3600–3607 Nucleic Acids Research, 2008, Vol. 36, No. 11 doi:10.1093/nar/gkn206 Published online 6 May 2008 Selection of a novel class of RNA–RNA interaction motifs based on the ligase ribozyme with defined modular architecture Shoji P. Ohuchi1, Yoshiya Ikawa2,3 and Yoshikazu Nakamura1,4,* 1Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, 2Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, 3PRESTO, Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Saitama 332-0012 and 4CREST, Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama 332-0012, Japan Received December 19, 2007; Revised March 5, 2008; Accepted April 7, 2008 ABSTRACT To develop molecular tools for the detection and control of RNA molecules whose functions rely on their 3D structures, we have devised a selection system to isolate novel RNA motifs that interact with a target RNA structure within a given structural context. In this system, a GAAA tetraloop and its specific receptor motif (11-ntR) from an artificial RNA ligase ribozyme with modular architecture (the DSL ribozyme) were replaced with a target structure and random sequence, respectively. Motifs recognizing the target structure can be identified by in vitro selection based on ribozyme activity. A model selection targeting GAAA-loop successfully identi- fied motifs previously known as GAAA-loop recep- tors. In addition, a new selection targeting a C-loop motif also generated novel motifs that interact with this structure. Biochemical analysis of one of the C-loop receptor motifs revealed that it could also function as an independent structural unit. INTRODUCTION Single-stranded RNAs can form deﬁned 3D structures based not only on Watson–Crick base-pairing interac- tions but also on speciﬁc, non-Watson–Crick tertiary interactions, which include additional hydrogen-bonds, hydrophobic interactions, electrostatic (metal-bridged) interactions and base-stacking. In nature, many RNA molecules and motifs exhibit speciﬁc functions that require the formation of a speciﬁc 3D structure, rather than simply a linear carrier of genetic code information. The most classical examples of such structural, nonprotein-coding RNAs (ncRNAs) are tRNA and rRNA, which play key roles in the central dogma of molecular biology (1). In addition, other structural ncRNAs are also known to play essential roles in the central dogma (1), since several regulatory elements on mRNA, like riboswitches and internal ribosome entry sites (IRESs), also function via their speciﬁc 3D structure (2). More recently, several structural ncRNAs have been discovered as speciﬁc modulators for intracellular proteins, and a signiﬁcant number of structural RNAs likely exist within the huge numbers of ncRNAs already found in higher eukaryote genomes (3). Thus, the development of new methods to detect and control structural dynamics of these RNAs is attractive areas of investigation (4–7). One elegant example of a molecular sensor for RNA dynamics is the P5abc RNA, which is a structural element of the Tetrahymena Group I intron ribozyme that acts to physiologically stabilize the core structure of the ribozyme to enhance its activity (8). Since the P5abc acts as an independently folded domain, the isolated P5abc RNA can also activate a truncated ribozyme lacking this region ( P5abc ribozyme) in trans. The P5abc has three small structural motifs (A-rich bulge, L5b and L5c loops), which independently interact with the ribozyme. These motifs are strictly located at deﬁned distances and orientations, with such a modular architecture enabling the highly speciﬁc and extraordinary strong recognition between P5abc RNA and P5abc ribozyme (9–11). Recently Johnson et al. have reported a new aspect of P5abc function whereby the P5abc RNA acts as a molecular sensor to discriminate between the native and misfolded conformations of the P5abc ribozyme (12). Therefore, RNA molecules that speciﬁcally recognized and bound a certain conformation of a target structural RNA could be designed in a modular manner. This approach, however, is far from practical since RNA *To whom correspondence should be addressed. Tel: +81 3 5449 5307; Fax: +81 3 5449 5415; Email: nak@ims.u-tokyo.ac.jp 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.