RNA nanoparticles come of age.

RNA has multiple functions in nature, including informational transfer (mRNA) [1], adaptor function (tRNAs) [2], guide functions (snRNAs, snoRNAs) [3,4] catalytic function (ribozymes and the large ribosomal RNA) [5–7], and environmental sensing (riboswitches) [8]. In contrast, DNA only serves as an information storage molecule, and proteins serve as structural and enzymatic molecules. Thus, no other class of macromolecules in living systems possesses the many functional attributes of RNA. How does one class of macromolecule possess so many different attributes? This is a key question that is addressed in a recent review on ‘the emerging field of RNA nanotechnology’ [9]. In this very relevant and up-to-date treatise on the real and potential applications of RNA as a nanocarrier for therapeutic and diagnostic application, Guo presents a comprehensive picture of the various chemical and structural features of RNA that make it an attractive nanoparticle platform. Nanotechnology has seen an explosion of new platforms within the past few years. The ability to fabricate nanometer-sized particles with desired shapes, chemistry, and sizes has resulted in a renewed interest of nano-sized particles for delivery of drugs, proteins, and nucleic acids [10–12]. Many of the particles are based on some form of synthetic polymer, which can be readily manufactured to a specific size and shape. These synthetic nanoparticles have the downside of being potentially contaminating and toxic to aquatic and other environments [13–16]. Thus, the attraction of RNA-based nanoparticles for therapeutic and industrial applications has merit. RNA as a nanoparticles platform has been a concept rooted in the development of RNA-based aptamers that can serve as platforms for biological catalysis as well as selective ligand-binding polymers [17,18]. The major chemical features of RNA that make it a nano-fabricating platform are those same features that provide the diversity of functions. These include the 2′-OH ribose moiety, the ability to form intramolecular structures using a combination of canonical and non-canonical base interactions, and the stability of RNA:RNA base pairs. In addition to the natural attributes, RNA can be backbone modified with a variety of simple chemistries such as 2′-fluoro or 2′-OMe, which impart protection against RNA degrading enzymes and increased thermodynamic stability of the Watson–Crick base pairs. Guo points out some important naturally occurring RNA nanoparticles structures that utilize loop–loop base pairing along with linear helical base pairing to form extremely stable nano-structures [9]. One of these is the dimerization domain of HIV-1, which is a parallel stranded structure that is triggered by loop–loop palindrome base pairing in the dimer-packaging signal domain. This forms a highly structured and tight nano-structure that ensures packaging of two 9-kb viral genomes into a single particle. For Guo, a more familiar RNA nanoparticle is the six RNA molecular component of the bacteriophage Phi29 packaging motor [19]. These RNAs self-assemble into a six-membered nanoparticle by a series of loop–loop interactions resembling a group of six people standing in a ring and contacting the persons to their left and right by holding the adjacent persons right hand with their left, and left hand with their right. Guo has creatively taken advantage of this self-assembly process to fabricate phi29 RNA-based nanoparticles that form various sized nanoparticles in which each RNA component is covalently attached to a functional sequence, such as an aptamer, ribozyme, or siRNA [20]. Depending on the number of phi29 RNA-based components; different sized and shaped nanoparticles can be developed. Thus, this simple bacteriophage system has provided a naturally occurring demonstration that RNA nanoparticles assembly can be systematically organized and controlled as one would find in the chemical compound nano-fabrication process. The strong loop–loop interactions of the phi29 system also provide a model for the ‘in silico’ design of RNA-based nanoparticles. With respect to the manufacturing of RNA nanoparticles, synthetic RNA chemistry has now become relatively straightforward and RNAs approaching 80 bases in length can be synthesized with high yield [21]. The backbone modified building blocks of 2′-fluoro and 2′-OMe RNA phosphoramidities are commercially available and are easily incorporated into an automated RNA synthesis regimen. Both of these modifications are useful for the biological applications of RNA nanoparticles since they both stabilize against RNAses and prolong the half-life of these molecules. This is a great advantage for RNA over other nanoparticle platforms since the bio-availability and stability can be modulated to the desired function. For instance, it is known that some motifs in RNA can trigger type I interferon responses inside of cells, but the simple addition of 2′-OMe can abrogate activation of this response [22,23]. Since 2′-OMe is a naturally occurring modification found in tRNAs and rRNAs, it is biologically tolerated and non-toxic. This brings forward the concern about the toxicity of some nanoparticle platforms, which are not biodegradable and hence can be potential environmental contaminants. RNA can be modified for stability and still maintain function in the absence of toxicity. Guo suggests in his review that RNA nanostructures can self-assemble in vivo following transcription [9]. This is certainly the case for the HIV-1 dimerization process. An important component of RNA folding in cells is the role played by RNA chaperones, proteins that bind to the RNA backbone and assist in base-pairing interactions [24,25] to assist in the proper folding of RNAs. Such chaperones do not discriminate on base sequence but simply assist in the formation of the most thermodynamically stable structures. Clearly, this is an advantage that RNA-based nanoparticles have over all other nanoparticles building materials. RNA also can be imported into cells by receptor-mediated endocytosis, which has formed the basis for RNA-based aptamer delivery of siRNAs [26,27]. Guo demonstrates that the phi29 RNAs can be covalently attached to aptamers and siRNAs, and then self-assemble into nanoparticles that function in the cell type, aptamer-directed delivery of the siRNAs [27]. In summary, this review by Guo brings to light the chemical and structural foundations for the exploitation of RNA as a platform for nanoparticles assembly [9]. He points out the self-assembly process and the ability to create a variety of shapes and sizes based on the size and interaction properties of the RNA building blocks. He also points out the many challenges that lie ahead in developing RNA as a major platform for nanoparticle fabrication. Perhaps the major challenge is in computational predictions of the shapes of the RNA nanoparticles. To date the RNA structural database is small relative to the protein or small molecule databases. Nevertheless, there are many solid structures derived from crystallographic or nuclear magnetic resonance analyses of biologically important RNAs, as well as some synthetic RNA motifs [28]. These known structural motifs can be used as the starting points or building blocks for homology modeling as is done for protein structure. Given the ever-increasing interest in RNA biology, there should be a continuing flow of new structural information that can be used to guide the design and assembly of RNA nanoparticles.