Nucleic Acids Research, 2008, Vol. 36, No. 11 3603 Figure 2. Schematic diagram of the in vitro selection. The RNA pool is incubated with the substrate RNA with 50 biotin moiety (a gray line with encircled ‘Bi’). Only the active variants with a desired RNA–RNA interaction (the right variant in the RNA pool) can react and be covalently ligated with the substrate. Thus, the active variants can be selectively recovered by aﬃnity puriﬁcation using streptavidin beads (‘SA’). After the reverse transcription, cDNAs encoding the active variants are selectively ampliﬁed by PCR using the selective primer with sequence identical to the substrate, followed by PCR using the nested primer with T7 promoter sequence (‘T7 prom’) to regenerate the enriched DNA pool for the next round of the selection. Selection of receptor motifs against the C-loop motif Next, we attempted to generate novel motifs recognizing an RNA structure whose natural receptor is completely unknown. We chose the C-loop motif from 23S rRNA of Haloarcula marismortui (C-50) as the ﬁrst target (Figure 1C). The C-loop motif is a class of asymmetric internal loops that locally increases the helical twist between neighboring stems, and typically, they do not directly interact with other RNA structures (19). To construct a library to isolate C-loop receptor motifs, the GAAA tetraloop of the GAAA-loop library was replaced with a C-loop motif with a neighboring single base pair, and the free end of the motif was capped with a UUCG tetraloop (Figure 1C). After seven rounds of selection, the ligation activity of the RNA pool was clearly detectable, suggesting restoration of ribozyme activity by binding to the new C-loop motif (Figure 4). Clones were randomly picked from the pool and their sequences determined (Table 2). Interestingly, 15 clones shared an apparent consensus sequence (underlined nucleotides in Table 2) and can be classiﬁed to three subgroups: Group 1, the major variant, consists of clone #05 (nine isolates) and its derivative clone #09; Group 2, the next frequent variant, consists of clone #10 (three clones) and its derivative clone #03; and Group 3 is clone #11. Next, we analyzed the ligation activity of these clones. Under the selection conditions we employed, all clones, except for clone #09, showed eﬃcient ligation activity (Table 2). Although the activities were one order of magnitude lower than the parental DSL-U5 ribozyme, their reaction rates are still 104-fold higher than the reported ligation eﬃciency of the nonenzymatic, template- dependent reaction under similar conditions (20). Analyses of a novel C-loop receptor motif In order to see whether the newly isolated motifs can act as independent structural units as intended, we examined the ability to place the most abundant sequence (hereafter #05 receptor) into a diﬀerent structural context. We trans- planted the #05 receptor into TectoRNA, an artiﬁcial RNA architecture developed by Jaeger and colleagues (21,22), since its self-dimerization properties are suitable to examine the modularity of the selected motif. The original TectoRNA (construct 1 of ref. 22), in which the GAAA- loop/11-ntR pair was connected by a linker helix of a suitable length, forms a homodimer in a concentration- dependent manner. The dissociation constant (Kd) for the dimerization can be determined by the titration of unlabeled RNA in the presence of trace amount of labeled RNA on EMSA. Under our assay conditions, the Kd value of the original TectoRNA with GAAA-loop/11-ntR pair was 34.5 3.4 nM (data not shown). The Kd value is one order of magnitude higher than that in the original report (22), probably due to the diﬀerences of EMSA conditions