RESUMO
Aptamers with fluorogenic ligands are emerging as useful tools to quantify and track RNA molecules. The RNA Mango family of aptamers have a useful combination of tight ligand binding, bright fluorescence, and small size. However, the simple structure of these aptamers, with a single base-paired stem capped by a G-quadruplex, can limit the sequence and structural modifications needed for many use-inspired designs. Here we report new structural variants of RNA Mango that have two base-paired stems attached to the quadruplex. Fluorescence saturation analysis of one of the double-stemmed constructs showed a maximum fluorescence that is â¼75% brighter than the original single-stemmed Mango I. A small number of mutations to nucleotides in the tetraloop-like linker of the second stem were subsequently analyzed. The effect of these mutations on the affinity and fluorescence suggested that the nucleobases of the second linker do not directly interact with the fluorogenic ligand (TO1-biotin), but may instead induce higher fluorescence by indirectly altering the ligand properties in the bound state. The effects of the mutations in this second tetraloop-like linker indicate the potential of this second stem for rational design and reselection experiments. Additionally, we demonstrated that a bimolecular mango designed by splitting the double-stemmed Mango can function when two RNA molecules are cotranscribed from different DNA templates in a single in vitro transcription. This bimolecular Mango has potential application in detecting RNA-RNA interactions. Together, these constructs expand the designability of the Mango aptamers to facilitate future applications of RNA imaging.
Assuntos
Aptâmeros de Nucleotídeos , Mangifera , Mangifera/genética , Mangifera/química , Mangifera/metabolismo , Aptâmeros de Nucleotídeos/química , Ligantes , Corantes Fluorescentes/química , RNA/químicaRESUMO
Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development.
Assuntos
Aptâmeros de Nucleotídeos , Trombina , Animais , Camundongos , Humanos , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/química , Trombina/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Distribuição Tecidual , RNA , Modelos Animais de Doenças , Fígado/metabolismo , Fígado/efeitos dos fármacos , Anticoagulantes/farmacologia , Anticoagulantes/química , Antitrombinas/farmacologia , Antídotos/farmacologia , Antídotos/químicaRESUMO
RNA therapeutics has advanced into the third milestone in pharmaceutical drug development, following chemical and protein therapeutics. RNA itself can serve as therapeutics, carriers, regulators, or substrates in drug development. Due to RNA's motile, dynamic, and deformable properties, RNA nanoparticles have demonstrated spontaneous targeting and accumulation in cancer vasculature and fast excretion through the kidney glomerulus to urine to prevent possible interactions with healthy organs. Furthermore, the negatively charged phosphate backbone of RNA results in general repulsion from negatively charged lipid cell membranes for further avoidance of vital organs. Thus, RNA nanoparticles can spontaneously enrich tumor vasculature and efficiently enter tumor cells via specific targeting, while those not entering the tumor tissue will clear from the body quickly. These favorable parameters have led to the expectation that RNA has low or little toxicity. RNA nanoparticles have been well characterized for their anticancer efficacy; however, little detail on RNA nanoparticle pathology and safety is known. Here, we report the in vitro and in vivo assessment of the pathology and safety aspects of different RNA nanoparticles including RNA three-way junction (3WJ) harboring 2'-F modified pyrimidine, folic acid, and Survivin siRNA, as well as the RNA four-way junction (4WJ) harboring 2'-F modified pyrimidine and 24 copies of SN38. Both animal models and patient serum were investigated. In vitro studies include hemolysis, platelet aggregation, complement activation, plasma coagulation, and interferon induction. In vivo studies include hematoxylin and eosin (H&E) staining, hematological and biochemical analysis as the serum profiling, and animal organ weight study. No significant toxicity, side effect, or immune responses were detected during the extensive safety evaluations of RNA nanoparticles. These results further complement previous cancer inhibition studies and demonstrate RNA nanoparticles as an effective and safe drug delivery vehicle for future clinical translations.
Assuntos
Nanopartículas , Neoplasias , Animais , Humanos , RNA Interferente Pequeno/genética , Sistemas de Liberação de Medicamentos , Neoplasias/metabolismo , Nanopartículas/química , PirimidinasRESUMO
Despite RNA's diverse secondary and tertiary structures and its complex conformational changes, nature utilizes a limited set of structural "motifs"-helices, junctions, and tertiary contact modules-to build diverse functional RNAs. Thus, in-depth descriptions of a relatively small universe of RNA motifs may lead to predictive models of RNA tertiary conformational landscapes. Motifs may have different properties depending on sequence and secondary structure, giving rise to subclasses that expand the universe of RNA building blocks. Yet we know very little about motif subclasses, given the challenges in mapping conformational properties in high throughput. Previously, we used "RNA on a massively parallel array" (RNA-MaP), a quantitative, high-throughput technique, to study thousands of helices and two-way junctions. Here, we adapt RNA-MaP to study the thermodynamic and conformational properties of tetraloop/tetraloop receptor (TL/TLR) tertiary contact motifs, analyzing 1,493 TLR sequences from different classes. Clustering analyses revealed variability in TL specificity, stability, and conformational behavior. Nevertheless, natural GAAA/11ntR TL/TLRs, while varying in tertiary stability by â¼2.5 kcal/mol, exhibited conserved TL specificity and conformational properties. Thus, RNAs may tune stability without altering the overall structure of these TL/TLRs. Furthermore, their stability correlated with natural frequency, suggesting thermodynamics as the dominant selection pressure. In contrast, other TL/TLRs displayed heterogenous conformational behavior and appear to not be under strong thermodynamic selection. Our results build toward a generalizable model of RNA-folding thermodynamics based on the properties of isolated motifs, and our characterized TL/TLR library can be used to engineer RNAs with predictable thermodynamic and conformational behavior.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Modelos Moleculares , TermodinâmicaRESUMO
RNA nanotechnology takes advantage of structural modularity to build self-assembling nano-architectures with applications in medicine and synthetic biology. The use of paranemic motifs, that form without unfolding existing secondary structure, allows for the creation of RNA nanostructures that are compatible with cotranscriptional folding in vitro and in vivo. In previous work, kissing-loop (KL) motifs have been widely used to design RNA nanostructures that fold cotranscriptionally. However, the paranemic crossover (PX) motif has not yet been explored for cotranscriptional RNA origami architectures and information about the structural geometry of the motif is unknown. Here, a six base pair-wide paranemic RNA interaction that arranges double helices in a perpendicular manner is introduced, allowing for the generation of a new and versatile building block: the paranemic-crossover triangle (PXT). The PXT is self-assembled by cotranscriptional folding and characterized by cryogenic electron microscopy, revealing for the first time an RNA PX interaction in high structural detail. The PXT is used as a building block for the construction of multimers that form filaments and rings and a duplicated PXT motif is used as a building block to self-assemble cubic structures, demonstrating the PXT as a rigid self-folding domain for the development of wireframe RNA origami architectures.
Assuntos
Nanoestruturas , RNA , RNA/química , Conformação de Ácido Nucleico , DNA/química , Nanotecnologia , Nanoestruturas/químicaRESUMO
In biological systems, conformational changes and allosteric modulation play pivotal roles in regulating biological functions, such as the dynamic change of protein molecules, in response to binding or interacting with other factors such as pH, voltage, salt, light, or ligand. RNA can be manipulated and tuned with a level of simplicity that is characteristic of DNA or polymers, while displaying versatility in structure, diversity in function, and adaptability in a configuration similar to proteins. In the past, the work on the investigation of conformational change mainly focused on protein. The induced-fit and conformational capture in RNA have also been explored, such as in the study of riboswitches. Herein, we report the engineering of three-dimensional RNA nanocubes and demonstrated the operation and regulation for its configuration. We demonstrate the operation of reconfigurable RNA nanocubes whose shapes change precisely and reversibly in response to a specific trigger strand. The shape, size, and conformation can be regulated precisely and reversibly in response to the specific triggering signals. The shape and conformational conversion were observed by cryo-EM and gel electrophoresis, respectively. Harnessing the size, shape, conformation, and self-assembly capabilities of the RNA nanocube can provide a new potential use of this technology as nanocarriers for the treatment of various diseases.
Assuntos
Imunomodulação/efeitos dos fármacos , Nanoestruturas/química , Nanotecnologia/métodos , Oligodesoxirribonucleotídeos/farmacologia , Riboswitch , Animais , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Engenharia Genética/métodos , Concentração de Íons de Hidrogênio , Interleucina-6/biossíntese , Interleucina-6/imunologia , Ligantes , Camundongos , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Células RAW 264.7 , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Design strategies for DNA and RNA nanostructures have developed along parallel lines for the past 30 years, from small structural motifs derived from biology to large 'origami' structures with thousands to tens of thousands of bases. With the recent publication of numerous RNA origami structures and improved design methods-even permitting co-transcriptional folding of kilobase-sized structures - the RNA nanotechnolgy field is at an inflection point. Here, we review the key achievements which inspired and enabled RNA origami design and draw comparisons with the development and applications of DNA origami structures. We further present the available computational tools for the design and the simulation, which will be key to the growth of the RNA origami community. Finally, we portray the transition from RNA origami structure to function. Several functional RNA origami structures exist already, their expression in cells has been demonstrated and first applications in cell biology have already been realized. Overall, we foresee that the fast-paced RNA origami field will provide new molecular hardware for biophysics, synthetic biology and biomedicine, complementing the DNA origami toolbox.
Assuntos
Nanoestruturas , Nanotecnologia , RNA/genética , RNA/química , Nanoestruturas/química , DNA/química , Simulação por Computador , Conformação de Ácido NucleicoRESUMO
Liver cancer such as hepatocellular carcinoma (HCC) poorly responds to chemotherapeutics as there are no effective means to deliver the drugs to liver cancer. Here we report GalNAc decorated exosomes as cargo for targeted delivery of Paclitaxel (PTX) and miR122 to liver tumors as an effective means to inhibit the HCC. Exosomes (Exos) are nanosized extracellular vesicles that deliver a payload to cancer cells effectively. GalNAc provides Exos targeting ability by binding to the asialoglycoprotein-receptor (ASGP-R) overexpressed on the liver cancer cell surface. A 4-way junction (4WJ) RNA nanoparticle was constructed to harbor 24 copies of hydrophobic PTX and 1 copy of miR122. The 4WJ RNA-PTX complex was loaded into the Exos, and its surface was decorated with GalNAc using RNA nanotechnology to obtain specific targeting. The multi-specific Exos selectively bind and efficiently delivered the payload into the liver cancer cells and exhibited the highest cancer cell inhibition due to the multi-specific effect of miR122, PTX, GalNAc, and Exos. The same was reflected in mice xenograft studies, the liver cancer was efficiently inhibited after systemic injection of the multi-specific Exos. The required effective dose of chemical drugs carried by Exos was significantly reduced, indicating high efficiency and low toxicity. The multi-specific strategy demonstrates that Exos can serve as a natural cargo vehicle for the targeted delivery of anticancer therapeutics to treat difficult-to-treat cancers.
Assuntos
Carcinoma Hepatocelular , Exossomos , Neoplasias Hepáticas , MicroRNAs , Humanos , Animais , Camundongos , Exossomos/química , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Ligantes , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Portadores de Fármacos/química , Paclitaxel , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
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.
Assuntos
Nanopartículas , Ácidos Nucleicos , Humanos , Ácidos Nucleicos/química , Monócitos , Nanopartículas/química , Interferons , Inteligência ArtificialRESUMO
The modular architecture of naturally occurring ribozymes makes them a promising class of structural platform for the design and assembly of three-dimensional (3D) RNA nanostructures, into which the catalytic ability of the platform ribozyme can be installed. We have constructed and analyzed RNA nanostructures with polygonal-shaped (closed) ribozyme oligomers by assembling unit RNAs derived from the Tetrahymena group I intron with a typical modular architecture. In this study, we dimerized ribozyme trimers with a triangular shape by introducing three pillar units. The resulting double-decker nanostructures containing six ribozyme units were characterized biochemically and their structures were observed by atomic force microscopy. The double-decker hexamers exhibited higher catalytic activity than the parent ribozyme trimers.
Assuntos
Nanoestruturas , RNA Catalítico , Tetrahymena , Íntrons , Nanoestruturas/química , Conformação de Ácido Nucleico , RNA/química , RNA Catalítico/metabolismo , Tetrahymena/metabolismoRESUMO
RNA interference (RNAi) mediated by small interfering RNA (siRNA) duplexes is a powerful therapeutic modality, but the translation of siRNAs from the bench into clinical application has been hampered by inefficient delivery inâ vivo. An innovative delivery strategy involves fusing siRNAs to a three-way junction (3WJ) motif derived from the phi29 bacteriophage prohead RNA (pRNA). Chimeric siRNA-3WJ molecules are presumed to enter the RNAi pathway through Dicer cleavage. Here, we fused siRNAs to the phi29 3WJ and two phylogenetically related 3WJs. We confirmed that the siRNA-3WJs are substrates for Dicer inâ vitro. However, our results reveal that siRNA-3WJs transfected into Dicer-deficient cell lines trigger potent gene silencing. Interestingly, siRNA-3WJs transfected into an Argonaute 2-deficient cell line also retain some gene silencing activity. siRNA-3WJs are most efficient when the antisense strand of the siRNA duplex is positioned 5' of the 3WJ (5'-siRNA-3WJ) relative to 3' of the 3WJ (3'-siRNA-3WJ). This work sheds light on the functional properties of siRNA-3WJs and offers a design rule for maximizing their potency in the human RNAi pathway.
Assuntos
Inativação Gênica , Humanos , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
RNA is an emerging platform for drug delivery, but the susceptibility of RNA to nuclease degradation remains a major barrier to its implementation inâ vivo. Here, we engineered flaviviral Xrn1-resistant RNA (xrRNA) motifs to host small interfering RNA (siRNA) duplexes. The xrRNA-siRNA molecules self-assemble inâ vitro, resist degradation by the conserved eukaryotic 5' to 3' exoribonuclease Xrn1, and trigger gene silencing in 293T cells. The resistance of the molecules to Xrn1 does not translate to stability in blood serum. Nevertheless, our results demonstrate that flavivirus-derived xrRNA motifs can confer Xrn1 resistance on a model therapeutic payload and set the stage for further investigations into using the motifs as building blocks in RNA nanotechnology.
Assuntos
Exorribonucleases/metabolismo , Flavivirus/metabolismo , Inativação Gênica , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Exorribonucleases/química , Flavivirus/química , Células HEK293 , Humanos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Viral/química , RNA Viral/genéticaRESUMO
The quest for artificial RNA viral complexes with authentic structure while being non-replicative is on its way for the development of viral vaccines. RNA viruses contain capsid proteins that interact with the genome during morphogenesis. The sequence and properties of the protein and genome determine the structure of the virus. For example, the Pariacoto virus ssRNA genome assembles into a dodecahedron. Virus-inspired nanotechnology has progressed remarkably due to the unique structural and functional properties of viruses, which can inspire the design of novel nanomaterials. RNA is a programmable biopolymer able to self-assemble sophisticated 3D structures with rich functionalities. RNA dodecahedrons mimicking the Pariacoto virus quasi-icosahedral genome structures were constructed from both native and 2'-F modified RNA oligos. The RNA dodecahedron easily self-assembled using the stable pRNA three-way junction of bacteriophage phi29 as building blocks. The RNA dodecahedron cage was further characterized by cryo-electron microscopy and atomic force microscopy, confirming the spontaneous and homogenous formation of the RNA cage. The reported RNA dodecahedron cage will likely provide further studies on the mechanisms of interaction of the capsid protein with the viral genome while providing a template for further construction of the viral RNA scaffold to add capsid proteins for the assembly of the viral nucleocapsid as a model. Understanding the self-assembly and RNA folding of this RNA cage may offer new insights into the 3D organization of viral RNA genomes. The reported RNA cage also has the potential to be explored as a novel virus-inspired nanocarrier.
Assuntos
Proteínas do Capsídeo/genética , Genoma Viral , Nanotecnologia/métodos , Nodaviridae/genética , RNA Viral/química , RNA Viral/genética , Proteínas Virais/genética , Proteínas do Capsídeo/metabolismo , Nodaviridae/metabolismo , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase , Inativação Gênica , Melanoma , Nanopartículas , Proteínas de Neoplasias , Ácidos Nucleicos , 1-Acilglicerofosfocolina O-Aciltransferase/antagonistas & inibidores , 1-Acilglicerofosfocolina O-Aciltransferase/biossíntese , Linhagem Celular Tumoral , Humanos , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Nanopartículas/química , Nanopartículas/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/biossíntese , Ácidos Nucleicos/química , Ácidos Nucleicos/farmacologiaRESUMO
The domains of DNA and RNA nanotechnology are steadily gaining in popularity while proving their value with various successful results, including biosensing robots and drug delivery cages. Nowadays, the nanotechnology design pipeline usually relies on computer-based design (CAD) approaches to design and simulate the desired structure before the wet lab assembly. To aid with these tasks, various software tools exist and are often used in conjunction. However, their interoperability is hindered by a lack of a common file format that is fully descriptive of the many design paradigms. Therefore, in this paper, we propose a Unified Nanotechnology Format (UNF) designed specifically for the biomimetic nanotechnology field. UNF allows storage of both design and simulation data in a single file, including free-form and lattice-based DNA structures. By defining a logical and versatile format, we hope it will become a widely accepted and used file format for the nucleic acid nanotechnology community, facilitating the future work of researchers and software developers. Together with the format description and publicly available documentation, we provide a set of converters from existing file formats to simplify the transition. Finally, we present several use cases visualizing example structures stored in UNF, showcasing the various types of data UNF can handle.
RESUMO
This Minireview discusses the design and applications of binary (also known as split) light-up aptameric sensors (BLAS). BLAS consist of two RNA or DNA strands and a fluorogenic organic dye added as a buffer component. When associated, the two strands form a dye-binding site, followed by an increase in fluorescence of the aptamer-bound dye. The design is cost-efficient because it uses short oligonucleotides and does not require conjugation of organic dyes with nucleic acids. In some applications, BLAS design is preferable over monolithic sensors because of simpler assay optimization and improved selectivity. RNA-based BLAS can be expressed in cells and used for the intracellular monitoring of biological molecules. BLAS have been used as reporters of nucleic acid association events in RNA nanotechnology and nucleic-acid-based molecular computation. Other applications of BLAS include the detection of nucleic acids, proteins, and cancer cells, and potentially they can be tailored to report a broad range of biological analytes.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Bactérias/química , Computadores Moleculares , DNA/análise , DNA/química , Humanos , Lógica , Compostos Orgânicos/análise , Proteínas/análise , RNA/análise , RNA/químicaRESUMO
The question of whether RNA is more stable or unstable compared to DNA or other nucleic acids has long been a subject of extensive scrutiny and public attention. Recently, thermodynamically stable and degradation-resistant RNA motifs have been utilized in RNA nanotechnology to build desired architectures and integrate multiple functional groups. Here we report the effects of phosphorothioate deoxyribonucleotides (PS-DNA), deoxyribonucleotides (DNA), ribonucleotides (RNA), 2'-F nucleotides (2'-F), and locked nucleic acids (LNA) on the thermal and in vivo stability of the three-way junction (3WJ) of bacteriophage phi29 motor packaging RNA. It was found that the thermal stability gradually increased following the order of PS-DNA/PS-DNA < DNA/DNA < DNA/RNA < RNA/RNA < RNA/2'-F RNA < 2'-F RNA/2'-F RNA < 2'-F RNA/LNA < LNA/LNA. This proposition is supported by studies on strand displacement and the melting of homogeneous and heterogeneous 3WJs. By simply mixing different chemically modified oligonucleotides, the thermal stability of phi29 pRNA 3WJ can be tuned to cover a wide range of melting temperatures from 21.2°C to over 95°C. The 3WJLNA was resistant to boiling temperature denaturation, urea denaturation, and 50% serum degradation. Intravenous injection of fluorescent LNA/2'-F hybrid 3WJs into mice revealed its exceptional in vivo stability and presence in urine. It is thus concluded that incorporation of LNA nucleotides, alone or in combination with 2'-F, into RNA nanoparticles derived from phi29 pRNA 3WJ can extend the half-life of the RNA nanoparticles in vivo and improve their pharmacokinetics profile.
Assuntos
Oligonucleotídeos/química , Oligonucleotídeos Fosforotioatos/química , RNA Viral/química , Animais , Fagos Bacilares , Pareamento de Bases , Portadores de Fármacos/farmacocinética , Meia-Vida , Cinética , Camundongos Endogâmicos BALB C , Nanopartículas/química , Estabilidade de RNA , RNA Viral/farmacocinética , Temperatura de TransiçãoRESUMO
Triple-negative breast cancer (TNBC) is an aggressive disease with a short median time from relapse to death. The increased aggressiveness, drug resistance, disease relapse, and metastasis are associated with the presence of stem cells within tumors. Several stem cell markers, such as CD24, CD44, CD133, ALDH1, and ABCG2, have been reported, but their roles in breast cancer tumorigenesis remain unclear. Herein, we apply RNA nanotechnology to deliver anti-microRNA (miRNA) for TNBC therapy. The thermodynamically and chemically stable three-way junction (3WJ) motif was utilized as the scaffold to carry an RNA aptamer binding to CD133 receptor and a locked nuclei acid (LNA) sequence for miRNA21 inhibition. Binding assays revealed the specific uptake of the nanoparticles to breast cancer stem cells (BCSCs) and TNBC cells. Functional assays showed that cancer cell migration was reduced, miR21 expression was inhibited, and downstream tumor suppressor PTEN and PDCD4 expressions were upregulated. In vitro and in vivo studies revealed that these therapeutic RNA nanoparticles did not induce cytokine secretion. Systemic injection of these RNA nanoparticles in animal trial demonstrated high specificity in TNBC tumor targeting and high efficacy for tumor growth inhibition. These results revealed the clinical translation potential of these RNA nanoparticles for TNBC therapy.
Assuntos
Antígeno AC133/metabolismo , Sistemas de Liberação de Medicamentos/métodos , MicroRNAs/genética , MicroRNAs/uso terapêutico , Nanopartículas/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Oligonucleotídeos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Células RAW 264.7 , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Sistemas de Liberação de Medicamentos , Exossomos , Nanopartículas/administração & dosagem , Ácidos Nucleicos/administração & dosagem , Linhagem Celular , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Microscopia de Força Atômica , NF-kappa B/genética , Nanopartículas/química , Interferência de RNARESUMO
The thermodynamic stabilities of four natural prohead or packaging RNA (pRNA) three-way junction (3WJ) nanomotifs and seven phi29 pRNA 3WJ deletion mutant nanomotifs were investigated using UV optical melting on a three-component RNA system. Our data reveal that some pRNA 3WJs are more stable than the phi29 pRNA 3WJ. The stability of the 3WJ contributes to the unique self-assembly properties of pRNA. Thus, ultrastable pRNA 3WJ motifs suggest new scaffolds for pRNA-based nanotechnology. We present data demonstrating that pRNA 3WJs differentially respond to the presence of metal ions. A comparison of our data with free energies predicted by currently available RNA secondary structure prediction programs shows that these programs do not accurately predict multibranch loop stabilities. These results will expand the existing parameters used for RNA secondary structure prediction from sequence in order to better inform RNA structure-function hypotheses and guide the rational design of functional RNA supramolecular assemblies.