(s)
Nanoparticles , Nucleic Acids , Osteoblasts , Osteoclasts , Staphylococcal Infections , Staphylococcus aureus , Staphylococcus aureus/drug effects , Staphylococcus aureus/immunology , Nanoparticles/chemistry , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteoclasts/drug effects , Osteoclasts/metabolism , Animals , Nucleic Acids/chemistry , Nucleic Acids/pharmacology , Staphylococcal Infections/immunology , Staphylococcal Infections/drug therapy , Mice , Interferon Type I/metabolism , Humans , Interferon-beta/metabolism , Adjuvants, Immunologic/pharmacologyABSTRACT
The intrinsic properties of RNA and DNA biopolymers emphasized by engineered nucleic acid nanoparticles (NANPs) offer accelerated development of next-generation therapies. The rational design of NANPs facilitates programmable architectures intended for regulated molecular and cellular interactions. The conventional bottom-up assembly of NANPs relies on the thermal annealing of individual strands. Here, we introduce a concept of nuclease-driven production of NANPs where selective digestion of functionally inert structures leads to isothermal self-assembly of liberated constituents. The working principles, morphological changes, assembly kinetics, and the retention of structural integrity for system components subjected to anhydrous processing and storage are assessed. We show that the assembly of precursors into a single structure improves stoichiometry and enhances the functionality of nuclease-driven products. Furthermore, the experiments with immune reporting cell lines show that the developed protocols retain the immunostimulatory functionality of tested NANPs. The presented approach enables exploitation of the advantages of conditionally produced NANPs and demonstrates that NANPs' stability, immunorecognition, and assembly can be regulated to allow for a more robust functional system.
Subject(s)
Nanoparticles , Nucleic Acids , Nucleic Acids/chemistry , RNA/chemistry , DNA/chemistry , Cell Line , Nanoparticles/chemistry , Nucleic Acid ConformationABSTRACT
The unbalanced coagulation of blood is a life-threatening event that requires accurate and timely treatment. We introduce a user-friendly biomolecular platform based on modular RNA-DNA anticoagulant fibers programmed for reversible extracellular communication with thrombin and subsequent control of anticoagulation via a "kill-switch" mechanism that restores hemostasis. To demonstrate the potential of this reconfigurable technology, we designed and tested a set of anticoagulant fibers that carry different thrombin-binding aptamers. All fibers are immunoquiescent, as confirmed in freshly collected human peripheral blood mononuclear cells. To assess interindividual variability, the anticoagulation is confirmed in the blood of human donors from the U.S. and Brazil. The anticoagulant fibers reveal superior anticoagulant activity and prolonged renal clearance in vivo in comparison to free aptamers. Finally, we confirm the efficacy of the "kill-switch" mechanism in vivo in murine and porcine models.
Subject(s)
Aptamers, Nucleotide , Nanoparticles , Nucleic Acids , Animals , Anticoagulants , Aptamers, Nucleotide/chemistry , Humans , Leukocytes, Mononuclear , Mice , Swine , Thrombin/chemistryABSTRACT
This Special Issue is intended to celebrate Professor Yechezkel Barenholz's distinguished achievements [...].
Subject(s)
Liposomes , NanotechnologyABSTRACT
Nucleic acid nanoparticles (NANPs) require a carrier to allow for their intracellular delivery to immune cells. Cytokine production, specifically type I and III interferons, allows for reliable monitoring of the carrier effect on NANP immunostimulation. Recent studies have shown that changes in the delivery platform (e.g., lipid-based carriers vs. dendrimers) can alter NANPs' immunorecognition and downstream cytokine production in various immune cell populations. Herein, we used flow cytometry and measured cytokine induction to show how compositional variations in commercially available lipofectamine carriers impact the immunostimulatory properties of NANPs with different architectural characteristics.
Subject(s)
Nanoparticles , Nucleic Acids , Lipids , Interferons , ImmunizationABSTRACT
Investigating and understanding novel antibacterial agents is a necessary task as there is a constant increase in the number of multidrug-resistant bacterial species. The use of nanotechnology to combat drug-resistant bacteria is an important research area. The laboratory experiment described herein demonstrates that changes in the nanostructure of a material lead to significantly different antibacterial efficacies. Silver has been known to be an effective antibacterial agent throughout history, but its therapeutic uses are limited when present as either the bulk material or cations in solution. Silver nanoparticles (AgNPs) and DNA-templated silver nanoclusters (DNA-AgNCs) are both nanostructured silver materials that show vastly different antibacterial activities when incubated with E. coli in liquid culture. This work aims to provide students with hands-on experience in the synthesis and characterization of nanomaterials and basic microbiology skills; moreover, it is applicable to undergraduate and graduate curricula.
ABSTRACT
Different therapeutic nucleic acids (TNAs) can be unified in a single structure by their elongation with short oligonucleotides designed to self-assemble into nucleic acid nanoparticles (NANPs). With this approach, therapeutic cocktails with precisely controlled composition and stoichiometry of active ingredients can be delivered to the same diseased cells for enhancing pharmaceutical action. In this work, an additional nanotechnology-based therapeutic option that enlists a biocompatible NANP-encoded platform for their controlled patient-specific immunorecognition is explored. For this, a set of representative functional NANPs is extensively characterized in vitro, ex vivo, and in vivo and then further analyzed for immunostimulation of human peripheral blood mononuclear cells freshly collected from healthy donor volunteers. The results of the study present the advancement of the current TNA approach toward personalized medicine and offer a new strategy to potentially address top public health challenges related to drug overdose and safety through the biodegradable nature of the functional platform with immunostimulatory regulation.
ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface spike glycoprotein - a major antibody target - is critical for virus entry via engagement of human angiotensin-converting enzyme 2 (ACE2) receptor. Despite successes with existing vaccines and therapies that primarily target the receptor binding domain (RBD) of the spike protein, the susceptibility of RBD to mutations provides escape routes for the SARS-CoV-2 from neutralizing antibodies. On the other hand, structural conservation in the spike protein can be targeted to reduce escape mutations and achieve broad protection. Here, we designed candidate stable immunogens that mimic surface features of selected conserved regions of spike protein through 'epitope grafting,' in which we present the target epitope topology on diverse heterologous scaffolds that can structurally accommodate the spike epitopes. Structural characterization of the epitope-scaffolds showed stark agreement with our computational models and target epitopes. The sera from mice immunized with engineered designs display epitope-scaffolds and spike binding activity. We also demonstrated the utility of the designed epitope-scaffolds in diagnostic applications. Taken all together, our study provides important methodology for targeting the conserved, non-RBD structural motifs of spike protein for SARS-CoV-2 epitope vaccine design and demonstrates the potential utility of 'epitope grafting' in rational vaccine design.
ABSTRACT
Nucleic acid nanoparticles, or NANPs, rationally designed to communicate with the human immune system, can offer innovative therapeutic strategies to overcome the limitations of traditional nucleic acid therapies. Each set of NANPs is unique in their architectural parameters and physicochemical properties, which together with the type of delivery vehicles determine the kind and the magnitude of their immune response. Currently, there are no predictive tools that would reliably guide the design of NANPs to the desired immunological outcome, a step crucial for the success of personalized therapies. Through a systematic approach investigating physicochemical and immunological profiles of a comprehensive panel of various NANPs, the research team developes and experimentally validates a computational model based on the transformer architecture able to predict the immune activities of NANPs. It is anticipated that the freely accessible computational tool that is called an "artificial immune cell," or AI-cell, will aid in addressing the current critical public health challenges related to safety criteria of nucleic acid therapies in a timely manner and promote the development of novel biomedical tools.
Subject(s)
Nanoparticles , Nucleic Acids , Humans , Nucleic Acids/chemistry , Monocytes , Nanoparticles/chemistry , Interferons , Artificial IntelligenceABSTRACT
Recent advances in nanotechnology now allow for the methodical implementation of therapeutic nucleic acids (TNAs) into modular nucleic acid nanoparticles (NANPs) with tunable physicochemical properties which can match the desired biological effects, provide uniformity, and regulate the delivery of multiple TNAs for combinatorial therapy. Despite the potential of novel NANPs, the maintenance of their structural integrity during storage and shipping remains a vital issue that impedes their broader applications. Cold chain storage is required to maintain the potency of NANPs in the liquid phase, which greatly increases transportation costs. To promote long-term storage and retention of biological activities at higher temperatures (e.g., +50 °C), a panel of representative NANPs is first exposed to three different drying mechanisms-vacuum concentration (SpeedVac), lyophilization (Lyo), and light-assisted drying (LAD)-and then rehydrated and analyzed. While SpeedVac primarily operates using heat, Lyo avoids temperature increases by taking advantage of pressure reduction and LAD involves a near-infrared laser for uniform drying in the presence of trehalose. This work compares and defines refinements crucial in formulating an optimal strategy for producing stable, fully functional NANPs and presents a forward advancement in their development for clinical applications.
Subject(s)
Nanoparticles , Nucleic Acids , Nanoparticles/chemistry , Nanotechnology , Nucleic Acids/chemistry , TemperatureABSTRACT
Nucleic acid nanoparticles (NANPs) have become powerful new platforms as therapeutic and diagnostic tools due to the innate biological ability of nucleic acids to identify target molecules or silence genes involved in disease pathways. However, the clinical application of NANPs has been limited by factors such as chemical instability, inefficient intracellular delivery, and the triggering of detrimental inflammatory responses following innate immune recognition of nucleic acids. Here, we have studied the effects of altering the chemical composition of a circumscribed panel of NANPs that share the same connectivity, shape, size, charge and sequences. We show that replacing RNA strands with either DNA or chemical analogs increases the enzymatic and thermodynamic stability of NANPs. Furthermore, we have found that such composition changes affect delivery efficiency and determine subcellular localization, effects that could permit the targeted delivery of NANP-based therapeutics and diagnostics. Importantly, we have determined that altering NANP composition can dictate the degree and mechanisms by which cell immune responses are initiated. While RNA NANPs trigger both TLR7 and RIG-I mediated cytokine and interferon production, DNA NANPs stimulate minimal immune activation. Importantly, incorporation of 2'F modifications abrogates RNA NANP activation of TLR7 but permits RIG-I dependent immune responses. Furthermore, 2'F modifications of DNA NANPs significantly enhances RIG-I mediated production of both proinflammatory cytokines and interferons. Collectively this indicates that off-target effects may be reduced and/or desirable immune responses evoked based upon NANPs modifications. Together, our studies show that NANP composition provides a simple way of controlling the immunostimulatory potential, and physicochemical and delivery characteristics, of such platforms.
Subject(s)
DNA/chemistry , Nanoparticles/chemistry , RNA/chemistry , Biological Transport , Cell Line , Cytokines/biosynthesis , DNA/metabolism , Humans , Interferon Regulatory Factors/metabolism , NF-kappa B/metabolism , Nanoparticles/metabolism , Oligonucleotides/chemistry , RNA/metabolism , ThermodynamicsABSTRACT
Nucleic acid-based assemblies that interact with each other and further communicate with the cellular machinery in a controlled manner represent a new class of reconfigurable materials that can overcome limitations of traditional biochemical approaches and improve the potential therapeutic utility of nucleic acids. This notion enables the development of novel biocompatible 'smart' devices and biosensors with precisely controlled physicochemical and biological properties. We extend this novel concept by designing RNA-DNA fibers and polygons that are able to cooperate in different human cell lines and that have defined immunostimulatory properties confirmed by ex vivo experiments. The mutual intracellular interaction of constructs results in the release of a large number of different siRNAs while giving a fluorescent response and activating NF-κB decoy DNA oligonucleotides. This work expands the possibilities of nucleic acid technologies by (i) introducing very simple design principles and assembly protocols; (ii) potentially allowing for a simultaneous release of various siRNAs together with functional DNA sequences and (iii) providing controlled rates of reassociation, stabilities in human blood serum, and immunorecognition.
Subject(s)
DNA/genetics , NF-kappa B/genetics , RNA/genetics , Transcription, Genetic , DNA/chemistry , Fluorescence Resonance Energy Transfer , Gene Expression Regulation/genetics , Humans , Oligodeoxyribonucleotides/genetics , Oligonucleotides/chemistry , Oligonucleotides/genetics , RNA/chemistry , RNA, Small Interfering/geneticsABSTRACT
Contractile forces within the planar interface between T cell and antigen-presenting surface mechanically stimulate T cell receptors (TCR) in the mature immune synapses. However, the origin of mechanical stimulation during the initial, i.e., presynaptic, microvilli-based TCR activation in the course of immune surveillance remains unknown and new tools to help address this problem are needed. In this work, we develop nucleic acid nanoassembly (NAN)-based technology for functionalization of hydrogels using isothermal toehold-mediated reassociation of RNA/DNA heteroduplexes. Resulting platform allows for regulation with NAN linkers of 3D force momentum along the TCR mechanical axis, whereas hydrogels contribute to modulation of 2D shear modulus. By utilizing different lengths of NAN linkers conjugated to polyacrylamide gels of different shear moduli, we demonstrate an efficient capture of human T lymphocytes and tunable activation of TCR, as confirmed by T-cell spreading and pY foci.
Subject(s)
Hydrogels/pharmacology , Lymphocyte Activation/drug effects , Lymphocytes/drug effects , Receptors, Antigen, T-Cell/genetics , Antigen-Presenting Cells/drug effects , DNA/chemistry , DNA/pharmacology , Humans , Hydrogels/chemistry , Lymphocyte Activation/genetics , Lymphocytes/metabolism , Nucleic Acid Heteroduplexes/chemistry , Nucleic Acid Heteroduplexes/genetics , Nucleic Acid Heteroduplexes/pharmacology , RNA/chemistry , RNA/genetics , Receptors, Antigen, T-Cell/drug effects , T-Lymphocytes/drug effectsABSTRACT
Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.
Subject(s)
1-Acylglycerophosphocholine O-Acyltransferase , Gene Silencing , Melanoma , Nanoparticles , Neoplasm Proteins , Nucleic Acids , 1-Acylglycerophosphocholine O-Acyltransferase/antagonists & inhibitors , 1-Acylglycerophosphocholine O-Acyltransferase/biosynthesis , Cell Line, Tumor , Humans , Melanoma/drug therapy , Melanoma/enzymology , Nanoparticles/chemistry , Nanoparticles/therapeutic use , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Nucleic Acids/chemistry , Nucleic Acids/pharmacologyABSTRACT
The relatively straightforward methods of designing and assembling various functional nucleic acids into nanoparticles offer advantages for applications in diverse diagnostic and therapeutic approaches. However, due to the novelty of this approach, nucleic acid nanoparticles (NANPs) are not yet used in the clinic. The immune recognition of NANPs is among the areas of preclinical investigation aimed at enabling the translation of these novel materials into clinical settings. NANPs' interactions with the complement system, coagulation systems, and immune cells are essential components of their preclinical safety portfolio. It has been established that NANPs' physicochemical properties-composition, shape, and size-determine their interactions with immune cells (primarily blood plasmacytoid dendritic cells and monocytes), enable recognition by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), and mediate the subsequent cytokine response. However, unlike traditional therapeutic nucleic acids (e.g., CpG oligonucleotides), NANPs do not trigger a cytokine response unless they are delivered into the cells using a carrier. Recently, it was discovered that the type of carrier provides an additional tool for regulating both the spectrum and the magnitude of the cytokine response to NANPs. Herein, we review the current knowledge of NANPs' interactions with various components of the immune system to emphasize the unique properties of these nanomaterials and highlight opportunities for their use in vaccines and immunotherapy.
Subject(s)
Immune System/drug effects , Immune System/immunology , Nanoparticles/administration & dosage , Nucleic Acids/immunology , Animals , Cytokines/immunology , Humans , Immunotherapy/methods , Nanostructures/administration & dosageABSTRACT
Silver has a long history of antibacterial effectiveness. The combination of atomically precise metal nanoclusters with the field of nucleic acid nanotechnology has given rise to DNA-templated silver nanoclusters (DNA-AgNCs) which can be engineered with reproducible and unique fluorescent properties and antibacterial activity. Furthermore, cytosine-rich single-stranded DNA oligonucleotides designed to fold into hairpin structures improve the stability of AgNCs and additionally modulate their antibacterial properties and the quality of observed fluorescent signals. In this work, we characterize the sequence-specific fluorescence and composition of four representative DNA-AgNCs, compare their corresponding antibacterial effectiveness at different pH, and assess cytotoxicity to several mammalian cell lines.
Subject(s)
Anti-Bacterial Agents/chemistry , DNA, Single-Stranded/chemistry , Metal Nanoparticles/chemistry , Silver/chemistry , Anti-Bacterial Agents/pharmacology , Cell Survival/drug effects , Escherichia coli/drug effects , Fluorescence , Humans , Metal Nanoparticles/toxicity , THP-1 CellsABSTRACT
Recent insights into the immunostimulatory properties of nucleic acid nanoparticles (NANPs) have demonstrated that variations in the shape, size, and composition lead to distinct patterns in their immunostimulatory properties. While most of these studies have used a single lipid-based carrier to allow for NANPs' intracellular delivery, it is now apparent that the platform for delivery, which has historically been a hurdle for therapeutic nucleic acids, is an additional means to tailoring NANP immunorecognition. Here, the use of dendrimers for the delivery of NANPs is compared to the lipid-based platform and the differences in resulting cytokine induction are presented.
Subject(s)
Cytokines/metabolism , Drug Carriers/chemistry , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Nucleic Acids/administration & dosage , Nucleic Acids/chemistry , Cell Line, Tumor , Drug Delivery Systems/methods , Humans , Lipids/chemistryABSTRACT
BACKGROUND: Bacterial meningitis and meningoencephalitis are associated with devastating neuroinflammation. We and others have demonstrated the importance of glial cells in the initiation of immune responses to pathogens invading the central nervous system (CNS). These cells use a variety of pattern recognition receptors (PRRs) to identify common pathogen motifs and the cytosolic sensor retinoic acid inducible gene-1 (RIG-I) is known to serve as a viral PRR and initiator of interferon (IFN) responses. Intriguingly, recent evidence indicates that RIG-I also has an important role in the detection of bacterial nucleic acids, but such a role has not been investigated in glia. METHODS: In this study, we have assessed whether primary or immortalized human and murine glia express RIG-I either constitutively or following stimulation with bacteria or their products by immunoblot analysis. We have used capture ELISAs and immunoblot analysis to assess human microglial interferon regulatory factor 3 (IRF3) activation and IFN production elicited by bacterial nucleic acids and novel engineered nucleic acid nanoparticles. Furthermore, we have utilized a pharmacological inhibitor of RIG-I signaling and siRNA-mediated knockdown approaches to assess the relative importance of RIG-I in such responses. RESULTS: We demonstrate that RIG-I is constitutively expressed by human and murine microglia and astrocytes, and is elevated following bacterial infection in a pathogen and cell type-specific manner. Additionally, surface and cytosolic PRR ligands are also sufficient to enhance RIG-I expression. Importantly, our data demonstrate that bacterial RNA and DNA both trigger RIG-I-dependent IRF3 phosphorylation and subsequent type I IFN production in human microglia. This ability has been confirmed using our nucleic acid nanoparticles where we demonstrate that both RNA- and DNA-based nanoparticles can stimulate RIG-I-dependent IFN responses in these cells. CONCLUSIONS: The constitutive and bacteria-induced expression of RIG-I by human glia and its ability to mediate IFN responses to bacterial RNA and DNA and nucleic acid nanoparticles raises the intriguing possibility that RIG-I may be a potential target for therapeutic intervention during bacterial infections of the CNS, and that the use of engineered nucleic acid nanoparticles that engage this sensor might be a method to achieve this goal.
Subject(s)
DNA, Bacterial/immunology , Microglia/immunology , RNA, Bacterial/immunology , Receptors, Pattern Recognition/immunology , Receptors, Retinoic Acid/immunology , Animals , Cells, Cultured , Humans , Interferon Regulatory Factor-3/biosynthesis , Interferons/biosynthesis , Mice , Mice, Inbred C57BLABSTRACT
RNAi-based technologies have shown biomedical potential; however, safe and efficient delivery of RNA remains a barrier for their broader clinical applications. Nucleic acid nanoparticles (NANPs) programmed to self-assemble and organize multiple therapeutic nucleic acids (TNAs) also became attractive candidates for diverse therapeutic options. Various synthetic nanocarriers are used to deliver TNAs and NANPs, but their clinical translation is limited due to immunotoxicity. Exosomes are cell-derived nanovesicles involved in cellular communication. Due to their ability to deliver biomolecules, exosomes are a novel delivery choice. In this study, we explored the exosome-mediated delivery of NANPs designed to target GFP. We assessed the intracellular uptake, gene silencing efficiency, and immunostimulation of exosomes loaded with NANPs. We also confirmed that interdependent RNA/DNA fibers upon recognition of each other after delivery, can conditionally activate NF-kB decoys and prevent pro-inflammatory cytokines. Our study overcomes challenges in TNA delivery and demonstrates future studies in drug delivery systems.