Defining the syntax for self-assembling RNA tertiary architectures

Abstract

Stable RNAs are modular and hierarchical three-dimensional (3D) architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions (1, 2). Using comparative sequence and structural analysis of known X-ray structures of RNAs, folding and assembly principles of RNA can presently be gathered to generate the syntax of a proto-language for rational design and prediction of RNA 3D shapes. RNA architectonics refers to the deciphering of this proto-language and to its use to build new functional RNA shapes with self-assembly properties (3-5). This approach can therefore contribute to the prediction and rational design of RNA tertiary structures for potential applications in nanotechnology, synthetic biology and medicine.

Introduction

The hierarchical and modular nature of RNA is encoded within its sequence. The recent identification of several versatile and small recurrent motifs suggests that a rather limited set of basic submotifs can account for the formation of most structural motifs uncovered in ribosomal and stable RNAs (6, 7). For instance, the “sequence network” that results from the sequence and structural relationships existing between most tertiary motifs identified to date (6-8) highlights the syntax of emerging folding and assembly rules that direct the stacking, orientation and positioning of RNA helices with respect to one another (7). Structural motifs can also act as structural scaffoldings for further structural expansion and can be functionally and topologically equivalent despite sequence and structural differences (7, 9). Our present motif structure network led to the refined prediction of the architecture for several natural RNA molecules including riboswitches (7) and group I introns (10). As exemplified below, it is also useful for rational design of self-assembling RNA architectures.

Results and Discussion

Beside theoretical understanding of the sequence and conformational space associated to RNA motifs (e.g (7, 11)), the in vitro biochemical and biophysical characterizations of RNA motifs has significantly increased their number of RNA motifs able to be used for nano-construction (12-14). For instance, we have gained new understanding of the rules governing the selectivity and thermodynamic of GNRA/receptor interactions (14). Recently, these tertiary interactions have been used to stack and stabilize contiguous helical elements of RNA to form 1D RNA filaments (15-17). By gaining new insight about the stability of T-loops, another class of RNA structural motifs that is involved in long-range interaction in RNA molecules (7, 11), new self-assembling RNA self-assembling units were designed to form “square-shaped” RNA particles (18). Additionally, RNA architectonics led to the reliable prediction and design of the 3D structure of several artificial RNA building blocks able to form various programmable 2D jigsaw puzzles (18, 19) and 3D nanoparticles (18) at the nano-scale level. More recently, as a proof of concept, we have demonstrated that structurally complex RNA structures based on a syntax involving a repertoire of several different RNA motifs can self-assemble into complex supra-molecular nano-particles (Geary, Chworos, Jaeger, in preparation).

These studies provide insights into self-assembly processes involving large populations of RNA molecules and demonstrate that small structural motifs can potentially code for the precise topology of an almost infinite variety of large molecular architectures. Ultimately, it is anticipated that RNA particles with the structural complexity of the ribosome could be generated through RNA architectonics. Our studies also offer the possibility to envision new materials that take advantage of RNA and other nucleic acid polymers to control the positioning and behavior of other molecular components associated to them (e.g: (3, 20-23)). Ultimately, the RNA architectonics approach will lead to the development of programmable materials based on nucleic acids with potential applications in nanotechnology, synthetic biology and medicine (18, 24, 25).