3604 Nucleic Acids Research, 2008, Vol. 36, No. 11 Figure 3. Clones obtained after the sixth round of selection from the GAAA-loop library. Consensus primary sequences and base pairings are highlighted in pink and purple, respectively. Point mutations at nonrandomized positions are indicated in red. between Jaeger’s and ours that is based on the condition employed in the in vitro selection. First, we grafted the C-loop motif and the #05 recep- tor into the corresponding positions of TectoRNA (Figure 5A). EMSA of this construct showed an apparent reduction in mobility in a concentration-dependent manner (Figure 5C). The mobility of low and high concentrations of the construct was close to those of the original TectoRNA, and the degree of the mobility change is consistent with a biphasic dimerization model with typical fast exchange kinetics (R2 = 0.956–0.998), suggesting that the construct dimerized, as is the case for the original TectoRNA (22). Its Kd value, determined as the kinetic equilibrium, was 168 nM (Figure 5C). In the library design, the free end of the stem neighboring the C-loop motif is capped with a UUCG tetraloop. To exclude the possibility that the selected motif might recognize this cap structure rather than the C-loop, Table 2. Clones obtained from the round 7 pool of C-loop library Clone Sequencea kobs [h 1]c FE [%]b Library NNNNNNNNNNNNNNNNNNN DSL-U5 TAAGGCAAACGCTAT #05 (x9)c GTGGCGATGCGAGCAGCAA #09 GTGGCGATGCGAGCAGCAG #10 (x3)c GTGGCGATTAGAGTAGCAA #03 GTGGCGATTAGAGTAGGAA #11 GTGGCGAGCAAGTTAGTAA ND 0.34 0.05 ND 0.02 0.06 0.04 ND 0.02 27 5 0.01 13 2 ND 0.00 18 3 0.01 10 3 0.00 15 2 aSequences originating from the randomized region are shown. None of the clones has point mutations within the nonrandomized regions. Consensus sequences are underlined. bThe reaction was carried out in the presence of 2 mM of the substrate RNA (S-1) under the same conditions as the in vitro selection. The kobs and FE values are calculated based on data ﬁtting with a single exponential curve. FE, ﬁnal extent of the reaction; ND, not detectable. cNumber of clones sharing the same sequence. Figure 4. Ligation reaction of the RNA pool from the C-loop library. Uniformly [32P]-labeled RNAs from round 0 or round 7 pool were incubated with or without substrate RNA (2 mM of S-1) in the buﬀer employed for the in vitro selection at 378C for 1 h. The reactions were terminated by adding sample-loading buﬀer (14), and these samples were separated on denaturing-PAGE. The ligated product is indicated with an arrowhead. we redesigned the TectoRNA construct to remove the UUCG-loop (Figure 5B). This new construct also dimer- ized with aﬃnity comparable to the construct with the UUCG-loop (Kd value of 264 nM as the kinetic equi- librium) (Figure 5D). As control experiments, substitution of the C-loop with 5 bp or the #05 receptor with 11-ntR was shown to abolish the self-dimerization ability (see Figure S1, Supplementary Data). These results indicate that the selected #05 receptor mainly recognizes the target C-loop motif and can be placed within a diﬀerent structural context as intended. In order to further analyze the interaction between the C-loop and #05 receptor motifs, we carried out chemical footprinting by using lead(II)-induced RNA cleavage (Figure 6). In the presence of suﬃcient concentration of other divalent cations, lead ions (Pb2+) induce the cleavage of the phosphate backbone of RNAs at nonbase pairing, solvent accessible sites (16). Phosphates around the C-loop motif were cleaved under monomeric conditions but protected under dimeric conditions (in the presence of a concentration of unlabeled RNA well above the Kd value), indicating that these phosphates, originally located at the