motifs recognizing local RNA structures are very limited. Therefore, it is important to develop a practical system to select for and identify novel RNA motifs that recognize a given RNA structure. In the present study, we have applied an artiﬁcial ligase ribozyme (designed-and-selected ligase; DSL) for the development of a selection system to generate novel RNA receptor motifs against a target RNA structure within a given structural context. The DSL ribozyme was con- structed by using a well-deﬁned and designed self-folding RNA as a structural scaﬀold. The features of this scaﬀold include: (i) a GAAA-tetraloop, ii) its receptor motif (11-nt receptor; hereafter 11-ntR) and (iii) stable, coaxially stacked base-triples known as the triple-helical scaﬀold (THS) found in the Tetrahymena ribozyme (13). After experimental validation of the scaﬀold structure, a catalytic modular unit was installed using in vitro selection (14). The resulting DSL ribozyme has a modular architecture that is amenable to module-based engineering, and the trans- acting ribozyme has been successfully redesigned by replac- ing the THS motif with GAAA-loop/11-ntR pair (14). To develop a DSL ribozyme-based selection system in this study, we have redesigned a shortened derivative of the DSL ribozyme by replacing its GAAA-loop and 11-ntR with a target structure and random sequence, respectively. Motifs recognizing the target structure can be identiﬁed by in vitro selection based on restoring ribozyme activity. MATERIALS AND METHODS Library construction Libraries were designed based on the DSL-U5 ribozyme (15). For the construction of GAAA-loop library, whose target motif is GAAA-loop, a synthetic template DNA (Figure 1A) (DSL-GAAA-N19; 50-AGGGAAGGAAA CTTCCCTGT GTCTTTTTTGAGCCCTCAAT CCNN NNNNNN NNNNNNNNNN NGGATCAATG GGT AGGACCA TCCGTTCCCT AGCAGGGTTC-30; where N stands for any nucleotide, and BanII site is indicated in italic) was ampliﬁed by PCR using ExTaq DNA polymerase (Takara-Bio, Japan) with forward and reverse primers (DSL-F3, 50-TAATACGACT CACTATAAGG GAAGGAAACT TCCCTGTGTC-30; and DSL-R2R3, 50-TCTGCCTAAG TGGGCAATGA GACTGGAACC CTGCTAGGGA ACGG-30, respectively. T7 promoter sequence is underlined). All oligodeoxynucleotides were purchased from Operon Biotechnologies (Japan). For the construction of a C-loop library whose target motif is the C-loop, the above GAAA-loop library was digested with the nonpalindromic restriction enzyme, BanII, and then, the 30-fragment containing the random sequence was puriﬁed by native PAGE and precipitated with ethanol. The fragment was ligated with a synthetic, 50-fragment containing the C-loop motif (C-50b-F1; 50-TAATA CGACT CACTATAAGG GAAAGCACTG GTTCG CCAGC TTTCCCTGTG GATTTTTGAG CC-30, where the T7 promoter sequence and the BanII site are underlined and italicized, respectively) in the presence of a splint DNA using T4 DNA ligase (Takara-Bio). The ligated product S-2 S-2 S-2 S-1 S-1 S-1 S-1 S-1 S-1 S-2 S-2 S-2 S-1 14 2 1 2 1 0.5 20 20 4 20 4 4 1 Nucleic Acids Research, 2008, Vol. 36, No. 11 3601 was ampliﬁed by PCR with C-50b-F1 and DSL-R2R3 as forward and reverse primers, respectively, and the ampli- ﬁed full-length fragment was puriﬁed by native PAGE. The PCR product was extracted by phenol/chloroform treat- ment, precipitated with ethanol and transcribed using T7 RNA polymerase (RiboMAX Large Scale RNA Production System-T7, Promega, WI, USA). After in vitro transcription, the template DNA was digested by RQ1 DNase (Promega), and the transcript was extracted by phenol/chloroform treatment, followed by puriﬁcation on Microcon YM-30 (Millipore, MA, USA) and MicroSpin G-25 columns (GE Healthcare, Uppsala, Sweden) to yield an RNA pool. Throughout the experi- ment, 1 1015 molecules in the RNA pool were used for the in vitro selection. Theoretically, 3.6 103 copies of 2.7 1011 (= 419) variants are included in the initial pool. In vitro selection In vitro selection was carried out essentially as described (14). Selection conditions were in 50 mM Tris–HCl (pH 7.5), (Figure 2) 100 mM NaCl and 20 mM MgCl2. Substrate RNAs [S-1 and S-2 (14)] were purchased from Gene Design Inc. (Japan). In order to exchange the guide sequence (complementary to the substrate RNA) of the libraries, and to eliminate undesired mutations at the nonrandomized regions, 50-fragments of the libraries were exchanged after every third round of the selection as follows. After the ﬁrst three rounds of selection from the GAAA-loop library, the 50-fragment was exchanged with DSL-F2 (50-TAATACGACT CACTATAAGG GAAGG AAACT TCCCTGTGGA TTTTTTGAGC C-30. T7 pro- moter sequence and BanII site are underlined and italicized, respectively) as described above. After the third and sixth rounds of the selection of C-loop library, the 50- fragment was exchanged with C-50b-F2 (50-TAAT ACGACT CACTATAAGG GAAAGCACTG GTTCG CCAGC TTTCCCTGTG TCTTTTTGAG CC-30. The T7 promoter sequence and BanII site are indicated by under- line and italic, respectively) and C-50b-F1, respectively. Other conditions for the in vitro selection are listed in Table 1, and the detailed selection procedure is described in the Supplementary Data (in vitro selection protocol). Table 1. Conditions for in vitro selection Substrate Reaction time (h) Round GAAA-loop library 1 2 3 4 5 6 C-loop library 1 2 3 4 5 6 7