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RNA is unique in nanoscale fabrication due to its amazing diversity of function and structure. RNA nanoparticles can be fabricated with a level of simplicity characteristic of DNA while possessing versatile tertiary structure and catalytic function similar to that of proteins. A large variety of single stranded loops are suitable for inter- and intramolecular interactions, serving as mounting dovetails in self-assembly without the need for external linking dowels. Novel properties of RNA nanoparticles have been explored for treatment and detection of diseases and various other realms. The higher thermodynamic stability, holding of noncanonical base pairing, stronger folding due to base stacking properties, and distinctive in vivo attributes make RNA unique in comparison to DNA. Indeed, the potential application of RNA nanotechnology in therapeutics is an exciting area of research. The use of RNAi in biomedical research has opened up new possibilities to silence or regulate the biological function of individual genes. Small interfering RNA (siRNA) has been extensively explored to genetically manipulate the expression in vitro and in vivo of particular genes identified to play a key role in cancerous or viral diseases. However, the efficient silencing of the desired gene depends upon efficient delivery of siRNA to targeted cells, as well as in vivo stability. In this chapter, we use the bacteriophage phi29 motor pRNA-derived nanocarrier as a polyvalent targeted delivery system, introduce the potential of RNA-based therapeutics using nanobiotechnology or nanotechnology methods with the fabrication and modification of pRNA nanoparticles, and highlight its potential to become a valuable research tool and viable clinical approach for gene therapy.

The 117-nucleotide (nt) RNA, called the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor, has been shown to be an efficient vector for the construction of RNA nanoparticles for the delivery of small interfering RNA (siRNA) into specific cancer or viral-infected cells. Currently, chemical synthesis of 117-nt RNA is not feasible commercially. In addition, labeling at specific locations on pRNA requires the understanding of its modular organization. Here, we report multiple approaches for the construction of a functional 117-base pRNA using two synthetic RNA fragments with variable modifications. The resulting bipartite pRNA was fully competent in associating with other interacting pRNAs to form dimers, as demonstrated by the packaging of DNA via the nanomotor and the assembly of phi29 viruses in vitro. The pRNA subunit assembled from bipartite fragments harboring siRNA or receptor-binding ligands were equally competent in assembling into dimers. The subunits carrying different functionalities were able to bind cancer cells specifically, enter the cell, and silence specific genes of interest. The pRNA nanoparticles were subsequently processed by Dicer to release the siRNA embedded within the nanoparticles. The results will pave the way toward the treatment of diseases using synthetic pRNA/siRNA chimeric nanoparticles.

Recent advances in RNA nanotechnology have led to the emergence of a new field and brought vitality to the area of therapeutics [P. Guo, The emerging field of RNA nanotechnology, Nat. Nanotechnol., 2010]. Due to the complementary nature of the four nucleotides and its special catalytic activity, RNA can be manipulated with simplicity characteristic of DNA, while possessing versatile structure and diverse function similar to proteins. Loops and tertiary architecture serve as mounting dovetails or wedges to eliminate external linking dowels. Unique features in transcription, termination, self-assembly, self-processing, and acid-resistance enable in vivo production of nanoparticles harboring aptamer, siRNA, ribozyme, riboswitch, or other regulators for therapy, detection, regulation, and intracellular computation. The unique property of noncanonical base-pairing and stacking enables RNA to fold into well-defined structures for constructing nanoparticles with special functionalities. Bacteriophage phi29 DNA packaging motor is geared by a ring consisting of six packaging RNA (pRNA) molecules. pRNA is able to form a multimeric complex via the interaction of two reengineered interlocking loops. This unique feature makes it an ideal polyvalent vehicle for nanomachine fabrication, pathogen detection, and delivery of siRNA or other therapeutics. This review describes methods in using pRNA as a building block for the construction of RNA dimers, trimers, and hexamers as nanoparticles in medical applications. Methods for industrial-scale production of large and stable RNA nanoparticles will be introduced. The unique favorable PK (pharmacokinetics) profile with a half life (T(1/2)) of 5-10h comparing to 0.25 of conventional 2'-F siRNA, and advantageous in vivo features such as non-toxicity, non-induction of interferons or non-stimulating of cytokine response in animals will also be reviewed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Both DNA and RNA can serve as powerful building blocks for bottom-up fabrication of nanostructures. A pioneering concept proposed by Ned Seeman 30 years ago has led to an explosion of knowledge in DNA nanotechnology. RNA can be manipulated with simplicity characteristic of DNA, while possessing noncanonical base-pairing, versatile function, and catalytic activity similar to proteins. However, standing in awe of the sensitivity of RNA to RNase degradation has made many scientists flinch away from RNA nanotechnology. Here we report the construction of stable RNA nanoparticles resistant to RNase digestion. The 2'-F (2'-fluoro) RNA retained its property for correct folding in dimer formation, appropriate structure in procapsid binding, and biological activity in gearing the phi29 nanomotor to package viral DNA and producing infectious viral particles. Our results demonstrate that it is practical to produce RNase-resistant, biologically active, and stable RNA for application in nanotechnology.