(RE67757), and dCsl4 (RE64677). They were amplified by PCR using the followingprimers: dRrp6 þ 1F and dRrp6 þ 579R; dMtr3 þ 1F and dMtr3 þ 326R; Rrp4 þ 1Fand Rrp4 þ 298R; dSki6 þ 1F and dSki6 þ 246R; dCsl4 þ 1F and dCsl4 þ 204R. Theywere digested with the appropriate restriction endonucleases and cloned into pMAL-C2(New England Biolabs) to create MBP–dRrp6N, MBP–dMtr3, MBP–dRrp4, MBP–dSki6and MBP–dCsl4. MBP fusions were expressed in Escherichia coli and recombinant proteinswere purified over amylose resin according to the manufacturer’s recommendations (NewEngland Biolabs). Recombinant proteins were injected into either rat or guinea-pig andantibodies were recovered in animal bleeds (Pocono Rabbit Farm and Laboratory Inc.).

We carried out immunoprecipitations in wash buffer (10 mM HEPES (pH 7.5), 10 mMTris-HCl (pH 7.5), 150 mM KCl, 150 mM NaCl, 3.5 mM MgCl2, 0.5 mM EDTA, 0.5%NP-40, 10% glycerol, 0.5 mg ml21 bovine serum albumin, 0.5 mM dithiothreitol (DTT)and 0.25 mM phenyl-methyl-sulphonyl fluoride). RNase A (Sigma) was used at afinal concentration of 100 mg ml21. Immune complexes were incubated with eitherprotein-A- or protein-G-conjugated agarose (Invitrogen) and washed with wash buffer.Beads were boiled with SDS loading dye and then analysed by SDS–PAGE and westernblotting.

Exoribonuclease assaysWe prepared substrate for in vitro exoribonuclease assays as follows. Plasmid pG2XM,containing the 5 0 end of hsp70 ORF, was digested with EcoRI to linearize the template, andthen 1 mg of linearized template was transcribed in vitro using the MEGAshortscript kit(Ambion). RNA was 5

0-end-labelled by incubating transcription reactions in the presence

of [g-32P]GTP (Amersham) for 3 min as the only source of GTP. Cold GTP was addedthereafter and reactions proceeded for 2 h. We extracted RNA with phenol/chloroform,precipitated it and passed it over a P6 resin (Bio-Rad) to remove unincorporated label.Each exoribonuclease reaction contained ,10 pmol of hsp70 5

0RNA, ,2 pmol of dSpt6–

exosome complex (2 ml of dSpt6FH or mock eluate) and reaction buffer (10 mM Tris,50 mM KCl, 5 mM MgCl2 and 10 mM DTT). Reactions were allowed to proceed for thedesired time and stopped by the addition of an equal volume of formamide dye. RNAspecies were separated on a 12% denaturing polyacrylamide gel and analysed byphosphoimaging.

ChIP and immunofluorescence analysesCrosslinked material was prepared from formaldehyde-fixed Kc cells. We carried outimmunoprecipitations with antibodies to Drosophila proteins and analysed them asdescribed5. Polytene immunofluorescence was done as described22.

Received 29 July; accepted 16 September 2002; doi:10.1038/nature01181.

1. Hirose, Y. & Manley, J. L. RNA polymerase II and the integration of nuclear events. Genes Dev. 14,

1415–1429 (2000).

2. Bentley, D. Coupling RNA polymerase II transcription with pre-mRNA processing. Curr. Opin. Cell

Biol. 11, 347–351 (1999).

3. Proudfoot, N. J., Furger, A. & Dye, M. J. Integrating mRNA processing with transcription. Cell 108,

501–512 (2002).

4. Kaplan, C. D., Morris, J. R., Wu, C. & Winston, F. Spt5 and spt6 are associated with active transcription

and have characteristics of general elongation factors in D. melanogaster. Genes Dev. 14, 2623–2634

(2000).

5. Andrulis, E. D., Guzman, E., Doring, P., Werner, J. & Lis, J. T. High-resolution localization of

Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and

transcription elongation. Genes Dev. 14, 2635–2649 (2000).

6. Hilleren, P., McCarthy, T., Rosbash, M., Parker, R. & Jensen, T. H. Quality control of mRNA 3 0 -end

processing is linked to the nuclear exosome. Nature 413, 538–542 (2001).

7. Butler, J. S. The yin and yang of the exosome. Trends Cell Biol. 12, 90–96 (2002).

8. Bousquet-Antonelli, C., Presutti, C. & Tollervey, D. Identification of a regulated pathway for nuclear

pre-mRNA turnover. Cell 102, 765–775 (2000).

9. van Hoof, A., Frischmeyer, P. A., Dietz, H. C. & Parker, R. Exosome-mediated recognition and

degradation of mRNAs lacking a termination codon. Science 295, 2262–2264 (2002).

10. Torchet, C. et al. Processing of 3 0 -extended read-through transcripts by the exosome can generate

functional mRNAs. Mol. Cell 9, 1285–1296 (2002).

11. Hartzog, G. A., Wada, T., Handa, H. & Winston, F. Evidence that Spt4, Spt5, and Spt6 control

transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. Genes Dev. 12, 357–369

(1998).

12. Bortvin, A. & Winston, F. Evidence that Spt6p controls chromatin structure by a direct interaction

with histones. Science 272, 1473–1476 (1996).

13. Gavin, A. C. et al. Functional organization of the yeast proteome by systematic analysis of protein

complexes. Nature 415, 141–147 (2002).

14. Allmang, C. et al. The yeast exosome and human PM-Scl are related complexes of 3 0 ! 5 0

exonucleases. Genes Dev. 13, 2148–2158 (1999).

15. Mitchell, P., Petfalski, E., Shevchenko, A., Mann, M. & Tollervey, D. The exosome: a conserved

eukaryotic RNA processing complex containing multiple 3 0 ! 5 0 exoribonucleases. Cell 91, 457–466

(1997).

16. Mitchell, P. & Tollervey, D. Musing on the structural organization of the exosome complex. Nature

Struct. Biol. 7, 843–846 (2000).

17. Lis, J. T., Mason, P., Peng, J. & Price, D. H. P-TFFb kinase recruitment and function at heat shock loci.

Genes Dev. 14, 792–803 (2000).

18. Erdjument-Bromage, H. et al. Examination of micro-tip reversed-phase liquid chromatographic

extraction of peptide pools for mass spectrometric analysis. J. Chromatogr. A 826, 167–181 (1998).

19. Geromanos, S., Freckleton, G. & Tempst, P. Tuning of an electrospray ionization source for maximum

peptide-ion transmission into a mass spectrometer. Anal. Chem. 72, 777–790 (2000).

20. Mann, M., Hojrup, P. & Roepstorff, P. Use of mass spectrometric molecular weight information to

identify proteins in sequence databases. Biol. Mass. Spectrom. 22, 338–345 (1993).

21. Fenyo, D., Qin, J. & Chait, B. T. Protein identification using mass spectrometric information.

Electrophoresis 19, 998–1005 (1998).

22. Park, J. M., Werner, J., Kim, J. M., Lis, J. T. & Kim, Y. J. Mediator, not holoenzyme, is directly recruited

to the heat shock promoter by HSF upon heat shock. Mol. Cell 8, 9–19 (2001).

23. Shopland, L. S. & Lis, J. T. HSF recruitment and loss at most Drosophila heat shock loci is coordinated

and depends on proximal promoter sequences. Chromosoma 105, 158–171 (1996).

Acknowledgements We thank members of the Lis laboratory for comments on the manuscript.

This work was supported by an NIH grant to J.T.L., a National Research Service Award to E.D.A.,

and a National Cancer Institute (NCI) Cancer Center Support Grant to P.T.

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to J.T.L.

(e-mail: jtl10@cornell.edu) or E.D.A.(e-mail: eda4@cornell.edu).

..............................................................

A ribozyme composed of onlytwo different nucleotidesJohn S. Reader & Gerald F. Joyce

Departments of Chemistry and Molecular Biology and The Skaggs Institute forChemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,La Jolla, California 92037, USA.............................................................................................................................................................................

RNA molecules are thought to have been prominent in the earlyhistory of life on Earth because of their ability both to encodegenetic information and to exhibit catalytic function1. Themodern genetic alphabet relies on two sets of complementarybase pairs to store genetic information. However, owing to thechemical instability of cytosine, which readily deaminates touracil2, a primitive genetic system composed of the bases A, U,G and C may have been difficult to establish. It has been suggestedthat the first genetic material instead contained only a singlebase-pairing unit3–7. Here we show that binary informationalmacromolecules, containing only two different nucleotide sub-units, can act as catalysts. In vitro evolution was used to obtainligase ribozymes composed of only 2,6-diaminopurine and uracilnucleotides, which catalyse the template-directed joining of twoRNA molecules, one bearing a 5

0-triphosphate and the other a

3 0 -hydroxyl. The active conformation of the fastest isolatedribozyme had a catalytic rate that was about 36,000-fold fasterthan the uncatalysed rate of reaction. This ribozyme is specificfor the formation of biologically relevant 3

0,50-phosphodiester

linkages.A good starting point for the evolution of a catalyst that contains

only two different subunits was the R3 ligase ribozyme, whichcontains only adenine, guanine and uracil nucleotides8,9. Thisribozyme catalyses attack of the 3 0-hydroxyl of an RNA substrateon the 5 0-triphosphate of the ribozyme, forming a 3 0 ,5 0-phospho-diester and releasing inorganic pyrophosphate. The chemistry ofthis reaction is identical to that catalysed by modern RNA poly-merase proteins. A templating region within the ribozyme isresponsible for binding the RNA substrate, and the sequences ofboth the template and substrate can be designed such that theycontain only adenine and uracil residues. In that format, both theribozyme and substrate are completely devoid of cytosine andundergo RNA ligation with a catalytic rate, k cat, of 0.013 min21

and Michaelis constant, K m, of 6.2 mM (ref. 9).The R3 ribozyme was found to be highly tolerant of base

substitutions involving replacement of every adenine by 2,6-diaminopurine (D). Three bonds are formed between 2,6-D anduracil in the context of a Waston–Crick base pair10, in comparisonwith adenine, which forms only two. Despite this difference, theD-substituted R3 ligase (Fig. 1a) retained a k cat of 0.001 min21 andK m of 12 mM, and reacted to a maximum extent of about 40%.

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Substituting diaminopurine 50-triphosphate (DTP) for ATP when

transcribing AT-containing DNA templates was found to have twoimportant advantages for in vitro evolution experiments. First, thissubstitution reduced ‘slippage’ of the RNA polymerase11 as itproceeded along the DNA template. Second, transcription couldbe initiated with a D residue at the 5 0 end of the RNA, allowingtranscripts to be produced in the complete absence of GTP and CTP.For all these reasons, the R3 ligase, modified to contain D, G and U,was chosen as the starting point for the evolution of ligaseribozymes that contain only D and U.

The first stage of the process to develop a DU-containing catalystinvolved substituting as many of the G residues as possible by eitherD or U, while still retaining some detectable activity. Substitutionswere tolerated throughout the stem-loop regions of the ribozyme,replacing G†U ‘wobble’ pairs with D†U pairs, and replacing G withD at most of the unpaired nucleotide positions. The final substi-tuted ribozyme contained only three of the 16 G residues that werepresent in the starting molecule. Two of the remaining G residueswere located at the ligation site, and the other was in the single-stranded region connecting the P4 and P2 stems (Fig. 1a).

The second stage of the development of a binary informationalcatalyst involved in vitro evolution to compensate for removal of thefinal G residues and improve catalytic activity. The sequence of theribozyme that contained only three G residues was modified byreplacing the remaining G residues with either D or U, thenintroducing random mutations (either D ! U or U ! D) at afrequency of 12% per nucleotide position. A population of 8 £ 1013

different randomized variants was constructed, containing only Dand U residues at nucleotide positions 1–66. Positions 66–74,located at the 3

0end of the ribozyme, are involved in binding the

oligonucleotide substrate. For purposes of in vitro evolution, thesubstrate contained the sequence of the T7 promoter element,which includes all four nucleotides. Thus the corresponding tem-plate region of the ribozyme was required, at least temporarily, tocontain all four nucleotides. Experience had shown, however, thattranscription in the presence of all four NTPs would invariably leadto a resurgence of G and C residues in the ribozyme. Thus a strategywas adopted whereby the 5

0portion of the ribozyme (positions

1–66) was transcribed in the presence of only DTP and UTP, afterwhich an oligonucleotide containing a constant substrate-bindingregion (positions 67–74) was attached by enzymatic ligationemploying T4 RNA ligase.

The population of ribozyme variants was given an opportunity toligate the promoter-containing substrate. The reacted moleculeswere purified by electrophoresis in a denaturing polyacrylamide gel,then reverse transcribed and PCR amplified in the presence of allfour standard deoxynucleoside 5

0-triphosphates. In principle, only

those molecules that contained D and U residues at positions 1–66and had catalysed the ligation reaction would be eligible forsubsequent transcription to generate progeny molecules. Afterfour rounds of selective amplification, a ligated product wasdetected in the polyacrylamide gel following the RNA-catalysedreaction. A fifth round was carried out and individual moleculeswere cloned from the population and sequenced. Only two of the 22sequenced clones contained a single contaminating G or C residueat positions 1–66. There was considerable sequence heterogeneityamong the clones. Only one sequence was found to occur repeat-edly, appearing in five of the clones that were examined. Individual

Figure 1 Sequence and secondary structure of ligase ribozymes containing either three

or two different nucleotide subunits. a, Ribozyme containing D, G and U residues, which

was made to react with a substrate containing only A and U. This structure is supported

by chemical modification and site-directed mutagenesis studies9. Bold G at positions 1,

58 and 63 indicates residues that could not be replaced by D or U without complete loss

of catalytic activity. b, Ribozyme containing only D and U, which was made to react with

a substrate containing only D and U. This structure is conjectural. Note that this

molecule is shortened by one nucleotide at the 50

end and lengthened by six

nucleotides at the 30

end compared with the ribozyme shown in a.

Figure 2 Time course of the cyclization reaction involving the final selected ribozyme,

which contained only D and U residues. The reaction mixture contained 20 nM RNA,

100 mM MgCl2, 0.01% SDS and 30 mM 2-(N-cyclohexylamino)ethanesulphonic acid

(CHES; pH 9.0), which was incubated at 23 8C. Products were separated in a 6%

denaturing polyacrylamide gel. a, Phosphorimager scan of selected time points.

b, Time course carried out to determine the maximum extent of reaction. Each data

point is the average of three separate experiments, with the error bars corresponding

to one standard deviation. The data were fitted to a single exponential to obtain a k obs

of 0.048 ^ 0.005 h21 and a maximum extent of reaction of 8.3%.

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cloned ribozymes were then prepared in a form that lacked G and Cresidues within the substrate-binding region. This was accom-plished by synthesizing DNA templates based on the sequence ofeach clone, but with modification of the substrate-binding region sothat it contained only D and U residues. The ribozymes containingonly D and U residues were then challenged to react with acomplementary substrate that contained only A and U residues.All eight of the clones that were tested in this fashion had somedetectable activity.

The sequences of the two most active clones were used as the basisto construct two separate pools of RNAs that were mutagenized at afrequency of 12% per nucleotide position. Five additional rounds ofselective amplification were performed, resulting in DU-containingribozymes that were improved with regard to their catalytic activity.Individual clones were again isolated from the final population; themost active of these clones is shown in Fig. 1b. This ribozymecontained many of the mutations that were present in the parentclone, but also contained new mutations, including several rever-sions. There was also a deletion at the 5 0 end of the ribozyme thatresulted in the 5

0-terminal residue being a uridine, located adjacent

to an unpaired adenosine at the 3 0 end of the substrate. Many othermutations were present throughout the final selected ribozyme,presumably resulting in substantial remodelling of its overallstructure and the detailed structure at the ligation junction.

The secondary structure of the final selected ribozyme that isdepicted in Fig. 1b is drawn by analogy to that of the D-substitutedstarting molecule, shown in Fig. 1a. Although the secondarystructure of the starting molecule is well established9, that of thefinal ribozyme must be regarded as conjectural, especially for the P2and P3 stems (see Fig. 1a). The final ribozyme can adopt severaldifferent structures, as suggested by the presence of multiple bandswhen it was analysed by non-denaturing polyacrylamide gel elec-trophoresis (data not shown). As discussed below, most of themolecules were not in an active conformation, which thwartedattempts to carry out meaningful analysis of the ribozyme’ssecondary structure.

The rate of reaction of the final selected ribozyme was examinedin a format in which the ribozyme and substrate contained only Dand U residues. Two different constructs were prepared, one inwhich the substrate was presented separately and another in which itwas tethered to the 3 0 end of the ribozyme by a stable hairpinstructure. The latter format allowed for a cyclization reaction

involving the 30-hydroxyl and 5

0-triphosphate of the same RNA,

enabling a more straightforward assessment of the fraction ofmolecules that were in a productive conformation. The observedrate of the cyclization reaction fitted well to a single exponentialmodel, with a kobs of 0.00080 min21 and a maximum extent ofabout 8% of the total RNA molecules (Fig. 2). A probable expla-nation for the substantial proportion of unreacted RNA moleculesis their propensity to misfold based on their extraordinary degree ofinternal self-complementarity.

The reaction with a separate RNA substrate was used to show thatthe ribozyme exhibits Michaelis–Menten saturation kinetics. Theribozyme was engineered to bind a separate 17-nucleotide RNAsubstrate having the sequence 5

0-UUDUUUUDDUUDUUDUD-3

0

(Fig. 1b). Experiments were performed in the presence of excessribozyme, employing a [5 0 -32P]-labelled substrate. On the basis ofthe initial rates of reaction in the presence of various concentrationsof ribozyme, and adjusting for a maximum extent of reaction of 6%,the apparent k cat was 0.0011 min21 and K m was 1.6 nM (Fig. 3). Theuncatalysed rate of ligation was measured under the same reactionconditions employing the same template and substrate sequences.That rate was 3 £ 1028 min21, which agrees well with previousmeasurements of uncatalysed template-directed RNA ligation12,and corresponds to a catalytic rate enhancement of about 36,000-fold. When the RNA-catalysed reaction was performed with asubstrate that was not complementary to the template, there wasno detectable reaction.

The ligation reaction catalysed by the DU-containing ribozymemay have resulted in the formation of either a 2

0,50- or 3

0,50-

phosphodiester linkage, owing to the lack of selective pressure tomaintain the 3

0,50-regiospecificity of the starting R3 ligase. The

regiospecificity of the reaction was analysed by employing the‘10-23’ DNA enzyme, which cleaves 3

0,50, but not 2

0,50

linkages ofRNA13. The ligated product was cleaved by the DNA enzyme togenerate two fragments of the expected size, demonstrating that theribozyme, itself composed of 3 0 ,5 0 -linked ribonucleotides, catalysesthe formation of a 3

0,50-phosphodiester linkage.

It has been suggested that the original genetic system containedonly two different nucleotides3–7, and subsequently evolved to itspresent, more complex form. A binary genetic system may havebeen advantageous during the early history of life on earth when theavailability of all four nucleotides might have been difficult tomaintain. Cytosine nucleotides are especially problematic becausethey undergo rapid deamination to uridylate, with a half-life of19 days at pH 7 and 100 8C (ref. 2). Thus the G†C pairing may nothave been sustainable until the invention of a mechanism forrestoring cytosine to uracil. By comparison, the half-life of adenineor diaminopurine at pH 7 and 100 8C is about one or two years,respectively2. The prebiotic synthesis of adenine or diaminopurineproceeds with comparable efficiency, both compounds beingobtained in good yield starting from aqueous ammonium cya-nide14,15.

Nucleic acid enzymes composed of only D and U residues pay aheavy price for their simplified composition in terms of bothcatalytic rate and the fraction of molecules that are in an activeconformation. Nonetheless, darwinian evolution can produce cat-alytically active structures even from such a severely restrictedchemical repertoire. The conformational plasticity of DU-contain-ing RNA may even offer an advantage with regard to the ability toexplore multiple structures for a given sequence16.

Ribozymes composed of other pairs of nucleotides, such as A andU, G and C, or even A and I (inosine), may be possible. It seems lesslikely that polymers composed of only two amino acids couldexhibit appreciable catalytic activity. Thus far a minimum of 14amino acids has been used to construct a catalytic polypeptide17,and as few as seven have been shown to be necessary to define afolded tertiary structure18. The absolute minimum number ofdistinct subunits that could be used to construct a functional

Figure 3 Saturation plot for the bimolecular reaction involving the final selected ribozyme

and an RNA substrate, both of which contained only D and U residues. Initial rates were

determined for the ligation reaction involving 0.25–25 nM ribozyme and a trace amount of

[50-32P]-labelled substrate. The reaction mixture contained 100 mM MgCl2, 0.01% SDS

and 30 mM CHES (pH 9.0), which was incubated at 23 8C. Each value for k obs represents

the average of three independent experiments, with the error bars corresponding to one

standard deviation. The data were fit to a Michaelis–Menten saturation plot to obtain a

k cat of 0.0041 ^ 0.0006 h21 and K m of 1.6 ^ 0.9 nM. The k cat was adjusted to

account for a 6% maximum extent of reaction, giving a value of 0.068 ^ 0.010 h21.

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informational macromolecule is two, as was the case in this study.Without at least two different subunits, there is no information andthus no basis for darwinian evolution. A

MethodsSynthesis of oligonucleotidesAll oligonucleotides were synthesized using an Applied Biosystems Expedite automatedDNA/RNA synthesizer, employing either standard DNA or 2 0 -O-triisopropyl-silyloxymethyl RNA phosphoramidites, which were purchased from Glen Research.Oligonucleotides were purified in a denaturing polyacrylamide gel, eluted from the gel,and desalted before use.

Construction of starting pool and in vitro evolutionA DNA template was synthesized on the basis of a modified form of the R3 ribozyme thatcontained only three G residues (shown in bold in Fig. 1a). The first and last residues of the66-nucleotide RNA transcript were fixed as D and U, respectively. The G residues atpositions 58 and 63 were converted to U and D, respectively, and the residues at positions2–65 were randomly mutagenized (D ! U or U ! D) at a frequency of 12% pernucleotide position. The DNA template was transcribed in the presence of 2 mM each ofDTP and UTP (but no GTP or CTP), then digested with RNase-free DNase I. Thetranscription products were purified in a 6% denaturing polyacrylamide gel, eluted fromthe gel, desalted, then ligated to chimaeric RNA/DNA molecules having the sequence50-CUAGUGAGGCTGGATTGGTACGGTC-3

0(RNA portion in bold; terminal

20, 3

0-dideoxycytidine in italics). The ligation reaction was carried out in a mixture

containing 5 mM transcript, 25 mM RNA/DNA chimaera, 0.9 U ml21 T4 RNA ligase, 20%(V/V) dimethyl sulphoxide (DMSO), 10 mM MgCl2, 10 mM DTT, 50 mM Tris-HCl (pH7.8), and 1 mM ATP, which was incubated at 17 8C for 16 h. The 90-nucleotide ligatedproducts were separated from unligated material in a 6% denaturing polyacrylamide gel,eluted from the gel, and desalted. The starting pool contained approximately 8 £ 1013

different molecules.RNA-catalysed RNA ligation was carried out in the presence of 0.5 mM pool RNA, 5 mM

substrate having the sequence 5 0 -GCCTCCGAACGCTCCTAATACGACTCACUAGA-3 0

(T7 RNA polymerase promoter sequence underlined: RNA portion in bold), 25 mMMgCl2, 50 mM KCl, 30 mM N-(2-hydroxyethyl)-piperazine-N

0-3-propanesulphonic acid

(EPPS; pH 8.5), 4 mM dithiothreitol (DTT), and 2 mM spermidine, which were incubatedat 23 8C for 17 h. It should be noted that the promoter sequence differs from the standardT7 promoter at the next-to-last position, which was found to be beneficial for initiatingtranscription with a nucleotide other than guanylate19. The ligated products wereseparated in a 6% denaturing polyacrylamide gel, eluted from the gel, and precipitatedwith ethanol in the presence of 20 pmol of a DNA primer having the sequence5 0 -GACCGTACCAATCCAGC-3 0 . The primer was used to initiate reverse transcriptionin the presence of all four dNTPs. The reverse transcripts were precipitated withethanol, then PCR-amplified using primers 5 0 -GACCGTACCAATCCAGC-3 0 and50-GCCTCCGAACGCTCC-3

0. The PCR products were purified using a Qiagen PCR

purification kit, then transcribed in the presence of only DTP and UTP to initiate the nextround of in vitro evolution. Subsequent rounds were performed similarly, except that theywere carried out on a smaller scale and employed progressively shorter incubation timesduring the RNA-catalysed reaction.

Kinetic analysisThe kinetic properties of the ribozyme containing D, G and U residues were measured inthe presence of 25 mM MgCl2, 50 mM NaCl and 25 mM EPPS (pH 8.5) at 23 8C,employing trace amounts of [a-32P]UTP-labelled ribozyme and excess RNA substratehaving the sequence 5

0-UUAAUAAAUAUA-3

0. A Michaelis–Menten saturation plot was

generated on the basis of the initial rates of reaction and correcting for the maximumextent of reaction as determined by long time points. The cyclization reaction involvingthe final selected ribozyme was carried out employing 20 nM of an RNA that containedboth the ribozyme and substrate domains, which was heated to 94 8C for 1 min, thenrapidly cooled on ice before initiating the reaction by addition of 100 mM MgCl2, 0.01%SDS, and 30 mM 2-(N-cyclohexylamino)ethanesulphonic acid (CHES; pH 9.0). Theproducts were separated in a 6% denaturing polyacrylamide gel, with a running buffer thatcontained 40 mM Tris-borate and 0.9 mM Na2EDTA, then quantified using aphosphorimager. The time course of the reaction was fit to a single exponential, with amaximum extent of 8.3%. The intermolecular reaction involving the final selectedribozyme was carried out under the same conditions as above, except that it employed0.25–25 nM ribozyme and a trace amount of [5 0 -32P]-labelled substrate. The maximumextent of reaction was determined in the presence of 1 mM ribozyme. A Michaelis–Mentensaturation plot was constructed based on the initial rates of reaction and used to obtainvalues for k cat and K m.

The uncatalysed rate of reaction was determined under the same conditions as above,employing 100 nM substrate having the sequence 5 0 -UUDUUUUDDUUDUUDUD-3 0

and either 1 or 5 mM 5 0 -triphosphorylated RNA having the sequence 5 0 -UDUDUDDUDDUDDDUUUUUUUDUUDUUDUDUDUDUDDUDDUUDDDDUDD-3

0

(template region underlined). The uncatalysed rate was the same in the presence of either 1or 5 mM template, demonstrating saturation of the template–substrate complex. Thereaction was carried out in quadruplicate over 91 h, with an observed linear rate of productformation of 3.0 ^ 1.6 £ 1028 min21 (r ¼ 0.89).

Analysis of regiospecificityA [5

0-32P]-labelled RNA substrate having the sequence 5

0-UUAAUAAAUAUA-3

0was

incubated for 16 h in the presence of excess ribozyme under the same conditions that wereemployed during in vitro evolution. The ligated products were isolated in a 6% denaturingpolyacrylamide gel, eluted from the gel, and precipitated with ethanol. A version of the10-23 DNA enzyme13, having the sequence 5 0 -TATTTATTATTATATAGGCTAGCTACAACGAATATTTATTAA-3

0, was directed to cleave

the phosphodiester linkage at the ligation junction. DNA-catalysed RNA cleavage wascarried out in the presence of a trace amount of 5

0-labelled ligated material, 60 mM DNA

enzyme, 25 mM MgCl2, 50 mM KCl and 50 mM EPPS (pH 8.5), which were incubated at37 8C for 90 min, then quenched by the addition of Na2EDTA. The digested products wereseparated in a 10% denaturing polyacrylamide gel and their mobility was compared tothat of authentic materials.

Received 30 August; accepted 17 September 2002; doi:10.1038/nature01185.

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Acknowledgements We thank J. Rogers for many discussions during the initial stages of this

project. We also thank the members of the Joyce laboratory for their advice and E. Tzima for

assistance in preparation of the manuscript. This work was supported by a grant from the

National Aeronautics and Space Administration and the Skaggs Institute for Chemical Biology.

J.S.R. was supported by a postdoctoral fellowship from the NASA Specialized Center for Research

and Training (NSCORT) in Exobiology.

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to G.F.J.

(e-mail: gjoyce@scripps.edu).

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