Isolation of Hammerhead Ribozymes with Altered Core Sequences by in Vitro Selection † Narendra K. Vaish, Paul A. Heaton, and Fritz Eckstein* Max-Planck-Institut fu ¨ r experimentelle Medizin, Hermann-Rein-Strasse 3, D-37075 Go ¨ ttingen, Germany ReceiVed December 23, 1996; ReVised Manuscript ReceiVed March 17, 1997 X ABSTRACT: The hammerhead ribozyme has an invariant nucleotide sequence in the core region. In order to search for alternative sequences which can support the cleavage after the triplet GUC, the core region of 10 nucleotides was randomized and subjected to in Vitro selection by repeated cycles of transcription, reverse transcription, and PCR. Active sequences were isolated after each transcription by denaturing PAGE, and after nine cycles of selection, two sequences dominated the pool. Both sequences conformed broadly to the consensus core region except that in one sequence a single A 9 U mutation was observed while in the other two mutations at A 9 U and U 7 A were seen. The catalytic efficiencies of these ribozymes were 6.4 and 14.1 μM -1 min -1 , respectively, as compared to 163 μM -1 min -1 for the consensus sequence. Interestingly, the consensus was not found in any of the selected sequences. This discrimination against the consensus sequence was attributed to the specificity of the enzymes used in the selection procedure. Hammerhead ribozymes are small self-cleaving RNAs that are found in certain virus and satellite RNAs that replicate via a rolling-circle mechanism [for a review, see Symons (1992)]. The ribozyme’s two-dimensional structure, depicted in Figure 1, was determined by sequence homology and consists of three base paired helices linked by a central core of conserved nucleotides. Further information on the sequence requirements of the hammerhead was determined by systematic mutation of the nucleotides in the core, which indicated that the closing base pair of stem II must be R 10.1 - Y 11.1 (where R and Y indicate purine and pyrimidine nucleotides, respectively) (Ruffner et al., 1990; Tuschl & Eckstein, 1993); variations are tolerated at position 7 in the core (Ruffner et al., 1990); and finally, the cleavage triplet, after which the ribozyme cleaves, has the general sequence NUH (where N is any nucleotide and H is either A, C, or U) (Ruffner et al., 1990; Shimayama et al., 1995; Zou- madakis & Tabler, 1995). Recently, the three-dimensional X-ray crystal structure has been determined (Pley et al., 1994; Scott et al., 1995), which has revealed a wealth of informa- tion on the secondary interactions of the nucleotides in the catalytic core. On the basis of the X-ray structures, the catalytic core has been divided into two regions, which form two distinct structural motifs: domain I, comprising nucle- otides C 3 -A 6 , contains a “uridine-turn”; and domain II, comprising nucleotides U 7 -A 9 and G 12 -A 14 , contains a GA tandem mismatch. Since the uridine-turn motif has the general sequence requirement UNR and other sequences have been found to adopt a structure similar to this motif (Jucker & Pardi, 1995), then it is not unreasonable to ask whether other sequences are possible in the hammerhead catalytic core. These alternative sequences could not, of course, be detected by single point mutations since two or more complementary mutations may be required to enable an active catalytic core to form. Thus, an alternative method, such as in Vitro selection, is required to search all possible core sequences. In Vitro selection is a technique where RNA or DNA sequences which exhibit certain properties are isolated from a large number of random sequences by repeated cycles of selection and amplification. The technique, which was first reported in 1990 (Ellington & Szostak, 1990; Tuerk & Gold, 1990), was initially used to select RNA motifs which were able to bind other molecules, such as proteins and small organic molecules [for reviews, see Abelson (1996); Famulok and Szostak (1993)], but has now developed sufficiently to select RNA and DNA sequences which have novel catalytic properties and to improve the catalytic activity of known ribozymes [for reviews, see Abelson (1996), Chapman and Szostak (1994), Famulok and Szostak (1993), and Kumar and Ellington (1995)]. The procedure described here to † This work was supported by the Deutsche Forschungsgemeinschaft and by a fellowship from the Alexander von Humbolt-Stiftung to N.K.V. * Corresponding author. Telephone: +49 551 3899 274. Fax: +49 551 3899 388. E-mail: eckstein@mail.mpiem.gwdg.de. X Abstract published in AdVance ACS Abstracts, May 1, 1997. 1 Abbreviations: PCR, polymerase chain reaction; PAGE, polyacryl- amide gel electrophoresis; RT, reverse transcription. FIGURE 1: Schematic representation of the hammerhead ribozyme. Nucleotides in boldface indicate the conserved catalytic core nucleotides that were randomized; italics indicate the cleavage triplet, and the arrow indicates the cleavage site. Ribozyme numbering is in accordance with Hertel et al. (1992). 6495 Biochemistry 1997, 36, 6495-6501 S0006-2960(96)03134-0 CCC: $14.00 © 1997 American Chemical Society