Proc. Natl. Acad. Sci. USA Vol. 94, pp. 391–396, January 1997 Biochemistry Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli SATOSHI FUJITA*†‡,TETSUHIKO KOGUMA*‡§,JUN OHKAWA*, KAZUYUKI MORI*§,TAKEO KOHDA*, HIDEO KISE†, SATOSHI NISHIKAWA*, MASAHIRO IWAKURA*, AND KAZUNARI TAIRA*§¶ *National Institute of Bioscience and Human Technology, Agency of Industrial Science & Technology, Ministry of International Trade and Industry, Tsukuba Science City 305, Japan; and Institutes of †Materials Science and §Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City 305, Japan Communicated by Stephen J. Benkovic, Pennsylvania State University, University Park, PA, November 4, 1996 (received for review December 20, 1995) ABSTRACT For use of ribozymes in vivo, it is desirable to (16, 17), it was postulated that this ribozyme might be much select functional ribozymes in the cellular environment (in the more effective than simple antisense molecules in several presence of inhibitory factors and limited concentrations of respects (16–20). However, because of their instability and mandatory Mg21 ions, etc.). As a first step toward this goal, their lower-than-expected activities in vivo, ribozymes have not we developed a new screening system for detection in vivo of an yet proven their superiority to antisense molecules. There active ribozyme from pools of active and inactive ribozymes seem to be several reasons for their low activity in vivo:(i) there using the gene for dihydrofolate reductase (DHFR) as a may be many cellular proteins in vivo that inhibit the ri- selective marker. In our DHFR expression vector, the se- bozymes’ catalytic activity (21, 22); (ii) the intracellular con- quence encoding either the active or the inactive ribozyme was centration of Mg21 ions is much lower than that used in vitro connected to the DHFR gene. The plasmid was designed such for testing the ribozyme activity (23–25); and (iii) several that, when the ribozyme was active, the rate of production of cellular RNases contribute to the ribozymes’ instability (26– DHFR was high enough to endow resistance to trimethoprim 31). To overcome some of these problems, many approaches (TMP). We demonstrated that the active ribozyme did indeed have been taken, including some attempts to select active cleave the primary transcript in vivo, whereas the inactive ribozymes in vitro (32–37). The drawback to selection in vitro ribozyme had no cleavage activity. Cells that harbored the is that the activity in vitro does not always reflect the activity active-ribozyme-coding plasmid grew faster in the presence of in vivo (38). Moreover, selection systems in vitro always involve a fixed concentration of TMP than the corresponding cells reverse transcription. The activity of the ribozyme is associated that harbored the inactive-ribozyme-coding plasmid. Conse- with its specific structure, but reverse transcriptase activity is quently, when cells were transformed by a mixture that known to be inhibited by some secondary structures (39). consisted of active- and inactive-ribozyme-coding plasmids at Therefore, there is always a risk of missing the most effective a ratio of 1:1, (i) mainly those cells that harbored active ribozymes during selection in vitro. ribozymes survived in the presence of TMP and (ii) both Because of these limitations of screening systems in vitro,we active- and inactive-ribozyme-harboring cells grew at an need to design a screening system in vivo whereby selection can identical rate in the absence of TMP, a demonstration of a be made under the cellular conditions under which ribozymes positive selection system in vivo. If the background ‘‘noise’’ can must be active. As a first step toward the development of a be removed completely in the future, the selection system screening system in vivo, we used the gene for dihydrofolate might usefully complement existing selection systems in vitro. reductase (DHFR) as a selective marker in Escherichia coli. The addition by DHFR of a methyl group to deoxyuridylic acid Ribozyme and antisense technologies appear to have potential to form thymidylic acid is an important reaction in DNA as methods for suppressing the expression of specific genes synthesis (40). Because DNA synthesis is required by all (1–9). Therefore, they could be powerful tools in gene therapy proliferating cells, inhibition of DNA synthesis is one of the for some diseases caused by aberrant gene expression, includ- most effective ways of controlling cell division. Several drugs, ing diseases caused by infectious agents such as HIV (7–10). such as trimethoprim (TMP) and methotrexate, are potent There are several strategies for inhibition of the expression of inhibitors of DHFR, and consequently, they inhibit DNA specific genes during transcription and translation (11–15). synthesis and the multiplication of cells (40–43). When an The hammerhead ribozyme belongs to the class of molecules inhibitor of DHFR, such as TMP, is present in the culture known as antisense RNAs (hereafter, the term ribozymes medium at a certain concentration, DHFR-producing clones, refers exclusively to hammerhead ribozymes unless otherwise which have had already been transfected by a DHFR- noted). However, because of short extra sequences that form expressing vector, are expected to survive and grow more the so-called catalytic loop, this ribozyme can act as an rapidly than non-expressing clones (44). Therefore, if we can enzyme. Since the substrate specificity of antisense and ri- control the level of expression of the DHFR gene by a bozyme molecules is high, antisense and ribozyme strategies ribozyme, we should be able to determine the activities of seem likely to have some value for therapeutic purposes (7). ribozymes in terms of resistance to TMP. Namely, TMP When the hammerhead ribozyme was engineered in such a resistance should be a function of ribozyme activity that can, way that it could cleave a specific RNA sequence ‘‘in trans’’ in turn, be estimated from the concentration of TMP in the culture medium. We report here that clones that survived at a The publication costs of this article were defrayed in part by page charge fixed concentration of TMP harbored mostly active ribozymes. payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: DHFR, dihydrofolate reductase; TMP, trimethoprim; Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA SD, Shine–Dalgarno. 0027-8424y97y94391-6$2.00y0 ‡S. Fujita and T. Koguma contributed equally to this work. PNAS is available online at http:yywww.pnas.org. ¶To whom reprint requests should be addressed at the § address. 391 Downloaded by guest on September 25, 2021 392 Biochemistry: Fujita et al. Proc. Natl. Acad. Sci. USA 94 (1997) Moreover, this selection system successfully identified a single reverse transcriptase (GIBCOyBRL) was added. The reverse base change in vivo and, therefore, to the best of our knowl- transcriptase reactions were carried out at 428C for 60 min to edge, this is the first report that suggests the possibility of avoid the influence of the secondary structure of the mRNA. positive selection in vivo of functional ribozymes. After the reverse transcriptase reaction, 2 ml of stop solution, containing 95% formamide, 20 mM EDTA, 0.05% bromo- MATERIALS AND METHODS phenol blue, and 0.05% xylene cyanol, was mixed with 3 mlof Bacterial Strains and Plasmids. E. coli HB101 (recA13, the reaction mixture, and the resulting sample was fractionated supE44; Takara Shuzo, Kyoto) was used as a recipient for ona7Mureay8% polyacrylamide gel. Four ddNTP sequenc- transformation. Several ribozyme expression vectors were ing reactions from the same 32P-labeled primer were fraction- constructed by modifying the DHFR expression vector ated together, creating sequencing ladders as markers. pTZDHFR20 (45). Synthesis of Oligonucleotides and Construction of Plas- RESULTS mids. Oligodeoxynucleotides [active-ribozyme linkers (for- Design and Construction of the Screening Vectors. We ward, 59-AGC TTA ACT AAT TGA ATT CCT GAT GAG designed a screening system in E. coli. In our screening vectors, TCC CTA GGG ACG AAA CCA TGG ACT AAC TAA CTA either an active or an inactive ribozyme sequence (Fig. 1) was AT-39; and the corresponding reverse sequence), pseudo-ATG connected upstream of the E. coli DHFR gene (Fig. 2). The linkers (forward, 59-CCG GAA AAG GAG GAA CTT CCA inactive ribozyme sequence differed from the active one by a TGG TCG AAT TCA ACC TAT ATG ATC AGT CTG ATT single G5 3 A (or A14 3 G) mutation within catalytic core of GCG GCG-39; and reverse), and 39-terminator linkers (for- the ribozyme. These mutations abolish ribozyme activity (20, ward, 59-TCG AGC GTC GTT AAA GCC CGC CTA ATG 46). If the ribozyme were targeted to the DHFR gene itself, the AGC GGG CTT TTT TTT TTA G-39; and reverse)] were growth of cells that had been transformed by active-ribozyme- synthesized with a DNA synthesizer (model 392; Applied coding plasmids should be slower in the presence of inhibitors Biosystems) and purified by chromatography. Single base of DHFR such as TMP and methotrexate. Then, clones change (G5 3 AorA14 3 G) was introduced within the surviving in the presence of TMP or methotrexate would turn active-ribozyme catalytic core (see Fig. 1). These changes had out to have inactive-ribozyme-coding sequences, with resultant already been shown to destroy cleavage activity (20, 46). Each negative selection. Since it is desirable to select colonies that linker was ‘‘tailed’’ with a recognition sequence for an appro- possess active ribozymes (positive selection), when we de- priate restriction endonuclease. Each oligonucleotide linker signed our vectors, the ribozyme was not targeted to the was denatured at 958C in a water bath and then gradually DHFR gene itself but to the region, designated the interspace, cooled to room temperature in TE buffer (10 mM TriszHCl, between two ATG codons (Fig.