Design, Construction, and Analysis of a Novel Class of Self-Folding RNA

Abstract

RNA can play multiple biological roles through use of its three-dimensional (3-D) structures. Recent advances in RNA structural biology have revealed that complex RNA 3D structures are assemblages of double-stranded helices with a variety of tertiary structural motifs. By employing RNA tertiary structural motifs together with the helices, we designed a novel class of self-folding RNA. In RNA composed of three helices (P1, P2, and P3), P1 interacts with P3 via a tetraloop-receptor interaction and P2 forms consecutive base-triples. Two designed RNAs of this class were prepared and their folding properties indicate that they form defined tertiary structures as designed. These RNAs may be used as modular units for constructing artificial ribozymes or nanometer-scale materials.

Generation of a catalytic module on a self-folding RNA

“…Three-dimensional structures of RNA structural units in such naturally occurring RNAs as the Tetrahymena ribozyme and RNA selection technique have enabled the construction of artificial RNAs with defined three-dimensional (3D) structures (Jaeger et al 2001;Ikawa et al 2002) and the acquisition of new functional RNA units (Williams et al 1994;Ohuchi et al 2002), respectively. The self-folding P4-P6 domain of the Tetrahymena ribozyme, which consists of 160 nucleotides, is nonessential for catalytic activity (Ikawa et al 2001, and references therein).…”

De novo synthesis and development of an RNA enzyme

“…2B) (7,13,(19)(20)(21). One or two mutations at the loop-receptor interaction and͞or consecutive base-triples significantly lowered or abolished the activity, respectively, indicating that the loss of the interactions destabilized the folded 3D structure (Fig.…”

“…The yield is higher than those of previously reported ligase ribozymes (65-70%; refs. 16 and 17) and comparable to that of a self-splicing intron RNA (18), indicating that the designed scaffold allows little misfolding (13). Presumably, a catalytic module that least interferes with the folding process was selected.…”

“…As one such example, we have previously reported the design of a self-folding RNA consisting of standard doublestranded helices connected by the two motifs: a tetraloopreceptor interaction and consecutive base-triples ( Fig. 1 A and B) (13). Results indicated that the constructed RNA folds compactly into the designed 3D structure.…”

“…Moreover, several commonly used RNA-RNA binding motifs in these RNAs were identified by phylogenetic comparison (6) and high-resolution structural analyses (7)(8)(9)(10). Consequently, it has become possible to design self-folding RNAs precisely by employing such motifs and mimicking the modular organization of natural RNAs (11)(12)(13). As one such example, we have previously reported the design of a self-folding RNA consisting of standard doublestranded helices connected by the two motifs: a tetraloopreceptor interaction and consecutive base-triples ( Fig.…”