Self-Assembling RNA Nanorings Based on RNA I/II Inverse Kissing Complexes with Associated Diceable siRNAsWe experimentally characterized by biochemical and biophysical methods the formation of thermostable and ribonuclease resistant RNA nanorings which were originally designed by us using computational methods. High yields of fully programmable nanorings were produced based on several RNAI/II kissing complex variants selected for their ability to promote polygon self-assembly. This self-assembly strategy relying on the particular geometry of bended kissing complexes has potential for developing multivalent interfering RNA delivery agents. This was verified by assembling the nanoring with 6 siRNAs. These constructs were then shown to be processed by Dicer, an enzyme that is part of the RNAi silencing pathway.Specification of Protocols for the Design and Self-Assembly of siRNA Functionalized RNA Particles for Use in Automated NanomedicineWe specified three assembly protocols to produce two different types of RNA self-assembling functional NPs using processes which are fully automatable. These NPs were engineered based on two of our nano-scaffold designs (nanoring and nanocube), which serve as carriers of multiple siRNAs. The NPs were functionalized by extension of up to 6 scaffold strands with siRNA duplexes. The assembly protocols yielded functionalized RNA NPs that we showed interacted in vitro with human recombinant Dicer to produce siRNAs. Our design strategies showed that we can provide fast, economical and easily controlled production of endotoxin-free therapeutic RNA NPs suitable for preclinical development.Using RNA Structural Flexibility Data in Nanostructure ModelingIn the emerging field of RNA-based nanotechnology there is a need for automation of the structure design process. Our goal is to develop computer methods for aiding in this process. Our RNAJunction data base contains thousands of RNA junctions that can be used as building blocks to construct RNA nanoparticles. Two programs we developed, NanoTiler and RNA2D3D, can combine such building blocks with idealized fragments of A-form helices to produce desired 3D nanostructures. Initially, the building blocks 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.Multistrand RNA Structure Prediction and Nanostructure Design including PseudoknotsOne of the steps required for determining the proper set of RNA strands that will self-assemble into a desired RNA nanostructure is to determine the sequences of these strands. This requires the prediction of the correct intra-strand and inter-strand interactions. We developed a program, NanoFolder, which accomplishes this task. It can include the prediction of pseudoknots, which is this case can be interpreted as inter-strand interactions. We showed that this algorithm, performs better than several other structure prediction methods when applied to RNA complexes with non-nested base-pairs. We also experimentally confirmed the self-assembly of a predictied 4-stranded RNA nanoparticle using this algorithm.Understanding the Effects of Carbocyclic Sugars Constrained to North and South Conformations on RNA NanodesignRelatively new types of modified nucleotides, namely carbocyclic sugars that are constrained to north or south conformations, can be used for RNA nanoparticle design to control their structures and stability by rigidifying nucleotides and altering the helical properties of RNA duplexes. Two RNA structures, an RNA dodecamer and an HIV kissing loop complex where several nucleotides were replaced with north or south constrained sugars, were studied by molecular dynamics (MD) simulations. The substituted south constrained nucleotides in the dodecamer widened the major groove and narrowed and deepened the minor groove thus inducing local conformational changes that resemble a B-form DNA helix. In the HIV kissing loop complex, north and south constrained nucleotides were substituted into flanking bases and stems. The modified HIV kissing loop complex showed a lower RMSD value than the normal kissing loop complex. The overall twist angle was also changed and its standard deviation was reduced. In addition, the modified RNA dodecamer and HIV kissing loop complex were characterized by principal component analysis (PCA) and steered molecular dynamics (SMD). PCA results showed that the constrained sugars stabilized the overall motions. The results of the SMD simulations indicated that as the backbone delta angles were increased by elongation, more force was applied to the modified RNA due to the constrained sugar analogues.Multiscale Modeling of Double-Helical DNA and RNA: A Unification through Lie GroupsThe modeling and characterization of RNA-based nanostructures is a difficult task given the size of such structures. From a practical stand point, all atom molecular dynamics studies of such molecules can obtain trajectories of several nanosecond durations, a limited time scale for a comprehensive characterization of such structures. Coarse-grained models have been developed to study the dynamics of RNA and also DNA structures. The models include different amounts and types of information. Such a treatment will ultimately allow us to study systems consisting of thousands of nucleotides, at time scales of microseconds and thus enable simulations of large RNA or DNA polymers in the context of bionanotechnology. In this research, a method that relies on Lie groups was used to describe motions in a coordinate free way or when necessary, coordinates are introduced in a way in which simplified equations result. What was considered here were double stranded RNA and DNA helices. Multilevel modeling was done. At the coarsest level worm-like chains with anisotropic bending stiffness were considered. It was then shown that bi-rod models converge to this for sufficiently long filament lengths. At yet finer levels elastic networks were considered and it was shown how they related to coarser levels. Finally it was shown how all atom molecular dynamics (fine grain) and AFM experimental results (coarse grain) relate to these models.First International Meeting on RNA NanotechnologyA meeting was held in which I was a co-organizer highlighting the recent advances in RNA nanotechnology as presented at the First International Conference of RNA Nanotechnology and Therapeutics, in Cleveland, OH. The conference was the first of its kind to bring together invited speakers in RNA nanotechnology from France, Sweden, South Korea, China, and throughout the United States to discuss RNA nanotechnology and its applications. It provided a platform for researchers from academia, government, and the pharmaceutical industry to share existing knowledge, vision, technology, and challenges in the field and promoted collaborations among researchers interested in advancing this emerging scientific discipline. The meeting covered a range of topics, including biophysical and single-molecule approaches for characterization of RNA nanostructures;structure studies on RNA nanoparticles by chemical or biochemical approaches, computation, prediction, and modeling of RNA nanoparticle structures;methods for the assembly of RNA nanoparticles;chemistry for RNA synthesis, conjugation, and labeling;and application of RNA nanoparticles in therapeutics.

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