3602 Nucleic Acids Research, 2008, Vol. 36, No. 11 Electrophoretic mobility shift assays (EMSA) For the preparation of uniformly labeled RNAs, in vitro transcription was carried out in the presence of [alpha-32P]-GTP. Synthetic single-stranded oligodeoxynu- cleotide templates annealed with a T7 promoter oligonu- cleotide were directly used for transcription. After RQ1 DNase treatment, phenol/chloroform extraction and ethanol precipitation, the transcripts were puriﬁed by denaturing PAGE, recovered by crush-and-soak method and precipitated with ethanol. The trace amount (0.2–0.4 nM) of the labeled RNAs and the indicated concentrations of unlabeled RNAs were folded separately. Initially, these RNAs were heated in water at 908C for 2 min, immediately cooled on ice, followed by folding for 10 min at 378C in the buﬀer employed for the in vitro selection. These separately folded, labeled and unlabeled RNAs were mixed and incubated for >30 min to form dimers. After the incubation, 1 ml of 50% glycerol was added to the 10 ml of the RNA samples, and these samples were run at 48C on 10% polyacrylamide gels containing 50 mM Tris–borate (pH 8.2) and 20 mM MgCl2. The RNA bands were quantiﬁed by FLA-5100 (Fujiﬁlm Life Science, Japan). Kd values were determined as the concentration at which half the RNA molecules form dimers. Lead(II)-induced cleavage In order to label the 50-end of RNA, unlabeled transcript was treated with alkaline phosphatase from Calf intestine (Takara-Bio) to dephosphorylate the 50-end, followed by phosphorylation with [gamma-32P]-ATP using T4 poly- nucleotide kinase (Takara-Bio). The labeled RNA was puriﬁed by denaturing PAGE as described above. The RNA folding and dimerization were carried out as described above in the presence of 50 mM HEPES-OAc (pH 7.5), 100 mM NaOAc and 20 mM Mg(OAc)2 instead of the selection buﬀer. It was conﬁrmed that the clone #05 ribozyme is also active under this buﬀer condition (data not shown). Lead(II)-induced cleavage analysis was carried out as described (16). RESULTS In order to isolate RNA receptor motifs for the construc- tion of molecular tools to detect and control structural dynamics of given RNAs, we have developed a selection system based on the DSL ribozyme. Although canonical SELEX (systematic evolution of ligands by exponential enrichment) is another method of choice (17), the motifs obtained by SELEX are not easily engineered within diﬀerent structural contexts without detailed biochemical and structural analyses. In contrast, due to the well-deﬁned architecture of the DSL ribozyme, the relative distance and orientation between the target structure and the isolated motif can be predicted without detailed experimental analyses, and thus, new receptor motifs isolated by this system may be directly employed in diﬀerent structural contexts. Figure 1. Secondary structures of the parental DSL-U5 ribozyme and its derived libraries. (A) The DSL-U5 ribozyme with the GAAA- tetraloop/11-ntR pair essential for the ribozyme activity highlighted in gray. (B) The GAAA-loop library with the target GAAA-tetraloop and the randomized nucleotides highlighted in gray. (C) The C-loop library with the target C-loop motif (C-50) with neighboring single base pairs and randomized nucleotides highlighted in gray. Selection of receptor motifs against GAAA tetraloop Previously, a mutational analysis of the DSL ribozyme showed that the THS motif is less important for ribozyme activity than the GAAA-loop/11-ntR pair. A miniaturized variant lacking the THS motif (DSL-U5; Figure 1A) is as active as the parental DSL ribozyme (14,15). We employed the DSL-U5 variant as a platform for our ribozyme-based selection system. We ﬁrst carried out the selection of motifs targeting the GAAA-loop (Figure 2). Since there is at least one deﬁned positive control sequence for this target, namely the 11-ntR motif, the GAAA loop is an ideal target to test our experimental design. Fifteen nucleotides constituting the 11-ntR motif as well as a capping stem– loop structure of the DSL-U5 ribozyme were replaced with 19 nt of random sequence (Figure 1B). Although two C-G base pairs adjacent to the catalytic unit are considered to be a part of the 11-ntR motif, they function to stabilize the catalytic unit structure, and thus these two base pairs were excluded from randomization. After six rounds of the selection/ampliﬁcation cycle based on ligation activity, the resulting RNA pool showed activity comparable to the parental DSL-U5 ribozyme (data not shown). Hence, we determined the sequences of clones randomly picked from the pool after round 6 of the selection (Figure 3). Out of 28 clones sequenced, 13 clones were highly similar (or even identical) to the 11-ntR motif. Fourteen additional clones were either identical, or highly similar to the previously reported C7.2 motif, which is an artiﬁcially selected receptor for the GAAA-loop (18). Together, these results demonstrate the proof-of-concept of this in vitro selection technology to generate receptor motifs against a given target RNA structure.