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Bioconjugate Chem. 2000, 11, 744−748
ARTICLES In Vitro Selection of a Ligase Ribozyme Carrying Alkylamino Groups in the Side Chains Naozumi Teramoto,† Yukio Imanishi,‡ and Yoshihiro Ito*,§ Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501, Japan, Graduate School of Materials Science, NAIST, Ikoma, 630-0101, Japan, and Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima, 770-8506, Japan. Received October 28, 1999; Revised Manuscript Received May 30, 2000
A novel ligase ribozyme was in vitro selected from a random-sequence RNA library including N6aminohexyl-modified adenine residues in place of natural adenine residues. This ribozyme mediated the formation of a phosphodiester bond with a DNA oligonucleotide through condensation with a 5′triphosphate moiety on the ribozyme. Among the clones isolated from this selection, one was shown to accelerate ligation about 250-fold compared to the original random-sequence RNA library. Almost no rate acceleration was observed when the N6-aminohexyl-groups on adenine residues were omitted. Furthermore, ligation was dependent on the presence of a template DNA oligonucleotide that bridged the two strands.
INTRODUCTION
MATERIALS AND METHODS
The in vitro selection technique is a powerful approach to isolate catalytic RNAs. Many novel catalytic RNAs have been isolated from large pools of random-sequence RNA libraries (1). Various chemical reactions, including the formation of a phosphodiester bond (2-4) and an amide bond (5), acyl-transfer (6-9), the metalation of porphyrin (10, 11), self-alkylation (12), Diels-Alder reaction (13), and the synthesis of a pyrimidine nucleotide (14) have been shown to be catalyzed by this approach. In the presence of the catalytic RNAs, some reaction rates were enhanced over 107 times above uncatalyzed reactions (15). These results are very interesting to consider in the context of the possibility of the RNA world (16). However, natural RNA molecules consist of only four components (adenosine, cytidine, guanosine, and uridine). They lack functional groups typically found in catalytic proteins, such as the imidazole of His, the carboxylate of Asp and Glu, the alkylamine of Lys, and the sulfhydryl of Cys, which are found in natural protein enzymes. If such functional groups are contained in the RNA molecules, the RNAs are considered to catalyze various chemical reactions. Dewey et al. (17) and Sakthivel et al. (18) as well as ourselves (19) have investigated the availability of modified nucleotides for in vitro selections of nucleic-acid ligands or catalytic polynucleotides. In the present study, a ligase ribozyme that contains nucleotides carrying a lysine-like side chain, N6-(6aminohexyl)adenosine, was in vitro selected from a random-sequence RNA library. We sought to investigate the possibility of including modified base analogues to accelerate the selection of catalytic RNAs.
Materials. Single-strand DNAs were synthesized by Sawady Technology (Tokyo, Japan), and the DNA for the initial template was purified by gel electrophoresis. Others were purified by HPLC. N6-(6-Aminohexyl)adenosine 5′-triphosphate (6-AmHxATP) was purchased from Life Technologies (Gaithersburg, MD) (20). Template for the Initial Pool of RNA. Twenty picomoles of gel-purified synthetic DNA containing 116 nucleotides of random sequence (5′-GGAACACTATCCGACTGGCACC-N116-CCTTGGTCATTAGGATCCCG3′) was amplified by PCR with primers (forward primer, T7P1, 5′-TTCTAATACGACTCACTATAGGAACACTATCCGACTGGCACC-3′; reverse primer, P2, 5′-CGGGATCCTAATGACCAAGG-3′). After amplification, PCR products were purified on 8% polyacrylamide gel, and 20 pmol of the purified product pool was used in the following transcription reaction (40 µL). We estimated that the initial pool contained ∼1013 different RNA molecules. General RNA Preparation. RNAs for the selection or ribozyme reaction assays were generally prepared by transcriptions of template DNAs. The transcription reactions were carried out using an AmpliScribe T7 Transcription Kit (Epicenter Technologies, Madison, WI). The reaction mixture contained approximately 0.5 pmol/µL of template DNA, 1× AmpliScribe T7 Reaction Buffer, AmpliScribe T7 Enzyme Solution, and 2 mM each of CTP, GTP, UTP, and 6-AmHxATP (or ATP for control assays). Full-length transcription products were purified on 50% urea/8% polyacrylamide gels. The yield of the transcription reaction using 6-AmHxATP was about 75% of that using ATP. Selection Protocol. The selection was performed as follows (Figure 1). In the first selection round, the initial RNA pool (pool 0, 150 pmol) was mixed with a substrate
* To whom correspondence should be addressed. † Kyoto University. ‡ NAIST. § The University of Tokushima.
10.1021/bc990146r CCC: $19.00 © 2000 American Chemical Society Published on Web 11/03/2000
Selection of a Ligase Ribozyme Carrying Alkylamino Groups
Bioconjugate Chem., Vol. 11, No. 6, 2000 745
Figure 1. Schematic representation of selection rounds. 6-AmHxATP was used instead of ATP.
oligodeoxynucleotide (5′-biotin-AAGCATCTAAGCATCTCAAGC-3′) and an external template (5′-CGGATAGTGTTCCGCTTGAGATGCTT-3′), denatured in water (85 °C, 3 min), and allowed to cool at room temperature (>5 min). The ligation reaction was started by addition of reaction buffer I (final concentration, 30 mM Tris-HCl, pH 7.4, 150 mM KCl, 0.1 mM EDTA, and 50 mM MgCl2). The final concentrations of the RNA pool, the substrate oligodeoxynucleotide, and the external template were 0.8, 1.6, and 0.8 µM, respectively. The mixture was incubated at 37 °C for 40 h. After the incubation, 50 µL of streptavidin-agarose beads (Pierce, Rockford, IL) suspended in 250 µL of a streptavidin binding buffer (30 mM Tris-HCl, pH 7.4, 500 mM NaCl, and 1 mM EDTA) was added to the ligation reaction. The suspension was gently shaken at room temperature for 30 min. The streptavidin-agarose beads were washed with the streptavidin binding buffer, water, and mild alkali (10 mM NaOH and 1 mM EDTA). RNA molecules bound to the streptavidinagarose beads were eluted twice by incubation at 94 °C for 8 min in 300 µL of biotin solution (10 mM biotin, 1 mM EDTA). The eluted RNA molecules were purified by ethanol precipitation.
The purified RNA molecules were reverse-transcribed in 100 µL of reaction using 10 units/µL of Superscript II RNase H- reverse transcriptase (Life Technologies, Gaithersburg, MD). The reverse transcription reaction was carried out at 45 °C for 2 h. First, the cDNA products were amplified by eight cycles of PCR with a selective primer (5′-AAGCATCTAAGCATCTCAAGC-3′) and the P2 primer. Second, the PCR products were amplified by PCR with the T7P1 primer and the P2 primer. PCR products were purified on 8% polyacrylamide gel and used in the transcription reaction for the second selection round. These procedures were repeated in the subsequent selection rounds (Figure 1). The ligation reaction time was reduced as the selection round proceeded (to 4 h at the round 7). In selection rounds 5-7, the DNA templates for the RNA pools were subjected to mutagenic PCR before transcription of the RNA pool according to Cadwell and Joyce (21). However, we used eight doubling times of mutagenic PCR, although they employed 30-60 doubling times. In our case, the doublings number exceeded eight times, and side products were found by electro-
746 Bioconjugate Chem., Vol. 11, No. 6, 2000
phoresis as a ladder pattern. Therefore, eight doubling times was chosen in the present selection process. Cloning and Sequencing. After seven rounds of selection, the RNA pool was transcribed from the DNA templates and purified on 50% urea/8% polyacrylamide gel. The RNA pool (100 pmol) was reacted with the substrate oligodeoxynucleotide in the presence of the external template at 37 °C for 32 h as described above. The reaction solution was mixed with one volume of gel loading buffer (95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue, 0.025% SDS, and 0.5 mM EDTA). The mixture was heated at 85 °C for 5 min and subsequently chilled on ice. The RNA reacted with the substrate oligodeoxynucleotide was separated from unreacted RNA by 50% urea/8% polyacrylamide gel electrophoresis. The gel piece containing the reacted RNA was excised, and the RNA was eluted, phenol-extracted, and ethanol-precipitated. The RNA was reverse-transcribed with P2 primer and amplified by PCR with the selective primer and P2 primer. The PCR products were purified on agarose gel and cloned into pT7Blue T-vector (Novagen, Madison, WI) at the T-cloning site. Individual clones were sequenced by ABI Prism 377 XL sequencer (Perkin-Elmer Biosystems, Foster City, CA). Self-Ligation Assays. RNAs for self-ligation assays were prepared as described in general RNA preparation. Gel-purified RNA (final; 1.0 µM), the substrate oligodeoxynucleotide modified with biotin at the 5′ end (final; 2.0 µM), and the external template (final, 2.0 µM) were annealed in water (85 °C, 3 min; rt > 5 min). The ligation reaction was started by addition of reaction buffer I, and the reaction mixture was incubated at 37 °C. The reaction was stopped by the addition of 2 vols of EDTA solution (120 mM EDTA, 71% formamide, 0.02% xylene cyanol, 0.02% bromophenol blue, and 0.02% SDS). The complex of the RNA, the substrate, and the external template was denatured (90 °C, 3 min), and each component was separated on 50% urea/8% polyacrylamide gel (20 × 20 cm). Varying amounts of single-strand DNAs modified with biotinyl groups at the 5′ ends were applied to other lanes of the gel as references for amounts of the biotinmodified polynucleotides. During electrophoresis, the substrate and the external template were immigrated out of the gel so that they would not hybridize to unreacted RNA in the subsequent biotin-detection process. RNA was transferred from the gel to a nylon membrane, and the biotin-modified polynucleotides were detected using Imaging highsChemilumi (Toyobo, Osaka, Japan). The chemiluminescence images were mainly photographed on several sheets of X-ray film (for various exposure times) and analyzed on a Macintosh computer using the publicdomain NIH Image program (developed at the U.S. National Institute of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/) or detected by Densitograph lumino CCD (Atto, Tokyo, Japan). Unreacted RNA and the substrate oligodeoxynucleotide were used as the control. The density of their lanes was measured and used as the background. Three experiments were performed on each sample and a mean value was obtained. The statistical significance of experimental data (P value) was determined by using t-test. The data are expressed as an average value ( standard deviation. RESULTS AND DISCUSSION
In Vitro Selection of Ligase Ribozymes. The selected RNA pool (pool 7) accelerated the self-ligation reaction with the substrate oligodeoxynucleotide (Figure 2). The observed rate constant, kobs, was 8.0 (( 1.4) ×
Teramoto et al.
Figure 2. Self-ligation reaction analyzed by Northern blotting. The arrow shows the self-ligation products carrying biotinyl groups. Table 1. Observed Ligation Activity of the Selected Clonea
sample
RNA
a b c d
clone clone clone clone random pool random pool
aminohexyl external n-butylgroups template amine + + + -
+ + + + +
+ (1 mM) -
kobs (h-1) 5.1 ((1.4) × 10-5 3.2 ((0.6) × 10-6 2.9 ((0.5) × 10-6 2.6 ((0.7) × 10-6 1.1 ((0.3) × 10-6 2.1 ((0.8) × 10-7
a a, b, c, and d correspond to a, b, c, and d in Figure 4, respectively. (+) Presence; (-) absence.
10-6 h-1, which was about 8-fold higher than that of the original random-sequence RNA library including aminohexyl-modified adenine residues (pool 0) (P < 0.003). When natural adenine residues were incorporated into the selected RNA sequence instead of aminohexyl-modified adenine residues, the RNA had almost no catalytic activity. This result demonstrated that the selected RNA catalyzed the self-ligation reaction with the assistance of the alkylamino groups. Cloning and Sequencing. Twelve clones from RNA pool 7 were sequenced. Five clones consisted of combinations of several primer sequences (P1, P2, or selective primer). Seven clones consisted of the same sequence. The secondary structure of the clone observed frequently was predicted by Mfold software [developed by Zuker et al. (22, 23) and available on the Internet at http://www.ibc.wustl.edu/∼zuker/rna/] (Figure 3), assuming that the program was applicable to nonnatural component-containing RNA. The following characteristics were found. A cluster composed of aminohexyl-modified adenosine residues was observed in one bulge loop. The cluster-containing region (bases 55-136), where several stems and loops are lined, is separated by unpaired bases (14-25 and 46-54) from the region forming complex with the substrate and the external template oligodeoxynucleotide. These unpaired bases might afford the ribozyme some degree of freedom as spacers. Catalytic Activity. The reaction rate of the clone was 5.1 (( 1.4) × 10-5 h-1 (kobs), which was about 50 times as high as that of the original random-sequence RNAs containing aminohexyl groups and about 250 times as high as that of random-sequence RNAs consisting of natural nucleic acid (P < 10-4) (Table 1 and line a in Figure 4). Previously, we performed other in vitro selections for the RNA-DNA ligation using natural RNAs or 2′-amino uridine. However, neither selection produced expected RNAs. In the case of natural RNAs, it was reported that
Selection of a Ligase Ribozyme Carrying Alkylamino Groups
Bioconjugate Chem., Vol. 11, No. 6, 2000 747
Figure 3. Predicted secondary structure of the selected clone with the oligodeoxynucleotide substrate and the external template. The circled region indicates the cluster formed by aminohexyl adenine residues.
Figure 4. Self-ligation activities of the selected clone. Ligation reaction of the clone carrying aminohexyl groups in the presence (a) and in the absence of the external template (b). Ligation reaction of the clone, which was transcribed with natural nucleotides, in the presence of the external template (c) and in the presence of 1 mM n-butylamine (d).
the presence of hydroxy group at 3′ end was needed in a self-ligation reaction of ribozyme with an RNA substrate (24), although Bartel and Szostak (1) found in vitroselected ribozymes catalyzed the self-ligation with RNA, and Cuenoud and Szostak (3) found an in vitro selected DNAzyme catalyzed self-ligation with DNA activated by phosphorimidazolide. On the other hand, the employment of 2′-amino uridine instead of uridine led to unexpected ligation products as reported previously (25). These results demonstrated that the incorporation of amino groups accelerated the selection of catalytic RNA and that the modificaiton site or pKa difference (aminohexyl, 1011; 2′-amino, ca. 6) played an important role. An external template was employed for positioning a substrate oligodeoxynucleotide into close proximity of the 5′ end of a ribozyme. It is known that external templates (DNA splints) improve the yields of ligation reaction of two RNA strands catalyzed by T4 DNA ligase (26) or T4 RNA ligase (27). The self-ligation reaction rate of the clone was reduced to 3.2 (( 0.6) × 10-6 h-1 by removing the external template (P < 0.001) (line b in Figure 4). This result suggested that the external template acted as an effector for substrate recognition in the reaction. The ribozyme interacted with the heteroduplex formed with the external template. The incorporated aminohexyl groups may aid this interaction, as Arg or Lys residues in DNA-interacting proteins (28), in addition to side-byside interactions between helices that was suggested in the group I ribozyme (29, 30), the hepatitis delta virus ribozyme (31), and the hairpin ribozyme (32). Formation of triplex between the heteroduplex and the single-strand region of the ribozyme is also taken into consideration
as suggested in the group I intron (33). Recently, Landweber and Pokrovskaya (34) selected a ligase ribozyme that catalyzes template-directed RNA ligation and found that the ribozyme consists of only one linear duplex with a substrate strand. This report indicated that the importance of DNA splint for ligation reaction. When the RNA clone was synthesized using unmodified adenosine, the reaction rate of the unmodified clone was reduced to 2.9 (( 0.5) × 10-6 h-1 (kobs), 15-20-fold lower than that of the aminohexyl-modified RNA (P < 0.001) (line c in Figure 4). In addition, the ligation activity of the unmodified clone was determined in the presence of 1 mM n-butylamine instead of aminohexyl side chains of modified RNA. However, n-butylamine had no effect on the catalytic reaction (P ) 0.6) (line d in Figure 4). These results suggest that the aminohexyl groups in the RNA played an important role in the catalytic behavior, although it is unknown whether the aminohexyl groups acted as the catalytic active site or maintained the threedimensional structure of modified RNA. Recently, Rogers and Joyce (35) showed even three nucleic-acid subunits other than cytidine are sufficient to obtain a ligase ribozyme. In the future, in vitro selection of nonnatural ribozyme will be important not only for investigation on RNA world but also for creation of new enzymes for industrial uses. LITERATURE CITED (1) Breaker, R. R. (1997) In Vitro Selection of Catalytic Polynucleotides. Chem. Rev. 97, 371-390. (2) Bartel, D. P., and Szostak, J. W. (1993) Isolation of New Ribozymes from a Large Pool of Random Sequences. Science 261, 1411-1418. (3) Hager, A. J., and Szostak, J. W. (1997) Isolation of Novel Ribozymes that Ligate AMP-activated RNA Substrates. Chem. Biol. 4, 607-617. (4) Cuenoud, B., and Szostak, J. W. (1995) A DNA Metalloenzyme with DNA Ligase Activity. Nature 375, 611-614. (5) Wiegand, T. W., Janssen, R. C., and Eaton, B. E. (1997) Selection of RNA Amide Synthases. Chem. Biol. 4, 675-683. (6) Illangasekare, M., Sanchez, G., Nickles, T., and Yarus, M. (1995) Aminoacyl-RNA Synthesis Catalyzed by an RNA. Science 267, 643-647. (7) Lohse, P. A., and Szostak, J. W. (1996) Ribozyme-catalyzed Amino-acid Transfer Reactions. Nature 381, 442-444. (8) Zhang, B., and Cech, T. R. (1997) Peptide Bond Formation by in Vitro Selected Ribozymes. Nature 390, 96-100. (9) Jenne, A., and Famulok, M. (1998) A Novel Ribozyme with Ester Transferase Activity. Chem. Biol. 5, 23-34. (10) Conn, M. M., Prudent, J. R., and Schlutz, P. G. (1996) Porphyrin Metalation Catalyzed by a Small RNA Molecule. J. Am. Chem. Soc. 118, 7012-7013. (11) Li, Y., and Sen, D. (1996) A Catalytic DNA for Porphyrin Metalation. Nat. Struct. Biol. 3, 743-747. (12) Wilson, C., and Szostak, J. W. (1995) In Vitro Evolution of a Self-alkylating Ribozyme. Nature 374, 777-782.
748 Bioconjugate Chem., Vol. 11, No. 6, 2000 (13) Tarasow, T. M., Tarasow, S. L., and Eaton, B. E. (1997) RNA-catalyzed Carbon-carbon Bond Formation. Nature 389, 54-57. (14) Unrau, P. J., and Bartel, D. P. (1998) RNA-catalyzed Nucleotide Synthesis. Nature 395, 260-263. (15) Pan, T. (1997) Novel and Variant Ribozymes Obtained through in Vitro Selection. Curr. Opin. Chem. Biol. 1, 1725. (16) Joyce, G. E., and Orgel, L. E. (1993) Prospects for Understanding the Origin of the RNA World. In The RNA World (R. F. Gesteland and J. F. Atkins, Eds.) pp 1-25, Cold Spring Harbor Laboratory, Plainview, NY. (17) Dewey, T. M., Mundt, A., Crouch, G. J., Zyzniewski, M. C., and Eaton, B. E. (1995) New Uridine Derivatives for Systematic Evolution of RNA Ligands by Exponential Enrichment. J. Am. Chem. Soc. 117, 8474-8475. (18) Sakthivel, K., and Barbas, C. F., III (1998) Expanding the Potential of DNA for Binding and Catalysis: Highly Functionalized dUTP Derivatives that are Substrates for Thermostable DNA Polymerase. Angew. Chem., Int. Ed. 37, 28722875. (19) Ito, Y., Teramoto, N., Kawazoe, N., Inada, K., and Imanishi, Y. (1998) Modified Nucleic Acid for Systematic Evolution of RNA Ligands by Exponential Enrichment. J. Bioact. Compat. Polym. 13, 114-123. (20) Folsom, V., Hunkeler, M. J., Haces, A., and Harding, J. D. (1989) Detection of DNA Targets with Biotinylated and Fluoresceinated RNA Probes. Anal. Biochem. 182, 309-314. (21) Cadwell, R. C., and Joyce, G. F. (1992) Randomization of Genes by PCR Mutagenesis. PCR Methods Appl. 2, 28-33. (22) Zuker, M. (1989) On Finding All Suboptimal Foldings of an RNA Molecule. Science 244, 48-52. (23) Jaeger, J. A., Turner, D. H., and Zuker, M. (1990) Predicting Optimal and Suboptimal Secondary Structure for RNA. Methods Enzymol. 183, 281-306. (24) Rohatgi, R., Bartel, D. P., and Szostak, J. W. (1996) Kinetics and Mechanistic Analysis of Nonenzymatic, Templatedirected Oligoribonucleotide Ligation. J. Am. Chem. Soc. 118, 3332-3339.
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