In Vitro Assembly of Cubic RNA-based Scaffolds Designed in Silico The organization of biological materials into versatile three dimensional assemblies could be used to build multifunctional therapeutic scaffolds for use in nanomedicine. We reported a strategy to computationally design and experimentally verify the formation of three-dimensional cubic nanoscale scaffolds that can self-assemble from RNA (also DNA and RNA/DNA hybrids) with precise control over their shape, size and composition. These cubic nanoscaffolds are only 13 nm in diameter and are composed of short oligonucleotides, making them amenable to chemical synthesis, point modifications and further functionalization. Nanocube assembly was verified by gel assays, dynamic light scattering and cryogenic electron microscopy. Formation of functional RNA nanocubes was also demonstrated by the use of a fluorescent RNA aptamer that was optimally active only upon full RNA assembly. We showed that the RNA nanoscaffolds can self-assemble in isothermal conditions (37 degrees C) during in vitro transcription, opening a route towards the construction of sensors, programmable packaging and cargo delivery systems for biomedical applications. Self-Assembling RNA Nanorings Based on RNA I/II Inverse Kissing Complexes with Associated Diceable siRNAs We 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 Nanomedicine We 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 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 Modeling In 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. Understanding the Effects of Carbocyclic Sugars Constrained to North and South Conformations on RNA Nanodesign Relatively 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. Coarse Graining of RNA Nanostructures for Molecular Dynamics Simulations The modeling and characterization of RNA-based nanostructures is a difficult task given the size of such structures. At best, all atom molecular dynamics studies of such molecules can obtain trajectories of a few nanoseconds duration, a limited time scale for a comprehensive characterization of such structures. A series of coarse-grained models have been developed for study of the molecular dynamics of RNA nanostructures. The models in the series have one to three beads per nucleotide and include different amounts of detailed structural information. Such a treatment allows us to reach, for systems of thousands of nucleotides, a time scale of microseconds and thus enable simulations of large RNA polymers in the context of bionanotechnology. We found that the three-beads-per-nucleotide models, described by a set of just a few universal parameters, were able to describe different RNA conformations and were comparable in structural precision to the models where detailed values of the backbone P-C4'dihedrals taken from a reference structure were included. First International Meeting on RNA Nanotechnology A 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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