Nanotiler To better facilitate the design of RNA based nanoparticles a software system, NanoTiler, was developed that permits RNA nanodesign at several different conceptual levels. A key feature of NanoTiler is its ability to accomplish combinatorial search of 3D RNA structure spaces by utilizing motifs derived from our RNAJunction database or designed de novo. Once a desired topology is realized, NanoTiler can then focus on producing a set of sequences that can be experimentally tested for the formation of the designed structure. The sequence optimization algorithm is applied to limit the amount of cross talk between the designed sequences. Ideally, each individually designed sequence should fold in an unobstructed fashion into its desired conformation (a target secondary structure representation of the sequence fragment. Sequences are repeatedly mutated, except for the portions that have to be maintained to preserve important motifs such as those obtained from our RNAJunction database, scoring each set of mutated sequences. NanoTiler in conjunction with other programs measures the degree of hybridization that occurs between the sequences and the degree of folding into the target secondary structure. Once an optimized set of sequences is generated, mutations are substituted back into the 3D structure. Once all fragments are designed, they are subjected to molecular mechanics to fix bond lengths and angles. Finally, if desired, the entire structure or portions of the structure are subjected to molecular dynamics to characterize the dynamical qualities of the designed nanostructure. Using the above described methodology RNA and RNA/DNA hybrid nanocubes were designed and experimentally proven to self-assemble. These cubes were composed of 6 short or 10 short oligonucleotides that make them amenable to chemical synthesis, point modifications and further functionalization. The nanocube assemblies were verified by gel assays, dynamic light scattering and cryogenic electron microscopy. In addition, the cubes were functionalized by the incorporation of fluorescent aptamers that would only light up upon proper cube assembly. It was also shown that the cubes can assemble under isothermal conditions at 37 degree centigrade during in vitro transcription which opens a route towards the construction of sensors, programmable packaging and cargo delivery systems. Molecular Dynamics Characterization of the Hexagonal RNA Nanoring The significant progress made over the past several years in understanding RNA structure, has led to research into RNA architectonics that deals with the self-assembly of RNA nanostructures of arbitrary size. My group designed an RNA hexagonal ring and nanotube composed of six A-form helical sides and six kissing loop motifs that approximate a 120 degree angle at each corner thus allowing the formation of hexagons. Formation of this ring has been demonstrated experimentally. An issue is the computational characterization of the nanoring. In conjunction with Roderick Melnik from the Wilfrid Laurier University in Ontario, Canada we have been performing all-atom molecular dynamics studies of the hexagonal ring. Because of its size such calculations take a long time and must be of limited duration. We wanted to determine how the stability of the nanoring was affected by temperature, counter-ions and solvent and how the nanoring is affected by external forces. Results indicate that the ring appears to be stable at 310 degrees K, while at 510 degrees K, as might be expected, the ring seems to be collapsing into a compact globular state on its way to unfolded single fragments. There was reduced hydration of the ring at the higher temperature. There was also a significant loss of hydrogen bond interactions in the ring at 510 degrees K. Phosphates became closer together at the higher temperature. We also found a surprising phenomenon where there was an uptake of ions as a function of increasing temperature. This may be due to the dependence of the water dielectric constant on temperature. We found that an estimate of the tensile elasticity of the nanoring against 2D in-plane compression was lower than a typical soft matter representative such as DNA. Mesoscopic model The modeling and characterization of RNA-based nanostructures is a difficult task given the size of such structures. This is exemplified by my groups previously designed RNA hexagonal nanoring and nanotube. 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. In conjunction with Roderick Melnik's group at the Wilfred Laurier University in Ontario, Canada we have been developing coarse-grained models of RNA that can be used to more easily characterize the large structures that are often found in RNA nanoparticles. A series of models based on 3 beads (each bead represents a group of atoms that contribute to the structural behavior of the system) were developed and simulated using molecular dynamics. The goal is to obtain universal parameters that can represent such large structures as coarse-grained entities that capture the interactions that exist between the beads. Such a coarse-grained treatment has allowed us to obtain microsecond time scales that are three orders of magnitude longer than all-atom molecular dynamics simulations. This methodology is allowing us to study the slowest conformational motions of the RNA. Parameterization of such bead models requires information obtained from experiments as well as from full atomistic molecular dynamics. What adds complexity is that RNA structures contain a wide variety of interactions beyond the commonly found Watson-Crick basing pairs. The nanoring exemplifies this issue because it not only contains A-form Watson-Crick interactions along its edges but also has non-canonical non-A-form interactions in the corners that are composed of kissing loop contacts. Our results indicate that variants of the 3-bead model perform reasonably well and that the inclusion of only the details about the Phosphate-C4 dihedral degrees of freedom is needed for a good representation. Rational Design of RNA Nanostructures In two recently invited published book chapters we laid out the basic principles for the rational design of RNA nanostructures. An understanding of how natural RNA molecules fold and assemble is an essential element. It is assumed that some RNA sequences have the ability to fold autonomously into precise 3-D structures outside of their natural context. These folds are called motifs and are often found in the database of RNA structures. However, not all solved structures are autonomous folding domains. Therefore it is still difficult to determine whether given sequences will fold and assemble as expected out of their natural context. Thus, several questions must be considered when designing RNA-based nanostructures. These include: 1) Is a given structure a motif? 2) Is the motif recurrent within multiple structures? 3) Is the motif able to form outside its natural context? 4) What is the stability of the motif outside its natural context? 5) what is the stability of the motif when associated with other motifs or helical connectors? 6) What is the relative flexibility of a motif within the desired framework? 7) How do environmental factors such as temperature and ion concentration affect stability? Approaches are discussed in the two chapters which attempt to answer some of these questions and layout an integrated approach of experimental and computational RNA nanodesign.
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