RNA nanoparticles can potentially be used to treat cancers and viral infection but their instability has hindered their therapeutic applications. The lack of covalent linkage or cross-linking in the nanoparticles causes dissociation in vivo. Here we show the assembly of thermodynamically stable three-way junction (3WJ) of the motor pRNA of bacteriophage phi29 from 3–6 pieces of RNA oligomers without the use of metal salts to form stable multifunctional nanoparticles. Each RNA oligomer contains either a receptor-binding ligand, aptamer, siRNA or ribozyme functional module. When mixed together, they self-assemble into tri-stars nanoparticles with a 3WJ core. The nanoparticles are resistant to 8M urea denaturation, stable in serum and remain intact at extremely low concentrations. The modules remain functional in vitro and in vivo, suggesting the 3WJ core can be used as a platform for building a variety of multifunctional nanoparticles. Of the 25 different 3WJ motifs evaluated, only one other motif in biological RNA shares similar characteristics as the phi29 3WJ.

Sensitivity and specificity are two most important factors to take into account for molecule sensing, chemical detection and disease diagnosis. A perfect sensitivity is to reach the level where a single molecule can be detected. An ideal specificity is to reach the level where the substance can be detected in the presence of many contaminants. The rapidly progressing nanopore technology is approaching this threshold. A wide assortment of biomotors and cellular pores in living organisms perform diverse biological functions. The elegant design of these transportation machineries has inspired the development of single molecule detection based on modulations of the individual current blockage events. The dynamic growth of nanotechnology and nanobiotechnology has stimulated rapid advances in the study of nanopore based instrumentation over the last decade, and inspired great interest in sensing of single molecules including ions, nucleotides, enantiomers, drugs, and polymers such as PEG, RNA, DNA, and polypeptides. This sensing technology has been extended to medical diagnostics and third generation high throughput DNA sequencing. This review covers current nanopore detection platforms including both biological pores and solid state counterparts. Several biological nanopores have been studied over the years, but this review will focus on the three best characterized systems including α-hemolysin and MspA, both containing a smaller channel for the detection of single-strand DNA, as well as bacteriophage phi29 DNA packaging motor connector that contains a larger channel for the passing of double stranded DNA. The advantage and disadvantage of each system are compared; their current and potential applications in nanomedicine, biotechnology, and nanotechnology are discussed.

Nanotechnology holds many advantages. Here we report another advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand-display on extracellular vesicle (EV) membranes for specific cell targeting, or to regulate intracellular trafficking of siRNA/miRNA. Placing membrane-anchoring cholesterol at the arrow-tail results in display of RNA aptamer or folate on EV outer surface. In contrast, placing the cholesterol at the arrow-head results in partial loading of RNA nanoparticles into the EVs. Taking advantage of the RNA ligand for specific targeting and EVs for efficient membrane fusion, the resulting ligand-displaying EVs were competent for specific delivery of siRNA to cells, and efficiently block tumor growth in three cancer models. PSMA aptamer-displaying EVs loaded with survivin siRNA inhibited prostate cancer xenograft. The same EV but displaying EGFR aptamer inhibited orthotopic breast cancer models. Likewise, survivin-loaded and folate-displaying EVs inhibited patient derived colorectal cancer xenograft.

Summary 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.