To achieve control over deliverable functionality and stability of RNA-based nanoparticles, the properties of DNA and RNA were merged in the development of computationally designed nanoparticles that were constructed from RNA/DNA hybrids. These molecules allowed higher stability in blood serum, attachment of fluorescent markers for tracking without interfering with RNA functionality, and the ability to split the components of functional elements inactivating them, but allowing later activation under the control of complementary toeholds by which the kinetics of re-association can be tuned. DS siRNAs (Diceable substrate siRNA) could be split into two components, each consisting of an RNA/DNA hybrid, where the DNA contains a complementary single-stranded toehold to its counterpart found in a complementary hybrid. The two hybrids, when transfected into cells recombine into two products due to the toeholds and the computationally determined thermodynamic difference between the hybrids and the products. The products, one consisting of a DNA duplex with its attached fluorophores induced a FRET affect, while the other product was a DS siRNA capable of silencing the targeted gene. The split functionality was extended to include multiple functionalites. A malachite green aptamer and DS siRNAs were split and incorporated in complementary hybrids. Experiments showed activation of both functionalities upon recombination of the strands with toeholds. In another experiment the silencing efficiency of hybrids containing 1-3 DS siRNAs targeting MDA-MB-231/GFP cell lines was measured. Silencing was proportional to the number of DS siRNA present in the hybrid with 3 DS siRNA showing the best silencing. We showed that long split functional hybrids can be produced by RNA polymerase II-dependent transcription using single-stranded DNA templates. The incorporation of transcription stop elements such as LNAs proved successful in generating hybrid constructs with the toeholds. Type I interferon response was tested and the results indicated that a minimal response was detected for hybrid reassociation of 3 DS siRNAs. However, the response was shown to be significantly higher for hybrid reassociations consisting of 7 components due to long DNA strands being reconstituted. Our work in RNA nanotechnology introduced novel nanoscaffolds e.g. nanorings. Besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous incorporation of assorted RNA aptamers, fluorescent dyes, proteins, as well as RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells. We showed how the nanoring design can achieve cell-targeting properties through incorporation of RNA aptamers specific for the human epidermal growth factor receptor. Also, since the incorporation of RNA functionalities such as DS RNAs into the nanoscaffolds presents difficulties for solid state chemical synthesis as RNA components generally cannot exceed 60 nucleotides in length, we solved this problem by annealing DS RNAs to nanoscaffolds using single-stranded toehold sites. Finally we showed how the therapeutic functionality of the nanoring can be triggered by the use of RNA-DNA hybrids. This new technique involves splitting the different functionalities between a RNA-DNA nanoring and cognate RNA-DNA hybrids with conditional intracellular activation of these functionalities. Various biochemical, biophysical, in vitro and in vivo methods were used to characterize and show the efficacy of these particles. This included knock down of HIV, and silencing of genes in xenograph mouse models. Importantly, interferon and pro-inflammatory cytokine activation assays indicated significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use. We used previously characterizedsix-stranded RNA nanocubes as scaffolds for the controlled delivery of multiple siRNAs. The RNA nanocubes were functionalized with six DS RNAs. Two other versions of the cube were made; one consisting of an RNA core with RNA-DNA hybrid DS RNA arms and the other consisting of a DNA core with RNA-DNA hybrid DS RNA arms. The arms in the latter two cases contained DNA toeholds which allowed for functional siRNA activation when presented with cognate RNA-DNA hybrid duplexes. Transfection experiments showed activation of functionality including down regulation of HIV. It was shown that DNA core cubes had the least interferon response, while all RNA cubes had the most, while the RNA core cube was in the middle. Since RNA is a flexible molecule it is important to consider the ramifications of this related to self-assembly of RNA nanoconstructs. Since MD is computationally time-consuming, we explored the use of a coarse-grained technique, Anisotropic Network Modeling (ANM), which can vary the coarseness of a molecule's representation from 1 bead per nucleotide, to a full atomic representation from 1 bead per atom. Forces and potential energies can be derived by assigning a spring constant to interactions that lie within a defined range of each bead. This approach shortens a simulation that would normally take weeks with MD to just a few hours. We focused on the low frequency c motions as an indicator of the most biologically relevant dynamic characteristics of the studied molecule. Our nanocubes were characterized with ANM, and results brought the computational and the experimental results into agreement. ANM also added insight into the observed assembly yields of the cube variants and their melting temperatures.We studied, using MD, the structural properties, Root Mean Square Deviation, the radius of gyration and radial distribution function (RDF) of RNA nanotubes up to the size of about 20nm in physological solutions. The concentration of ions around the tube as a function of time at a particular temperature were characterized. We found that when the temperature increases, the number of ions increased within a certain distance of the tube. Also, the number of ions within this distance around the tube decreases in quenched runs. RDF plots also demonstrated a similar trend with temperature in the case of RNA nanorings.The delivery of RNA-based nanoconstructs in cell culture and in vivo is essential for the development of therapeutic methodologies using these agents. Non-modified naked RNAs have short half-lives in blood serum due to nucleases and have difficulty crossing cell membranes due to their inherent negative charge. To counter some of these issues we evaluated oxime ether lipids (OELs) containing modifications in the hydrophobic domains and hydrophilic head groups for complex formation with siRNA molecules and siRNA delivery efficiency of resulting complexes. The potential of OELs to deliver nucleic acids and silence the green fluorescent protein gene was analyzed using MDA-MB-231 and MDA-MB-231/GFP cells, respectively. We found that the introduction of hydroxyl groups to the polar domain of the OELs and unsaturation into the hydrophobic domain favor higher transfection and gene silencing in a cell cultures. There is a need for simple, efficient assembly assays of RNA-based nanoparticles. Common methods for tracking RNA assemblies such as native polyacrylamide gels and atomic force microscopy are often time-intensive. We developed a technique for rapid analysis of RNA NP assembly stages using the formation of fluorescent silver nanoclusters (Ag NC). This method exploits the single-stranded specificity and sequence dependence of Ag NC formation to produce unique optical readouts for each stage of RNA NP assembly. Invited review papers and book chapters were also written on the above described subjects.
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