3606 Nucleic Acids Research, 2008, Vol. 36, No. 11 between these two sequences, the #05 receptor is likely to recognize the C-loop motif by speciﬁc, non-Watson–Crick tertiary interactions. However, it cannot be excluded at present that the receptor binding may cause the structural change of the C-loop motif. Therefore, structural and physicochemical studies of the RNA–RNA interaction are necessary to determine whether the receptor motif recog- nizes the native structure of the C-loop motif or not. DISCUSSION In this study, we have developed a selection system that enables the identiﬁcation of novel RNA motifs that interact with a target RNA structure within a desired structural context. After successful selection against the GAAA tetraloop, used as a proof-of-concept model, the work aimed to generate novel receptor motifs against the C-loop motif. Although the C-loop is not considered as an RNA–RNA interaction motif, we successfully identiﬁed a novel class of receptor motifs against it. In the model selection experiment against the GAAA- loop, we isolated two previously known GAAA-loop receptors, a naturally occurring 11-ntR and an artiﬁcial C7.2 motif generated previously by Costa and Michel, via in vitro selection based on the activity of T4 td Group I intron ribozyme (18). Interestingly, their selection also generated a third class of GAAA-loop receptors (C7.34 motif), which was not obtained in the present work [Note that clone #27 has CCC/GGG base pairings similar to the C7.34 motif but is expected to form the secondary structure diﬀerent from the consensus structure of the C7.34 motif family (18,23,24)]. Possible explanations for not having obtained a C7.34-like motif include diﬀerences in experimental conditions, library design (i.e. the length and the position of the random sequences introduced), and most importantly, the diﬀerence of the parental ribozymes as platforms for the two selection systems. The two ribozymes may have distinct structural contexts to the common target motif (GAAA-loop). For example, if steric hindrance around the GAAA-loop diﬀers between the two ribozymes, it may provide selective pressure since one receptor motif may be bulky, requiring much more void space around the GAAA-loop while the other receptor motif may be very compact. The ability of the ribozyme to maintain activity despite diﬀerences in orientation and/or physical aﬃnity between the GAAA-loop and its receptor might also be possible factor(s). These factors may result in diﬀerent selective pressures, under which diﬀerent motifs can be adapted and isolated even though they are independent structural units. Thus, parallel and/or sequential selections based on diﬀerent contexts (for example, refs. 18,25–28) may be required to produce motifs truly independent from the structural context. Importantly, the experiment aiming to generate novel receptor motifs against the C-loop motif, which is not considered as an RNA–RNA interaction motif, also identiﬁed novel receptor motifs. The most abundant sequence (#05) was further investigated to determine if it can function within a diﬀerent structural context. By grafting the target/receptor motif pair into a previously reported RNA architecture (TectoRNA), structural and functional independency of the new receptor motif was clearly demonstrated. Therefore, we believe that RNA motifs isolated via this selection system can be directly employed for RNA engineering, such as the design of artiﬁcial RNA architectures (29) or novel molecular tools for desired target RNAs including structured ncRNAs, regulatory mRNA elements, as well as RNA components of large, complicated ribonucleoprotein complexes like the ribosome and the spliceosome. After submission of this manuscript, we became aware of a recent study that warrants mention. Geary et al. (30) have reported the isolation of artiﬁcial receptor motifs against the GGAA-tetraloop by the selection method based on TectoRNA dimerization. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank C.G. Crist for critical reading of the manuscript. This work was supported in part by grants from The Ministry of Education, Sports, Culture, Science and Tech- nology of Japan (MEXT), Precursory Research for Embry- onic Science and Technology (PRESTO) grant (Y.I.) and Core Research for Evolution Science and Technology (CREST) grant (Y.N.) from the Japan Science and Tech- nology Agency. Funding to pay the Open Access publica- tion charges for this article was provided by the CREST Japan Science and Technology Agency. Conﬂict of interest statement. None declared. REFERENCES 1. Gesteland,R.F., Cech,T.R. and Atkins, J.F. (eds) (2006) The RNA World, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Batey,R.T. (2006) Structures of regulatory elements in mRNAs. Curr. Opin. Struct. Biol., 16, 299–306. 3. Mattick,J.S. and Makunin,I.V. (2006) Non-coding RNA. Hum. Mol. Genet., 15, R17–R29. 4. Sosnick,T.R. and Pan,T. (2003) RNA folding: models and perspectives. Curr. Opin. Struct. Biol., 13, 309–316. 5. 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