Proc. Nati. Acad. Sci. USA Vol. 89, pp. 3985-3989, May 1992 Biochemistry

Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme DONG-JING FU AND LARRY W. MCLAUGHLIN Department of Chemistry, 140 Commonwealth Drive, Boston College, Chestnut Hill, MA 02167

Communicated by Peter B. Dervan, January 15, 1992 (received for review October 8, 1991)

tion of the 2'-hydroxyl with the release of the 5'-hydroxyl and generation of a 2',3'-cyclic phosphodiester. In this respect the mechanism is similar to the first step of the hydrolysis reaction catalyzed by pancreatic ribonuclease (14). By using substrates containing chiral phosphorothioate diesters, three phosphodiester residues have been identified that appear to be critical for efficient cleavage (15, 16). Studies employing two phosphorothioate diastereomers at the cleavage site suggest that the Mg2+ cofactor is bound to the pro-R oxygen in the unmodified complex and that hydrolysis occurs with an in-line mechanism (17-19). The structure adopted by the ribozyme and its substrate in the active catalytic complex is presently unknown. Modeling studies of the Lucerne transient streak virusoid (20) based on energy minimization and computational dynamics have suggested that the cytidine residue on the 3' side of the scissile phosphodiester is on the surface of the complex and does not interact with other bases. This conformation forces the ribose-phosphate backbone to make an abrupt turn as it bridges the helical stems I and III, which in turn directs the pro-R and pro-S oxygens of the 3'-phosphodiester toward the inward side of the hammerhead where complexation with the Mg2+ cofactor can occur. There are no x-ray studies to date, and only preliminary NMR studies have been reported (21). Studies of a series of sequence mutations of the conserved nucleotides present in the core hammerhead structure indicate that all nine of the conserved single-stranded nucleotide residues are critical for cleavage activity (7, 10, 11, 13, 22). In all cases, mutations of the nine nonhelical conserved residues did not alter the ability of the hammerhead complex to form, but cleavage activity was eliminated or rates were reduced by >1 order of magnitude. These results can be interpreted to reflect a structure in which the conserved residues orient the position(s) of specific functional groups in the active site such that base-base interactions or the ability of the complex to bind and position the Mg2+ cofactor are optimized. Sequence mutations can be expected to alter this organization of the active site, similar to that observed for specific amino acid mutations in the active sites of certain enzymes, and reduce the catalytic competence of the ribozyme. The devastating effects observed for base substitutions within the nine conserved nonhelical residues suggest that the location of specific interbase or Mg2+-nucleoside interactions will be best accomplished by mutagenesis at the atomic level (that is, by specifically removing individual base or carbohydrate functional groups without complete base

Eight modified ribozymes of 19 residues have ABSTRACT been prepared with individual purine amino or hydroxyl groups excised. The modified ribozymes were chemically synthesized with the substitution of a single 2'-deoxyadenosine, 2'-deoxyguanosine, inosine, or purine riboside for residues G10, A"l, G3, or A". Five of the modified ribozymes cleaved the 24-mer substrate with little change in rate as monitored by simple first-order kinetics. However, deletion of the 2-amino group at G10 (replacement with inosine) or deletion of either of the 2'-hydroxyls at G10 or G13 (replacement with 2'-deoxyguanosine) resulted in ribozymes with a drastic decrease in cleavage efficiency. Increasing the concentration of the Mg2+ cofactor from 10 mM to 50 mM significantly enhanced cleavage efficiency by these three derivatives. Steady-state kinetic assays for these three ribozymes indicated that the modifications result in both an increase in K. and a decrease in kct,. These results suggest that the exocyclic amino group at G1' and the hydroxyls at G"' and G13 are important both for ribozymesubstrate binding and for the Mg2+-catalyzed cleavage reaction.

RNA-processing reactions that involve the cleavage of phosphodiester bonds are critical steps for the production of many mature RNAs from corresponding precursors and appear to be important in the replication of several plant satellite RNAs (for reviews see refs. 1-3). Autolytic self-cleavage regions are present in certain virusoid RNAs and occur in a common structural domain termed a "hammerhead" (4). The consensus structure of a self-cleaving hammerhead contains 13 conserved nucleotides held together by three helical regions (4, 5). Nine of the conserved nucleotides occur in nonhelical regions and are critical for the observed cleavage reaction. Sequence variations in the helical regions can alter the rate of the reaction (6), but only the A-U and possibly the C-G base pair at the base of stem III, at the 5' side of the cleavage site, appear to be critical for activity (7). In addition to the hammerhead structural domain, a metal cofactor is required for the observed processing reaction. Mg2" is usually employed, but Mn2+ appears to function equally well in some cases (8). Cleavage occurs at the phosphodiester bond 3' to the residue located at the bend between two of the helices (this is most commonly cytidine) (6). The in vivo reaction takes place in a unimolecular complex, but cleavage can be observed in vitro from complexes formed from two (or even three) oligonucleotides (9-12), as long as the conserved nucleotide residues and the hammerhead structure are maintained. Hammerheads composed of two RNA fragments can exist in three distinct complexes differing in the location of the respective 3' and 5' termini and the hairpin loop. All three complexes exhibit the self-cleavage reaction but the rates of cleavage (as measured by tl/2 values) can differ significantly (13). The RNA cleavage reaction, catalyzed by Mg2+ (or Mn2+), proceeds as a transesterifica-

substitution). A number of modified ribozymes have been prepared in which specific (or multiple) 2'-deoxyribonucleoside residues have been incorporated in place of the native ribo derivatives (this excises the 2'-hydroxyl at single or multiple sites) (8, 23-26). The substitution of multiple dA (or 2'-deoxy-2'fluoroadenosine) residues appears to result in a cumulative decrease in activity without implicating a specific critical residue. The use of 2'-fluoro or 2'-amino derivatives provides nuclease-resistant ribozymes (26).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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In the present work we describe the effects on catalytic efficiency that result from the deletion of individual purine nucleobase amino groups or purine 2'-hydroxyl groups in a hammerhead ribozyme.

MATERIALS AND METHODS Phosphoramidites. The nucleosides inosine and purine riboside were obtained from Sigma and converted to the corresponding 5'-O-(4,4'-dimethoxytrityl)-2'-O-t-butyldi-

methylsilyl-3'-O-[(N,N-diisopropylamino)(,B-cyanoethoxy)-

phosphinyl] derivative essentially by the procedures of Ogilvie and coworkers (27), Sung and Narang (28), and Usman and coworkers (29).* These procedures will be described in detail elsewhere. The 2'-deoxynucleoside phosphoramidites were obtained from Cruachem (Sterling, VA) and the ribo derivatives were products of MilliGen/Biosearch (Woburn, MA). Oligonucleotide Synthesis. The oligonucleotides were synthesized on controlled-pore glass supports in an Applied Biosystems 380A DNA synthesizer. After deprotection by base (concentrated ammonium hydroxide/ethanol, 3:1, for 6 hr at 500C) and anhydrous fluoride (1.0 M tetrabutylammonium fluoride in tetrahydrofuran for 16 hr at ambient temperature) the oligonucleotides were desalted and then isolated by chromatography on a Mono Q column (Pharmacia; 0.5 x 5 cm) at a flow rate of 1.5 ml/min in 5 mM sodium cacodylate (pH 6.0) with a gradient of NaCl (0-0.45 M over 30 ml followed by 0.45-0.55 M over 60 ml). The 19-mers typically eluted in the range of 40-47 ml, whereas the 24-mer eluted at the end of the gradient (90 ml). After isolation, the fragments were desalted (Sephadex G-10) and lyophilized to dryness. Nucleoside Analysis. Nucleoside composition was determined after S1 nuclease/bacterial alkaline phosphatase hydrolysis. A 10-,ul reaction mixture containing 0.5 A260 unit of oligomer in 200 mM NaCl/5 mM MgCl2/0.1 mM ZnSO4/25 mM sodium acetate, pH 5.5, was incubated for 5 min at ambient temperature. To this solution was added 5 ,ul of 100 mM Tris-HCl (pH 8.0) and 1 unit of bacterial alkaline phosphatase. After an additional 60 min incubation at ambient temperature, a 5-,ul aliquot was analyzed by HPLC on an ODS-Hypersil column (4.6 x 250 mm) in 20 mM sodium phosphate (pH 5.5) and a gradient of 0-70% methanol (60 min). Radioisotopic Labeling. The 24-mer was 5'-end-labeled with [y-32P]ATP as follows. A 50-Al reaction mixture containing 1 A260 unit of 24-mer (-0.1 mM), 10 mM MgCl2, 10 mM dithiothreitol, 0.2 mM Na2EDTA, 0.1 mM ATP, 300-600 ,uCi of [y-32P]ATP (1 ,Ci = 37 kBq), and 20 units of T4 polynucleotide kinase was incubated for 60 min at 37°C. The product was isolated by adsorption on a C18 Sep-Pak cartridge (Waters). The cartridge was washed with water and then with 40-50% methanol in water to elute the product. The labeled 24-mer was repurified by electrophoresis in a 20% polyacrylamide/7 M urea gel. The product band was excised, extracted with 0.1 M ammonium acetate, pH 7.0, and desalted with a C18 Sep-Pak cartridge. The specific activity of the 24-mer was typically 0.01 ,Ci/pmol. *We had some difficulty in monitoring the final reaction (conversion of the protected nucleoside to the 3'-phosphoramidite) by thin-layer chromatography as described for the common nucleoside derivatives (29). However, by observing the H1, resonance in the 1H NMR spectrum, we could confirm that complete formation of the phosphoramidite had taken place. For example, 5'-O-dimethoxytrityl2'-O-t-butyldimethylsilylpurine riboside exhibited a single H1, doublet centered at 6.14 ppm, while the corresponding N,Ndiisopropyl-,8-cyanoethylphosphoramidite derivative exhibited two H1, doublets centered at 6.21 and 6.27 ppm with no remaining signal at 6.14 ppm.

Proc. Natl. Acad. Sci. USA 89

(1992)

Stoichiometric Cleavage Analysis. Two 50-,p1 solutions containing either 0.6 ,uM ribozyme or 0.4 AM substrate in 50 mM Tris-HCl (pH 8.0) with 10 mM or 50 mM MgCl2 were each heated briefly to 90'C and cooled to 370C. The reaction was initiated by mixing the two solutions. Aliquots of 10 ,u1 were withdrawn, and the reaction was quenched by the addition of 1 volume of 50 mM Na2EDTA/7 M urea/10% glycerol/0.05%

xylene cyanol/0.05% bromphenol blue. The extent of cleavage was analyzed by electrophoresis in denaturing polyacrylamide gels (14 x 16 cm). After autoradiography, the substrate and product bands were excised and lyophilized to dryness, and the radioactivity was determined by scintillation counting. Catalytic Cleavage Analysis. These reactions were performed and monitored as described above at 550C in 40 1.l of 10 mM MgCl2/50 mM Tris-HCI, pH 8.0. The ribozyme concentration in these reactions was 0.1 ,M (native sequence) or 0.2 ,uM (110, dG'0, and dG13 sequences), and from four to eight substrate concentrations were used that varied from 0.4 to 40 ,uM depending on the individual sequence. Aliquots of 4 ,ul were taken from the reaction mixture at various times and quenched and analyzed as described above. Kinetic parameters were obtained from linear Lineweaver-Burk and Eadie-Hofstee plots and by curve fitting to the hyperbolic plots of velocity vs. substrate concentration. RESULTS AND DISCUSSION To examine the role of specific purine amino and hydroxyl functional groups in ribozyme activity, we prepared a number of oligonucleotides with base analogues in which specific functional groups had been excised. The deletion of the purine exocyclic amino groups of the bases adenine and guanine was accomplished by the introduction of purine and hypoxanthine. The corresponding nucleosides [purine riboside (P) and inosine (I)] were converted to the phosphor-

amidite building blocks by standard procedures (27, 28) in which the 2'-hydroxyl groups were protected as the t-butyldimethylsilyl ethers. The ribozyme complex, identical with that described by Uhlenbeck (ref. 4; see also Fig. 3), was formed by the synthesis of two RNA fragments of 19 and 24 nucleotides in length. The use of these relatively short RNA fragments simplified analyses for purity and for the presence of the nucleoside analogue. This complex is identical with that used by Ruffner et al. (7) for a corresponding study of a series of sequence mutations. The 24-mer substrate and the 19-mer native and modified ribozymes were synthesized on controlled-pore glass supports. After deprotection and purification of the ribozyme fragments (see Materials and Methods) the sequences were analyzed for purity by HPLC (as illustrated in Fig. la for the P14-containing 19-mer) and by polyacrylamide gel electrophoresis. A small amount of each RNA sequence was degraded with S1 nuclease and bacterial alkaline phosphatase. HPLC analysis of the hydrolysate could be used to confirm the presence of the appropriate base analogue, as shown for the Pl4-containing 19-mer (Fig. lb). Eight ribozyme-substrate complexes were formed by substitution of the four conserved purine nucleoside residues present within the ribozyme sequence. The guanosine residues at positions 10 and 13 were each replaced by 2'deoxyguanosine (dG10 and dG13) and by inosine (110 and 113). In similar fashion, the adenosine residues at positions 11 and 14 were replaced by 2'-deoxyadenosine (dA1" and dA14) and by purine riboside (P"- and pl4). We examined the melting temperatures (Tm) for all eight modified complexes as well as the native complex at a concentration of 1.5 gM in the absence of Mg2+ (1 M NaCI/10 mM sodium phosphate, pH 7.0) and observed that all Tm values for the modified com-

Biochemistry: Fu and McLaughlin

Proc. NatL. Acad. Sci. USA 89 (1992)

a

0 (1-4

10

5

15

20

b G

,0

C U

A

0

5

10

15

20

Retention Time (min) FIG. 1. (a) HPLC analysis of the P'4-containing 19-mer after purification by Mono Q FPLC. Column, 4.6 x 250-mm ODSHypersil (5 tim); buffer, 20 mM KH2PO4 (pH 5.5) with a linear gradient of 0-35% methanol in 30 min. (b) HPLC analysis of the P14-containing 19-mer after hydrolysis with S1 nuclease and bacterial alkaline phosphatase. P. purine. HPLC conditions were as described for a.

plexes were within 1.50C of that of the native complex (Tm =

56.1-C). The cleavage reaction was examined initially under stoichiometric conditions at 370C, pH 8.0. This incubation temperature is less than the reported optimal value (550C) but is identical to that used previously for the study of a series of sequence mutations (7). The stoichiometric complex was studied with a slight excess of ribozyme in comparison to the substrate sequence. Under these conditions the substrate should be fully complexed and any effects due to product release eliminated. The reactions were initiated by the addition of ribozyme to substrate in the presence of Mg2', and substrate cleavage was monitored by polyacrylamide gel electrophoresis. First-order rate constants (kf) were determined from the half-lives of the reactions after normalizing the extent of reaction to account for small amounts of

3987

uncleaved substrate (=5%) that are present even after an extended incubation time, and the amount of uncleaved substrate was plotted as a function of time (Fig. 2). The half-life of the reaction for the native sequence (til2 = 6.2 min, kf = 0.11 min') obtained under these conditions is similar to that reported by Uhlenbeck and coworkers (4, 7) (til2 = 5 min, kf = 0.14 min-). The results for the deletion-modified ribozyme sequences were compared with those of the native sequence under identical conditions; the relative rates of cleavage (k,,) are noted with the corresponding analogue substitution in Fig. 3. The deletion of some individual purine amino and hydroxyl groups resulted in significant variations in the rate of the cleavage reaction. For example, the deletion of either of the adenine amino groups at positions 11 and 14 (P11 and p14) did not alter the relative rate of cleavage significantly (krel = 0.79 and 0.90, respectively). Substitution of hypoxanthine for guanine at position 13 (krl = 0.36) had only a moderate effect, but the same substitution by hypoxanthine at position 10 resulted in a 25-fold reduction in rate (krel = 0.043). The adenine amino groups would be present in the major groove of a duplex structure, while the guanine amino groups would appear in the minor groove. The observed relative rates suggest that of the residues probed, only a single minorgroove substituent, the 2-amino group of guanine at position 10, is critical for efficient cleavage. The results obtained for the substitution of hypoxanthine for guanine at position 10 are similar to earlier qualitative observations (30). Koizumi and Otsuka (19) had observed that the presence of guanosine at the cleavage site dramatically reduced cleavage activity. They have substituted inosine for cytidine at the cleavage site and observed significant changes in activity (19) and have theorized that the presence of the guanine 2-amino group at the cleavage site may induce significant steric hindrance. In contrast, the guanine amino group at position 10 does not interfere with the reaction but may be involved in a critical hydrogen-bonding interaction to assist in stabilizing the complex or to precisely position the metal cofactor. Deletion of the 2'-hydroxyl residues from adenine at positions 11 and 14 did not significantly alter the rate of reaction (krel = 0.79 and 1.91, respectively). However, similar deletions at positions 10 (dG'0) and 13 (dG13) reduced the rate of reaction by =60-fold (krei = 0.015) and =20-fold (krel = 0.046), respectively. The dramatic differences in relative rates of reaction for the dA substitutions vs. the dG substitutions suggest that two hydroxyls at positions 10 and 13 play a critical role in the reaction whereas those at 11 and 14 are less important. The results obtained from the substitutions at

C 0

CZ

0.1

0

100

200

300 400 Time (min)

500

600

FIG. 2. Plots of the logarithm of the concentration of uncleaved substrate vs. time for four selected ribozymes: the native sequence (e), the 1l'-containing sequence (A), the dG3-containing sequence (o), and the dG'0-containing sequence (o).

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Proc. Natl. Acad. Sci. USA 89 (1992)

III c C

G

A U C-G A-U

Cleavage Site

C UCGAGC I AGCUCGG AC ,U ,G 5,

A A

5,

GCGCC U

II

d6

G

A1'4

c1o

G13U A1

FIG. 3. Illustration of the relative first-order cleavage rates (kre,) and the corresponding nucleoside analogue. The nine conserved single-stranded nucleoside residues are represented by "outlined" letters.

positions 11 and 14 are consistent with those reported by Olsen et al. (8) for a series of dA and 2'-deoxy-2'-fluoroadenosine substitutions in a related ribozyme-substrate complex (studied in the presence of Mn'+). Substitution of dA or the corresponding fluoro derivative for A'4, or the double substitution at A"l and A14, did not drastically alter the efficiency of the reaction. Perreault et al. (25) have examined a series of ribozymes that have been singly or multiply substituted by deoxynucleosides (in the presence of Mg2+) and have reported a 20-fold decrease in activity for the A14 -* dA14 substitution. We observed no such effects for this substitution in the present complex. A similar 20-fold decrease in cleavage rate was observed with deletion of the neighboring 2'-hydroxyl (at G"), while Perreault et al. observed little effect of the 2'-hydroxyl at this position. It is difficult to resolve this discrepancy. It is possible that similar but alternative conformations may be available for a given ribozyme-substrate complex depending on the choice of substrate, ribozyme, and metal ion cofactor, so that adjacent hydroxyls may be available interchangeably for key interactions. The 60-fold decrease in cleavage rate for the G10 dG10 substitution is similar to but larger than that reported by Perreault et al. (25) for a related ribozyme-substrate complex. This may also reflect the differences in complexes studied. The decrease in reaction rate for the I -, dG13-, and dG10-containing sequences could result from the inability of the modified complexes to effectively position the Mg2+ cofactor. To examine this possibility, we monitored the stoichiometric reactions in the presence of a 5-fold higher concentration of Mg2+ (50 mM). This increase in cofactor concentration resulted in a small (2-fold) reduction in the half-life for the native complex (t1/2 = 3.2 min). By compar-

ison, the half-lives for the three modified sequences were significantly reduced. At high Mg2' concentration the relative rate for I" complex increased from 0.039 to 0.16, only a factor of 6 slower than the native complex. Similar increases in relative rates from 0.046 to 0.13 and 0.015 to 0.088 were obtained for the dG" and dG10 complexes, respectively. The increase in cleavage rates by 1 order of magnitude at higher Mg2+ concentration suggests that all three functional groups may be involved in positioning the Mg2` ion for efficient hydrolysis. However, we cannot at this time exclude the possibility that the increased Mg2' exerts its effect in a purely structural manner to assist in making a more productive complex. The three complexes that appear to have lost a critical functional group (the I10-, dG13-, and dG10-containing oligonucleotides) were further analyzed under catalytic conditions. At 37°C the rate of reaction for these three modified oligonucleotides was reduced to the point that it was difficult to obtain reproducible kinetic parameters. The complexes are all stable at higher temperatures (Tm = 56.1 1.5°C) and the catalytic parameters have been determined for the native sequence previously at 55°C (4). At this temperature we were able to derive Michaelis-Menten parameters by using a ribozyme concentration of 0.1 ,M and substrate concentrations from 0.4 to 30 ,uM at pH 8.0 and 10 mM Mg2 Under these conditions, the native complex exhibited a Km of0.88 ,uM and a kcat of0.94 min-' (Table 1). These values are in reasonable agreement with those reported by Uhlenbeck (4) for the identical complex (Km = 0.62 ,uM, kcat = 0.5 min-'). The I10- dG'3-, and dGl'-containing fragments under similar conditions (0.2 ,M ribozyme and 0.4-30 ,uM substrate) all exhibited higher Km values and lower kcat values than the native complex (Table 1). The poor catalytic efficiency for the three modified complexes ±

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Biochemistry:

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McLaughlin

Table 1. Kinetic parameters of native and deletionmodified ribozyme 103-kcat/Km Ribozyme kcat, mind Kmi, JM 1100 Native 0.88 0.94 110 3.3 4.2 0.014 0.82 6.9 0.0057 dG1O 3.5 dG13 13 0.048 Reactions were conducted in 50 mM Tris HCI, pH 8.0/10 mM MgCl2 with 0.2 uM ribozyme at various substrate concentrations, 550C. Parameters were derived from values obtained during the initial 10%o of substrate cleavage.

is the result of an =10-fold increase in Km with the same relative decrease in kcat. This results in apparent bimolecular rate constants (kcat/Km) that are >2 orders of magnitude smaller than that observed for the native sequence (Table 1). The decrease in catalytic efficiency with the deletion of a single functional group is more dramatic for this bimolecular hammerhead complex than observed with other complexes, and this observation may reflect the choice of complex in which a relatively large 24-mer substrate is employed with the similarly sized (19-mer) ribozyme. It is unclear how closely the Km value approximates the dissociation constant for the complex, since we have no data for substrate or product on and off rates. The k, at value may reflect the rate of the chemical reaction but could also be related to product release. However, the simplest interpretation of these changes in parameters suggests that the two 2'-hydroxyls and the guanine amino group are important for both substrate binding and efficient chemical catalysis. Hydrogen-bonding interactions with a hydrated Mg2+ cofactor could thus be important for both ribozymesubstrate binding and the positioning of the metal cofactor to function as an efficient electrophilic catalyst. Note Added in Proof. After this manuscript was accepted for publication, Williams et al. (31) reported that deletion of the 2'-OH from the guanosine corresponding to G13 in a related ribozyme complex results in a catalytic activity that is reduced by a factor of at least 150.

This work was supported by the National Science Foundation (DMB-8904306). L.W.M. is the recipient of an American Cancer Society Faculty Research Award (FRA-384). 1. Symons, R. H. (1989) Trends Biochem. Sci. 14, 445-450. 2. Bruening, G. (1989) in RNA Processing, eds. Dahlberg, J. E. & Abelson, J. N. (Academic, New York), pp. 546-558.

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3. Sheldon, C. C. & Symons, R. H. (1989) Nucleic Acids Res. 18, 5433-5411. 4. Uhlenbeck, 0. C. (1987) Nature (London) 328, 596-600. 5. Forster, A. C. & Symons, R. H. (1987) Cell 50, 9-16. 6. Fedor, M. J. & Uhlenbeck, 0. C. (1990) Proc. Natl. Acad. Sci. USA 87, 1668-1672. 7. Ruffner, D. E., Stormo, G. D. & Uhlenbeck, 0. C. (1990) Biochemistry 29, 10695-10702. 8. Olsen, D. B., Benseler, F., Aurup, H., Pieken, W. A. & Eckstein, F. (1991) Biochemistry 30, 9735-9741. 9. Haseloff, J. & Gerlach, W. L. (1988) Nature (London) 334, 585-591. 10. Koizumi, M., Ewai, S. & Otsuka, E. (1988) FEBS Lett. 239, 285-288. 11. Jefferies, A. C. & Symons, R. H. (1989) Nucleic Acids Res. 17, 1371-1377. 12. Koizumi, M., Hayase, Y., Iwai, S., Kamiya, H., Inoue, H. & Otsuka, E. (1989) Nucleic Acids Res. 17, 7059-7070. 13. Ruffner, D. L., Dahm, S. C. & Uhlenbeck, 0. C. (1989) Gene 82, 31-41. 14. Usher, D. A., Erenrich, E. S. & Eckstein, F. (1972) Proc. Natl. Acad. Sci. USA 69, 115-119. 15. Buzayan, J. M., Tol, H. v., Feldstein, P. A. & Bruening, G. (1990) Nucleic Acids Res. 18, 4447-4451. 16. Ruffner, D. E. & Uhlenbeck, 0. C. (1990) Nucleic Acids Res. 18, 6025-6033. 17. van Tol, H., Buzayan, J. M., Feldstein, P. A., Eckstein, R. & Bruening, G. (1990) Nucleic Acids Res. 18, 1971-1975. 18. Slim, G. & Gait, M. J. (1991) NucleicAcids Res. 19, 1183-1188. 19. Koizumi, M. & Otsuka, E. (1991) Biochemistry 30, 5145-5150. 20. Mei, H.-Y., Kaaret, T. W. & Bruice, T. C. (1989) Proc. Natl. Acad. Sci. USA 86, 9727-9731. 21. Heus, H. A. & Pardi, A. (1991) J. Mol. Biol. 217, 113-124. 22. Sampson, J. R., Direnzo, A. B., Behlen, L. S. & Uhlenbeck, 0. C. (1990) Biochemistry 29, 2523-2532. 23. Perreault, J.-P., Wu, T.-F., Cousineau, B., Ogilvie, K. K. & Cedergren, R. (1990) Nature (London) 344, 565-567. 24. Yang, J.-H., Perreault, J.-P., Labuda, D., Usman, N. & Cedergren, R. (1990) Biochemistry 29, 11156-11160. 25. Perreault, J.-P., Labuda, D., Usman, N., Yang, J.-H. & Cedergren, R. (1991) Biochemistry 30, 4020-4025. 26. Pieken, W. A., Olsen, D. B., Benseler, F., Aurup, H. & Eckstein, F. (1991) Science 253, 314-316. 27. Hakimelahi, G. H., Proba, Z. A. & Ogilvie, K. K. (1982) Can. J. Chem. 60, 1106-1113. 28. Sung, W. L. & Narang, S. A. (1982) Can. J. Chem. 60, 111120. 29. Scaringe, S. A., Francklyn, C. & Usman, N. (1990) Nucleic Acids Res. 18, 5433-5441. 30. Odai, O., Hiroaki, H., Sakata, T., Tanaka, T. & Uesugi, S. (1990) FEBS Lett. 267, 150-152. 31. Williams, D. M., Pieken, W. A. & Eckstein, F. (1992) Proc. Natl. Acad. Sci. USA 89, 918-921.

Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.

Eight modified ribozymes of 19 residues have been prepared with individual purine amino or hydroxyl groups excised. The modified ribozymes were chemic...
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