Proc. Natl. Acad. Sci. USA Vol. 89, pp. 918-921, February 1992 Biochemistry
Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications (RNA enzyme/2'-modified guanosine/critical nuceotides)
DAVID M. WILLIAMS, WOLFGANG A. PIEKEN, AND FRITZ ECKSTEIN* Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, Hermann-Rein Strasse 3, W-3400 G6ttingen, Federal Republic of Germany
Communicated by Leslie E. Orgel, October 15, 1991 (receivedfor review August 12, 1991)
G-5
The importance of the 2'-hydroxyl group of ABSTRACT several guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. Five ribozymes in which single guanosine residues were substituted with 2'amino-, 2'-fluoro-, or 2'-deoxyguanosine were chemically synthesized. The comparison of the catalytic activity of the three 2' modifications at a specific position allows conclusions about the functional role of the parent 2'-hydroxyl group. Substitutions of nonconserved nucleotides within the ribozyme caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, when either of the guanosines within the single-stranded loop between stem I and stem H of the ribozyme was replaced by 2'-deoxyguanosine or 2'-fluoro-2'-deoxyguanosine, the catalytic activities of the resulting ribozymes were reduced by factors of at least 150. The catalytic activities of the corresponding ribozymes containing 2'-amino-2'-deoxyguanosine substitutions at these positions, however, were both reduced by factors of 15. These effects resulted from decreases in the respective k,,t values, whereas variations in the Km values were comparatively small. A different pattern of reactivity of the three 2' modifications was observed at the guanosine immediately 3' to stem II of the ribozyme. Whereas both 2'-deoxyguanosine and 2'-amino-2'deoxyguanosine at this position showed catalytic activity similar to that of the unmodified ribozyme, the activity of the corresponding 2'-fluoro-2'-deoxyguanosine-containing ribozyme was reduced by a factor of 15. The implications ofthese substitution-specific reactivities on the functional role of the native 2'-hydroxyl groups are discussed.
"
-
G
C --I G
III
U -- A
//~ A
22
U U 20
G
S
G
A C- rC3 A
C C G G
U
.'..
k
5
U
.,
iG
CG
---
18 17
11
A
FIG. 1. Structure of the hammerhead ribozyme-substrate complex. Boxed regions contain the invariant nucleotides; shaded guanosines are those under study here; E, ribozyme; S, substrate; arrow, position of cleavage; Roman numerals, position of stems.
replacement of 2'-hydroxyl functions at selected ribonucleotide positions has on the catalytic efficiency of the ribozyme. The substitution of ribonucleotides by 2'-deoxyribonucleotides can be valuable in identifying specific hydroxyl groups of conserved nucleotides that are essential to catalysis. Perreault et al. (8, 9) demonstrated that when the nucleotides G9 or A13t in their hammerhead ribozyme were replaced by their 2'-deoxynucleotide counterparts, the kcat values were reduced by factors of 14 and 20, respectively. These authors have proposed that at least these two hydroxyl groups of the ribozyme and a hydroxyl group of the uridine next to the cleavage site in the substrate may be involved in binding magnesium, which is required for efficient catalytic activity
The processing of several RNAs associated with a number of plant viruses involves a self-cleavage reaction within a structural domain consisting of three helices and 13 conserved nucleotides known as a hammerhead (for reviews, see refs. 1 and 2). Although this hammerhead structure can be encompassed within a single RNA strand, it may also comprise an enzyme part and a substrate part (3) as shown in Fig. 1. Such hammerhead ribozymes can be designed to cleave almost any target RNA as long as it contains certain trinucleotides of the sequence NUX (where N is any nucleotide and X is preferably a cytidine but cannot be a guanosine) (4, 5). The cleavage reaction is facilitated by divalent cations such as Mg2+ or Mn2' and takes place on the 3' side of the third nucleotide (X) producing a 5'-hydroxyl and a 2',3'cyclic phosphate product (3). The scissile phosphodiester linkage is cleaved with inversion of configuration at phosphorus (6, 7). However, it still remains unclear why and how the catalytic activity of the ribozyme is superior with oligoribo- rather than oligodeoxyribonucleotides (8). One approach toward answering this question is to determine what effect the
(8, 9).
We have examined the catalytic efficiencies of hammerhead ribozymes (as shown in Fig. 1) containing either 2'amino-2'-deoxy- or 2'-fluoro-2'-deoxynucleotides at selected positions (10, 11). Substitutions by these analogues are potentially informative for two reasons. For one, they differ markedly in their preference of 3'-endo, which is characteristic for ribonucleosides, over 2'-endo conformation, with *To whom reprint requests should be addressed. tThe positions G9 and A13 in the hammerhead ribozyme described by Perreault et al. (8, 9) correspond to positions G10 and A14 in the ribozymes investigated here.
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.
918
Biochemistry: Williams et al. 2'-fluoro-2'-deoxynucleosides displaying a 3'-endo/2'-endo ratio greater than that of ribonucleosides and 2'-amino-2'deoxynucleosides favoring the 2'-endo conformation even more than 2'-deoxynucleosides do (12, 13). Second, these 2' modifications differ in their ability to participate in hydrogen bonding. Whereas the 2'-fluorine atom can serve only as hydrogen-bond acceptor, the 2'-amino group can act as both donor and acceptor, similar to the 2'-hydroxyl group of ribose. A pKa value of 6.2 has been reported for the amino group of 2'-amino-2'-deoxyuridine (14) and the corresponding value in polymers is =6.0 (15). Thus, at pH 7.5 used in the cleavage reactions in the present study, this group would be expected to be unprotonated. The 2'-deoxy moiety, of course, does not participate in hydrogen bonding. We have shown that any one of the adenosine residues of the hammerhead shown in Fig. 1 can be replaced by either the 2'-fluoro or the 2'-deoxy analogues without loss of activity (11), indicating that none of these 2'-hydroxyl groups are critical for activity. When all the cytidine residues were replaced by their 2'-fluoro counterparts, again, no significant decrease of activity was observed and the same substitution of all the uridines led to only a 5-fold reduction (10). This also demonstrates that the pyrimidine nucleoside 2'-hydroxyl groups are not essential for activity. We describe here the systematic replacement of five guanosines (positions 10, 13, 19, 24, and 27) in a ribozyme with 2'-deoxy-, 2'-fluoro-2'-deoxy-, or 2'-amino-2'-deoxyguanosines (Fig. 1) and compare the effects of the various 2' modifications on the kinetic parameters.
MATERIALS AND METHODS T4 polynucleotide kinase was obtained from United States Biochemical. [y-32P]ATP (10 ,uCi/,l; 1 Ci = 37 GBq) was obtained from Amersham. X-ray film (X-Omat) was purchased from Kodak. Preparation of Oligoribonucleotides. 2'-Fluoro-2'-deoxyguanosine was synthesized using diethylaminosulfur trifluoride by a procedure similar to that described for 2'-fluoro2'-deoxyadenosine (11). 2'-Amino-2'-deoxyguanosine was synthesized as described (16). The 2'-amino group of 2'amino-2'-deoxyguanosine was protected by trifluoro acetylation (16), followed by protection of the N2 amino group as the dimethylformamidine derivative (17, 18). The 3'-O-(2cyanoethyl N,N-diisopropylphosphoramidites) of the analogues were prepared as described for 2'-fluorothymidine (19). The 2'-O-(t-butyldimethylsilyl)-3 '-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) monomers of normal ribonucleotides were obtained from Milligen/Biosearch. Oligoribonucleotides were prepared on a 1-,umol scale and purified as described (10), being 5'-32P-labeled prior to purification by PAGE. Oligonucleotide concentrations were determined by assuming a residue extinction coefficient at 260 nm of 6.6 x 103 M-1-cm-1 (20). Characterization of Oligoribonucleotides. The homogeneity of both ribozyme enzyme and ribozyme substrate was confirmed by PAGE of the 5'-32P-labeled oligoribonucleotides followed by autoradiography. The positions of 2'-amino-, 2'-fluoro-, or 2'-deoxyguanosine within the ribozymes were verified by partial alkaline hydrolysis (10, 21). Determination of Ribozyme Steady-State Parameters. Cleavage reactions were performed in the presence of Mg2+, and kinetic constants were determined essentially as described (10). Stock solutions of 100 nM ribozyme and 2 ,uM substrate were prepared in 50 mM Tris-HCI (pH 7.5), preheated separately at 95°C for 1 min, and cooled to 25°C for 15 min. The ribozyme stock solution was adjusted to 10 mM MgCl2 and incubated at 250C for 15 min. The cleavage reactions were performed in 10 mM MgCl2 and 50 mM Tris HCl (pH 7.5) using a ribozyme concentration of 10 nM,
Proc. Natl. Acad. Sci. USA 89 (1992)
919
and the 5'-32P-labeled substrate was supplied at five concentrations between 100 and 700 nM. For the catalytically less-efficient ribozymes, which possessed either a 2'-fluoro2'-deoxy- or 2'-deoxyguanosine at position 10 or 13, the concentrations were 20-50 nM and 200-1000 nM, respectively. The cleavage reactions were performed at 250C in 50 pLI and were initiated by adding substrate to the solutions containing ribozyme and briefly vortex mixing. Samples of 10 A.l were removed at appropriate times between 1 and 180 min and quenched with 20 MAI of stop mixture (7 M urea/50 mM EDTA/0.04% bromophenol blue/0.04% xylene cyanol)/ water, 1:1 (vol/vol). The cleavage-reaction samples (20 1l) were analyzed by PAGE on 20% denaturing gels, and the autoradiographs were subsequently quantitated using scanning laser densitometry as described (10). All kinetic parameters were determined from EadieHofstee plots. For any given ribozyme, kcat and Km were found to vary by a factor of -2. These values increase or decrease by the same factor between experiments, so that the kcat/Km values of different ribozymes give the most reliable assessment of their catalytic properties (22). RESULTS The hammerhead chosen for these studies (Fig. 1) was as described (10, 11), since it has been shown by Fedor and Uhlenbeck (20) to have a high turnover number and thus be particularly suitable for kinetic studies. Both the modified and unmodified hammerhead oligoribonucleotides were obtained by automated chemical synthesis, and the locations of the analogues were confirmed by alkaline hydrolysis (10, 21). The effects of single 2' modifications of the guanosines (Fig. 1) at positions 10 and 13 of the invariant region are displayed in Table 1. A dramatic reduction in catalytic efficiency of ==150-fold for ribozymes containing 2'deoxyguanosines at these positions was observed. This reduction in efficiency is predominantly due to a decrease of nearly three orders of magnitude of kcat, whereas the Km values for these two modified ribozymes deviate only by a factor of 3 from that of the unmodified ribozyme. Only when the enzyme concentration was increased to 20 and 40 nM could the catalytic activity of these ribozyme analogues be measured. A qualitatively similar result was obtained when these guanosines were substituted by 2'-fluoro-2'-deoxyguanosines (Table 1). However, the presence of 2'-amino-2'deoxyguanosine at position 10 or 13 led to reductions in catalytic efficiencies by factors of only 10, compared to the unmodified ribozyme. The cleavage rates of ribozymes with 2'-amino modifications at these positions are at least an order of magnitude higher than those of ribozymes carrying 2'Table 1. Kinetic parameters of hammerhead ribozymes carrying 2' modifications at positions 10 and 13
Ribozyme Native
E-dG10
E-FG1o E-NG1o
kcat,
Ki,
kcat/Km,
min' 2.31 0.006 0.04 0.8 0.005
nM 154 41 308 153 51 227 190
/AM-'-min' 15.0 0.1* 0.2* 1.2 0.1*
E-dG13 0.008 E-FG13 0.04* 0.16 0.8 E-NG13 E-dG", ribozyme carrying 2'-deoxyguanosine at position n; E-NGn, ribozyme carrying 2'-amino-2'-deoxyguanosine at position n; E-FGn, ribozyme carrying 2'-fluoro-2'-deoxyguanosine at position n.
*Values determined with higher enzyme concentrations than the standard reaction.
920
Biochemistry: Williams et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
Table 2. Kinetic parameters of hammerhead ribozymes carrying 2' modifications at positions 19 and 24
Ribozyme Native
E-dG19 E-FG19 E-NG19 E-dG24 E-FG24 E-NG24 Abbreviations
kcat,
min2.31 3.29 2.8 1.21 1.30 2.8 0.83 are as in Table 1.
Km, nM 154 158 176 95 87 116 66
kcat/Km,
jM-1 mink 15.0 20.8 15.9 12.7 14.9 24 12.6
deoxy or 2'-fluoro modifications. Thus, this modification can at least in part fulfill the role of the hydroxyl group. Essentially no change in catalytic efficiency was seen when 2'-deoxyguanosines or 2'-fluoro-2'-deoxyguanosines were incorporated at position 19 or 24 (Table 2). The same was true for 2'-amino-2'-deoxyguanosines at these positions. Therefore, the 2'-hydroxyl groups of G19 and G24 are not crucial to the function of the hammerhead ribozyme. At position 27 again no effect on cleavage efficiency was observed by the introduction of the 2'-deoxy modification. However, a significant difference was seen for the 2'-fluoro derivative, which displayed a kcat of 0.3 min1 compared to the value of 2.3 min' for the unmodified ribozyme and, interestingly, was also slower than the 2'-deoxy derivative (Table 3). The activity of the ribozyme containing the 2'amino analogue at position 27 was e5-fold higher than that of the corresponding 2'-fluoro derivative and only -2-fold lower than that of the corresponding 2'-deoxyguanosine and guanosine-containing ribozymes. Again, these decreased activities resulted principally from changes in the respective kcat values.
DISCUSSION The structural and conformational requirements of a hammerhead ribozyme necessary for catalytic activity are still not adequately defined. Indeed, previous studies have demonstrated that the hammerhead ribozyme does not have to be composed exclusively of ribonucleotides. Thus, the pyrimidine nucleosides and the adenosines can be replaced by the 2'-fluoro derivatives without significant loss of activity (10, 11). In addition, certain nucleotides in the invariant region can be replaced by their 2'-deoxy analogues with some retention of activity. Thus, Perreault et al. (8, 9), for a slightly different construct than ours, have reported that 2'deoxynucleotide substitution of G9t led to a reduction in catalytic activity by a factor of -20 relative to the unmodified ribozyme. A small decrease was also noted when G12 (corresponding to G13 in the present study) was replaced by 2'-deoxyguanosine. In each case, these values were the consequences of a decrease in the kcat; the Km values remained essentially unchanged. The results presented here identify the 2'-hydroxyl groups of guanosines at positions 10 and 13 of the chosen hammerhead as critical for activity. These are the guanosines that Table 3. Kinetic parameters of hammerhead ribozymes carrying 2' modifications at position 27
kcats
Ribozyme Native E-dG27 E-FG27 E-NG27 Abbreviations are as
min-' 2.31 4.78 0.3 0.92 in Table 1.
Km, nM 154 242 210 136
kcat/Km,
AM-'-min' 15.0 19.8 1.4 6.8
upon replacement of their ribose moiety by 2'-deoxyribose display dramatic decreases of =150-fold in catalytic activity. Replacement of these two guanosines by 2'-fluoro-2'deoxyguanosine results in ribozymes that have comparably low activities. However, replacement of the 2'-deoxyribonucleoside by the 2'-amino-2'-deoxynucleoside restores activity to a level of il~ot of the unmodified ribozyme. The decrease in activity found by Perreault et al. (9) for the ribozyme containing a 2'-deoxyguanosine substitution at the position corresponding to G10t was about an order of magnitude smaller than the decrease observed in this investigation. The larger effect observed upon replacing this guanosine by 2'-deoxyguanosine (or also by 2'-fluoro-2'-deoxyguanosine) in our case may be due to slight differences between the two ribozyme systems, such as small differences in sequence, lengths of stems, and, possibly most significantly, the presence of the trinucleotide CUC rather than GUC at the cleavage site. A mutagenesis study of another hammerhead system revealed that such changes to the sequence on the 5' side of the cleavage site can dramatically affect the cleavage rate (5). Since the differences in reaction temperature employed for the two studies might be responsible for the difference in results, we additionally determined the kinetic parameters at 370C for both the unmodified ribozyme and the ribozyme containing 2'-deoxyguanosine at position 10 (data not shown). At this temperature, the catalytic activity of the 2'-deoxyguanosine-containing ribozyme was reduced by a factor of ==180 relative to the unmodified ribozyme. This value is of the same order of magnitude as the differences in catalytic activities determined for the two ribozymes at 250C (Table 1). However, Perreault et al. (9) have reported a difference of 14 in the catalytic activities of the corresponding ribozymes in their study. Therefore, it seems likely that the variation in base sequence between the two ribozyme systems is responsible for the observed relative differences in catalytic activity between the unmodified and 2'-deoxyguanosine-substituted ribozymes. The fact that we see a comparatively large effect at G13 may also be a consequence of sequence differences between the two systems. A comparison of these results suggests that there is some flexibility in the formation of a catalytically active hammerhead structure. Furthermore, it should be pointed out that no single 2' substitution, except at the site of cleavage, abolishes catalysis completely. The maximal decreases in activity of two orders of magnitude are small compared to effects typically seen for substitutions of catalytically crucial residues in active sites of proteins (23). Therefore, the present analysis of the role of specific 2'-hydroxyl groups is based on differences in catalytic activity rather than all-or-none effects. The difference in reactivity at positions 10 and 13 of 2'-deoxy and 2'-fluoro modifications compared to that of the 2'-amino group allows some conclusion about the function of the 2'-hydroxyl groups at these positions in the native ribozyme. One role this group could play would lie in maintaining a particular conformation of the nucleosides in question. A comparative study of the conformations of uridine, 2'-deoxyuridine, 2'-fluoro-2'-deoxyuridine, and 2'-amino-2'deoxyuridine (13) in aqueous solution shows that the percent of 3'-endo conformer is 58%, 40%, 87%, and 25%, respectively. If conformation were the critical feature, little loss in catalytic activity would be expected for the 2'-fluoro analogues as these nucleoside analogues have predominantly the 3'-endo conformation, very much like the ribonucleosides. The conformation of the 2'-amino-2'-deoxynucleosides, on the other hand, is predominantly 2'-endo and thus more akin to that of 2'-deoxynucleosides (13). On a purely conformational argument, the ribozyme containing this modification would be expected to have an activity similar to that of the 2'-deoxyguanosine-containing ribozyme, both of which
Biochemistry: Williams et al. would be dissimilar to the activity of the 2'-fluoro-modified species. This, however, is not the case. If, however, hydrogen bonding is the prevalent function of the 2'-hydroxyl group, then one may distinguish between its function as a hydrogen-bond donor or acceptor. Although the riboside, the 2'-amino analogue, and the 2'-fluoro analogue share the capability to function as hydrogen-bond acceptors, only the former two can function as hydrogen-bond donors. Thus, for a role as acceptor, no pronounced difference in catalytic activity for these three modifications is predicted. However, as the ribozymes modified with a 2'-amino group at position 10 or 13 are much more active than the 2'-fluoromodified counterparts, hydrogen-donor capability might be the quality that is most important for activity. This analysis does not contradict the suggested role of the 2'-hydroxyl group at position 10 in binding of the Mg2e cofactor (9), as long as it is hydrated. However, an alternate explanation for the observed reactivity pattern may be the direct coordination of the 2'-hydroxyl group to a Mg2+ ion. At position G27 the pattern of reactivity is quite different. Here the 2'-fluoro-modified analogue reacts an order of magnitude slower than the unmodified and the 2'-deoxymodified ribozyme. The reactivity of the 2'-amino-modified species is only slightly decreased. Since the 2'-deoxy modification is tolerated at this position, the native 2'-hydroxyl group does not seem to be involved in catalytically critical hydrogen bonding. The fact that the 2'-fluoro modification, which favors the 3'-endo conformation, is the least tolerated may reflect the conformational restraints imposed on this guanosine by its location adjacent to stem II. This study demonstrates that the two guanosines in positions 10 and 13 in the ribozyme under study are the most sensitive to replacement of the 2'-hydroxyl group by a variety of substituents and that 2'-amino groups can partially fulfill their role. The results provide some suggestions as to the possible function of these hydroxyl groups. We are greatly indebted to F. Benseler for his expertise in nucleoside analogue and oligonucleotide synthesis and U. Kutzke for expert technical assistance. We thank the Deutsche Forschungsgemeinschaft for support (Ec 28/14-1).
Proc. Natl. Acad. Sci. USA 89 (1992)
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1. Sheldon, C. C., Jeffries, A. C., Davies, C. & Symons, R. H. (1990) in Nucleic Acids and Molecular Biology, eds. Eckstein, F. & Lilley, D. M. J. (Springer, Berlin), Vol. 4, pp. 227-242. 2. Symons, R. H. (1989) Trends Biochem. Sci. 14, 445-450. 3. Uhlenbeck, 0. C. (1987) Nature (London) 328, 596-600. 4. Haseloff, J. & Gerlach, W. L. (1988) Nature (London) 334, 585-591. 5. Ruffner, D. E., Stormo, G. D. & Uhlenbeck, 0. C. (1990) Biochemistry 29, 10695-10702. 6. van Tol, H., Buzayan, J. M., Feldstein, P. A., Eckstein, F. & Bruening, G. (1990) Nucleic Acids Res. 18, 1971-1975. 7. Slim, G. & Gait, M. J. (1991) Nucleic Acids Res. 19, 1183-1188. 8. Perreault, J.-P., Wu, T., Cousineau, B., Ogilvie, K. K. & Cedergren, R. (1990) Nature (London) 344, 565-567. 9. Perreault, J.-P., Labuda, D., Usman, N., Yang, J.-H. & Cedergren, R. (1991) Biochemistry 30, 4020-4025. 10. Pieken, W. A., Olsen, D. B., Benseler, F., Aurup, H. & Eckstein, F. (1991) Science 253, 314-317. 11. Olsen, D. B., Benseler, F., Aurup, H., Pieken, W. A. & Eckstein, F. (1991) Biochemistry 30, 9735-9741. 12. Saenger, W. (1984) in Principles ofNucleic Acid Structure, ed. Saenger, W. (Springer, New York), pp. 51-178. 13. Guschlbauer, W. & Jankowski, K. (1980) Nucleic Acids Res. 8, 1421-1433. 14. Verheyden, J. P. H., Wagner, D. & Moffatt, J. G. (1971) J. Org. Chem. 36, 250-254. 15. Hobbs, J., Sternbach, H., Sprinzl, M. & Eckstein, F. (1973) Biochemistry 12, 5138-5145. 16. Imazawa, M. & Eckstein, F. (1979) J. Org. Chem. 44, 20392041. 17. McBride, L. J., Kierzek, R., Beaucage, S. L. & Caruthers, M. H. (1986) J. Am. Chem. Soc. 108, 2040-2048. 18. Vu, H., McCollum, C., Jacobson, K., Theisen, P., Vinayak, R., Spiess, E. & Andrus, A. (1990) Tetrahedron Lett. 31, 72697272. 19. Williams, D. M., Benseler, F. & Eckstein, F. (1991) Biochemistry 30, 4001-4009. 20. Fedor, M. J. & Uhlenbeck, 0. C. (1990) Proc. Natl. Acad. Sci. USA 87, 1668-1672. 21. Beijer, B., Sulston, I., Sproat, B., Rider, P., Lamond, A. I. & Neuner, P. (1990) Nucleic Acids Res. 18, 5143-5151. 22. Fersht, A. (1985) Enzyme, Structure and Mechanism (Freeman, New York), 2nd Ed., pp. 98-120. 23. Gerlt, J. A. (1987) Chem. Rev. 87, 1079-1105.
Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications (RNA enzyme/2'-modified guanosine/critical nuceotides)
DAVID M. WILLIAMS, WOLFGANG A. PIEKEN, AND FRITZ ECKSTEIN* Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, Hermann-Rein Strasse 3, W-3400 G6ttingen, Federal Republic of Germany
Communicated by Leslie E. Orgel, October 15, 1991 (receivedfor review August 12, 1991)
G-5
The importance of the 2'-hydroxyl group of ABSTRACT several guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. Five ribozymes in which single guanosine residues were substituted with 2'amino-, 2'-fluoro-, or 2'-deoxyguanosine were chemically synthesized. The comparison of the catalytic activity of the three 2' modifications at a specific position allows conclusions about the functional role of the parent 2'-hydroxyl group. Substitutions of nonconserved nucleotides within the ribozyme caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, when either of the guanosines within the single-stranded loop between stem I and stem H of the ribozyme was replaced by 2'-deoxyguanosine or 2'-fluoro-2'-deoxyguanosine, the catalytic activities of the resulting ribozymes were reduced by factors of at least 150. The catalytic activities of the corresponding ribozymes containing 2'-amino-2'-deoxyguanosine substitutions at these positions, however, were both reduced by factors of 15. These effects resulted from decreases in the respective k,,t values, whereas variations in the Km values were comparatively small. A different pattern of reactivity of the three 2' modifications was observed at the guanosine immediately 3' to stem II of the ribozyme. Whereas both 2'-deoxyguanosine and 2'-amino-2'deoxyguanosine at this position showed catalytic activity similar to that of the unmodified ribozyme, the activity of the corresponding 2'-fluoro-2'-deoxyguanosine-containing ribozyme was reduced by a factor of 15. The implications ofthese substitution-specific reactivities on the functional role of the native 2'-hydroxyl groups are discussed.
"
-
G
C --I G
III
U -- A
//~ A
22
U U 20
G
S
G
A C- rC3 A
C C G G
U
.'..
k
5
U
.,
iG
CG
---
18 17
11
A
FIG. 1. Structure of the hammerhead ribozyme-substrate complex. Boxed regions contain the invariant nucleotides; shaded guanosines are those under study here; E, ribozyme; S, substrate; arrow, position of cleavage; Roman numerals, position of stems.
replacement of 2'-hydroxyl functions at selected ribonucleotide positions has on the catalytic efficiency of the ribozyme. The substitution of ribonucleotides by 2'-deoxyribonucleotides can be valuable in identifying specific hydroxyl groups of conserved nucleotides that are essential to catalysis. Perreault et al. (8, 9) demonstrated that when the nucleotides G9 or A13t in their hammerhead ribozyme were replaced by their 2'-deoxynucleotide counterparts, the kcat values were reduced by factors of 14 and 20, respectively. These authors have proposed that at least these two hydroxyl groups of the ribozyme and a hydroxyl group of the uridine next to the cleavage site in the substrate may be involved in binding magnesium, which is required for efficient catalytic activity
The processing of several RNAs associated with a number of plant viruses involves a self-cleavage reaction within a structural domain consisting of three helices and 13 conserved nucleotides known as a hammerhead (for reviews, see refs. 1 and 2). Although this hammerhead structure can be encompassed within a single RNA strand, it may also comprise an enzyme part and a substrate part (3) as shown in Fig. 1. Such hammerhead ribozymes can be designed to cleave almost any target RNA as long as it contains certain trinucleotides of the sequence NUX (where N is any nucleotide and X is preferably a cytidine but cannot be a guanosine) (4, 5). The cleavage reaction is facilitated by divalent cations such as Mg2+ or Mn2' and takes place on the 3' side of the third nucleotide (X) producing a 5'-hydroxyl and a 2',3'cyclic phosphate product (3). The scissile phosphodiester linkage is cleaved with inversion of configuration at phosphorus (6, 7). However, it still remains unclear why and how the catalytic activity of the ribozyme is superior with oligoribo- rather than oligodeoxyribonucleotides (8). One approach toward answering this question is to determine what effect the
(8, 9).
We have examined the catalytic efficiencies of hammerhead ribozymes (as shown in Fig. 1) containing either 2'amino-2'-deoxy- or 2'-fluoro-2'-deoxynucleotides at selected positions (10, 11). Substitutions by these analogues are potentially informative for two reasons. For one, they differ markedly in their preference of 3'-endo, which is characteristic for ribonucleosides, over 2'-endo conformation, with *To whom reprint requests should be addressed. tThe positions G9 and A13 in the hammerhead ribozyme described by Perreault et al. (8, 9) correspond to positions G10 and A14 in the ribozymes investigated here.
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.
918
Biochemistry: Williams et al. 2'-fluoro-2'-deoxynucleosides displaying a 3'-endo/2'-endo ratio greater than that of ribonucleosides and 2'-amino-2'deoxynucleosides favoring the 2'-endo conformation even more than 2'-deoxynucleosides do (12, 13). Second, these 2' modifications differ in their ability to participate in hydrogen bonding. Whereas the 2'-fluorine atom can serve only as hydrogen-bond acceptor, the 2'-amino group can act as both donor and acceptor, similar to the 2'-hydroxyl group of ribose. A pKa value of 6.2 has been reported for the amino group of 2'-amino-2'-deoxyuridine (14) and the corresponding value in polymers is =6.0 (15). Thus, at pH 7.5 used in the cleavage reactions in the present study, this group would be expected to be unprotonated. The 2'-deoxy moiety, of course, does not participate in hydrogen bonding. We have shown that any one of the adenosine residues of the hammerhead shown in Fig. 1 can be replaced by either the 2'-fluoro or the 2'-deoxy analogues without loss of activity (11), indicating that none of these 2'-hydroxyl groups are critical for activity. When all the cytidine residues were replaced by their 2'-fluoro counterparts, again, no significant decrease of activity was observed and the same substitution of all the uridines led to only a 5-fold reduction (10). This also demonstrates that the pyrimidine nucleoside 2'-hydroxyl groups are not essential for activity. We describe here the systematic replacement of five guanosines (positions 10, 13, 19, 24, and 27) in a ribozyme with 2'-deoxy-, 2'-fluoro-2'-deoxy-, or 2'-amino-2'-deoxyguanosines (Fig. 1) and compare the effects of the various 2' modifications on the kinetic parameters.
MATERIALS AND METHODS T4 polynucleotide kinase was obtained from United States Biochemical. [y-32P]ATP (10 ,uCi/,l; 1 Ci = 37 GBq) was obtained from Amersham. X-ray film (X-Omat) was purchased from Kodak. Preparation of Oligoribonucleotides. 2'-Fluoro-2'-deoxyguanosine was synthesized using diethylaminosulfur trifluoride by a procedure similar to that described for 2'-fluoro2'-deoxyadenosine (11). 2'-Amino-2'-deoxyguanosine was synthesized as described (16). The 2'-amino group of 2'amino-2'-deoxyguanosine was protected by trifluoro acetylation (16), followed by protection of the N2 amino group as the dimethylformamidine derivative (17, 18). The 3'-O-(2cyanoethyl N,N-diisopropylphosphoramidites) of the analogues were prepared as described for 2'-fluorothymidine (19). The 2'-O-(t-butyldimethylsilyl)-3 '-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) monomers of normal ribonucleotides were obtained from Milligen/Biosearch. Oligoribonucleotides were prepared on a 1-,umol scale and purified as described (10), being 5'-32P-labeled prior to purification by PAGE. Oligonucleotide concentrations were determined by assuming a residue extinction coefficient at 260 nm of 6.6 x 103 M-1-cm-1 (20). Characterization of Oligoribonucleotides. The homogeneity of both ribozyme enzyme and ribozyme substrate was confirmed by PAGE of the 5'-32P-labeled oligoribonucleotides followed by autoradiography. The positions of 2'-amino-, 2'-fluoro-, or 2'-deoxyguanosine within the ribozymes were verified by partial alkaline hydrolysis (10, 21). Determination of Ribozyme Steady-State Parameters. Cleavage reactions were performed in the presence of Mg2+, and kinetic constants were determined essentially as described (10). Stock solutions of 100 nM ribozyme and 2 ,uM substrate were prepared in 50 mM Tris-HCI (pH 7.5), preheated separately at 95°C for 1 min, and cooled to 25°C for 15 min. The ribozyme stock solution was adjusted to 10 mM MgCl2 and incubated at 250C for 15 min. The cleavage reactions were performed in 10 mM MgCl2 and 50 mM Tris HCl (pH 7.5) using a ribozyme concentration of 10 nM,
Proc. Natl. Acad. Sci. USA 89 (1992)
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and the 5'-32P-labeled substrate was supplied at five concentrations between 100 and 700 nM. For the catalytically less-efficient ribozymes, which possessed either a 2'-fluoro2'-deoxy- or 2'-deoxyguanosine at position 10 or 13, the concentrations were 20-50 nM and 200-1000 nM, respectively. The cleavage reactions were performed at 250C in 50 pLI and were initiated by adding substrate to the solutions containing ribozyme and briefly vortex mixing. Samples of 10 A.l were removed at appropriate times between 1 and 180 min and quenched with 20 MAI of stop mixture (7 M urea/50 mM EDTA/0.04% bromophenol blue/0.04% xylene cyanol)/ water, 1:1 (vol/vol). The cleavage-reaction samples (20 1l) were analyzed by PAGE on 20% denaturing gels, and the autoradiographs were subsequently quantitated using scanning laser densitometry as described (10). All kinetic parameters were determined from EadieHofstee plots. For any given ribozyme, kcat and Km were found to vary by a factor of -2. These values increase or decrease by the same factor between experiments, so that the kcat/Km values of different ribozymes give the most reliable assessment of their catalytic properties (22). RESULTS The hammerhead chosen for these studies (Fig. 1) was as described (10, 11), since it has been shown by Fedor and Uhlenbeck (20) to have a high turnover number and thus be particularly suitable for kinetic studies. Both the modified and unmodified hammerhead oligoribonucleotides were obtained by automated chemical synthesis, and the locations of the analogues were confirmed by alkaline hydrolysis (10, 21). The effects of single 2' modifications of the guanosines (Fig. 1) at positions 10 and 13 of the invariant region are displayed in Table 1. A dramatic reduction in catalytic efficiency of ==150-fold for ribozymes containing 2'deoxyguanosines at these positions was observed. This reduction in efficiency is predominantly due to a decrease of nearly three orders of magnitude of kcat, whereas the Km values for these two modified ribozymes deviate only by a factor of 3 from that of the unmodified ribozyme. Only when the enzyme concentration was increased to 20 and 40 nM could the catalytic activity of these ribozyme analogues be measured. A qualitatively similar result was obtained when these guanosines were substituted by 2'-fluoro-2'-deoxyguanosines (Table 1). However, the presence of 2'-amino-2'deoxyguanosine at position 10 or 13 led to reductions in catalytic efficiencies by factors of only 10, compared to the unmodified ribozyme. The cleavage rates of ribozymes with 2'-amino modifications at these positions are at least an order of magnitude higher than those of ribozymes carrying 2'Table 1. Kinetic parameters of hammerhead ribozymes carrying 2' modifications at positions 10 and 13
Ribozyme Native
E-dG10
E-FG1o E-NG1o
kcat,
Ki,
kcat/Km,
min' 2.31 0.006 0.04 0.8 0.005
nM 154 41 308 153 51 227 190
/AM-'-min' 15.0 0.1* 0.2* 1.2 0.1*
E-dG13 0.008 E-FG13 0.04* 0.16 0.8 E-NG13 E-dG", ribozyme carrying 2'-deoxyguanosine at position n; E-NGn, ribozyme carrying 2'-amino-2'-deoxyguanosine at position n; E-FGn, ribozyme carrying 2'-fluoro-2'-deoxyguanosine at position n.
*Values determined with higher enzyme concentrations than the standard reaction.
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Proc. Natl. Acad. Sci. USA 89 (1992)
Table 2. Kinetic parameters of hammerhead ribozymes carrying 2' modifications at positions 19 and 24
Ribozyme Native
E-dG19 E-FG19 E-NG19 E-dG24 E-FG24 E-NG24 Abbreviations
kcat,
min2.31 3.29 2.8 1.21 1.30 2.8 0.83 are as in Table 1.
Km, nM 154 158 176 95 87 116 66
kcat/Km,
jM-1 mink 15.0 20.8 15.9 12.7 14.9 24 12.6
deoxy or 2'-fluoro modifications. Thus, this modification can at least in part fulfill the role of the hydroxyl group. Essentially no change in catalytic efficiency was seen when 2'-deoxyguanosines or 2'-fluoro-2'-deoxyguanosines were incorporated at position 19 or 24 (Table 2). The same was true for 2'-amino-2'-deoxyguanosines at these positions. Therefore, the 2'-hydroxyl groups of G19 and G24 are not crucial to the function of the hammerhead ribozyme. At position 27 again no effect on cleavage efficiency was observed by the introduction of the 2'-deoxy modification. However, a significant difference was seen for the 2'-fluoro derivative, which displayed a kcat of 0.3 min1 compared to the value of 2.3 min' for the unmodified ribozyme and, interestingly, was also slower than the 2'-deoxy derivative (Table 3). The activity of the ribozyme containing the 2'amino analogue at position 27 was e5-fold higher than that of the corresponding 2'-fluoro derivative and only -2-fold lower than that of the corresponding 2'-deoxyguanosine and guanosine-containing ribozymes. Again, these decreased activities resulted principally from changes in the respective kcat values.
DISCUSSION The structural and conformational requirements of a hammerhead ribozyme necessary for catalytic activity are still not adequately defined. Indeed, previous studies have demonstrated that the hammerhead ribozyme does not have to be composed exclusively of ribonucleotides. Thus, the pyrimidine nucleosides and the adenosines can be replaced by the 2'-fluoro derivatives without significant loss of activity (10, 11). In addition, certain nucleotides in the invariant region can be replaced by their 2'-deoxy analogues with some retention of activity. Thus, Perreault et al. (8, 9), for a slightly different construct than ours, have reported that 2'deoxynucleotide substitution of G9t led to a reduction in catalytic activity by a factor of -20 relative to the unmodified ribozyme. A small decrease was also noted when G12 (corresponding to G13 in the present study) was replaced by 2'-deoxyguanosine. In each case, these values were the consequences of a decrease in the kcat; the Km values remained essentially unchanged. The results presented here identify the 2'-hydroxyl groups of guanosines at positions 10 and 13 of the chosen hammerhead as critical for activity. These are the guanosines that Table 3. Kinetic parameters of hammerhead ribozymes carrying 2' modifications at position 27
kcats
Ribozyme Native E-dG27 E-FG27 E-NG27 Abbreviations are as
min-' 2.31 4.78 0.3 0.92 in Table 1.
Km, nM 154 242 210 136
kcat/Km,
AM-'-min' 15.0 19.8 1.4 6.8
upon replacement of their ribose moiety by 2'-deoxyribose display dramatic decreases of =150-fold in catalytic activity. Replacement of these two guanosines by 2'-fluoro-2'deoxyguanosine results in ribozymes that have comparably low activities. However, replacement of the 2'-deoxyribonucleoside by the 2'-amino-2'-deoxynucleoside restores activity to a level of il~ot of the unmodified ribozyme. The decrease in activity found by Perreault et al. (9) for the ribozyme containing a 2'-deoxyguanosine substitution at the position corresponding to G10t was about an order of magnitude smaller than the decrease observed in this investigation. The larger effect observed upon replacing this guanosine by 2'-deoxyguanosine (or also by 2'-fluoro-2'-deoxyguanosine) in our case may be due to slight differences between the two ribozyme systems, such as small differences in sequence, lengths of stems, and, possibly most significantly, the presence of the trinucleotide CUC rather than GUC at the cleavage site. A mutagenesis study of another hammerhead system revealed that such changes to the sequence on the 5' side of the cleavage site can dramatically affect the cleavage rate (5). Since the differences in reaction temperature employed for the two studies might be responsible for the difference in results, we additionally determined the kinetic parameters at 370C for both the unmodified ribozyme and the ribozyme containing 2'-deoxyguanosine at position 10 (data not shown). At this temperature, the catalytic activity of the 2'-deoxyguanosine-containing ribozyme was reduced by a factor of ==180 relative to the unmodified ribozyme. This value is of the same order of magnitude as the differences in catalytic activities determined for the two ribozymes at 250C (Table 1). However, Perreault et al. (9) have reported a difference of 14 in the catalytic activities of the corresponding ribozymes in their study. Therefore, it seems likely that the variation in base sequence between the two ribozyme systems is responsible for the observed relative differences in catalytic activity between the unmodified and 2'-deoxyguanosine-substituted ribozymes. The fact that we see a comparatively large effect at G13 may also be a consequence of sequence differences between the two systems. A comparison of these results suggests that there is some flexibility in the formation of a catalytically active hammerhead structure. Furthermore, it should be pointed out that no single 2' substitution, except at the site of cleavage, abolishes catalysis completely. The maximal decreases in activity of two orders of magnitude are small compared to effects typically seen for substitutions of catalytically crucial residues in active sites of proteins (23). Therefore, the present analysis of the role of specific 2'-hydroxyl groups is based on differences in catalytic activity rather than all-or-none effects. The difference in reactivity at positions 10 and 13 of 2'-deoxy and 2'-fluoro modifications compared to that of the 2'-amino group allows some conclusion about the function of the 2'-hydroxyl groups at these positions in the native ribozyme. One role this group could play would lie in maintaining a particular conformation of the nucleosides in question. A comparative study of the conformations of uridine, 2'-deoxyuridine, 2'-fluoro-2'-deoxyuridine, and 2'-amino-2'deoxyuridine (13) in aqueous solution shows that the percent of 3'-endo conformer is 58%, 40%, 87%, and 25%, respectively. If conformation were the critical feature, little loss in catalytic activity would be expected for the 2'-fluoro analogues as these nucleoside analogues have predominantly the 3'-endo conformation, very much like the ribonucleosides. The conformation of the 2'-amino-2'-deoxynucleosides, on the other hand, is predominantly 2'-endo and thus more akin to that of 2'-deoxynucleosides (13). On a purely conformational argument, the ribozyme containing this modification would be expected to have an activity similar to that of the 2'-deoxyguanosine-containing ribozyme, both of which
Biochemistry: Williams et al. would be dissimilar to the activity of the 2'-fluoro-modified species. This, however, is not the case. If, however, hydrogen bonding is the prevalent function of the 2'-hydroxyl group, then one may distinguish between its function as a hydrogen-bond donor or acceptor. Although the riboside, the 2'-amino analogue, and the 2'-fluoro analogue share the capability to function as hydrogen-bond acceptors, only the former two can function as hydrogen-bond donors. Thus, for a role as acceptor, no pronounced difference in catalytic activity for these three modifications is predicted. However, as the ribozymes modified with a 2'-amino group at position 10 or 13 are much more active than the 2'-fluoromodified counterparts, hydrogen-donor capability might be the quality that is most important for activity. This analysis does not contradict the suggested role of the 2'-hydroxyl group at position 10 in binding of the Mg2e cofactor (9), as long as it is hydrated. However, an alternate explanation for the observed reactivity pattern may be the direct coordination of the 2'-hydroxyl group to a Mg2+ ion. At position G27 the pattern of reactivity is quite different. Here the 2'-fluoro-modified analogue reacts an order of magnitude slower than the unmodified and the 2'-deoxymodified ribozyme. The reactivity of the 2'-amino-modified species is only slightly decreased. Since the 2'-deoxy modification is tolerated at this position, the native 2'-hydroxyl group does not seem to be involved in catalytically critical hydrogen bonding. The fact that the 2'-fluoro modification, which favors the 3'-endo conformation, is the least tolerated may reflect the conformational restraints imposed on this guanosine by its location adjacent to stem II. This study demonstrates that the two guanosines in positions 10 and 13 in the ribozyme under study are the most sensitive to replacement of the 2'-hydroxyl group by a variety of substituents and that 2'-amino groups can partially fulfill their role. The results provide some suggestions as to the possible function of these hydroxyl groups. We are greatly indebted to F. Benseler for his expertise in nucleoside analogue and oligonucleotide synthesis and U. Kutzke for expert technical assistance. We thank the Deutsche Forschungsgemeinschaft for support (Ec 28/14-1).
Proc. Natl. Acad. Sci. USA 89 (1992)
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