letters to nature
(RE67757), and dCsl4 (RE64677). They were amplified by PCR using the following
primers: dRrp6 þ 1F and dRrp6 þ 579R; dMtr3 þ 1F and dMtr3 þ 326R; Rrp4 þ 1F
and Rrp4 þ 298R; dSki6 þ 1F and dSki6 þ 246R; dCsl4 þ 1F and dCsl4 þ 204R. They
were digested with the appropriate restriction endonucleases and cloned into pMAL-C2
(New England Biolabs) to create MBP–dRrp6N, MBP–dMtr3, MBP–dRrp4, MBP–dSki6
and MBP–dCsl4. MBP fusions were expressed in Escherichia coli and recombinant proteins
were purified over amylose resin according to the manufacturer’s recommendations (New
England Biolabs). Recombinant proteins were injected into either rat or guinea-pig and
antibodies 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 mM
Tris-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 a
final concentration of 100 mg ml21. Immune complexes were incubated with either
protein-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 western
blotting.
Exoribonuclease assays
We 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, and
then 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 added
thereafter 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 0 RNA, ,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 the
desired time and stopped by the addition of an equal volume of formamide dye. RNA
species were separated on a 12% denaturing polyacrylamide gel and analysed by
phosphoimaging.
ChIP and immunofluorescence analyses
Crosslinked material was prepared from formaldehyde-fixed Kc cells. We carried out
immunoprecipitations with antibodies to Drosophila proteins and analysed them as
described5. 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).
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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: [email protected]) or E.D.A.(e-mail: [email protected]).
..............................................................
A ribozyme composed of only
two different nucleotides
John S. Reader & Gerald F. Joyce
Departments of Chemistry and Molecular Biology and The Skaggs Institute for
Chemical 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 early
history of life on Earth because of their ability both to encode
genetic information and to exhibit catalytic function1. The
modern genetic alphabet relies on two sets of complementary
base pairs to store genetic information. However, owing to the
chemical instability of cytosine, which readily deaminates to
uracil2, a primitive genetic system composed of the bases A, U,
G and C may have been difficult to establish. It has been suggested
that the first genetic material instead contained only a single
base-pairing unit3–7. Here we show that binary informational
macromolecules, containing only two different nucleotide subunits, can act as catalysts. In vitro evolution was used to obtain
ligase ribozymes composed of only 2,6-diaminopurine and uracil
nucleotides, which catalyse the template-directed joining of two
RNA molecules, one bearing a 5 0 -triphosphate and the other a
3 0 -hydroxyl. The active conformation of the fastest isolated
ribozyme had a catalytic rate that was about 36,000-fold faster
than the uncatalysed rate of reaction. This ribozyme is specific
for the formation of biologically relevant 3 0 ,5 0 -phosphodiester
linkages.
A good starting point for the evolution of a catalyst that contains
only two different subunits was the R3 ligase ribozyme, which
contains only adenine, guanine and uracil nucleotides8,9. This
ribozyme catalyses attack of the 3 0 -hydroxyl of an RNA substrate
on the 5 0 -triphosphate of the ribozyme, forming a 3 0 ,5 0 -phosphodiester and releasing inorganic pyrophosphate. The chemistry of
this reaction is identical to that catalysed by modern RNA polymerase proteins. A templating region within the ribozyme is
responsible for binding the RNA substrate, and the sequences of
both the template and substrate can be designed such that they
contain only adenine and uracil residues. In that format, both the
ribozyme and substrate are completely devoid of cytosine and
undergo 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 2,6diaminopurine (D). Three bonds are formed between 2,6-D and
uracil in the context of a Waston–Crick base pair10, in comparison
with adenine, which forms only two. Despite this difference, the
D-substituted R3 ligase (Fig. 1a) retained a k cat of 0.001 min21 and
K m of 12 mM, and reacted to a maximum extent of about 40%.
© 2002 Nature Publishing Group
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letters to nature
Substituting diaminopurine 5 0 -triphosphate (DTP) for ATP when
transcribing AT-containing DNA templates was found to have two
important advantages for in vitro evolution experiments. First, this
substitution reduced ‘slippage’ of the RNA polymerase11 as it
proceeded along the DNA template. Second, transcription could
be initiated with a D residue at the 5 0 end of the RNA, allowing
transcripts 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 ligase
ribozymes that contain only D and U.
The first stage of the process to develop a DU-containing catalyst
involved substituting as many of the G residues as possible by either
D or U, while still retaining some detectable activity. Substitutions
were tolerated throughout the stem-loop regions of the ribozyme,
replacing G†U ‘wobble’ pairs with D†U pairs, and replacing G with
D at most of the unpaired nucleotide positions. The final substituted ribozyme contained only three of the 16 G residues that were
present in the starting molecule. Two of the remaining G residues
were located at the ligation site, and the other was in the singlestranded region connecting the P4 and P2 stems (Fig. 1a).
The second stage of the development of a binary informational
catalyst involved in vitro evolution to compensate for removal of the
final G residues and improve catalytic activity. The sequence of the
ribozyme that contained only three G residues was modified by
replacing the remaining G residues with either D or U, then
introducing random mutations (either D ! U or U ! D) at a
frequency of 12% per nucleotide position. A population of 8 £ 1013
different randomized variants was constructed, containing only D
and U residues at nucleotide positions 1–66. Positions 66–74,
located at the 3 0 end of the ribozyme, are involved in binding the
oligonucleotide substrate. For purposes of in vitro evolution, the
substrate contained the sequence of the T7 promoter element,
which includes all four nucleotides. Thus the corresponding template region of the ribozyme was required, at least temporarily, to
contain all four nucleotides. Experience had shown, however, that
transcription in the presence of all four NTPs would invariably lead
to a resurgence of G and C residues in the ribozyme. Thus a strategy
was adopted whereby the 5 0 portion of the ribozyme (positions
1–66) was transcribed in the presence of only DTP and UTP, after
which an oligonucleotide containing a constant substrate-binding
region (positions 67–74) was attached by enzymatic ligation
employing T4 RNA ligase.
The population of ribozyme variants was given an opportunity to
ligate the promoter-containing substrate. The reacted molecules
were purified by electrophoresis in a denaturing polyacrylamide gel,
then reverse transcribed and PCR amplified in the presence of all
four standard deoxynucleoside 5 0 -triphosphates. In principle, only
those molecules that contained D and U residues at positions 1–66
and had catalysed the ligation reaction would be eligible for
subsequent transcription to generate progeny molecules. After
four rounds of selective amplification, a ligated product was
detected in the polyacrylamide gel following the RNA-catalysed
reaction. A fifth round was carried out and individual molecules
were cloned from the population and sequenced. Only two of the 22
sequenced clones contained a single contaminating G or C residue
at positions 1–66. There was considerable sequence heterogeneity
among the clones. Only one sequence was found to occur repeatedly, 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 5 0 end and lengthened by six
nucleotides at the 3 0 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%.
842
© 2002 Nature Publishing Group
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letters to nature
cloned ribozymes were then prepared in a form that lacked G and C
residues within the substrate-binding region. This was accomplished by synthesizing DNA templates based on the sequence of
each clone, but with modification of the substrate-binding region so
that it contained only D and U residues. The ribozymes containing
only D and U residues were then challenged to react with a
complementary substrate that contained only A and U residues.
All eight of the clones that were tested in this fashion had some
detectable activity.
The sequences of the two most active clones were used as the basis
to construct two separate pools of RNAs that were mutagenized at a
frequency of 12% per nucleotide position. Five additional rounds of
selective amplification were performed, resulting in DU-containing
ribozymes that were improved with regard to their catalytic activity.
Individual clones were again isolated from the final population; the
most active of these clones is shown in Fig. 1b. This ribozyme
contained many of the mutations that were present in the parent
clone, but also contained new mutations, including several reversions. There was also a deletion at the 5 0 end of the ribozyme that
resulted in the 5 0 -terminal residue being a uridine, located adjacent
to an unpaired adenosine at the 3 0 end of the substrate. Many other
mutations were present throughout the final selected ribozyme,
presumably resulting in substantial remodelling of its overall
structure and the detailed structure at the ligation junction.
The secondary structure of the final selected ribozyme that is
depicted in Fig. 1b is drawn by analogy to that of the D-substituted
starting molecule, shown in Fig. 1a. Although the secondary
structure of the starting molecule is well established9, that of the
final ribozyme must be regarded as conjectural, especially for the P2
and P3 stems (see Fig. 1a). The final ribozyme can adopt several
different structures, as suggested by the presence of multiple bands
when it was analysed by non-denaturing polyacrylamide gel electrophoresis (data not shown). As discussed below, most of the
molecules were not in an active conformation, which thwarted
attempts to carry out meaningful analysis of the ribozyme’s
secondary structure.
The rate of reaction of the final selected ribozyme was examined
in a format in which the ribozyme and substrate contained only D
and U residues. Two different constructs were prepared, one in
which the substrate was presented separately and another in which it
was tethered to the 3 0 end of the ribozyme by a stable hairpin
structure. The latter format allowed for a cyclization reaction
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
[5 0 -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.
NATURE | VOL 420 | 19/26 DECEMBER 2002 | www.nature.com/nature
involving the 3 0 -hydroxyl and 5 0 -triphosphate of the same RNA,
enabling a more straightforward assessment of the fraction of
molecules that were in a productive conformation. The observed
rate of the cyclization reaction fitted well to a single exponential
model, with a k obs of 0.00080 min21 and a maximum extent of
about 8% of the total RNA molecules (Fig. 2). A probable explanation for the substantial proportion of unreacted RNA molecules
is their propensity to misfold based on their extraordinary degree of
internal self-complementarity.
The reaction with a separate RNA substrate was used to show that
the ribozyme exhibits Michaelis–Menten saturation kinetics. The
ribozyme was engineered to bind a separate 17-nucleotide RNA
substrate having the sequence 5 0 -UUDUUUUDDUUDUUDUD-3 0
(Fig. 1b). Experiments were performed in the presence of excess
ribozyme, employing a [5 0 -32P]-labelled substrate. On the basis of
the initial rates of reaction in the presence of various concentrations
of 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). The
uncatalysed rate of ligation was measured under the same reaction
conditions employing the same template and substrate sequences.
That rate was 3 £ 1028 min21, which agrees well with previous
measurements of uncatalysed template-directed RNA ligation12,
and corresponds to a catalytic rate enhancement of about 36,000fold. When the RNA-catalysed reaction was performed with a
substrate that was not complementary to the template, there was
no detectable reaction.
The ligation reaction catalysed by the DU-containing ribozyme
may have resulted in the formation of either a 2 0 ,5 0 - or 3 0 ,5 0 phosphodiester linkage, owing to the lack of selective pressure to
maintain the 3 0 ,5 0 -regiospecificity of the starting R3 ligase. The
regiospecificity of the reaction was analysed by employing the
‘10-23’ DNA enzyme, which cleaves 3 0 ,5 0 , but not 2 0 ,5 0 linkages of
RNA13. The ligated product was cleaved by the DNA enzyme to
generate two fragments of the expected size, demonstrating that the
ribozyme, itself composed of 3 0 ,5 0 -linked ribonucleotides, catalyses
the formation of a 3 0 ,5 0 -phosphodiester linkage.
It has been suggested that the original genetic system contained
only two different nucleotides3–7, and subsequently evolved to its
present, more complex form. A binary genetic system may have
been advantageous during the early history of life on earth when the
availability of all four nucleotides might have been difficult to
maintain. Cytosine nucleotides are especially problematic because
they undergo rapid deamination to uridylate, with a half-life of
19 days at pH 7 and 100 8C (ref. 2). Thus the G†C pairing may not
have been sustainable until the invention of a mechanism for
restoring cytosine to uracil. By comparison, the half-life of adenine
or diaminopurine at pH 7 and 100 8C is about one or two years,
respectively2. The prebiotic synthesis of adenine or diaminopurine
proceeds with comparable efficiency, both compounds being
obtained in good yield starting from aqueous ammonium cyanide14,15.
Nucleic acid enzymes composed of only D and U residues pay a
heavy price for their simplified composition in terms of both
catalytic rate and the fraction of molecules that are in an active
conformation. Nonetheless, darwinian evolution can produce catalytically active structures even from such a severely restricted
chemical repertoire. The conformational plasticity of DU-containing RNA may even offer an advantage with regard to the ability to
explore multiple structures for a given sequence16.
Ribozymes composed of other pairs of nucleotides, such as A and
U, G and C, or even A and I (inosine), may be possible. It seems less
likely that polymers composed of only two amino acids could
exhibit appreciable catalytic activity. Thus far a minimum of 14
amino acids has been used to construct a catalytic polypeptide17,
and as few as seven have been shown to be necessary to define a
folded tertiary structure18. The absolute minimum number of
distinct subunits that could be used to construct a functional
© 2002 Nature Publishing Group
843
letters to nature
informational macromolecule is two, as was the case in this study.
Without at least two different subunits, there is no information and
thus no basis for darwinian evolution.
A
Methods
Synthesis of oligonucleotides
All oligonucleotides were synthesized using an Applied Biosystems Expedite automated
DNA/RNA synthesizer, employing either standard DNA or 2 0 -O-triisopropylsilyloxymethyl 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 evolution
A DNA template was synthesized on the basis of a modified form of the R3 ribozyme that
contained only three G residues (shown in bold in Fig. 1a). The first and last residues of the
66-nucleotide RNA transcript were fixed as D and U, respectively. The G residues at
positions 58 and 63 were converted to U and D, respectively, and the residues at positions
2–65 were randomly mutagenized (D ! U or U ! D) at a frequency of 12% per
nucleotide position. The DNA template was transcribed in the presence of 2 mM each of
DTP and UTP (but no GTP or CTP), then digested with RNase-free DNase I. The
transcription products were purified in a 6% denaturing polyacrylamide gel, eluted from
the gel, desalted, then ligated to chimaeric RNA/DNA molecules having the sequence
5 0 -CUAGUGAGGCTGGATTGGTACGGTC-3 0 (RNA portion in bold; terminal
2 0, 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 (pH
7.8), and 1 mM ATP, which was incubated at 17 8C for 16 h. The 90-nucleotide ligated
products 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 mM
MgCl2, 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 incubated
at 23 8C for 17 h. It should be noted that the promoter sequence differs from the standard
T7 promoter at the next-to-last position, which was found to be beneficial for initiating
transcription with a nucleotide other than guanylate19. The ligated products were
separated in a 6% denaturing polyacrylamide gel, eluted from the gel, and precipitated
with ethanol in the presence of 20 pmol of a DNA primer having the sequence
5 0 -GACCGTACCAATCCAGC-3 0 . The primer was used to initiate reverse transcription
in the presence of all four dNTPs. The reverse transcripts were precipitated with
ethanol, then PCR-amplified using primers 5 0 -GACCGTACCAATCCAGC-3 0 and
5 0 -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 next
round of in vitro evolution. Subsequent rounds were performed similarly, except that they
were carried out on a smaller scale and employed progressively shorter incubation times
during the RNA-catalysed reaction.
Kinetic analysis
The kinetic properties of the ribozyme containing D, G and U residues were measured in
the 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 substrate
having 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 maximum
extent of reaction as determined by long time points. The cyclization reaction involving
the final selected ribozyme was carried out employing 20 nM of an RNA that contained
both the ribozyme and substrate domains, which was heated to 94 8C for 1 min, then
rapidly 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). The
products were separated in a 6% denaturing polyacrylamide gel, with a running buffer that
contained 40 mM Tris-borate and 0.9 mM Na2EDTA, then quantified using a
phosphorimager. The time course of the reaction was fit to a single exponential, with a
maximum extent of 8.3%. The intermolecular reaction involving the final selected
ribozyme was carried out under the same conditions as above, except that it employed
0.25–25 nM ribozyme and a trace amount of [5 0 -32P]-labelled substrate. The maximum
extent of reaction was determined in the presence of 1 mM ribozyme. A Michaelis–Menten
saturation plot was constructed based on the initial rates of reaction and used to obtain
values for k cat and K m.
844
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 -UDUDU
DDUDDUDDDUUUUUUUDUUDUUDUDUDUDUDDUDDUUDDDDUDD-3 0
(template region underlined). The uncatalysed rate was the same in the presence of either 1
or 5 mM template, demonstrating saturation of the template–substrate complex. The
reaction was carried out in quadruplicate over 91 h, with an observed linear rate of product
formation of 3.0 ^ 1.6 £ 1028 min21 (r ¼ 0.89).
Analysis of regiospecificity
A [5 0 -32P]-labelled RNA substrate having the sequence 5 0 -UUAAUAAAUAUA-3 0 was
incubated for 16 h in the presence of excess ribozyme under the same conditions that were
employed during in vitro evolution. The ligated products were isolated in a 6% denaturing
polyacrylamide gel, eluted from the gel, and precipitated with ethanol. A version of the
10-23 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 was
carried 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 at
37 8C for 90 min, then quenched by the addition of Na2EDTA. The digested products were
separated in a 10% denaturing polyacrylamide gel and their mobility was compared to
that of authentic materials.
Received 30 August; accepted 17 September 2002; doi:10.1038/nature01185.
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Laboratory Press, Cold Spring Harbor, 1999).
2. Levy, M. & Miller, S. L. The stability of the RNA bases: implications for the origin of life. Proc. Natl
Acad. Sci. USA 95, 7933–7938 (1998).
3. Rich, A. Horizons in Biochemistry (eds Kasha, M. & Pullman, B.) 103–126 (Academic, New York,
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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: [email protected]).
© 2002 Nature Publishing Group
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