© 1991 Oxford University Press Nucleic Acids Research, Vol. 19, No. 3 547

Phosphorothioate-containing RNAs show mRNA activity inthe prokaryotic translation systems in vitro

Takuya Ueda, Hideki Tohda, Nobutoshi Chikazumi, Fritz Eckstein1 and Kimitsuna WatanabeDepartment of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute ofTechnology, Nagatsuta, Midori-ku, Yokohama 227, Japan and 1Abteilung Chemie, Max-Planck Institutfur experimentelle Medizin, Hermann-Rein strasse 3, D-3400, Gottingen, FRG

Received October 18, 1990; Revised and Accepted January 3, 1991

ABSTRACT

Phosphorothioate-containing RNAs were generated bytranscription of coliphage T7 ONA using the Spdiastereomers of ribonucleoside 5'-O-(1-thiotriphos-phates) and T7 RNA polymerase. RNAs In which asingle nucleotide was substituted by the correspondingnucleoside phosphorothioate functioned as mRNA inthe cell-free translation systems prepared fromEscherichla coli and from an extreme theimophilicbacterium, Thermus thermophllus. This substitutionincreased the efficiency of protein synthesis bystabilizing the mRNAs in these systems. As theproportion of substituted nucleotides was increased,their mRNA activity was decreased accordingly. Asjudged from the analysis by SDS-polyacrylamide gel-electrophoresis, the proteins synthesized usingphosphorothioate-containing mRNAs as template wereidentical to those obtained with unsubstituted mRNAs.However, larger proteins which were barely detectablewhen unsubstituted mRNA was used were wellrepresented when phosphorothioate-RNA was usedinstead. The advantages in using thephosphorothioate-mRNAs in the in vitro translationsystems are discussed.

INTRODUCTION

One of the problems encountered in using translational systemsreconstructed in vitro, is the instability of the mRNA. As mRNAis easily degraded by nucleases the translational efficiency is veryoften not satisfactory for preparing proteins in amounts sufficientfor structural and functional analysis as well as their utilization.Thus, the stabilization of mRNA against degradation wouldgreatly increase the usefulness of cell-free translation systems.

Phosphorothioate-containing nucleic acids in which one of theoxygens in the internucleotidic linkage is replaced by sulfur havefound considerable application in molecular biology (1). Thephosphorothioate groups can easily be introduced into DNA andRNA as the Sp diastereomers of dNTPaS and NTPaS,respectively, are readily accepted as substrates by DNA as wellas RNA polymerases (2). Several reports have described that thepresence of these phosphorothioate groups increases the stabilityof oligodeoxynucleotides, DNA and RNA (2). In particular ithas been shown that phosphorothioate-containing

polyribonucleotides are considerably more stable against RNasesand other nucleases than the unmodified polymers and that thiseffect can result in an enhanced biological activity (3).

It stands to reason, therefore, to explore the possibility ofincorporating phosphorothioate groups into mRNA and toexamine the consequences of their presence with respect tostabilization against nucleases and to template properties intranslational systems. Moreover our previous studies havesuggested the usefulness of protein synthesis system of T.thermophilus for such a study (4,5). Thus, we decided to examinethe effect of phosphorothioate substitution in the T. thermophilusand E. coli. translation systems.

MATERIALS AND METHODSMaterialsT7 RNA polymerase was purified from its overproducing strainof E. coli kindly provided by Dr. J. Studier, according to themethod reported previously (6,7). The Sp diastereomers of thefour nucleoside 5'-O-(l-thiotriphosphates) were synthesizedchemically as described previously (8), or purchased fromAmersham, Japan. Radioactive amino acids, [U-l4C]-aminoacids (protein hydrolysate, mixture of 16 amino acids except forasparagine, glutamine, tryptophan cysteine; 2.11 GBq/milliatom)and [35S] methionine (55.5 TBq/mmol), were purchased fromAmersham, Japan. Pyruvate kinase from Bacillus stearothermo-philus was a gift from Dr. H. Sakai, the University of Tokyo.Sephadex G-50 was purchased from Pharmacia, RNase-freeDNase I, NTPs, pyruvate kinase from rabbit muscle andphosphoenolpyruvate from Boehringer Mannheim, T7 coliphageDNA, spermidine, spermine and phosphodiesterase from Sigma,RNase inhibitor from human placenta and agarose for gelelectrophoresis from Takara, cold amino acids from WakoChemicals, and nuclease P] from Seikagaku Kogyou andacrylamide for gel electrophoresis from Bio-Rad.

The components for the cell-free protein synthesis system

Components used for the in vitro translation were prepared fromE. coli A 19 (9) or T. thermophilus HB 27 (10) by the sameprocedures as reported previously (4).

The tRNA fraction was prepared according to the Zubay'smethod (11), as reported previously (4).

548 Nucleic Acids Research, Vol. 19, No. 3

Enzymatic synthesis of RNAs by T7 RNA polymerase

The enzymatic synthesis of the phosphorothioate-containingRNAs was performed according to the method by Sampson andUhlenbeck (12) with a slight modification, where T7 coliphageDNA was used as a DNA template. The reaction mixture (totalvolume, 50 /AI) contained 40mM Tris-HCl (pH8.0), 15mMMgCl2, 5mM dithiothreitol, 50/tg/ml bovine serum albumin,lmM spermidine, 100 units of RNase inhibitor of humanplacenta, 1/tg of T7 phage DNA and 2mM each of nucleoside5'-triphosphates or any of the Sp-NTPaS. The reaction wasinitiated by addition of 2/tg of T7 RNA polymerase, and wascarried out at 42°C for 60 minutes. It was terminated by additionof 5/tl of 500mM EDTA (pH 8.0). The template DNA wasremoved by treatment with RNase-free DNase I. The resultingsolution was applied onto a Sephadex G-50 gel-filtration column,and the fractions in the void volume were collected. It was stockedat —20°C until use. By these procedures, RNAs could beseparated from the template DNA as well as the free nucleotides.The synthesized RNAs were analyzed by 1% agarose gelelectrophoresis and staining with ethidium bromide to determinetheir chain lengths.

In vitro translation systemsThe reaction mixture used for T. thermophilus translation assays(total volume 25 /tl) consisted of 50mM Tris-HCl (pH7.5), 60mMNH4CI, 5mM Mg(CH3COO)2, 6mM 2-mercaptoethanol, 3mMspermine, lmM ATP, 0.2mM GTP, 5mM phosphoenolpyruvate,0.08/tg pyruvate kinase of Bacillus stearothermophilus, 9 /tg ofT. thermophilus ribosomes, 8 ng tRNA fraction, 100/tg proteinequivalent of S100 fraction and 0.5 /tg S30 fraction. The S30fraction was preincubated at 65 °C for 7 min in the presence oflmM ATP, 0.2mM GTP, 5mM phosphoenolpyruvate and 0.08/tgpyruvate kinase for activation of factors involved in the S30

Transcriptlonal efficiency (%)

Fig. 1. Relative efficiencies of the synthesis of RNAs for transcription of T7 DNAwith T7 RNA polymerase using NTPs and NTPorS as substrates. The amountsof RNAs, purified by gel filtration as described in Materials and Methods, wereestimated by measuring the absorbance at 260nm. The efficiency of each RNAproduct was calculated assuming a 100% efficiency for the unsubstituted RNAs.The substituted nucleotides are shown under each bar. N represents unsubstitutedRNAs.

fractions. The radioactive amino acids and mRNAs were addedto start the reaction at 65°C.

For the single nucleotide-substituted RNAs, [I4C] amino acids(mixture of 16 amino acids; total radioactivity, 9.25 kBq) at aconcentration of 5 nM for each amino acid and the remaining4 nonradioactive amino acids (asparagine, glutamine, tryptophanand cysteine) at a concentration of 50 nM each were added tothe reaction mixture. For analysis of the synthesized proteins bygel electrophoresis, 28.0 kBq [35S] methionine and 50 nM eachof the remaining 19 amino acids were added to the reactionmixture. After incubation for appropriate time periods, aliquotsof the reaction mixtures were withdrawn onto the Whatman 3MMfilter disks, and which were boiled in 10% trichloroacetic acid(TCA) for 10 min. The remaining hot TCA-insolubleradioactivity on the disks was measured in a liquid scintilationcounter. The translation assay for the E. coli system was carriedout in a similar manner as above, except that the reaction wascarried out at 37°C and the buffer without spermine was usedat pH 7.8 and that ribosomes (9 /ig), S100 (100 ng) and S30(25 /tg) fractions obtained from E. coli were used instead of thosefrom T. thermophilus, and pyruvate kinase was from rabbitmuscle instead of B. stearothermophilus.

Analysis of the synthesized proteinsThe reaction mixtures used for the translation assays wereanalyzed by 12% SDS polyacrylamide gel electrophoresis underreduced conditions (13).

RESULTSEnzymatic synthesis of phosphorothioate-containing RNAsT7 coliphage DNA consists of 39,936 base-pairs with 17promoters and one termination site for T7 RNA polymerase. Thelength of RNAs synthesized in vitro by T7 RNA polymerase is

crrStCD

nc0leo

tide

Nu •..; . - %.,C [ : . . . ...A .... ,,,..G . : . :

u c |itiiiiiiiii)iiiiiiii«iiii)(it)itiiiiiiiiiiiin«tiiiniiit(iiiiiiiiiiUG \fmmmmmmmmmmm\mfi\mm^G c [imp iMiiHMiiimuiiimi«MHiiiiimiiniHiBiiiiiAU MmmmmMMMmmHi\mmm\m

AGCk^m^^^^^^^^

A uc pmmmsmm^mA UG kmmmmsmmm

AUGC ///////////A

".'* -:-• '".:.. 1

"* "i ...1minimumINHHUI

nmmI

**

***

(ntd.

5000

20001000

500

N A U G C S

Fig. 2. Electrophoretk analysis of phosphorothioate RNAs. 3 jig each ofunsubstituted (N), single phosphorothioate-substituted (A, U, G, O and fourphosphorothioate-substituted (S) RNAs were analyzed by 1% agarose gel-electrophoresis and stained by ethidium bromide. Asterisks indicate the bandscorresponding to the mRNAs predicted from the sequence of T7 phage DNA(13). RNA length was estimated by using an RNA marker (0.24-9.5 Kb RNALadder purchased from BRL).

Nucleic Acids Research, Vol. 19, No. 3 549

expected to range between 500 and 36,000 nucleotides (14).Transcription of such long RNA molecules by T7 RNApolymerase using NTPaS as substrates has not been reported sofar. Thus, the transcriptional efficiency of these RNA moleculeswas first examined.

Fig. 1 shows the efficiency with which variously substitutedRNAs are synthesized from T7 DNA by T7 RNA polymerasein a period of 1 hour, estimated by measuring the amount of RNAproducts purified by Sephadex G-50 column chromatography.When only one NTP was replaced by the corresponding NTPaS,RNAs were synthesized almost with an equal efficiency asunsubstituted RNAs. With pyrimidine nucleosidephosphorothioates as substrates the amounts of RNA producedwere about 96% of that with all NTPs as substrates, whereaswith the purine nucleoside phosphorothioates it was 84 - 91 %.When the four NTPs were substituted by the correspondingNTPaS, the efficiency gradually decreased to 33%, the valueobserved when all four NTPs were replaced by NTPaS. In thesereactions, 16 - 48 ng of the substituted RNAs could be obtainedin 50 /tl reaction mixtures, sufficient amounts for examining theirmRNA activities in the in vitro translation systems.

Analysis of the phosphorothioates incorporated into RNAsT7 DNA dependent RNA polymerase incorporates the Sp-NTPaS with inversion of configuration at phosphorus, thusyielding an internucleotidic phosphorothioate linkage of the Rpconfiguration (1,15). Snake venom phosphodiesterase cleaves theRp phosphorothioate linkage (1). As expected, digestion ofaliquots of the phosphorothioate transcripts with this enzymegenerated the nucleoside 5'-phosphorothioates in combinationwith the nucleoside 5'-phosphates as detected by HPLC analysis(data not shown). This analysis confirms that thephosphorothioates have indeed been incorporated.

Characterization of the synthesized RNAsThe chain-lengths of the phosphorothioate RNAs were analyzedby agarose gel electrophoresis. They were in the range from

several hundred to ten thousand nucleotides in all cases as shownin Fig. 2. Their electrophoretic patterns were similar to that ofthe unsubstituted RNAs. In addition, RNA bands correspondingto the sizes predicted from the DNA sequence of the T7 coliphage(14) (marked by asterisks) were detected in all cases. This analysisshows that neither the incorporation process was prematurelyterminated nor were there any unusual pause sites.

Protein synthesis directed by the phosphorothioate RNAsThe mRNA activity of phosphorothioate RNAs was examinedin the in vitro translation systems of T. thermophilus and E. coli.The results obtained with phosphorothioate RNA in which onlyone nucleotide was replaced by a phosphorothioate are reportedin Fig. 3 a and b. In the T. thermophilus system each of the fourphosphorothioate RNAs was used more efficiently as templatethan the unsubstituted RNA.

However, there are differences. Phosphorothioate substitutionin pyrimidine nucleotides enhanced mRNA activity by a factorof approximately two, whereas that of purine nucleotides showedonly a slight enhancement. The situation was quite different inthe E. coli system, where the phosphorothioate substitutions ofguanosine and uridine showed a slight enhancement of mRNAactivities but that of adenosine had no effect, and that of cytosineresulted in a somewhat lower activity than the unsubstitutedRNAs.

When two, three and all four nucleotides in RNAs werereplaced by phosphorothioate ribonucleotides, the mRNA activitywas scarcely detected using [I4C] labeled amino acids. Toincrease the sensitivity of the system [35S] methionine was usedfor the assay. Incorporation of [35S] radioactivity was indeeddetectable at higher concentration of RNA even when all fournucleotides were replaced by phosphorothioates (data not shown).

Stability of phosphorothioate RNAsThe stabilities of RNAs in both translation systems weredetermined. After preincubation of the RNAs in the translationreaction mixture without amino acids for various time periods,

(a) (b)

a.o

«•o

ocaO

2

Iooc

1500

1000 -

500

10 20

Incubation time (mln)

5000

4000

Ids

8gE(B

o

ie1

3000

2000

1000

010 20

Incubation time (min)

Fig. 3. Time course of the translation activities of unsubstituted and phosphorothioate RNAs using the in viirv translation systems of T. thermophilus (a) and E.coli (b). 1.5 fig each of unsubstituted RNAs (O), RNAs substituted with either of adenosine ( • ) , uridine ( • ) , guanosine ( • ) and cytosine (A) phosphorothioateswere translated with [l4C]-amino acids. The control curve without template RNAs is presented as ( • ) .

550 Nucleic Acids Research, Vol. 19, No. 3

(a) (b)

H 60 "

CO

mQC

5 10 15Pre-lncubation time (min)

5 10 15Pre-incubation time (min)

Fig. 4. Relative stabilities of RNAs in the translation system of T. thermophilus (a) and E. coli (b). The RNAs were preincubated in the reaction mixture withoutamino acids for the times incubated, and following the addition of amino acids to the reaction mixture, the incorporation of radioactivity into hot TCA-insolublematerial was determined after reaction for 7 min. The symbols are the same as in Fig. 3.

the protein synthesis reaction was initiated by addition of aminoacids. The amount of protein synthesis was determined afterreaction for 7 minutes. The decrease in incorporation of [I4C]amino acid mixture was taken as a measure for the degradationof the RNAs (Fig. 4).

Approximately 80% of the unsubstituted RNAs were degradedduring the first 5 min of preincubation in the T. thermophilussystem, but only between 5 and 45% of the RNAs with a singlenucleotide substitution depending on the nucleotidephosphorothioate present. The relative remaining activity wasthe highest in the RNA with G-substitution and the lowestwith U-substitution in the order G > C > A > U. After pre-incubation these differences disappeared but the phosphorothioateRNAs were still more active than the unsubstituted RNAs.

In the E. coli systems, the stabilizing effect of thephosphorothioate substitution was not as pronounced. The relativeremaining activity of the unsubstituted RNA was 33% in the5 min preincubation, whereas those of the single substitutedRNAs were between 33—62% in the order ofA > G > C > U , the U-substitution showing no stabilizationeffect in this systems.

Electrophoretic analysis of synthesized proteinThe proteins synthesized in the translation systems of T.thermophilus and E. coli were analyzed by SDS-polyacrylamidegel-electrophoresis (Fig. 5). The molecular weights of theproducts obtained with the phosphorothioate RNAs were verysimilar to those obtained with the unsubstituted RNAs in bothsystems. Several proteins synthesized in both systems (the bandswith asterisks in Fig. 5) are similar to those known to beexpressed in E. coli cells infected by T7 coliphage (14). Theseresults strongly suggest that the translation system of this extremethermophilic bacterium is equivalent to that of E. coli as hadpreviously been demonstrated by Ohno-Iwashita et al. (16). Thesimilar sizes of the proteins synthesized by either the unsubstitutedRNA Qane N) or the substituted RNAs (lanes A, U, G and C)also suggests that phosphorothioate RNAs are translated in a

W

(kDa)

92.566.245.0

31.0

21.5

14.4

(kDa)

92.566.2

45.0

• 31.0

21.5

14.4

N A U G C N A U G C

Fig. 5. Autoradiograph of [35S]-labeled proteins synthesized with 1.5 /ig ofphosphorothioate RNAs in the in vitro translation system of T. thermophilus (a)and E. coli (b). Unsubstituted RNAs (N) and RNAs substituted with either ofadenosine (A), uridine (U), guanosine (G) or cytidine (Q phosphorothioates weretranslated with [33S]-methionine for 30 min. These reaction mixtures weredirectly subjected to 12% SDS-polyacrylamide gel electrophoresis. The asteriskslocated at the left side of the autoradiograms show the bands which are also foundin the in vivo expression system of E. coli infected by T7 coliphage (14). Themolecular weights of proteins were estimated by using SDS-PAGE molecularweight standards-low (Bio-Rad Laboratories, US).

similar way as the unsubstituted RNAs in these systems, includingboth proper initiation and termination in the translation process.

In the T. thermophilus system some proteins produced by thephosphorothioate RNAs are more distinct than those producedby the unsubstituted RNAs. In particular, the bands of the largestproteins (around 40 kDa) were scarcely detected with theunsubstituted RNA, but can clearly be seen with all the substitutedRNAs (Fig. 5a). RNAs substituted with pyrimidine nucleosidephosphorothioates (lanes U and C in Fig. 5a) produced a largeramount of proteins than those with purine nucleosidephosphorothioates (lanes A and G). Any of the phosphorothioateRNAs led to the production of more protein than the unsubstitutedRNA. These results are consistent with the results shown in

Nucleic Acids Research, Vol. 19, No. 3 551

fig. 3a. This effect is not so distinct in the £ coli system (Fig. 5b)which is also consistent with the results shown in fig. 3b. Thisdifference might result from the presence of different nucleasesin the two systems.

DISCUSSION

There are numerous reports in the literature describing thetranscription with T7 RNA polymerase in the presence of NTPaS(15,17-23). In all of these examples not more than 1000nucleotides were transcribed. We show here that coliphage T7DNA can be transcribed in the presence of any one of the fourNTPaS. These transcripts cover a wide range from a few hundredup to several thousand nucleotides in length. Substitution of oneof the NTPs by the corresponding NTPaS had only a negligibleeffect on the amount of RNA produced in a period of 1 hour.However, replacing more than one NTP by the correspondingNTPaS up to all four nucleotides gradually decreased the amountof RNA synthesized. Gel electrophoretic analysis of thetranscripts indicated that the population of RNAs was the sameindependent of whether the transcription was carried outexclusively with NTPs or any mixture of NTPs and NTPaSs oreven in the presence of the four NTPaS. This indicates that thesmaller yield of RNA transcripts obtained in the presence of allfour NTPaS as substrates is due to a decrease in rate rather thanpremature termination of transcription.

The phosphorothioate-RNAs were tested as mRNA in twoprokaryotic translation systems, that prepared from E. coli andthat from T. thermophilus. All the monophosphorothioate-RNAsserved as better templates for the translation than the unmodifiedRNA in the T. thermophilus system. The RNAs prepared withthe two pyrimidine nucleoside phosphorothioates CTPaS andUTPaS were approximately twice as active as the parent RNA,whereas those prepared with the two purine nucleosidephosphorothioates GTPaS and ATPaS only about 1.2 times. Inthe E. coli system the effects of the phosphorothioate substitutionwas not as pronounced and there was not such a distinct differencebetween the pyrimidine and the purine phosphorothioate-RNAs.In fact, the cytidine phosphorothioate-RNA was even a somewhatpoorer template than the parent RNA.

In comparing the results obtained in the two systems it hasto be borne in mind that the incubation temperature for the T.thermophilus system is 65 °C whereas in the E. coil system itis 37°C. At the higher temperature the flexibility of the RNAis much greater. It might be that at this temperatureconformational constraints in the phosphorothioates are not assevere and allow adoption of more favourable conformations fortranslation.

In order to see whether the phosphorothioate-RNAs wereindeed more stable than the unmodified RNA in the twotranslation systems, the RNAs were preincubated in the reactionmixture for up to 15 min. in the absence of the amino acid. Thetranslation reaction was then started by the addition of the aminoacids and the activities of the various RNAs compared. The mostpronounced differences were again detected in the 7! thermophilussystem and there particularly after a preincubation for 5 min.Again all the phosphorothioate-RNAs were more active than theparent RNA. The guanosine phosphorothioate-RNA was 5 timesand the uridine phosphorothioate 3 times as active as the parentRNA. The other two phosphorothioate-RNAs were ofintermediate activity.

In order to interpret these data it is important to realize thatthe introduction of the phosphorothioates can have at least two

effects. First, there will be an inhibitory effect on nucleases. Ithas been shown previously that RNase A, RNase T! and calfserum nucleases are inhibited by the presence of phosphorothioategroups in alternating and homopolyribonucleotides (24,25). Inaddition, there is ample evidence that the presence ofphosphorothioates in oligodeoxynucleotides increases theirstability against nucleases (26).

That nucleases indeed are responsible for low translationalactivity was shown in an experiment with unmodified RNA inthe E.coli system where placental RNase inhibitor was added.The translational activity was indeed increased about twofold (datanot shown). This inhibitor, however, is not active in the T.thermophilus system. Thus, the observation in the T. thermophilussystem that the phosphorothioate-RNAs prepared with CTPaSand UTPaS directed protein synthesis more efficiently than thoseprepared with GTPaS and ATPaS might be taken as an indicationthat pyrimidine-specific RNases are either more prevalent orinhibited better by phosphorothioates than those cleaving at purinenucleotides.

However, a second effect has to be considered. Milligan andUhlenbeck (21) have found that the binding of R17 coat proteinto its RNA is weakened by the presence of phosphorothioategroups in 3 out of 21 positions and enhanced by that in oneposition. Results reported by Buzayan et al. (18) demonstratethat autocatalytic cleavage of satellite RNA of tobacco ringspotvirus is reduced by a phosphorothioate substitution at oneparticular position. Similar observations were made by Waring(22) for group I intron splicing. All these results suggest thata few phosphate groups in a given RNA might be moresusceptible to changes introduced by a phosphorothioate groupthan others leading to interference with the proper functioningof the RNA. The translational process involves numerousinteractions of the mRNA with proteins and other RNAs. Thus,the lower template activity of the purine phosphorothioate-RNAscould be caused by a particular weakening of an interaction whichdoes not occur with the pyrimidine phosphorothioates. At presentno distinction between these two possibilities can be made.

Gel analysis of the mixture of proteins produced with variousRNAs in the T. thermophilus system shows that in the presenceof the monophosphorothioate-RNAs a larger amount of proteinsis produced in agreement with the radioactive incorporation data.This is particularly evident for the proteins with larger molecularweights. Thus proteins with a molecular weight >40 kDa arebarely visible when unmodified RNA is the template but are verypronounced with all the phosphorothioate-RNAs. This clearlyis an advantage in using the phosphorothioate-RNAs in theT. thermophilus system.

Moreover, the T. thermophilus system has generally someadvantages in comparison with E. coli system for the in vitroprotein synthesis (4,5) such as stability for longer time, easiercontrol of temperature and greater resistance toward nucleases.

Recently a continuous flow system using an ultrafiltrationmembrane for the in vitro protein synthesis has been developed(27,28) which allows to obtain proteins on a preparative scale.We expect that the use of phosphorothioate-RNAs described inthis report will be useful in constructing such a flow system onthe basis of the T. Thermophilus translation system.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. F.Studier for providing strain BL21harboring the plasmid pAR1219 and Dr. H.Himeno for purifyingT7 RNA polymerase.

552 Nucleic Acids Research, Vol. 19, No. 3

REFERENCES

1. Eckstein, F. and Gish, G. (1989) TIBS, 14, 97-100.2. Eckstein, F. (1985) Annu. Rev. Biochem., 54, 367-402.3. Black, D. R., Eckstein, F., DeClercq, E. and Merigan, T. C.(1972)

Antimicrob. Agents Chemolher., 3, 198-206.4. Watanabe, K., Ohshima, T., Iijima, K., Yamaizumi, Z. and Nishimura,

S. (1980)7. Biochem., 87, 1-13.5. Yokoyama, S., Watanabe, K. and Miyazawa, T. (1987) Adv.Biophys., 23,

115-147.6. Davanloo, P., Rosenberg, A. H., Dunn, J. J. and Studier, F. (1984) Proc.

Nail. Acad. Sci. USA, 81, 2035-2039.7. Grodberg, J. and Dunn, J. J. (1988) J. Bacterial., 170, 1245-1253.8. Ludwig, J. and Eckstein, F. (1989) / . Org. Chem., 54, 631-635.9. Takanami, M. (1967) / . Mol. Biol, 23, 135-148.

10. Ohshima, T. and Imahori K. (1971), J. Gen. Appi, 17, 513-517.11. Zubay, G. (1962) J. Mol. Biol., 4, 347-356.12. Sampson, J. R. and UWenbeck, O. C. (1988) Proc. Noll. Acad. Sci. USA,

85, 1033-1037.13. Laemmli, U. K. (1970) Nature, 227, 680-685.14. Dunn, J. J. and Studier, S. (1983) J. Mol. Biol., 166, 477-535.15. Griffiths, A. D., Potter, B. V. L. and Eperon, I. C.(1987) Nucleic Acids

Res., 15, 4145-4162.16. Ohnolwashita Y., Oshima T. and Imahon K. (1975) Zeiischrift JOr AUg.

Mikrobiologie, 15, 131-134.17. Griffiths, A. D., Potter, B. V. L. and Eperon.l. C.(1988)/. Biol. Chem.,

263, 12295-12304.18. Buzayan, J. M., van Tol, H., Feldstein, P. A. and Breuning, G. (1990)

Nucleic Acids Res., 18, 4447-4451.19. Buzayan, J. M., Feldstein, P. A., Breuning, G. and Eckstein, F. (1988)

Biochem. Biophys. Res. Commun., 156, 340-347.20. Gish, G. and Eckstein, F. (1988) Science , 240, 1520-1522.21. Milligan, J. F. and Uhlenbeck, O. C. (1989) Biochemistry, 28, 2849-2855.22. Waring, R. B. (1989) Nucleic Acids Res., 17, 10281-10293.23. van Tol, H., Buzayan, J. M., Feldstein, P. A., Eckstein, F. and Bruening,

G. (199OJ Nucleic Acids Res., 18, 1971-1975.24. De Clercq, E., Eckstein, F., Sternbach, H. and Merigan, T. C. (1970)

Virology, 42, 421-428.25. Eckstein, F. and Gindl, H. (1970) Ear. J. Biochem., 13, 558-564.26. Spitzer, S. and Eckstein, F. (1988) Nucleic Acids Res., 16, 11691 -11704.27. Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Yu. and Aiakhov,

Yu. B. (1988) Science, 242, 1162-1164.28. Baranov, V. I., Morozov, I. Yu., Ortlepp, S. A. and Spirin, A. S. (1989)

Gene, 84, 463-466.