Proc. Nat. Acad. Sci. USAVol. 72, No. 1, pp. 122-126, January 1975

T4-Induced RNA Ligase Joins Single-Stranded Oligoribonucleotides(RNA synthesis/RPC-5 chromatography/gradient sievorptive chromatography)

GRAHAM C. WALKER*, OLKE C. UHLENBECK, ELLIOTT BEDOWSt, AND RICHARD I. GUMPORTtDepartment of Biochemistry, School of Chemical Sciences and School of Basic Medical Sciences, and t Department of Microbiology,School of Life Sciences, University of Illinois, Urbana, Ill. 61801

Communicated by N. J. Leonard, October 9, 1974

ABSTRACT RNA ligase isolated from Escherichia coliinfected with bacteriophage T4 will catalyze the formationof an intermolecular 3' -- 5' phosphodiester linkage be-tween an oligoribonucleotide with a free 3'-hydroxyl andanother oligoribonucleotide with a 5'-phosphate. Uponreaction with (Ap)5C, nearly quantitative conversion ofthe hexamer [5'-"2Plp(Up)iU to the dodecamer (Ap)5C[3' -5'-"Plp(Up)5U was observed. The product was identifiedby its mobility on RPC-5 column chromatography, itsresistance to alkaline phosphatase, and the appearance ofthe expected radiolabeled products on hydrolysis withalkali, ribonuclease A, snake venom phosphodiesterase,and spleen phosphodiesterase. The coupling of other pairsof single-stranded oligoribonucleotides has also beendemonstrated. The intermolecular joining reaction isprobably mechanistically similar to the intramolecularcyclization activity previously reported for T4 RNA ligase.It is expected that this enzyme will be useful for the syn-thesis of RNA fragments of defined sequence.

RNA ligase from Escherichia coli infected with bacteriophageT4 catalyzes the ATP-dependent formation of phosphodiesterbonds in RNA (1-3). Two structurally distinct classes ofmolecules have been reported to be polynucleotide substratesfor this enzyme. First, RNA ligase has been shown to form aphosphodiester bond between the 3'-hydroxyl and the 5'-phosphate termini of an oligoribonucleotide, producing asingle-stranded circular product (1, 2). Second, in analogy toT4 DNA ligase (4), T4 RNA ligase also appears capable ofjoining the 3'-hydroxyl end of one ribo-oligomer to the 5'-phosphate of another when the two molecules are aligned in adouble helix by hydrogen-bonding to a third, complementary"splint" strand (3). Although the enzyme has not been puri-fied to homogeneity, it appears likely that the two activitiesare catalyzed by the same protein. The intramolecular cycli-zation reaction ofRNA ligase, which occurs in the absence of a.splint strand, suggests that the critical requirement for ac-tivity of the enzyme is not double helical structure, but simplya high concentration of 3'-hydroxyl with respect to 5'-phosphate groups. If one end of an oligonucleotide binds tothe enzyme and the chain is not too long, the other end wouldbe in very high local concentration, facilitating an intra-molecular reaction. This suggests that an intermolecular reac-tion without a splint strand might be achieved (a) by pre-venting the intramolecular reaction through the use of a 5'-phosphate terminated oligomer too short to cyclize and (b)by using a very high concentration of another oligomer with

hydroxyl groups on both the 3'- and 5'-termini. In this paperwe present evidence for such an intermolecular reaction withT4 RNA ligase and examine the effect of a variety of parame-ters on the efficiency of the reaction. Since high yields ofproduct may be obtained, RNA ligase should be useful in thesynthesis of oligoribonucleotides of defined sequence.

MATERIALS AND METHODS

Materials. Nucleoside 5'-diphosphates were purchased fromSigma Chemical Co., and all other nucleotides were obtainedfrom P-L Biochemicals, Inc. (Ip)5I and endonuclease fromNeuroepora crassa were purchased from Miles Laboratories.Bacterial alkaline phosphatase, ribonuclease A, ribonucleaseT1, and spleen phosphodiesterase were purchased fromWorthington Biochemical Corp. Polynucleotide kinase was agift from C. Richardson. 32Pi was purchased from NewEngland Nuclear Corp. ['y-'2P]ATP was prepared by themethod of Glynn and Chappell (5). Bacteriophage and bac-terial strains were obtained from J. Drake.

Preparation of Oligoribonucleotides. The polyribonucleotidespoly(U), poly(A), poly(A,C), poly(A,U), and poly(A,G) weresynthesized from the corresponding ribonucleoside diphos-phates with polynucleotide phosphorylase from Micrococcusluteus. Oligomers with 3'-phosphate termini and numberaverage chain length (in) = 5-10 were prepared from thepolynucleotides by controlled alkaline hydrolysis or by RNaseA or RNase T1 digestion (6). Poly(A) was digested withNeurospora crassa endonuclease to give a solution of oligo-riboadenylates with a 5'-terminal phosphate and ii = 6 (7).Oligonucleotides in each homologous series were separatedon DEAE-Sephadex A-25 (in the bicarbonate form) by alinear gradient (0.1-1.2 M) of triethylammonium bicarbonate(pH 7.5). When an oligonucleotide was needed without aterminal phosphate, it was treated with bacterial alkalinephosphatase and repurified on a DEAE-Sephadex A-25column.

Oligomers were labeled on the 5'-terminus with [32P]phos-phate, using polynucleotide kinase as described by Silberet al. (1). The labeled products were isolated by thin-layerchromatography on cellulose-coated glass plates, followed bygradient elution from DEAE-Sephadex columns with tri-ethylammonium bicarbonate.

Separation of Oligoribonucleotides by RPC-6 Column Chro-matography. The resin was prepared by mixing Plaskon withAndogen 464 in chloroform as described by Pearson et al. (8).The resin was washed extensively with 0.2 M KC1, 0.01 Mimidazole*HCl (pH 7.0), and 0.7 X 10-cm jacketed columns

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* Current address: Department of Biochemistry, University ofCalifornia, Berkeley, Calif. 94720.t To whom correspondence should be addressed.

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PT4 RNA Ligase Intermolecular Reaction 123

were packed at 370 under pressure from a peristaltic pump.Columns were eluted with 150-ml, 0.2-1.0 M KCl lineargradients in 0.01 M imidazole-HCl (pH 7.0) at the fastestpossible flow rate (about 40 ml/hr), and the effluent wasmonitored at 260 nm on a LKB Uvicord system. As may beseen in Fig. 2B, these conditions are adequate to resolveindividual members of a homologous series of oligoribonucleo-tides to a chain length of twenty. If the gradient is extendedto 2.0 M KCl, more than sixty peaks may be resolved. Fordetermination of [82P]-labeled products, fractions of 1.8 mlwere collected into plastic vials and the Cerenkov irradiationfor the whole fraction was determined.

Assay of RNA Ligase. The intramolecular cyclization reac-tion catalyzed by RNA ligase was used as the routine methodof assay during the purification of the enzyme. The assaymixture was that of Silber et al. (1), except that about 0.021.Ci of [5'-32P]poly(A) (a mixture of oligomers from 22 to27 nucleotide residues in length) at a concentration of from0.02 to 0.74 MAM in termini was the substrate and the totalassay volume was 50 ,l. The reaction mixtures were incubatedat 370 for 30 min and at 1000 for 2 min, and then cooled.Water (50 ul) and 0.1 unit of bacterial alkaline phosphatasewere added, and the mixtures were incubated at 650 for 30min. A 50-IAI aliquot of the cooled reaction mixture was trans-ferred to a 2-cm-square piece of Whatman DEAE-paper(DE81). The papers were dried under an infrared lamp,washed three times by gentle agitation in 50 ml of 0.3 Mammonium formate (pH 8.2), and processed as described (9).One unit is arbitrarily' defined as 1 pmol of [5'-32P]terminusrendered resistant to phosphatase in 30 min at a substrateconcentration of 0.025,uM.The intermolecular reaction of RNA ligase was assayed in a

mixture (25 ul) containing 50 mM Tris HCl (pH 7.5), 0.15mM ATP, bovine serum albumin (10 Mg/ml), 10 mM MgCl2,1.3 mM dithiothreitol, RNA ligase (about 1 pug), and variousRNA oligomers as substrates. A hexaribonucleotide with a5'-terminal [82Pjphosphate (5-20 Ci/mmol) was present inthe reactions at a concentration of about 1 MAM and wascalled the donor molecule. The other RNA oligomer, calledthe acceptor molecule, varied from three to six residues inlength, was unlabeled, had a 5'-hydroxyl group, and waspresent at a concentration of 3-5 mM. Both oligomers had3'-hydroxyl termini unless otherwise stated. The mixture wasincubated at 370 for 15 min and at 100° for 1 min and thencooled. Ten A2w0 units of (Ap)nU markers were added and themixture was analyzed by chromatography on RPC-5 asdescribed.

Purification of RNA Ligase. E. coli B was grown to 5 X108 cells per ml at 370 in 1.5 liters of modified Lbroth (10)and infected with bacteriophage T4r+ at a multiplicity ofinfection of 5. After 16 min of further shaking at 370, themixture was rapidly chilled to 00 by addition of ice. The cellswere collected by centrifugation, resuspended in 50 mM Tris -

HCl (pH 7.5), 1 mM dithiothreitol, 1 mM EDTA (Buffer I),and disrupted by five 15-sec bursts of sonication. The dis-rupted suspension was centrifuged at 27,000 X g at 40 for 10min, and the supernatant was diluted to an A260 of 110 withBuffer I (35.6 ml, 720 total units, 3.7 units/mg). Five percentstreptomycin sulfate was added to a final concentration of0.8% (w/v) with stirring at 00 over a 20-min period and theprecipitate, which contained polynucleotide kinase activity

FRACTION NUMBER

FIG. 1. Purification of RNA ligase. (A) Gradient sievorptiveprofile of the first column. The A2s0 peak (solid curve) appearingat the void volume is primarily protein, the second A28o peak ap-pearing at the total column volume is primarily nucleic acid.RNA ligase activity (dotted curve) was assayed by the intra-molecular cyclization assay described in Materials and Methods.The KC1 concentration (dashed curve) was monitored by conduc-tivity. (B) Sephadex G-75 column profile. The major A28o peak(solid curve) appears at the void volume. RNA ligase activity isassayed by the intramolecular cyclization (dotted curve) and inter-molecular joining (-) assays described in Materials and Methods.

(11), was removed by centrifugation at 27,000 X g for 10min at 4°. The protein in the supernatant was precipitatedwith 70% ammonium sulfate.Gradient sievorptive chromatography (12) was performed

at room temperature by applying a 27.5-ml linear gradientof 0.i75-0.400 M KCl in 0.1 M Tris - HCl (pH 7.5), 1 mM 2-mercaptoethanol, 0.1 mM EDTA to a 1.8 X 38-cm (100 ml)DEAE-Sephadex A-25 column that had been equilibratedwith 0.175 M KCl in the same buffered mixture. The ammo-nium sulfate pellet, which had been dissolved in 3.0 ml of 0.75M KCl, was then applied to the column, washed on with 5ml of 1 M KCl in 50% glycerol, and eluted with 1 M KCl.In order to avoid mixing solutions, liquids were run from thebottom upwards in all the above steps. The flow rate of 40ml/hr was maintained by a peristaltic pump, and 2.1-mlfractions were collected. The absorbance at 280 nm and theRNA ligase activity were determined and their profiles areshown in Fig. 1A. The fractions containing RNA ligase ac-tivity were pooled (GS-I, 19 ml, 990 total units, 151 units/mg) and precipitated with 70% ammonium sulfate. The pre-cipitate was dissolved in 0.8 ml of 0.75 M KCl, and appliedto the bottom of a 0.9 X 30-cm (20 ml) DEAE-SephadexA-25 column which had had a 5.5-ml linear gradient of 0.175-0.400 M KCl in 0.1 M Tris * HCl (pH 7.5), 1 mM 2-mercapto-ethanol, 0.1 mM EDTA pumped onto it as described for thefirst column. The protein was washed onto the column with1 ml of 1 M KCl in 50% glycerol and was eluted with 1 MKCl. Fractions of 0.65 ml were collected at 8 ml/hr. Thetubes containing RNA ligase activity were pooled (GS-II,4.5 ml, 980 total units, 168 units/mg) and precipitated withammonium sulfate as described.

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124 Biochemistry: Walker et al.

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FIG. 2. RPC-5 column chromatography of oligoribonucleo-tides. The percent transmission of the series of (Ap)nU markeroligomers was recorded (dashed curves). 32P-Labeled oligomerswere detected by determining Cerenkov radiation of individualfractions (solid curves). (A) [5'_32Pjp(Up)5U incubated with RNAligase. (B) Intermolecular reaction of [5'-32P]p(Up)5U with(Ap)5C. (C) RNase A digestion product of major 32p peak inpanel B (solid curve) chromatographed with nonradioactive(Ap)5Cp (dashed curve). (D) Intermolecular reaction of [5'-32P]p(Up)5U with (Ap)2C. (E) Intermolecular reaction of [5'-32P]p(Ap)5G with (Ap)5C.

Gel filtration chromatography on Sephadex G-75 was per-

formed at 4° by applying the ammonium sulfate pellet, whichhad been dissolved in 0.5 ml of 20 mM Tris HCl (pH 7.5),100 mM KCl, 1 mM 2-mercaptoethanol, 0.1 mM EDTA, toa 1.8 X 75-cm column which had been equilibrated with thesame buffer. The profiles of the An0 and RNA ligase activityare shown in Fig. 1B. Both the intramolecular cyclizationand intermolecular joining activities appeared to comigrate,suggesting that the two activities were caused by the same

protein. Tubes 39 and 40 were pooled and used for the studiesreported (G-75, 4.5 ml, 261 total units, 549 units/mg). TheG-75 fraction is stable for 2 months or more at 4°.

RESULTS

Intermolecular Reaction and Product Characterization. RNAligase catalyzed the formation of a phosphodiester bond be-tween the 3'-hydroxyl group of (Ap)5C (acceptor) and the 5'-phosphate of [5'-_2P]p(Up)5U (donor).When a reaction mixture containing only the [5' 32P]p-

(Up)5U donor (1 ,M) was incubated with RNA ligase andthen chromatographed on an RPC-5 column, the resultsshown in Fig. 2A were obtained. More than 95% of the inputradioactivity eluted as a single peak at the same salt concen-

tration as unincubated [5'3-2P]p(Up)5U. All of the radioactivity

in the peak could be hydrolyzed by bacterial alkaline phos-phatase, indicating that the [32P]phosphate was in a terminalposition. These control experiments demonstrated that noreaction had occurred, since an intramolecular reaction wouldhave rendered the 5'-phosphate resistant to alkaline phos-phatase and would have reduced the net charge by one,thereby altering the elution position. The base compositiondependence of RPC-5 separations is illustrated by the fact that[5'-32P]p(Up)5U, a hexamer with a terminal phosphate,migrated with (Ap)4U, a pentamer without a terminal phos-phate. The relatively small amount of degradation of the[5'-32P]p(Up)5U observed indicated that the ligase prepara-tion was fairly free of nuclease activity.When a reaction mixture containing both 1 ,uM [5'-32P]p-

(Up)5U (donor) and 3.9 mM (Ap)5C (acceptor) was incubatedwith RNA ligase and chromatographed, the results shown inFig. 2B were obtained. More than 90% of the input radio-activity eluted as a single sharp peak at a slightly higher saltconcentration than the (Ap)8U marker, indicating that anoligonucleotide product significantly larger than [5' 32P]p-(Up)5U had been formed. Since there is a base compositiondependence of the mobility of oligomers on RPC-5, the exactchain length cannot be assigned by comparison with the(Ap)0U markers. It will be shown that the most likely identifi-cation of the single larger product is (Ap)5C [3'-* 5' 32P]pp(Up)5U.

All of the radioactivity in the major peak from Fig. 2B wasresistant to alkaline phosphatase, indicating that the 5'-terminal phosphate of the [5'-32P]p(Up)5U had been convertedto an internal position. When the new oligomer was treatedwith RNase A and was rechromatographed on RPC-5, asingle 32P-labeled product was obtained which migratedexactly with an unlabeled (Ap)5Cp marker (Fig. 2C). Sincethe [32P]phosphate of the material was again sensitive toalkaline phosphatase, the identity of the RNase A product as(Ap)5C ['-32P]p was confirmed. Thus, the sequential treat-ment of the donor and acceptor with RNA ligase and ribo-nuclease resulted in the transfer of the [5'-32P]phosphate from[5'-32P]p(Up)5U to the 3'-hydroxyl of (Ap)5C to yield (Ap)5-C[s'-32P]p, indicating that a 3' to 5' phosphodiester bondhad been formed in the RNA ligase product.The requirement for a free 3'-hydroxyl on the acceptor was

checked by repeating the reaction described in Fig. 2B, butwith an equal concentration of (Ap)5Cp substituted for the(Ap)5C. Upon chromatography on RPC-5, all the radio-activity eluted as unchanged [5'-32P]p(Up)5U, as in Fig. 2A.This observation supported the RNA ligase-catalyzed joiningof the 3'-hydroxyl of (Ap)5C to the 5'-terminal phosphateof [5'-82P]p(Up)6U.The identification of the labeled oligomers displayed in

Fig. 2A and B was confirmed by hydrolysis to nucleotides andanalysis by high voltage paper electrophoresis (13). Whenthe major 32P-labeled compound eluting from RPC-5 (Fig.2A) at the same salt concentration as [5'1-2P]p(Up)5U was

hydrolyzed with alkali, 2'(3'),5'-UDP was obtained as theonly radioactive nucleotide. Hydrolysis with snake venom

phosphodiesterase yielded 5'-UMP, and the compound was

not susceptible to degradation by spleen phosphodiesterase,indicating that it had a 5'-phosphate terminus. These data,taken together with the RPC-5 chromatography profile, indi-cate that RNA ligase does not alter [5'-32P]p(Up)5U in theabsence of an acceptor molecule.

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T4 RNA Ligase Intermolecular Reaction 125

TABLE 1. Intermolecular ligase reactions

Donor Acceptor Yield (%)*

[6'532P]p(Up)5U (Ap)5C 96[5' 32P]p(Up)6U (Ap)3C 46[5'-32P]p(Up)5U (Ap)2C 48[5at32P]p(Ap)5G (Ap)5C 54[6'-32P]p(Ap)5A (Ap)5C 25[6'_32P]p(Ap)4A (Ap)5C 11[5at32P]p(Ip)65 (Ap)5C 22

* Yield was calculated on the basis of % of 32p incorporatedinto the intermolecular product. Since the reaction conditionswere not identical in each case and were not optimized, differencesin yield do not necessarily reflect the specificity of the enzyme.

When the major peak from Fig. 2B, which contained theintermolecular reaction product, was hydrolyzed witbl alkali,the only radioactive product obtained was 2'(3')-CMP.Hydrolysis with spleen phosphodiesterase yielded the sameproduct, but degradation with snake venom phosphodiesterasegave 5'-UMP as the only radioactive nucleotide. These ob-servations confirm that RNA ligase had catalyzed the forma-tion of a phosphodiester bond between the 3'-hydroxyl of acytidyl residue and the 5'-phosphate of a uridyl residue. Theseresults, along with the RPC-5 chromatographic behavior andRNase A susceptibility of the oligomer, are entirely consistentwith the assignment of the structure, (Ap)5C[3'-'5'-32P]p-(Up)5U, to the product resulting from the intermolecularreaction.

Variation of Substrates. The chain length dependence ofthe acceptor in the intermolecular reaction was examined byincubating the donor [5'-32P]p(Up)5U with the acceptors(Ap)3C or (Ap)2C and RNA ligase under the same conditionsdescribed in Fig. 2B. The elution point of the product onRPC-5 chromatography was dependent on the chain lengthof the acceptor. With (Ap)5C the new peak eluted just afterthe (Ap)8U marker, but with the acceptors (Ap)3C and(Ap)2C the products eluted with the (Ap)7U and just after the(Ap)6U markers, respectively (Fig. 2D). The product peakswere identified as the intermolecular adducts as in the previoussection. In each case, substitution of the corresponding(Ap).Cp for the (Ap)nC eliminated any reaction and [5'-32P]p(Up)5U was the only radioactive product recovered.Several 5'-phosphate donors besides [5'-32P]p(Up)5U have

been tested as substrates for intermolecular reactions. Reac-tion mixtures containing the acceptor (Ap)5C and one of thedonors [5'-32P]p(Ap)5G, [5'-32P]p(Ap)5A, [5'-32P]p(Ap)4A,or [5'-32P]p(Ip)5I were incubated in the presence of RNAligase as described in Fig. 2B and then were chromatographedon RPC-5. In each case a new radioactive product was formedwhich eluted at a higher salt concentration than the unreacteddonor, as shown in Fig. 2E for the reaction between [5'-32PJp(Ap)5G and (Ap)5C. In this case the donor and theproduct had similar base compositions to the (Ap)nU chroma-tographic markers used so that they eluted at the approximatepositions expected for a hexamer and a dodecamer that con-tain predominantly adenosine residues. In addition to its be-havior on RPC-5, the new product was characterized as(Ap)5C [3'-*5'_32P]p(Ap)5G by digestion with alkali andnucleases in a manner similar to that described. The yields ofproducts resulting from the intermolecular reaction between

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FIG. 3. Extent of intermolecular reaction between [5'-32P]p-(Up)5U and (Ap)5C as a function of time and enzyme concentra-tion. Each 25-A, reaction mixture, containing 1 ug (-), 0.3 ug (N),or 0.1 ug (A) of RNA ligase, was analyzed on an RPC-5 columnafter termination at the indicated time.

(Ap)5C and the other donors observed under a fixed set ofconditions are shown in Table 1.

Reaction Kinetics Between (Ap)5C and p(Up)5U. Inter-molecular reactions between (Ap)5C and [5'-32P]p(Up)5Uwere carried out at three different enzyme concentrations forvarious times. The products were analyzed by RPC-5 columnchromatography as described. The amount of intermolecularproduct increased with both time and enzyme concentration(Fig. 3). However, the rate of the reaction was not linear withrespect to enzyme concentration under these conditions. Forexample, under conditions where 1 jug of enzyme convertedall the hexamer to dodecamer, 0.3 jg of enzyme catalyzedonly 18% conversion. This lack of proportionality betweenthe rate and enzyme concentration was also observed with theintramolecular cyclization reaction, with the same enzymepreparation and [5'-32P]p(Ap)21,26A as a substrate (datanot shown). The inability to obtain an activity proportionalto enzyme concentration at any stage of preparation of RNAligase has made it difficult to obtain a useful specific activity.

Dependence ofActivity on Donor and Acceptor Concentrations.The rate of the intermolecular reaction showed a strong con-centration dependence of both the donor and acceptor oligo-mers. A series of reactions were run at varying (Ap)5C accep-tor concentrations and 1 IATI [5'-32P]p(Up)5U with a constantamount of enzyme for a given time of incubation, and analyzedby RPC-5 column chromatography (Fig. 4A). As the acceptorconcentration increased from 0.03 mM to 0.8 mM, the rateincreased by a factor of about ten. When 4 mi\I (Ap)5C wasused, the rate was even greater, but all the (pU)6 was con-verted to product during the reaction time. In a similar seriesof experiments, the amount of (Ap)5C(pA)6 product wasmeasured as a function of (pA)6 donor concentration and 4mM (Ap)5C with a constant amount of enzyme for a giventime of incubation (Fig. 4B). As the donor concentrationincreased from 6 ,ulI to 200 zi\I, the rate increased about 18-fold. These results suggest that further increases in reactantconcentrations would increase the reaction rates.

DISCUSSION

The ability of T4 RNA ligase to catalyze the joining of twosingle-stranded oligoribonucleotides should not be considereda separate activity of the enzyme. The activity reported herehas chemical and structural requirements quite similar to thatcatalyzing the intramolecular joining of the 3'- and 5'-

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ligase. (A) Varying concentrations of (Ap)5C with 1 ,uM [5'-

'2P]p(Up)5U. (B) Varying concentrations of [5'-32Plp(Ap)5A

with 4 mM (Ap)5C.

ends of the same molecule, which leads to the formation of

single-stranded circles reported earlier (1-3). Higher concen-

trations of the Eaceptor must be used in the intermolecularreaction to permit the binding of two oligomers to the enzyme.

The relation of this activity to the reported nick-sealing

activity of RNA ligase (3) is less clear. If both activities are

catalyzed by the same protein at similar rates, the implicationis that the complementary splint strand is only required to

bring the 3'-terminus of one oligomer into close proximitywith the 5'-terminus of another. It is interesting to note thatT4 DNA ligase does not appear to have both activities with

corresponding deoxyoligomers (4, 14). Thus, although intra-molecular cyclization of oligo- (15) and poly- (16) deoxy-nucleotides has been reported with E. coli DNA ligase, itonly occurs when the single-stranded oligomer can loop backto make a nick. Careful studies with defined substrates shouldaid in relating the nick-sealing and single-strand-joining reac-

tions of T4 RNA ligase.The ability of T4 RNA ligase to catalyze the joining of two

single-stranded oligoribonucleotides will have considerableuse in the synthesis of defined RNA sequences. Current en-

zymatic methods of oligoribonucleotide synthesis (6) usuallyinvolve the addition of single residues or blocks of identicalresidues to the end of a growing chain. The absence of a generalmethod for the synthesis of a 3'-'.5'-phosphodiester linkagebetween ribo-oligomers prevents the synthesis of long se-

quences in adequate yield. The nearly quantitative yield ob-tained for the (Ap)5C plus p(Up)5U joining reaction, theability of oligomers as short as trimers to be joined, and theapparent lack of any absolute base specificity, at least as far as

donor molecule is concerned, indicate the potential utility ofRNA ligase in synthetic work. In their synthesis of a tRNAgene, Khorana and his coworkers have demonstrated thepractical effectiveness of a DNA ligase activity in couplingoligodeoxyribonucleotides (17). DNA ligase, however, re-

quires a complementary splint strand and, therefore, thesynthesis of that strand as well as the desired strand. RNA

ligase has the advantage of not requiring the complementarystrand, thereby simplifying the synthetic task.

In this work, very low concentrations of the 32P-labeleddonor molecules were used in order to avoid the possibility ofself-reaction. As a result, the absolute rate of reaction wasquite low. However, the strong dependence of rate upon thesubstrate concentrations suggests that synthetic applicationsmay be carried out with practically obtainable amounts ofenzyme if both the donor and acceptor oligomers are presentin equal concentrations in the millimolar range. In order toprevent self-reaction, the 3'-hydroxyl group of the donorwould have to be blocked to ensure a unique product. Thiscould be accomplished either by a 3'-phosphate or by the2'(3')-O-(a-methoxylethyl) ether (18) or the 2'(3')-O-iso-valeryl ester (19, 20) derivatives, which are intermediates inthe stepwise syntheses of oligoribonucleotides. It, therefore,appears feasible to develop an alternative synthetic scheme forthe synthesis of oligoribonucleotides of defined sequence for avariety of physical and biochemical studies.

We thank J. Wachsman for helpful discussions and J. FentonWilliams for running many columns. This work was supported bygrants from the NIH (GM19059 to O.C.U. and GM 19442 toR.I.G.). O.C.U. and R.I.G. are NIH Career DevelopmentAwardees. G.C.W. is a National Research Council of CanadaPostdoctoral Fellow.

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