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

Importance of specific purine amino and hydroxyl groups forefficient cleavage by a hammerhead ribozymeDONG-JING FU AND LARRY W. MCLAUGHLINDepartment 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)

ABSTRACT Eight modified ribozymes of 19 residues havebeen prepared with individual purine amino or hydroxylgroups excised. The modified ribozymes were chemically syn-thesized with the substitution of a single 2'-deoxyadenosine,2'-deoxyguanosine, inosine, or purine riboside for residuesG10, A"l, G3, or A". Five of the modified ribozymes cleavedthe 24-mer substrate with little change in rate as monitored bysimple first-order kinetics. However, deletion of the 2-aminogroup at G10 (replacement with inosine) or deletion of either ofthe 2'-hydroxyls at G10 or G13 (replacement with 2'-deoxy-guanosine) resulted in ribozymes with a drastic decrease incleavage efficiency. Increasing the concentration of the Mg2+cofactor from 10mM to 50mM significantly enhanced cleavageefficiency by these three derivatives. Steady-state kinetic assaysfor these three ribozymes indicated that the modificationsresult in both an increase in K. and a decrease in kct,. Theseresults suggest that the exocyclic amino group at G1' and thehydroxyls at G"' and G13 are important both for ribozyme-substrate binding and for the Mg2+-catalyzed cleavage reac-tion.

RNA-processing reactions that involve the cleavage of phos-phodiester bonds are critical steps for the production ofmanymature RNAs from corresponding precursors and appear tobe important in the replication of several plant satellite RNAs(for reviews see refs. 1-3). Autolytic self-cleavage regions arepresent in certain virusoid RNAs and occur in a commonstructural domain termed a "hammerhead" (4). The consen-sus structure of a self-cleaving hammerhead contains 13conserved nucleotides held together by three helical regions(4, 5). Nine of the conserved nucleotides occur in nonhelicalregions and are critical for the observed cleavage reaction.Sequence variations in the helical regions can alter the rate ofthe reaction (6), but only the A-U and possibly the C-G basepair at the base of stem III, at the 5' side of the cleavage site,appear to be critical for activity (7). In addition to thehammerhead structural domain, a metal cofactor is requiredfor the observed processing reaction. Mg2" is usually em-ployed, but Mn2+ appears to function equally well in somecases (8). Cleavage occurs at the phosphodiester bond 3' tothe 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 formedfrom two (or even three) oligonucleotides (9-12), as long asthe conserved nucleotide residues and the hammerheadstructure are maintained. Hammerheads composed of twoRNA fragments can exist in three distinct complexes differingin the location of the respective 3' and 5' termini and thehairpin loop. All three complexes exhibit the self-cleavagereaction 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-

tion ofthe 2'-hydroxyl with the release ofthe 5'-hydroxyl andgeneration of a 2',3'-cyclic phosphodiester. In this respectthe mechanism is similar to the first step of the hydrolysisreaction catalyzed by pancreatic ribonuclease (14). By usingsubstrates containing chiral phosphorothioate diesters, threephosphodiester residues have been identified that appear tobe critical for efficient cleavage (15, 16). Studies employingtwo phosphorothioate diastereomers at the cleavage sitesuggest that the Mg2+ cofactor is bound to the pro-R oxygenin the unmodified complex and that hydrolysis occurs with anin-line mechanism (17-19).The structure adopted by the ribozyme and its substrate in

the active catalytic complex is presently unknown. Modelingstudies of the Lucerne transient streak virusoid (20) based onenergy minimization and computational dynamics have sug-gested that the cytidine residue on the 3' side of the scissilephosphodiester is on the surface of the complex and does notinteract with other bases. This conformation forces theribose-phosphate backbone to make an abrupt turn as itbridges the helical stems I and III, which in turn directs thepro-R and pro-S oxygens ofthe 3'-phosphodiester toward theinward side of the hammerhead where complexation with theMg2+ 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 conservednucleotides present in the core hammerhead structure indi-cate that all nine of the conserved single-stranded nucleotideresidues are critical for cleavage activity (7, 10, 11, 13, 22).In all cases, mutations of the nine nonhelical conservedresidues did not alter the ability of the hammerhead complexto form, but cleavage activity was eliminated or rates werereduced by >1 order of magnitude. These results can beinterpreted to reflect a structure in which the conservedresidues orient the position(s) of specific functional groups inthe active site such that base-base interactions or the abilityof the complex to bind and position the Mg2+ cofactor areoptimized. Sequence mutations can be expected to alter thisorganization of the active site, similar to that observed forspecific amino acid mutations in the active sites of certainenzymes, and reduce the catalytic competence of the ri-bozyme. The devastating effects observed for base substitu-tions within the nine conserved nonhelical residues suggestthat the location of specific interbase or Mg2+-nucleosideinteractions will be best accomplished by mutagenesis at theatomic level (that is, by specifically removing individual baseor carbohydrate functional groups without complete basesubstitution).A number of modified ribozymes have been prepared in

which specific (or multiple) 2'-deoxyribonucleoside residueshave 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 cumulativedecrease in activity without implicating a specific criticalresidue. The use of 2'-fluoro or 2'-amino derivatives providesnuclease-resistant ribozymes (26).

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3986 Biochemistry: Fu and McLaughlin

In the present work we describe the effects on catalyticefficiency that result from the deletion of individual purinenucleobase amino groups or purine 2'-hydroxyl groups in ahammerhead ribozyme.

MATERIALS AND METHODSPhosphoramidites. The nucleosides inosine and purine ri-

boside were obtained from Sigma and converted to thecorresponding 5'-O-(4,4'-dimethoxytrityl)-2'-O-t-butyldi-methylsilyl-3'-O-[(N,N-diisopropylamino)(,B-cyanoethoxy)-phosphinyl] derivative essentially by the procedures of Ogil-vie and coworkers (27), Sung and Narang (28), and Usmanand coworkers (29).* These procedures will be described indetail elsewhere. The 2'-deoxynucleoside phosphoramiditeswere obtained from Cruachem (Sterling, VA) and the riboderivatives were products of MilliGen/Biosearch (Woburn,MA).

Oligonucleotide Synthesis. The oligonucleotides were syn-thesized on controlled-pore glass supports in an AppliedBiosystems 380A DNA synthesizer. After deprotection bybase (concentrated ammonium hydroxide/ethanol, 3:1, for 6hr at 500C) and anhydrous fluoride (1.0 M tetrabutylammo-nium fluoride in tetrahydrofuran for 16 hr at ambient tem-perature) the oligonucleotides were desalted and then iso-lated 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 sodiumcacodylate (pH 6.0) with a gradient of NaCl (0-0.45 M over30 ml followed by 0.45-0.55 M over 60 ml). The 19-merstypically eluted in the range of 40-47 ml, whereas the 24-mereluted at the end of the gradient (90 ml). After isolation, thefragments were desalted (Sephadex G-10) and lyophilized todryness.

Nucleoside Analysis. Nucleoside composition was deter-mined after S1 nuclease/bacterial alkaline phosphatase hy-drolysis. A 10-,ul reaction mixture containing 0.5 A260 unit ofoligomer in 200 mM NaCl/5 mM MgCl2/0.1 mM ZnSO4/25mM sodium acetate, pH 5.5, was incubated for 5 min atambient temperature. To this solution was added 5 ,ul of 100mM Tris-HCl (pH 8.0) and 1 unit of bacterial alkaline phos-phatase. After an additional 60 min incubation at ambienttemperature, a 5-,ul aliquot was analyzed by HPLC on anODS-Hypersil column (4.6 x 250 mm) in 20 mM sodiumphosphate (pH 5.5) and a gradient of 0-70% methanol (60min).

Radioisotopic Labeling. The 24-mer was 5'-end-labeledwith [y-32P]ATP as follows. A 50-Al reaction mixture con-taining 1 A260 unit of 24-mer (-0.1 mM), 10 mM MgCl2, 10mM dithiothreitol, 0.2 mM Na2EDTA, 0.1 mM ATP, 300-600,uCi of [y-32P]ATP (1 ,Ci = 37 kBq), and 20 units of T4polynucleotide kinase was incubated for 60 min at 37°C. Theproduct was isolated by adsorption on a C18 Sep-Pak car-tridge (Waters). The cartridge was washed with water andthen with 40-50% methanol in water to elute the product. Thelabeled 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 de-salted with a C18 Sep-Pak cartridge. The specific activity ofthe 24-mer was typically 0.01 ,Ci/pmol.

*We had some difficulty in monitoring the final reaction (conversionof the protected nucleoside to the 3'-phosphoramidite) by thin-layerchromatography as described for the common nucleoside deriva-tives (29). However, by observing the H1, resonance in the 1H NMRspectrum, we could confirm that complete formation of the phos-phoramidite had taken place. For example, 5'-O-dimethoxytrityl-2'-O-t-butyldimethylsilylpurine riboside exhibited a single H1, dou-blet centered at 6.14 ppm, while the corresponding N,N-diisopropyl-,8-cyanoethylphosphoramidite derivative exhibited twoH1, doublets centered at 6.21 and 6.27 ppm with no remaining signalat 6.14 ppm.

Stoichiometric Cleavage Analysis. Two 50-,p1 solutions con-taining either 0.6 ,uM ribozyme or 0.4 AM substrate in 50 mMTris-HCl (pH 8.0) with 10 mM or 50 mM MgCl2 were eachheated briefly to 90'C and cooled to 370C. The reaction wasinitiated by mixing the two solutions. Aliquots of 10 ,u1 werewithdrawn, and the reaction was quenched by the addition of1 volume of50mM Na2EDTA/7 M urea/10% glycerol/0.05%xylene cyanol/0.05% bromphenol blue. The extent of cleav-age was analyzed by electrophoresis in denaturing polyacryl-amide gels (14 x 16cm). After autoradiography, the substrateand product bands were excised and lyophilized to dryness,and the radioactivity was determined by scintillation count-ing.

Catalytic Cleavage Analysis. These reactions were per-formed and monitored as described above at 550C in 40 1.l of10 mM MgCl2/50 mM Tris-HCI, pH 8.0. The ribozymeconcentration in these reactions was 0.1 ,M (native se-quence) or 0.2 ,uM (110, dG'0, and dG13 sequences), and fromfour to eight substrate concentrations were used that variedfrom 0.4 to 40 ,uM depending on the individual sequence.Aliquots of 4 ,ul were taken from the reaction mixture atvarious times and quenched and analyzed as describedabove. Kinetic parameters were obtained from linear Line-weaver-Burk and Eadie-Hofstee plots and by curve fitting tothe hyperbolic plots of velocity vs. substrate concentration.

RESULTS AND DISCUSSIONTo examine the role of specific purine amino and hydroxylfunctional groups in ribozyme activity, we prepared a numberof oligonucleotides with base analogues in which specificfunctional groups had been excised. The deletion of thepurine exocyclic amino groups of the bases adenine andguanine was accomplished by the introduction of purine andhypoxanthine. The corresponding nucleosides [purine ribo-side (P) and inosine (I)] were converted to the phosphor-amidite building blocks by standard procedures (27, 28) inwhich the 2'-hydroxyl groups were protected as the t-bu-tyldimethylsilyl ethers.The ribozyme complex, identical with that described by

Uhlenbeck (ref. 4; see also Fig. 3), was formed by thesynthesis of two RNA fragments of 19 and 24 nucleotides inlength. The use of these relatively short RNA fragmentssimplified analyses for purity and for the presence of thenucleoside analogue. This complex is identical with that usedby Ruffner et al. (7) for a corresponding study of a series ofsequence mutations. The 24-mer substrate and the 19-mernative and modified ribozymes were synthesized on con-trolled-pore glass supports. After deprotection and purifica-tion of the ribozyme fragments (see Materials and Methods)the sequences were analyzed for purity by HPLC (as illus-trated in Fig. la for the P14-containing 19-mer) and bypolyacrylamide gel electrophoresis. A small amount of eachRNA sequence was degraded with S1 nuclease and bacterialalkaline phosphatase. HPLC analysis of the hydrolysatecould be used to confirm the presence of the appropriate baseanalogue, as shown for the Pl4-containing 19-mer (Fig. lb).

Eight ribozyme-substrate complexes were formed by sub-stitution of the four conserved purine nucleoside residuespresent within the ribozyme sequence. The guanosine resi-dues 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 and14 were replaced by 2'-deoxyadenosine (dA1" and dA14) andby purine riboside (P"- and pl4). We examined the meltingtemperatures (Tm) for all eight modified complexes as well asthe native complex at a concentration of 1.5 gM in theabsence of Mg2+ (1 M NaCI/10 mM sodium phosphate, pH7.0) and observed that all Tm values for the modified com-

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a

(1-4

,0

0 5 10 15 20

bG

C

U

A

0 5 10 15 20

Retention Time (min)

FIG. 1. (a) HPLC analysis of the P'4-containing 19-mer afterpurification by Mono Q FPLC. Column, 4.6 x 250-mm ODS-Hypersil (5 tim); buffer, 20 mM KH2PO4 (pH 5.5) with a lineargradient of 0-35% methanol in 30 min. (b) HPLC analysis of theP14-containing 19-mer after hydrolysis with S1 nuclease and bacterialalkaline phosphatase. P. purine. HPLC conditions were as describedfor a.

plexes were within 1.50C of that of the native complex (Tm =56.1-C).The cleavage reaction was examined initially under stoi-

chiometric conditions at 370C, pH 8.0. This incubation tem-perature is less than the reported optimal value (550C) but isidentical to that used previously for the study of a series ofsequence mutations (7). The stoichiometric complex wasstudied with a slight excess of ribozyme in comparison to thesubstrate sequence. Under these conditions the substrateshould be fully complexed and any effects due to productrelease eliminated. The reactions were initiated by the addi-tion of ribozyme to substrate in the presence of Mg2', andsubstrate cleavage was monitored by polyacrylamide gelelectrophoresis. First-order rate constants (kf) were deter-mined from the half-lives of the reactions after normalizingthe extent of reaction to account for small amounts of

uncleaved substrate (=5%) that are present even after anextended incubation time, and the amount of uncleavedsubstrate was plotted as a function of time (Fig. 2). Thehalf-life of the reaction for the native sequence (til2 = 6.2 min,kf = 0.11 min') obtained under these conditions is similar tothat reported by Uhlenbeck and coworkers (4, 7) (til2 = 5 min,kf = 0.14 min-). The results for the deletion-modifiedribozyme sequences were compared with those of the nativesequence under identical conditions; the relative rates ofcleavage (k,,) are noted with the corresponding analoguesubstitution in Fig. 3.The deletion of some individual purine amino and hydroxyl

groups resulted in significant variations in the rate of thecleavage reaction. For example, the deletion of either of theadenine amino groups at positions 11 and 14 (P11 and p14) didnot alter the relative rate of cleavage significantly (krel = 0.79and 0.90, respectively). Substitution of hypoxanthine forguanine at position 13 (krl = 0.36) had only a moderate effect,but the same substitution by hypoxanthine at position 10resulted in a 25-fold reduction in rate (krel = 0.043). Theadenine amino groups would be present in the major grooveof a duplex structure, while the guanine amino groups wouldappear in the minor groove. The observed relative ratessuggest that of the residues probed, only a single minor-groove substituent, the 2-amino group of guanine at position10, is critical for efficient cleavage. The results obtained forthe substitution of hypoxanthine for guanine at position 10are similar to earlier qualitative observations (30). Koizumiand Otsuka (19) had observed that the presence of guanosineat the cleavage site dramatically reduced cleavage activity.They have substituted inosine for cytidine at the cleavage siteand observed significant changes in activity (19) and havetheorized that the presence of the guanine 2-amino group atthe cleavage site may induce significant steric hindrance. Incontrast, the guanine amino group at position 10 does notinterfere with the reaction but may be involved in a criticalhydrogen-bonding interaction to assist in stabilizing the com-plex or to precisely position the metal cofactor.

Deletion of the 2'-hydroxyl residues from adenine at po-sitions 11 and 14 did not significantly alter the rate ofreaction(krel = 0.79 and 1.91, respectively). However, similar dele-tions at positions 10 (dG'0) and 13 (dG13) reduced the rate ofreaction by =60-fold (krei = 0.015) and =20-fold (krel = 0.046),respectively. The dramatic differences in relative rates ofreaction for the dA substitutions vs. the dG substitutionssuggest that two hydroxyls at positions 10 and 13 play acritical role in the reaction whereas those at 11 and 14 are lessimportant. The results obtained from the substitutions at

C0

CZ

0.10 100 200 300 400 500 600

Time (min)

FIG. 2. Plots of the logarithm of the concentration of uncleavedsubstrate 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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3988 Biochemistry: Fu and McLaughlin

IIIc

C GA U Cleavage Site

A

GCGCCd6 G A1'

C-G

A-UA C UCGAGC

AGCUCGGAC ,U ,GU

4 c1o

G13U A1

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

positions 11 and 14 are consistent with those reported byOlsen et al. (8) for a series of dA and 2'-deoxy-2'-fluoro-adenosine substitutions in a related ribozyme-substrate com-plex (studied in the presence of Mn'+). Substitution of dA orthe corresponding fluoro derivative for A'4, or the doublesubstitution at A"l and A14, did not drastically alter theefficiency of the reaction. Perreault et al. (25) have examineda series of ribozymes that have been singly or multiplysubstituted 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 thissubstitution in the present complex. A similar 20-fold de-crease in cleavage rate was observed with deletion of theneighboring 2'-hydroxyl (at G"), while Perreault et al. ob-served little effect of the 2'-hydroxyl at this position. It isdifficult to resolve this discrepancy. It is possible that similarbut alternative conformations may be available for a givenribozyme-substrate complex depending on the choice ofsubstrate, ribozyme, and metal ion cofactor, so that adjacenthydroxyls may be available interchangeably for key interac-tions. The 60-fold decrease in cleavage rate for the G10dG10 substitution is similar to but larger than that reported byPerreault et al. (25) for a related ribozyme-substrate com-plex. This may also reflect the differences in complexesstudied.The decrease in reaction rate for the I -, dG13-, and

dG10-containing sequences could result from the inability ofthe modified complexes to effectively position the Mg2+cofactor. To examine this possibility, we monitored thestoichiometric reactions in the presence of a 5-fold higherconcentration of Mg2+ (50 mM). This increase in cofactorconcentration resulted in a small (2-fold) reduction in thehalf-life for the native complex (t1/2 = 3.2 min). By compar-

ison, the half-lives for the three modified sequences weresignificantly reduced. At high Mg2' concentration the rela-tive rate for I" complex increased from 0.039 to 0.16, only afactor of 6 slower than the native complex. Similar increasesin relative rates from 0.046 to 0.13 and 0.015 to 0.088 wereobtained for the dG" and dG10 complexes, respectively. Theincrease in cleavage rates by 1 order of magnitude at higherMg2+ concentration suggests that all three functional groupsmay be involved in positioning the Mg2` ion for efficienthydrolysis. However, we cannot at this time exclude thepossibility that the increased Mg2' exerts its effect in a purelystructural manner to assist in making a more productivecomplex.The three complexes that appear to have lost a critical

functional group (the I10-, dG13-, and dG10-containing oligo-nucleotides) were further analyzed under catalytic conditions.At 37°C the rate of reaction for these three modified oligonu-cleotides was reduced to the point that it was difficult to obtainreproducible kinetic parameters. The complexes are all stableat higher temperatures (Tm = 56.1 ± 1.5°C) and the catalyticparameters have been determined for the native sequencepreviously at 55°C (4). At this temperature we were able toderive Michaelis-Menten parameters by using a ribozymeconcentration of0.1 ,M and substrate concentrations from 0.4to 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 of0.94min-' (Table 1). These values are in reasonable agreementwith those reported by Uhlenbeck (4) for the identical complex(Km = 0.62 ,uM, kcat = 0.5 min-'). The I10- dG'3-, anddGl'-containing fragments under similar conditions (0.2 ,Mribozyme and 0.4-30 ,uM substrate) all exhibited higher Kmvalues and lower kcat values than the native complex (Table 1).The poor catalytic efficiency for the three modified complexes

5,

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Table 1. Kinetic parameters of native and deletion-modified ribozyme

Ribozyme Kmi, JM kcat, mind 103-kcat/KmNative 0.88 0.94 1100110 4.2 0.014 3.3dG1O 6.9 0.0057 0.82dG13 13 0.048 3.5Reactions 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 initial10%o of substrate cleavage.

is the result of an =10-fold increase in Km with the samerelative decrease in kcat. This results in apparent bimolecularrate constants (kcat/Km) that are >2 orders of magnitudesmaller than that observed for the native sequence (Table 1).The decrease in catalytic efficiency with the deletion ofa singlefunctional group is more dramatic for this bimolecular ham-merhead complex than observed with other complexes, andthis observation may reflect the choice of complex in which arelatively large 24-mer substrate is employed with the similarlysized (19-mer) ribozyme. It is unclear how closely the Kmvalue approximates the dissociation constant for the complex,since we have no data for substrate or product on and offrates.The k, at value may reflect the rate of the chemical reaction butcould also be related to product release. However, the sim-plest interpretation of these changes in parameters suggeststhat the two 2'-hydroxyls and the guanine amino group areimportant for both substrate binding and efficient chemicalcatalysis. Hydrogen-bonding interactions with a hydratedMg2+ cofactor could thus be important for both ribozyme-substrate binding and the positioning of the metal cofactor tofunction as an efficient electrophilic catalyst.

Note Added in Proof. After this manuscript was accepted for publi-cation, Williams et al. (31) reported that deletion of the 2'-OH fromthe guanosine corresponding to G13 in a related ribozyme complexresults 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 CancerSociety Faculty Research Award (FRA-384).

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