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 allow higher stability in blood serum, attachment of fluorescent markers for tracking, 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, a DNA duplex with its attached fluorophores induced a FRET affect, while the other was a DS siRNA capable of silencing the targeted gene. This concept was extended for our previously characterized six-stranded RNA nanocubes 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. To allow for multiple functionalities to be delivered and activated simultaneously, previously designed RNA rings were used as scaffolds carrying six hybrids. However, the hybrids now contained RNA toeholds rather than DNA. Toeholds of 2, 4, 6, and 8 nucleotides were investigated for their efficiency of re-association. FRET revealed that longer toeholds led to improved re-association rates. The functionality of these particles was confirmed in different human cell lines. From the perspective of thermodynamics, the use of RNA toeholds is advantageous as it reduces the length of the single stranded ends required to unzip the hybrids and generate the functional RNA element. From a design perspective, the RNA toehold can be part of the functional DS RNA, or other potential RNA moiety, reducing the size and minimizing the design constraints of the resulting hybrid duplexes. Conditional hybrids that contain ssRNA toeholds also prove advantageous for incorporation into more complex RNA nanoparticles. It was shown that RNA nanorings functionalized with RNA toeholded hybrids exhibited increased yields from enzymatic co-transcriptional synthesis, as well as reduced overall nanoparticle size, compared to nanorings functionalized with DNA-toeholded hybrid duplexes. An additional scheme has also been exploited using RNA-RNA interactions. Here, an RNA strand is designed to interact with specific mRNA strands in cells. The RNA strand contains both therapeutic and trigger components that are designed to dissociate from each other in the presence of a trigger mRNA and form a byproduct as well as a short hairpin-like RNA which can be processed by dicer to form functional siRNA. The conformational change takes place due to the presence of an extended ssRNA toehold in the trigger-binding strand which allows for the specific binding to an mRNA in diseased cells. This approach allows for the conditional activation of therapeutic RNAs only where a designated trigger strand is present. For potential use in treatment of diseases, such as cancer, this can reduce off target silencing and allow for more precise treatment. Because the self-assembly of RNA complexes is an interplay of several RNA strands, a new algorithm, HyperFold was developed that predicts the folding properties of all possible combinations of strand complexes. Also, the general problem of finding the lowest free energy base pairing is difficult for computational algorithms. Because the number of possible base pair combinations grows exponentially with sequence length, searching through all possible base pair combinations quickly becomes prohibitive for nucleic acid structures possessing long strands and pseudoknotted structures. HyperFold solves this problem using a tunable heuristic that depends on a single parameter. By default the search utilizes a middle-ground strategy. The resulting search strategy is a tunable approach for predicting the interactions between multiple RNA and DNA strands with possibly complex knotted structures. Designing self-assembling RNA ring structures based on known 3D structural elements connected via linker helices is a challenging task due to the immense number of motif combinations, many of which do not lead to ring-closure. We developed an in silico solution to this design problem by combinatorial assembly of RNA 3-way junctions, bulges, and kissing loops, and tabulating the cases that lead to ring formation. The solutions found are made available in the form of a web Ring Catalog. As an example of a potential use of this resource, we chose a predicted RNA square structure consisting of five RNA strands and demonstrated experimentally that the self-assembly of those five strands leads to the formation of a square-like complex. 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. 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. A new class of cationic lipids - bolaamphiphiles or bolas was studied (GLH-58 and GLH-60) for their ability to efficiently deliver siRNAs to cancer cells. Both bolas have similar hydrophobic domains and contain either one, in GLH-58, or two, in GLH-60 positively charged head groups at each end of the hydrophobic core. We computationally predicted and experimentally validated that GLH-58 formed more stable nano sized micelles and performed significantly better in comparison to GLH-60 for siRNA delivery. 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. Several invited review papers and book chapters were also written on the above described subjects.
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