Engineered RNA nanodesigns for applications in RNA nanotechnology: Invited reviews (paper and chapter) compare and contrast some of the differences between RNA and DNA nanotechnologies emphasizing the advantages to be attained from the use of RNA as a modality for developing RNA-based nanoparticles. Years of experience in understanding RNAs'biological structure and function has enabled the design of RNA based functional nanoparticles and have brought these particles to preclinical levels. A multitude of RNA motifs from nature and de novo designs can be used for self-assembling various RNA shapes. Functionalities can be gleaned from siRNAs, riboswitches, ribozymes and RNA aptamers to name a few. Two major design approaches are discussed, RNA architectonics and a single-stranded tiles approach. RNA architectonics relies on secondary structure formation of the RNA building blocks while the single-stranded tiles approach attempts to reduce RNA secondary structure to a minimum but relies on relatively short inter-strand helical interactions. Computational approaches to RNA nanodesign are summarized, including our software, NanoTiler, RNA2D3D, and the RNAjunction database. Our methods for co-transcriptional assembly of RNA nanoparticles that may contain modified bases for nuclease protection is summarized (see below). Functionalization of our RNA nanorings and nanocubes with siRNAs targeting various genes are discussed as well as a new methodology involving the use of RNA/DNA hybrids with split functionalities is summarized (also below). Using RNA structural flexibility Data in Nanostructure Modeling: In the emerging field of RNA-based nanotechnology there is a need for automation of the structure design process. Initially, the building blocks used to build our RNA nanostructures were treated as rigid objects. Experimental data, however, shows that RNA accommodates its shape to the constraints of larger structural contexts. We included the flexibility of our building blocks into the full design process. By using an experimentally proven system, the RNA tectosquare, we showed that considering the flexibility of its kissing loop motifs as well as distortions in its helical regions appears to be necessary to achieve a realistic design. In addition, we describe a coarse grained method using anisotropic network modeling to significantly speed up the process of determining the main motions that are inherent in RNA nanostructures. The procedure is able to account for structure size variations compared to dynamic light scattering experiments and the thermodynamic stabilities associated with different subtle sequence variations. Co-transcriptional assembly of chemically modified RNA nanostructures functionalized with siRNAs: We developed a one-pot co-transciptional assembly technique that uses T7 RNA polymerase operating on DNA templates in conjunction with manganese ions to produce functionalized RNA nanoparticles that can include modified bases that protect the particles from nucleases found in human blood serum. The methodology effectively permits the inexpensive assembly of relatively large RNA nanoconstructs. Assemblies done under physiological conditions of complex nanostructures (some containing as many as 22 strands) functionalized with multiple siRNAs were accomplished. The siRNAs were associated with the scaffolds. Nuclease protection was significantly improved, and the different synthesized particles were shown to silence well with and without the modified bases. Activation of split functionalities on reassociation of RNA-DNA hybrids: Using initially inactive RNA-DNA hybrid duplexes containing split functionalities, we developed a methodology that enables the activation of the functionalites by reassociation of the DNA and RNA into their associated RNA and DNA duplex products. Reassociation is triggered by the independent delivery of complementary hybrids that each contain complementary DNA toeholds. Upon reassociationfunctional RNA complexes and functional DNA complexes are produced. These products are more thermodynamically stable than their starting hybrid duplex reactants. This novel approach has several advantages 1) conditional activation of spit functionalities;2) protection against nucleases in the blood;3) DNA and RNA can be functionalized;4) kinetics of reassociation can be controlled by toehold length;5)real time tracking can be accomplished by attaching molecular beacons to the DNA strands;6) use of multiple split functionalities i.e. DNA and RNA split functionalities can become activated upon reassociation and;7) hybrid duplexes can be targeted independently. Using this approach we showed that the methodology works in vitro and in vivo where reassociation of diceable siRNAs occurred in cells with concomitant silencing of the targeted genes. Genes targeted, besides eGFP in a breast cancer cell line, were the protease and the envelope genes in HIV-1, which showed very significant reductions in virus levels and associated viral proteins in infected cells. In addition toxicity levels were low. Also, a cancer gene (GSTP1) in A549 lung adenocarcinoma cells showed a significant decrease in GTSP1 protein using appropriately designed hybrids individually co-tranfected on two different days. Biodistribution studies of the hybrids showed significant tumor uptake in xenograph breast cancer mouse models as well as significant silencing of eGFP. Hybrids were also resistant to mouse nucleases in the bloodstream. As a further proof of principle, a split inactive malachite green aptamer was shown to be activated upon hybrid recombination. In silico, in vivo and in vitro studies of bolaamphiphiles for potential therapeutic siRNA delivery: To overcome obstacles in siRNA delivery, such as crossing cellular membranes and short half-lives in blood, we studied the use of bolaamphiles complexed with siRNA as a delivery agent. Bolaamphiphiles consist of 2 positively charged head-groups that flank an internal hydrophobic chain. They have relatively low toxicities, long persistence in the blood and have the ability to form poly-cationic micelles under aqueous conditions that associate with siRNAs. Two different bolaamphiphiles with acetylcholine head groups attached to an alkyl chain in two distinct configurations, GLH-19 and GLH-20, were compared for their abilities to complex with siRNAs and deliver them into the cells inducing gene silencing. Molecular dynamics simulations showed that bolaamphiphiles associate with siRNAs due to electrostatic, hydrogen bonding, and hydrophobic interactions. These in silico studies were supported by various in vitro, cell culture and in vivo studies. MD simulations predicted better protection against nuclease degradation for siRNAs associated with the GLH-19 micelles. MM-PBSA and MM-GBSA methods predicted a higher binding affinity for the GLH-19/siRNA complex. Consistent with these computational results, gel experiments indicated stronger binding and more stable interactions for the GLH-19/siRNA complexes, which in addition showed almost no degradation in the presence of nucleases. cryo-EM studies characterized the bolaamphiphile/siRNA complexes indicating that GLH-19/siRNA and GLH-20/siRNA have different relative hydrophobicities with GLH-19/siRNA being less hydrophobic. Based on cell culture transfections and the in vivo live fluorescence imaging results, we concluded that both GLH-19 and GLH-20 bolaamphiphiles have great potential to be used as carriers for siRNA delivery with GLH-19 being a better candidate. Bola/siRNA complexes significantly increase the chemical stability of siRNAs and provide excellent intracellular uptake followed by specific gene silencing. Moreover, depending on the application, the extent of chemical protection of the siRNA can be altered by simply changing the carrier.
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