High-Throughput Explorations of RNA Structural Modularity

A levels of RNA structure influence the diverse biological functions of these biomolecules. The primary sequence of an RNA folds into a base-paired secondary structure, which may then form a complex intramolecular tertiary structure, which in turn may interact with an RNA, protein, or smallmolecule ligand partner. Much is known about the folding and unfolding energetics of RNA secondary structure, but our understanding of the thermodynamic stability and folding of RNA tertiary structure remains incomplete. That our understanding of RNA tertiary folding remains so incomplete is perplexing. The principles that underlie RNA folding ought to be simple. There are only four primary RNA nucleotide building blocks, and there are relatively few basic kinds of secondary structures (helices, loops, and junctions). Moreover, the number of ways that RNAs interact to form complex higher-order structures, as visualized to date in highresolution structures, is also fairly limited. Pioneering biochemical and biophysical studies investigating RNA tertiary structure and folding have established numerous principles regarding energetics and folding. These foundational studies have generally focused on one or several variations in sequence, topology, or ionic conditions within a model higher-order RNA structure such as tRNA, ribozymes, other noncoding RNAs, or the small and large ribosomal subunits. Stochastic and molecular dynamics simulations of model RNAs have further defined how topological constraints influence tertiary structure folding and dynamics. A major insight from all of these studies is that RNA structure is modular, meaning that one local structure can be readily substituted with another. For example, a helix can be substituted with another of a different sequence as long as the substituted element is roughly the same “size”. In addition, different kinds of through-space tertiary interactions can often be substituted for one another as long as steric and connectivity features are compatible with the overall RNA fold. Indeed, this substitutability is the foundation for the phylogenetic studies that led to the first, largely accurate, models of the ribosomal RNAs. But how modular is RNA? How many different structural assemblies can be formed by a given class of RNA? How is the number of distinct local sequence combinations (which scales as N and can rapidly become very large) related to the total number of distinct structural conformations? To what extent can diverse RNA sequences and topologies accomplish similar tertiary folds? Recently, Denny et al. began to address these fundamental questions and to develop a predictive model for the modularity of RNA structure by creating a high-throughput biophysical screen to examine the folding energetics of RNA two-way junctions (Figure 1, box). Two-way junctions, also called bulges or internal loops, are key contributors to RNA dynamic behavior and comprise the simplest level of structure more complex than a helix. The approach taken by Denny et al. uses a cleverly repurposed high-throughput sequencing instrument to measure the conformational effects of diverse RNA junction variations on the thermodynamic binding equilibria between two structured RNAs. The authors made use of a known pseudosymmetric tertiary assembly comprised of two RNA tetraloops and their two tetraloop receptors (Figure 1, blue). To form, the two sets of tertiary interactions must be connected by an intervening, mostly helical, linker of the right geometry. To test the effect of RNA two-way junctions on tertiary structure formation, the authors inserted various junction sequences into one half of the tetraloop−receptor motif (Figure 1, orange), which was immobilized on a surface. A second tetraloop−receptor element was free in solution and modified with a fluorophore to enable measurement of the extent of assembly formation. Three RNA helix lengths were used on the “flow” side of the tertiary structure assembly and, notably, ∼1700 RNA junctions were evaluated on the “chip”