Self-assembling RNA Research Articles

RNA Origami Functions as a Self-Adjuvanted Nanovaccine Platform for Cancer Immunotherapy.

Peptide-based vaccines have been widely investigated in cancer immunotherapy. Despite their high specificity, safety, and low production cost, these vaccines have shown limited success in clinical studies, owing to their poor immunogenicity. Extensive efforts have been devoted to increasing the immunogenicity of peptide vaccines by mixing peptides with adjuvants and/or promoting their delivery to tumor-draining lymph nodes (TdLNs) for better antigen presentation by and maturation of dendritic cells. Among these efforts, the exploration of various nanoparticles has been at the forefront of the rational design and construction of peptide-based vaccines. Here, we present a nanovaccine platform that is built on a self-assembled RNA origami (RNA-OG) nanostructure. As previously reported, this RNA-OG nanostructure is a potent toll-like receptor (TLR)3 agonist. In addition, due to its robust synthesis and versatility in modification, RNA-OG could be readily linked to peptides of interest. Thus, these RNA-OG nanostructures function as adjuvanted nanocarriers to construct RNA-OG-peptide nanovaccines that are uniform in size, consistent in peptide loading, and highly stable. Here, we demonstrate that the assembled RNA-OG-peptide nanovaccines induced dendritic cell maturation, reduced tumor-mediated immunosuppression, and mobilized tumor-specific CD8+ T cell responses at the tumor site. Together, these actions led to the elicitation of an effective antitumor immunity that increased the survival of tumor-bearing mice. The combination of RNA-OG-based nanovaccines with the α-PD-1 immune checkpoint blockade further enhanced the immunity. Hence, our RNA-OG nanostructures represent a robust, simple, and highly effective platform to empower peptide-based vaccines for cancer immunotherapy.

Diversity of Self-Assembled RNA Complexes: From Nanoarchitecture to Nanomachines.

New tool development for various nucleic acid applications is an essential task in RNA nanotechnology. Here, we determined the ability of self-limited complex formation by a pair of oligoribonucleotides carrying two pairwise complementary blocks connected by a linker of different lengths in each chain. The complexes were analyzed using UV melting, gel shift assay analysis, and molecular dynamics (MD) simulations. We have demonstrated the spontaneous formation of various self-limited and concatemer complexes. The linear concatemer complex is formed by a pair of oligomers without a linker in at least one of them. Longer linkers resulted in the formation of circular complexes. The self-limited complexes formation was confirmed using the toehold strand displacement. The MD simulations indicate the reliability of the complexes' structure and demonstrate their dynamics, which increase with the rise of complex size. The linearization of 2D circular complexes into 1D structures and a reverse cyclization process were demonstrated using a toehold-mediated approach. The approach proposed here for the construction and directed modification of the molecularity and shape of complexes will be a valuable tool in RNA nanotechnology, especially for the rational design of therapeutic nucleic acids with high target specificity and the programmable response of the immune system of organisms.

In vivo self-assembled small RNA targets H19 lncRNA for the treatment of colorectal cancer.

The majority of molecularly targeted therapies in clinical use target disease-related proteins, but only a small fraction (∼1.5%) of human genome is protein-coding region. Considering that ∼70% of human genome is transcribed to noncoding RNAs, targeting noncoding RNAs rather than protein-coding RNAs can significantly expand the proportion of human genome that can be manipulated. H19 long noncoding RNA (lncRNA) is aberrantly expressed in a variety of cancer types and actively contributes to multiple steps of tumorigenesis. Therefore, we selected H19 as a representative target and designed synthetic anti-H19 construct for the self-assembly and delivery of anti-H19 small RNA (sRNA) to prevent colorectal cancer development and metastasis based on the natural ability of the host liver to package sRNA-encapsulating small extracellular vesicles (sEVs) and the endogenous circulating sEVs to transfer sRNA. As anticipated, the synthetic anti-H19 construct successfully generated anti-H19 sRNA-encapsulating sEVs and exhibited high silencing efficiency on H19 lncRNA in an ex vivo model. In orthotopic and lung metastasis mouse models of colorectal cancer, the anti-H19 construct exhibited significantly superior therapeutic efficacy over 5-fluorouracil (5-Fu) in preventing primary tumor growth and lung metastasis. Particularly, the anti-H19 sRNA-encapsulating sEVs were generated in a nontoxic, nonimmunogenic and biocompatible manner. In summary, this study demonstrates that the in vivo self-assembled anti-H19 sRNA can serve as a new therapeutic agent for colorectal cancer.

RNA origami as a vaccine assembly platform to promote CD8 T Cell mediated anti-tumor immunity

Abstract CD8 T cell mediated immunity is critical for controlling tumor growth. However, constructing synthetic vaccines that can effectively induce this arm of the immunity has been challenging. With the advancement of nanotechnologies, various nanostructures have been explored to link antigens and adjuvants together for increased stability, lymph node delivery, cellular uptake, and activation of innate immunity, all of which may help elicit potent T cell responses against tumors. Here, we explored a self-assembling RNA origami (RNA-OG) nanostructure as a vaccine platform to assemble tumor antigens. In addition to acting as an antigen carrier, RNA-OG is an intrinsic TLR3 agonist and therefore functions as a potent adjuvant. Thus, these RNA-OG-antigen vaccines offer simultaneous delivery of antigens and adjuvants for better initiation of T-cell mediated immunity. We demonstrated enhanced antigen cross-presentation of RNA-OG-linked antigens by dendritic cells and subsequent induction of antigen-specific CD8-T cell responses. Using B16-OVA (ovalalbumin) murine melanoma tumor model, RNA-OG-OVA vaccines induced an anti-tumor immune response that contributed to long-term tumor immunity and increased survival of the animals. Overall, our data demonstrates that RNA-OG can function as a potent anti-tumor vaccine platform.

Assembly of RNA Nanostructures from Double-Crossover Tiles.

Artificial self-assembling RNA scaffolds can be produced from many types of RNA motifs that are rationally designed. These scaffolds are of interest as nanoscale organizers, with applications in drug delivery and synthetic cells. Here we describe design strategies, production methods, and imaging of micrometer-sized RNA nanotubes and lattices that assemble from RNA tiles comprising multiple distinct strands.

Rational design of self-assembled RNA nanostructures for HIV-1 virus assembly blockade.

Many pathological processes are driven by RNA-protein interactions, making such interactions promising targets for molecular interventions. HIV-1 assembly is one such process, in which the viral genomic RNA interacts with the viral Gag protein and serves as a scaffold to drive Gag multimerization that ultimately leads to formation of a virus particle. Here, we develop self-assembled RNA nanostructures that can inhibit HIV-1 virus assembly, achieved through hybridization of multiple artificial small RNAs with a stem–loop structure (STL) that we identify as a prominent ligand of Gag that can inhibit virus particle production via STL-Gag interactions. The resulting STL-decorated nanostructures (double and triple stem–loop structures denoted as Dumbbell and Tribell, respectively) can elicit more pronounced viral blockade than their building blocks, with the inhibition arising as a result of nanostructures interfering with Gag multimerization. These findings could open up new avenues for RNA-based therapy.

RNA Hydrogel Combined with MnO2 Nanoparticles as a Nano-Vaccine to Treat Triple Negative Breast Cancer.

Hypoxia is not only the reason of tumor metastasis but also enhances the spread of cancer cells from the original tumor site, which results in cancer recurrence. Herein, we developed a self-assembled RNA hydrogel that efficiently delivered synergistic DNA CpG and short hairpin RNA (shRNA) adjuvants, as well as MnO2 loaded-photodynamic agent chlorine e6 (MnO2@Ce6), and a chemotherapy drug doxorubicin (DOX) into MDA-MB-231cells. The RNA hydrogel consists of one tumour suppressor miRNA (miRNA-205) and one anti-metastatic miRNA (miRNA-182), both of which showed an outstanding effect in synergistically abrogating tumours. The hydrogel would be dissociated by endogenous Dicer enzyme to release loaded therapeutic molecules, and in the meantime induce decomposition of tumor endogenous H2O2 to relieve tumor hypoxia. As a result, a remarkable synergistic therapeutic effect is achieved through the combined chemo-photodynamic therapy, which simultaneously triggers a series of anti-tumor immune responses. Besides, the hydrogel as the carrier which modified aptamer to targeted MDA-MB-231 has the advantages of good biocompatibility and low cytotoxicity. This strategy could be implemented to design any other microRNA (miRNA) as the carrier, combined with other treatment methods to treat human cancer, thereby overcoming the limitations of current cancer therapies.

Self-assembled RNA nanocarrier-mediated chemotherapy combined with molecular targeting in the treatment of esophageal squamous cell carcinoma

BackgroundEsophageal cancer is the fifth most common cancer affecting men in China. The primary treatment options are surgery and traditional radio-chemotherapy; no effective targeted therapy exists yet. Self-assembled RNA nanocarriers are highly stable, easily functionally modified, and have weak off-tumor targeting effects. Thus, they are among the most preferred carriers for mediating the targeted delivery of anti-tumor drugs. miR-375 was found to be significantly down-regulated in esophageal squamous cell carcinoma (ESCC) tissues and its overexpression effectively inhibits the proliferation, migration, and invasion of ESCC cells. Moreover, epidermal growth factor receptor (EGFR) was overexpressed in ESCC cells, and accumulation of RNA nanoparticles in ESCC tumors was enhanced by EGFR-specific aptamer (EGFRapt) modification.ResultsHerein, a novel four-way junction RNA nanocarrier, 4WJ-EGFRapt-miR-375-PTX simultaneously loaded with miR-375, PTX and decorated with EGFRapt, was developed. In vitro analysis demonstrated that 4WJ-EGFRapt-miR-375-PTX possesses strong thermal and pH stabilities. EGFRapt decoration facilitated tumor cell endocytosis and promoted deep penetration into 3D-ESCC spheroids. Xenograft mouse model for ESCC confirmed that 4WJ-EGFRapt-miR-375-PTX was selectively distributed in tumor sites via EGFRapt-mediating active targeting and targeted co-delivery of miR-375 and PTX exhibited more effective therapeutic efficacy with low systemic toxicity.ConclusionThis strategy may provide a practical approach for targeted therapy of ESCC.Graphical

Self-assembled small messenger RNA nanospheres for efficient therapeutic apoptin expression and synergistic Gene-Chemotherapy of breast cancer

In this work, small self-assembled messenger RNA nanospheres (mRNA-NSs) were successfully prepared by rolling circle transcription on a constructed apoptin plasmid. The self-assembled mRNA-NSs have a uniform diameter of approximately 65 nm, good dispersity in solution, and efficient therapeutic apoptin expression ability. In addition, the mRNA-NSs have a high loading capacity of 8.2% for the antitumor drug doxorubicin (Dox), which can effectively deliver the loaded Dox into 4 T1 cells. Cellular experiments show that Dox-loaded self-assembled messenger RNA nanospheres (mRNA-NSs@Dox) can reduce the viability of 4 T1 breast cancer cells by significantly upregulating Bax protein, thereby inducing the activation of Caspase 3 in 4 T1 cells. In vivo experiments show that mRNA-NSs@Dox can effectively increase the necrosis of tumor tissue, reduce the expression of Ki67, and exhibit a synergistic gene-chemotherapy effect in breast cancer-bearing mice. Taken together, this study successfully prepared self-assembled apoptin messenger RNA nanospheres (mRNA-NSs), which can improve the expression of the therapeutic protein apoptin and exhibit excellent synergistic antitumor effects after loading Dox, providing new ideas for the gene treatment and chemotherapy of breast cancer.

Development of a Novel Class of Self-Assembling dsRNA Cancer Therapeutics: A Proof-of-Concept Investigation

Cancer has proven to be an extremely difficult challenge to treat. Several fundamental issues currently underlie cancer treatment, including differentiating self from nonself, functional coupling of the recognition and therapeutic components of various therapies, and the propensity of cancerous cells to develop resistance to common treatment modalities via evolutionary pressure. Given these limitations, there is an increasing need to develop an all-encompassing therapeutic that can uniquely target malignant cells, decouple recognition from treatment, and overcome evolutionarily driven cancer resistance. We describe herein a new class of programmable self-assembling double-stranded RNA (dsRNA)-based cancer therapeutics that uniquely targets aberrant genetic sequences and in a functionally decoupled manner, undergoes oncogenic RNA-activated displacement (ORAD), initiating a therapeutic cascade that induces apoptosis and immune activation. As a proof of concept, we show that RNA strands targeting the EWS/Fli1 fusion gene in Ewing sarcoma cells that are end blocked with phosphorothioate bonds and additionally sealed with a 2′-deoxyuridine (2′-U)-modified DNA protector can be used to induce specific and potent killing of cells containing the target oncogenic sequence but not wild type.

RNA Origami Nanostructures for Potent and Safe Anticancer Immunotherapy.

Rapid developments in nucleic acid nanotechnology have enabled the rational design and construction of self-assembling DNA and RNA nanostructures that are highly programmable. We recently developed a replicable single-stranded RNA origami (RNA-OG) technology that allows a long RNA molecule to be programmed to self-assemble into nanostructures of various shapes. Here, we show that such RNA-OG is highly stable in serum/plasma, and we thus exploited its immunostimulatory potential. We demonstrated that the RNA-OG stimulates a potent innate response primarily through a Toll-like receptor 3 (TLR3) pathway. In a murine peritoneal metastatic colon cancer model, intraperitoneally injected RNA-OG induced significant tumor retardation or regression by activating NK- and CD8-dependent antitumor immunity and antagonizing the peritoneal immunosuppressive environment. Unlike polyinosinic/polycytidylic acid (PolyIC), a well-known double-stranded RNA analogue, the RNA-OG treatment did not cause a high level of type-I interferons in the blood nor apparent toxicity upon its systemic administration in the animals. This work establishes the function of RNA-OG as a potent line of TLR3 agonists that are safe and effective for cancer immunotherapy.

Synthetic bionanotechnology: synthetic biology finds a toehold in nanotechnology.

Enabled by its central role in the molecular networks that govern cell function, RNA has been widely used for constructing components used in biological circuits for synthetic biology. Nucleic acid nanotechnology, which exploits predictable nucleic acid interactions to implement programmable molecular systems, has seen remarkable advances in in vitro nanoscale self-assembly and molecular computation, enabling the production of complex nanostructures and DNA-based neural networks. Living cells genetically engineered to execute nucleic acid nanotechnology programs thus have outstanding potential to significantly extend the current limits of synthetic biology. This perspective discusses the recent developments and future challenges in the field of synthetic bionanotechnology. Thus far, researchers in this emerging area have implemented dozens of programmable RNA nanodevices that provide precise control over gene expression at the transcriptional and translational levels and through CRISPR/Cas effectors. Moreover, they have employed synthetic self-assembling RNA networks in engineered bacteria to carry out computations featuring up to a dozen inputs and to substantially enhance the rate of chemical synthesis. Continued advancement of the field will benefit from improved in vivo strategies for streamlining nucleic acid network synthesis and new approaches for enhancing network function. As the field matures and the complexity gap between in vitro and in vivo systems narrows, synthetic bionanotechnology promises to have diverse potential applications ranging from intracellular circuits that detect and treat disease to synthetic enzymatic pathways that efficiently produce novel drug molecules.

Immunostimulatory Effects Triggered by Self‐Assembled Microspheres with Tandem Repeats of Polymerized RNA Strands

Self-assembled RNA particles have been exploited widely to maximize the therapeutic potential of RNA. However, the immune response via RNA particles is not fully understood. In addition, the investigation of the immunogenicity from RNA-based particles is required owing to inherent immunostimulatory effects of RNA for clinical translation. To examine the immune stimulating potency, rationally designed microsized RNA particles, called RNA microspheres (RMSs), are generated with single or double strands via rolling circle transcription. The RMSs show an exceptional stability in the presence of serum, while they are selectively degraded under endolysosomal conditions. With precisely controlled size, both RMSs are successfully taken up by macrophages. Unlike the nature of RNA fragments, RMSs induce only basal-level expression of inflammatory cytokines as well as type I interferon from macrophages, suggesting that RMSs are immunocompatible in the therapeutic dose range. Taken together, this study could help accelerate clinical translation and broaden the applicability of the self-assembled RNA-based particles without being limited by their potential immunotoxicity, while a systematic controllability study observing the release of RNA fragments from RMSs would provide self-assembled RNA-based structures with a great potential for immunomodulation.

Self-Assembling RNA Nanoparticle for Gene Expression Regulation in a Model System.

In the search for enzymatically processed RNA fragments, we found the novel three-way junction motif. The structure prediction suggested the arrangement of helices at acute angle approx.60°. This allows the design of a trimeric RNA nanoparticle that can be functionalized with multiple regulatory fragments. Such RNA nano-object of equilateral triangular shape was applied for gene expression regulation studies in two independent cellular systems. Biochemical and functional studies confirmed the predicted shape and structure of the nanoparticle. The regulatory siRNA fragments incorporated into the nanoparticle were effectively released and triggered gene silencing. The regulatory effect was prolonged when induced with structuralized RNA compared to unstructured siRNAs. In these studies, the enzymatic processing of the motif was utilized for function release from the nanoparticle, enabling simultaneous delivery of different regulatory functions. This methodology of sequence search, RNA structural prediction, and application for rational design opens a new way for creating enzymatically processed RNA nanoparticles.

Synthetic small RNAs: Current status, challenges, and opportunities.

Small regulatory RNAs act at the levels of transcription, posttranscription, and translation, with numerous roles that include binding to protein targets, protein modification, binding to messenger RNA targets, and regulation of gene expression. We discuss the development of a number of riboregulators and riboswitches, highlighting their use in metabolic engineering and genetic control. Riboregulators and riboswitches are self-assembled RNA molecules that can dynamically change their conformation, acting as regulatory switches that affect biological changes. They have currently been designed, characterized, and implemented in a wide range of organisms and cell types, including bacteria, yeast, and mammalian cells. We have identified and examined the recent advances in RNA synthetic biology, underlining the potential future development of their use and capabilities, noting how these can be ultimately expanded and improved into novel biotechnological, biomedical, and industrial applications.

Floxuridine-containing nucleic acid nanogels for anticancer drug delivery

Herein, we report the self-assemblies of floxuridine-containing DNA and RNA nanogels with a precise drug loading ratio as effective drug delivery systems. Based on the structural similarity between the nucleoside analogue floxuridine (F) and the natural nucleoside thymidine (T), F can be incorporated into nucleic acid strands via either solid-phase synthesis or enzyme-mediated transcription. With the retained property of molecular recognition, the synthesized F-integrated DNA or RNA strands can be used as building units and further assembled into nucleic acid based spherical nanogels, which can be efficiently taken up by cancer cells and then release the therapeutic agents. As such, the drug-containing nucleic acid nanogels exhibit excellent inhibitory activity against cancer cells.

RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity.

In response to injuries to the CNS, astrocytes enter a reactive state known as astrogliosis, which is believed to be deleterious in some contexts. Activated astrocytes overexpress intermediate filaments including glial fibrillary acidic protein (GFAP) and vimentin (Vim), resulting in entangled cells that inhibit neurite growth and functional recovery. Reactive astrocytes also secrete inflammatory molecules such as Lipocalin 2 (Lcn2), which perpetuate reactivity and adversely affect other cells of the CNS. Herein, we report proof-of-concept use of the packaging RNA (pRNA)-derived three-way junction (3WJ) motif as a platform for the delivery of siRNAs to downregulate such reactivity-associated genes. In vitro, siRNA-3WJs induced a significant knockdown of Gfap, Vim, and Lcn2 in a model of astroglial activation, with a concomitant reduction in protein expression. Knockdown of Lcn2 also led to reduced protein secretion from reactive astroglial cells, significantly impeding the perpetuation of inflammation in otherwise quiescent astrocytes. Intralesional injection of anti-Lcn2-3WJs in mice with contusion spinal cord injury led to knockdown of Lcn2 at mRNA and protein levels in vivo. Our results provide evidence for siRNA-3WJs as a promising platform for ameliorating astroglial reactivity, with significant potential for further functionalization and adaptation for therapeutic applications in the CNS.

A homodimer interface without base pairs in an RNA mimic of red fluorescent protein.

Corn, a 28-nucleotide RNA, induces yellow fluorescence of its cognate ligand (3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, DFHO) by >1000-fold. It was selected in vitro to overcome limitations of other fluorogenic RNAs, particularly rapid photobleaching. We now report the Corn-DFHO co-crystal structure, discovering that the functional species is a quasisymmetric homodimer. Unusually, the dimer interface, where six unpaired adenosines break overall 2-fold symmetry, lacks any intermolecular base pairs. The homodimer encapsulates one DFHO at its inter-protomer interface, sandwiching it with a G-quadruplex from each protomer. Corn and the green-fluorescent Spinach RNA are structurally unrelated. Their convergent use of G-quadruplexes underscores the usefulness of this motif for RNA-induced small-molecule fluorescence. The asymmetric dimer interface of Corn could form the basis for the development of mutants that only fluoresce as heterodimers. Such variants would be analogous to Split GFP, and may be useful in analyzing RNA co-expression or association, or in designing self-assembling RNA nanostructures.

Computational Generation of RNA Nanorings.

A variety of designed RNA ring structures (ranging from triangles to hexagonal rings) have been reported in the scientific literature. Designing self-assembling RNA ring structures from structural motifs is, however, a nontrivial problem as there are many combinations of motifs and linking helices. Moreover, most combinations of motifs and linker helices will not lead to ring closure. A solution to this problem was recently published using a "design-by-catalog" approach where motif combinations that lead to rings are precomputed and tabulated. Here we present a web-browser based workflow for creating RNA rings using Galaxy, a web-based platform that can be used for workflow management. An example of how these RNArings are generated and processed to create a 3D model of the ring is discussed.

A self-assembling RNA aptamer-based graphene oxide sensor for the turn-on detection of theophylline in serum

To date, few effective fluorescent biosensors based on RNA aptamers have been developed because the intrinsic instability of RNA in the presence of nucleases precludes the application of RNA aptamers for the analysis of biological fluids. In this study, we developed a simple, sensitive, selective turn-on fluorescent aptasensor for theophylline detection in serum, utilizing ligand-induced self-assembling RNA aptamers and two different interaction stages of the aptamer fragments with graphene oxide (GO). A single strand of the theophylline RNA aptamer (33-mer) was split at the end loop region into two shorter fragments, one of which was labeled with a fluorophore (FAM). In the absence of theophylline, the adsorption of the two individual fragments on GO brought the fluorophore in close proximity to the GO surface, resulting in highly efficient quenching of fluorescence. The system showed very low background fluorescence. Conversely, the fragments self-assembled into an RNA aptamer/theophylline complex and were dissociated from GO. The quenched fluorescence was significantly recovered, and theophylline could be detected at a wide range of concentrations from 1 to 100μM, with a detection limit of 0.155μM and good selectivity in serum. Moreover, because of the shorter RNA fragments and the effective protection ability of GO from nuclease cleavage, the RNA sequences remained stable during the experiments. This design may serve as an example for the application of RNA aptasensors in the clinical setting.