Cody Geary

Artificial DNA and RNA structures have been used as scaffolds for a variety of nanoscale devices. In comparison to DNA structures, RNA structures have been limited in size, but they also have advantages: RNA can fold during transcription and thus can be genetically encoded and expressed in cells. We introduce an architecture for designing artificial RNA structures that fold from a single strand, in which arrays of antiparallel RNA helices are precisely organized by RNA tertiary motifs and a new type of crossover pattern. We constructed RNA tiles that assemble into hexagonal lattices and demonstrated that lattices can be made by annealing and/or cotranscriptional folding. Tiles can be scaled up to 660 nucleotides in length, reaching a size comparable to that of large natural ribozymes.

The structural information encoding specific conformations of natural RNAs can be implemented within artificial RNA sequences to control both three-dimensional (3D) shape and self-assembling interfaces for nanotechnology and synthetic biology applications. We have identified three natural RNA motifs known to direct helical topology into approximately 90 degrees bends: a five-way tRNA junction, a three-way junction, and a two-helix bend. These three motifs, embedded within rationally designed RNAs (tectoRNA), were chosen for generating square-shaped tetrameric RNA nanoparticles. The ability of each motif to direct the formation of supramolecular assemblies was compared by both native gel assays and atomic force microscopy. While there are multiple structural solutions for building square-shaped RNA particles, differences in the thermodynamics and molecular dynamics of the 90 degrees motif can lead to different biophysical behaviors for the resulting supramolecular complexes. We demonstrate via structural assembly programming how the different 90 degrees motifs can preferentially direct the formation of either 2D or 3D assemblies.

RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with nanoscale precision. The design of genetically expressible RNA origami, which must fold cotranscriptionally, requires modelling and design tools that simultaneously consider thermodynamics, the folding pathway, sequence constraints and pseudoknot optimization. Here, we describe RNA Origami Automated Design software (ROAD), which builds origami models from a library of structural modules, identifies potential folding barriers and designs optimized sequences. Using ROAD, we extend the scale and functional diversity of RNA scaffolds, creating 32 designs of up to 2,360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparisons of optimized and non-optimized structures validate that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify the construction of custom RNA scaffolds for nanomedicine and synthetic biology. RNA origami can be used for the modular design of RNA nanoscaffolds but can be challenging to design. Newly developed computer-aided design software has now been shown to improve the folding yield of kilobase-sized RNA origami. These structures fold from a single strand during transcription by an RNA polymerase, and are able to position small molecules and protein components with nanoscale precision.

Specific recognitions of GNRA tetraloops by small helical receptors are among the most widespread long-range packing interactions in large ribozymes. However, in contrast to GYRA and GAAA tetraloops, very few GNRA/receptor interactions have yet been identified to involve GGAA tetraloops in nature. A novel in vitro selection scheme based on a rigid self-assembling tectoRNA scaffold designed for isolation of intermolecular interactions with A-minor motifs has yielded new GGAA tetraloop-binding receptors with affinity in the nanomolar range. One of the selected receptors is a novel 12 nt RNA motif, (CCUGUG ... AUCUGG), that recognizes GGAA tetraloop hairpin with a remarkable specificity and affinity. Its physical and chemical characteristics are comparable to those of the well-studied '11nt' GAAA tetraloop receptor motif. A second less specific motif (CCCAGCCC ... GAUAGGG) binds GGRA tetraloops and appears to be related to group IC3 tetraloop receptors. Mutational, thermodynamic and comparative structural analysis suggests that natural and in vitro selected GNRA receptors can essentially be grouped in two major classes of GNRA binders. New insights about the evolution, recognition and structural modularity of GNRA and A-minor RNA-RNA interactions are proposed.