Programmable formation of catalytic RNA triangles and squares by assembling modular RNA enzymes

Abstract

RNA is a biopolymer that is attractive for constructing nano-scale objects with complex structures. Three-dimensional (3D) structures of naturally occurring RNAs often have modular architectures. The 3D structure of a group I (GI) ribozyme from Tetrahymena has a typical modular architecture, which can be separated into two structural modules (ΔP5 and P5abc). The fully active ribozyme can be reconstructed by assembling the two separately prepared modules through highly specific and strong assembly between ΔP5 ribozyme and P5abc RNA. Such non-covalent assembly of the two modules allows the design of polygonal RNA nano-structures. Through rational redesign of the parent GI ribozyme, we constructed variant GI ribozymes as unit RNAs for polygonal-shaped (closed) oligomers with catalytic activity. Programmed trimerization and tetramerization of the unit RNAs afforded catalytically active nano-sized RNA triangles and squares, the structures of which were directly observed by atomic force microscopy (AFM).

Artificial RNA Motifs Expand the Programmable Assembly between RNA Modules of a Bimolecular Ribozyme Leading to Application to RNA Nanostructure Design

“…To design artificial RNA nanostructures with new P5b/P6 and P5c/P2 interacting pairs through modular engineering of the group I ribozyme, we chose the GAAC-R(GAAC) pair and the M5 pair as new modular parts for a closed RNA tetramer with square shape. Monomer units of this square tetramer are engineered variants of the group I ribozyme ( Figure 4 A) that we described previously [ 17 ]. In the monomer units, the P5a region of the P5abc module and the P8 region of the ΔP5 module were covalently bonded with a duplex serving as a rigid spacer ( Figure 1 C).…”

“…Electrophoretic mobility shift assays (EMSA) for bimolecular P5abc/ΔP5 complexes were performed according to the procedure reported by Tanaka et al [ 20 ]. EMSAs were carried out to analyze the assembly of Lα RNA, Lβ RNA, and F RNA according to the procedure reported by Oi et al [ 17 ].…”

“…These properties suggest that artificial RNA nanostructures designed by RNA origami (a methodology analogous to DNA origami) could be constructed in vivo [ 16 ]. To expand the methodologies of RNA nanostructure construction beyond RNA origami, we have recently reported the engineering of naturally occurring RNA structures [ 17 ]. This methodology allows us to install the biological functions of naturally occurring ribozymes and riboswitches into assembled RNA nanostructures, in which the biological functions can be expressed depending on the assembly of unit-RNA molecules.…”

“…Through a rational strategy, we connected P5abc RNA and ΔP5 RNA to design a unit RNA that can exhibit self-oligomerization. In the unit RNA, the two modules were joined by a connector element to allow the resulting unit to form closed oligomers with polygonal shapes, such as triangular trimers and square tetramers [ 17 ]. Self-oligomerization of the unit RNA yielded a mixture of assembled structures containing both triangular trimers and square tetramers.…”

“…Self-oligomerization of the unit RNA yielded a mixture of assembled structures containing both triangular trimers and square tetramers. The selective formation of the desired structure was achieved by designing orthogonal interfaces between P5abc and ΔP5 modules [ 17 , 20 ]. In the orthogonal interfaces, we employed a non-natural RNA–RNA interacting motif, i.e., GGAA/R(GGAA), which belongs to the same RNA motif family (GNRA/receptor motif family) as the natural counterpart GAAA/R(GAAA) [ 21 , 22 ].…”