- [Show abstract] [Hide abstract] ABSTRACT: The significance of bionanomotors in nanotechnology is analogous to mechanical motors in daily life. Here the principle and approach for designing and constructing biomimetic nanomotors with continuous single-directional motion are reported. This bionanomotor is composed of a dodecameric protein channel, a six-pRNA ring, and an ATPase hexamer. Based on recent elucidations of the one-way revolving mechanisms of the phi29 double-stranded DNA (dsDNA) motor, various RNA and protein elements are designed and tested by single-molecule imaging and biochemical assays, with which the motor with active components has been constructed. The motor motion direction is controlled by three operation elements: (1) Asymmetrical ATPase with ATP-interacting domains for alternative DNA binding/pushing regulated by an arginine finger in a sequential action manner. The arginine finger bridges two adjacent ATPase subunits into a non-covalent dimer, resulting in an asymmetrical hexameric complex containing one dimer and four monomers. (2) The dsDNA translocation channel as a one-way valve. (3) The hexameric pRNA ring geared with left-/right-handed loops. Assessments of these constructs reveal that one inactive subunit of pRNA/ATPase is sufficient to completely block motor function (defined as K = 1), implying that these components work sequentially based on the principle of binomial distribution and Yang Hui's triangle.
- [Show abstract] [Hide abstract] ABSTRACT: RNA tetrahedral nanoparticles with two different sizes are successfully assembled by a one-pot bottom-up approach with high efficiency and thermal stability. The reported design principles can be extended to construct higher order polyhedral RNA architectures for various applications such as targeted cancer imaging and therapy.
- [Show abstract] [Hide abstract] ABSTRACT: Both siRNA and miRNA can serve as powerful gene silencing reagents but their specific delivery to cancer cells in vivo without collateral damage to healthy cells remains challenging. We report here the application of RNA nanotechnology for specific and efficient delivery of anti-miRNA seed-targeting sequence to block the growth of prostate cancer in mouse models. Utilizing the thermodynamically ultra-stable three-way junction of the pRNA of phi29 DNA packaging motor, RNA nanoparticles were constructed by bottom-up self-assembly containing the anti-PSMA aptamer as targeting ligand and anti-miR17 or anti-miR21 as therapeutic modules. The 16 nm RNase resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection in mice and strongly bound to tumors with little or no accumulation in healthy organs 8 hr post-injection, and subsequently repressed tumor growth at low doses with high efficiency.Molecular Therapy (2016); doi:10.1038/mt.2016.85.
- [Show abstract] [Hide abstract] ABSTRACT: MicroRNAs play important roles in regulating the gene expression and life cycle of cancer cells. In particular, miR-21, an oncogenic miRNA is a major player involved in tumor initiation, progression, invasion and metastasis in several cancers, including triple negative breast cancer (TNBC). However, delivery of therapeutic miRNA or anti-miRNA specifically into cancer cells in vivo without collateral damage to healthy cells remains challenging. We report here the application of RNA nanotechnology for specific and efficient delivery of anti-miR-21 to block the growth of TNBC in orthotopic mouse models. The 15 nm therapeutic RNA nanoparticles contains the 58-nucleotide (nt) phi29 pRNA-3WJ as a core, a 8-nt sequence complementary to the seed region of miR-21, and a 39-nt epidermal growth factor receptor (EGFR) targeting aptamer for internalizing RNA nanoparticles into cancer cells via receptor mediated endocytosis. The RNase resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection into mice and strongly bound to tumors with little or no accumulation in healthy organs 8 h postinjection, and subsequently repressed tumor growth at low doses. The observed specific cancer targeting and tumor regression is a result of several key attributes of RNA nanoparticles: anionic charge which disallows nonspecific passage across negatively charged cell membrane; "active" targeting using RNA aptamers which increases the homing of RNA nanoparticles to cancer cells; nanoscale size and shape which avoids rapid renal clearance and engulfment by lung macrophages and liver Kupffer cells; favorable biodistribution profiles with little accumulation in healthy organs, which minimizes nonspecific side effects; and favorable pharmacokinetic profiles with extended in vivo half-life. The results demonstrate the clinical potentials of RNA nanotechnology based platform to deliver miRNA based therapeutics for cancer treatment.
- [Show abstract] [Hide abstract] ABSTRACT: Gastric cancer is the second leading cause of cancer-related death worldwide. RNA nanotechnology has recently emerged as an important field due to recent finding of its high thermodynamic stability, favorable and distinctive in vivo attributes. Here we reported the use of the thermostable three-way junction (3WJ) of bacteriophage phi29 motor pRNA to escort folic acid, a fluorescent image marker and BRCAA1 siRNA for targeting, imaging, delivery, gene silencing and regression of gastric cancer in animal models. In vitro assay revealed that the RNA nanoparticles specifically bind to gastric cancer cells, and knock-down the BRCAA1 gene. Apoptosis of gastric cancer cells was observed. Animal trials confirmed that these RNA nanoparticles could be used to image gastric cancer in vivo, while showing little accumulation in crucial organs and tissues. The volume of gastric tumors noticeably decreased during the course of treatment. No damage to important organs by RNA nanoparticles was detectible. All the results indicated that this novel RNA nanotechnology can overcome conventional cancer therapeutic limitations and opens new opportunities for specific delivery of therapeutics to stomach cancer without damaging normal cells and tissues, reduce the toxicity and side effect, improve the therapeutic effect, and exhibit great potential in clinical tumor therapy.
- [Show abstract] [Hide abstract] ABSTRACT: To find methods for potent drug development by targeting to biocomplex with high copy number. Phi29 DNA packaging motor components with different stoichiometries were used as model to assay virion assembly with Yang Hui's Triangle [Formula: see text], where Z = stoichiometry, M = drugged subunits per biocomplex, p and q are the fraction of drugged and undrugged subunits in the population. Inhibition efficiency follows a power function. When number of drugged subunits to block the function of the complex K = 1, the uninhibited biocomplex equals q(z), demonstrating the multiplicative effect of stoichiometry on inhibition with stoichiometry 1000 > 6 > 1. Complete inhibition of virus replication was found when Z = 6. Drug inhibition potency depends on the stoichiometry of the targeted components of the biocomplex or nanomachine. The inhibition effect follows a power function of the stoichiometry of the target biocomplex.
- [Show abstract] [Hide abstract] ABSTRACT: Radiation reagents that specifically target tumors are in high demand for the treatment of cancer. The emerging field of RNA nanotechnology might provide new opportunities for targeted radiation therapy. This study investigates whether chemically modified RNA nanoparticles derived from the packaging RNA (pRNA) three-way junction (3WJ) of phi29 DNA-packaging motor are resistant to potent I-125 and Cs-131 radiation, which is a prerequisite for utilizing these RNA nanoparticles as carriers for targeted radiation therapy. pRNA 3WJ nanoparticles were constructed and characterized, and the stability of these nanoparticles under I-125 and Cs-131 irradiation with clinically relevant doses was examined. RNA nanoparticles derived from the pRNA 3WJ targeted tumors specifically and they were stable under irradiation of I-125 and Cs-131 with clinically relevant doses ranging from 1 to 90 Gy over a significantly long time up to 20 days, while control plasmid DNA was damaged at 20 Gy or higher.
- [Show abstract] [Hide abstract] ABSTRACT: RNA nanotechnology is an emerging field at the interface of biochemistry and nanomaterials that shows immense promise for applications in nanomedicines, therapeutics and nanotechnology. Noncoding RNAs, such as siRNA, miRNA, ribozymes, and riboswitches, play important roles in the regulation of cellular processes. They carry out highly specific functions on a compact and efficient footprint. The properties of specificity and small size make them excellent modules in the construction of multifaceted RNA nanoparticles for targeted delivery and therapy. Biological activity of RNA molecules, however, relies on their proper folding. Therefore their thermodynamic and biochemical stability in the cellular environment is critical. Consequently, it is essential to assess global fold and intracellular lifetime of multifaceted RNA nanoparticles to optimize their therapeutic effectiveness. Here, we describe a method to express and assemble stable RNA nanoparticles in cells, and to assess the folding and turnover rate of RNA nanoparticles in vitro as well as in vivo in real time using a thermostable core motif derived from pRNA of bacteriophage Phi29 DNA packaging motor and fluorogenic RNA modules.
- [Show abstract] [Hide abstract] ABSTRACT: Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues.
Dataset: Supplementary Material
- [Show abstract] [Hide abstract] ABSTRACT: RNA nanoparticles derived from the three-way junction (3WJ) of the pRNA of bacteriophage phi29 DNA packaging motor were previously found to be thermodynamically stable. As the nanoparticles could have potential in ocular drug delivery, the objectives in the present study were to investigate the distribution of pRNA nanoparticles after subconjunctival injection and examine the feasibility to deliver the nanoparticles to the cells of cornea and retina. Alexa647-labeled pRNA nanoparticles (pRNA-3WJ and pRNA-X) and double-stranded RNA (dsRNA) were administered via subconjunctival injection in mice. Alexa647 dye was a control. Topical administration was performed for comparison. Ocular clearance of pRNA nanoparticles and dsRNA after the injection was assessed using whole-body fluorescence imaging of the eyes. The numbers of cells in the ocular tissues with nanoparticle cell internalization were determined in fluorescence microscopy of dissected eye tissues. After subconjunctival injection, pRNA nanoparticles and dsRNA were observed to distribute into the eyes and cleared through the lymph. pRNA-3WJ, pRNA-X, and dsRNA were found in the cells of the conjunctiva, cornea, and sclera, but only pRNA-X was in the cells of the retina. Topical administration was not effective in delivering the nanoparticles to the eye. The pRNA nanoparticles were delivered to the cells in the eye via subconjunctival injection, and cell internalization was achieved in the cornea with pRNA-3WJ and pRNA-X and in the retina with pRNA-X. Only the X-shape pRNA-X could enter the retina.
- [Show abstract] [Hide abstract] ABSTRACT: Human genome sequencing reveals that only 1.5% of the DNA sequence codes for protein. More and more evidence reveals that a substantial part of the 98.5% so-called "junk" DNAs actually code for small noncoding RNAs. Two milestones, chemical drugs and protein drugs, have appeared in the history of drug development, and it is expected that a third milestone in drug development will be RNA drug or chemical drugs that target RNA. This review focuses on the development of RNA therapeutics for potential cancer treatment by applying RNA nanotechnology. A therapeutic RNA nanoparticle is so unique that in this particle the scaffold, the ligand, and the therapeutic can all be composed of RNA. The special physicochemical properties lend to delivery of siRNA, miRNA, ribozymes, and fluogenenic RNA as imaging agents or RNA aptamer as ligands for targeting. With recent advances in solving the RNA chemical, enzymatic, and thermodynamic stability issues, RNA nanoparticles have been found to be advantageous for in vivo applications due to their uniform nano-scale size, precise stoichiometry, polyvalent nature, low immunogenicity, low toxicity, and target specificity. In vivo animal studies have revealed that RNA nanoparticles can specifically target tumors without unwanted accumulation in normal organs. We summarize the key studies that led to the detailed understanding of RNA particle formation, as well as chemical and thermodynamic stability. We discuss methods and "toolkits" for RNA nanoparticle construction. The current challenges in clinical application of RNA nanotechnology, such as endosome trapping and production cost, will also be discussed.
- [Show abstract] [Hide abstract] ABSTRACT: Misfolding and associated loss of function are common problems in constructing fusion RNA complexes due to changes in energy landscape and the nearest-neighbor principle. Here we report the incorporation and application of the pRNA-3WJ motif of the phi29 DNA packaging motor into fusion RNA with controllable and predictable folding. The motif included three discontinuous ∼18 nucleotide (nt) fragments, displayed a distinct low folding energy (Shu D et al., Nature Nanotechnology, 2011, 6:658-667), and folded spontaneously into a leading core that enabled the correct folding of other functionalities fused to the RNA complex. Three individual fragments dispersed at any location within the sequence allowed the other RNA functional modules to fold into their original structures with authentic functions, as tested by Hepatitis B virus ribozyme, siRNA, and aptamers for malachite green (MG), spinach, and streptavidin (STV). Only nine complementary nucleotides were present for any two of the three ∼18-nt fragments, but the three 9 bp branches were so powerful that they disrupted other double strands with more than 15 bp within the fusion RNA. This system enabled the production of fusion complexes harboring multiple RNA functionalities with correct folding for potential applications in biotechnology, nanomedicine and nanotechnology. We also applied this system to investigate the principles governing the folding of RNA in vivo and in vitro. Temporal production of RNA sequences during in vivo transcription caused RNA to fold into different conformations that could not be predicted with routine principles derived from in vitro studies.
- [Show abstract] [Hide abstract] ABSTRACT: RNA nanotechnology is a term that refers to the design, fabrication and use of nanoparticles that are mainly composed of RNAs via bottom-up self-assembly. The packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor has been developed into a nanodelivery platform. This protocol describes the synthesis, assembly and functionalization of pRNA nanoparticles on the basis of three 'toolkits' derived from pRNA structural features: interlocking loops for hand-in-hand interactions, palindrome sequences for foot-to-foot interactions and an RNA three-way junction for branch extension. siRNAs, ribozymes, aptamers, chemical ligands, fluorophores and other functionalities can also be fused to the pRNA before the assembly of the nanoparticles, so as to ensure the production of homogeneous nanoparticles and the retention of appropriate folding and function of the incorporated modules. The resulting self-assembled multivalent pRNA nanoparticles are thermodynamically and chemically stable, and they remain intact at ultralow concentrations. Gene-silencing effects are progressively enhanced with increasing numbers of siRNAs in each pRNA nanoparticle. Systemic injection of the pRNA nanoparticles into xenograft-bearing mice has revealed strong binding to tumors without accumulation in vital organs or tissues. The pRNA-based nanodelivery scaffold paves a new way for nanotechnological application of pRNA-based nanoparticles for disease detection and treatment. The time required for completing one round of this protocol is 3-4 weeks when including in vitro functional assays, or 2-3 months when including in vivo studies.
- [Show abstract] [Hide abstract] ABSTRACT: Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed "toolkits" utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.
- [Show abstract] [Hide abstract] ABSTRACT: One of the advantages of nanotechnology is the feasibility to construct therapeutic particles carrying multiple therapeutics with defined structure and stoichiometry. The field of RNA nanotechnology is emerging. However, controlled assembly of stable RNA nanoparticles with multiple functionalities which retain their original role is challenging due to refolding after fusion. Herein, we report the construction of thermodynamically stable X-shaped RNA nanoparticles to carry four therapeutic RNA motifs by self-assembly of reengineered small RNA fragments. We proved that each arm of the four helices in the X-motif can harbor one siRNA, ribozyme, or aptamer without affecting the folding of the central pRNA-X core, and each daughter RNA molecule within the nanoparticle folds into their respective authentic structures and retains their biological and structural function independently. Gene silencing effects were progressively enhanced as the number of the siRNA in each pRNA-X nanoparticles gradually increased from one to two, three, and four. More importantly, systemic injection of ligand-containing nanoparticles into the tail-vein of mice revealed that the RNA nanoparticles remained intact and strongly bound to cancers without entering the liver, lung or any other organs or tissues, while remaining in cancer tissue for more than 8 h.
- [Show abstract] [Hide abstract] ABSTRACT: RNA nanoparticles have applications in the treatment of cancers and viral infection; however, the instability of RNA nanoparticles has hindered their development for therapeutic applications. The lack of covalent linkage or crosslinking in nanoparticles causes dissociation in vivo. Here we show that the packaging RNA of bacteriophage phi29 DNA packaging motor can be assembled from 3-6 pieces of RNA oligomers without the use of metal salts. Each RNA oligomer contains a functional module that can be a receptor-binding ligand, aptamer, short interfering RNA or ribozyme. When mixed together, they self-assemble into thermodynamically stable tri-star nanoparticles with a three-way junction core. These nanoparticles are resistant to 8 M urea denaturation, are stable in serum and remain intact at extremely low concentrations. The modules remain functional in vitro and in vivo, suggesting that the three-way junction core can be used as a platform for building a variety of multifunctional nanoparticles. We studied 25 different three-way junction motifs in biological RNA and found only one other motif that shares characteristics similar to the three-way junction of phi29 pRNA.
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