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INTRODUCTION |
The RNA world theory assumes that modern life arose from molecular
ancestors in which RNA molecules both stored genetic information and
catalyzed chemical reactions (1-4). According to this scenario, ribozymes of the RNA world would have been able to self-replicate (5)
and to control complex metabolisms with an expanded chemical repertoire
(6, 7). Until recently, RNA catalysis was believed to be restricted to
phosphate chemistry, but in vitro selection experiments and
recent discoveries concerning natural ribozymes have demonstrated that
the catalytic capacities of RNA are far more promising and exciting
than previously anticipated (8-13). However, in comparison with
proteins, the chemical spectrum of ribozymes remains limited because of
the limited chemical diversity of RNA, which is composed of only four
different building blocks.
Yet RNA could increase its range of functionalities by incorporating
catalytic building blocks such as imidazole, thiol, and functional
amino and carboxylate groups (14, 15). Moreover, primeval nucleotides
were not necessarily restricted to standard nucleotides; modified
nucleotides may have played a role in catalysis in the RNA world (16,
17).
Another way for RNA to increase its chemical diversity would consist in
the binding of exogenous molecules carrying reactive groups and
handling them as catalytic cofactors. We recently reported the
isolation of new RNA aptamers able to bind adenine in a novel mode of
purine recognition (18). Adenine is a likely prebiotic analog of
histidine. Its catalytic capabilities are equivalent to histidine
because of the presence of a free imidazole moiety (19-21). It was
previously shown that when adenine is placed in a favorable
microenvironment, its catalytic efficiency is strongly enhanced
(22-24). Such favorable microenvironments could result from adenine
binding to RNA and thereby providing catalytic sites. In this
perspective, it is significant that abasic hairpin and hammerhead
ribozymes can be rescued by the addition of exogenous bases that
restore activity (25, 26) and that imidazole and cytosine can
rescue a cytosine mutation in a self-cleaving hepatitis delta virus
ribozyme (27, 28). Also significant is the sequence homology between
loop B of the hairpin ribozyme and the XBA aptamer (29), which was
selected on the basis of its capacity to bind xanthine and guanine.
Hence, the hairpin ribozyme may constitute a good starting point for
the search of new ribozymes that require catalytic organic cofactors.
We thus designed a SELEX procedure (systematic
evolution of ligands by exponential
enrichment) to select inactive hairpin variants capable of recovering
activity by the addition of exogenous free adenine. The selection was
started from hairpin ribozyme with randomized sequences located in
regions previously reported to be required for catalysis. Using this
approach, our aim was to maximize the opportunities of selecting new
hairpin ribozymes in which adenine restores the activity by its direct
involvement in catalysis. Here we report the discovery of two new
hairpin ribozymes that require adenine as catalytic cofactor for their reversible self-cleavage.
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MATERIALS AND METHODS |
Preparation of the Starting RNA Pool--
Single-stranded DNA
template and primers were synthesized chemically (Genset). The sequence
of primer P1 (promoter primer) is
5'-TAATACGACTCACTATAGGGTACGCTGAAACAG-3' (T7 promoter sequence in bold), and that of primer P2 (reverse primer) is
5'-CCTCCGAAACAGGACTGTCAGGG-GGTACCAG-3'. The 80-nucleotide-long
template consists of the minus strand that allows the synthesis of a
hairpin ribozyme randomized in 20 critical positions. Its entire
sequence is
5'-CCTCCGAAACAGGACTGTCAGGGGGTACCAGNNNNNNNNNCACAACGTGNNNNNNNCTGGTTGACNNNNCTGTTTCAGCG-3'. The two primer binding regions are located in the 5'- and 3'-termini. The 20 randomized nucleotides are all located within loops A and B
(Fig. 1). The mutations are expected to perturb the geometry of the
active site and hence the activity of the ribozyme. The variants to be
selected are those that are inactive but whose activity is rescued by
free adenine. In contrast to loop B, which is entirely randomized, only
the first four unpaired nucleotides of loop A are randomized, because
the 3'-unpaired sequence of the loop A is located in the reverse primer
binding region that must be kept constant.
A 6-ml PCR (Invitrogen) reaction with 1 µM each primer
(P1 and P2) and 0.1 µM template (about 3.4 × 1014 random molecules) amplified by 10 cycles was
performed. The random double-stranded DNA pool was then
ethanol-precipitated and submitted to in vitro transcription
(6 ml) using T7 RNA polymerase (Fermentas). The reaction mixture
contained 2.5 mM each rNTP, 2 mM spermidine, 10 mM dithiothreitol, 10 mM NaCl, 6 mM MgCl2, 1 µM randomized DNA and
24,000 units of T7 RNA polymerase. After overnight incubation at
37 °C, the reaction mixture was purified on a 10% denaturing polyacrylamide gel (PAGE), ethanol-precipitated, and resuspended in
distilled water, yielding ~10 nmol of randomized RNA.
Selection Procedure--
Each selection round consisted in the
alternation of negative and positive selection steps followed by
reamplification of the selected material. Negative selection steps were
performed to eliminate molecules that can self-cleave in the absence of adenine. The RNA was incubated in cleavage buffer (40 mM
HEPES, 6 mM MgCl2, pH 7.5) without adenine and
purified by 10% denaturing PAGE. Positive selection was performed by
incubating uncleaved RNA in cleavage buffer supersaturated with adenine
(20-30 mM). The cleaved fragments were purified by
denaturing PAGE and amplified by
RT-PCR.1 Additionally, during
the last two selection rounds, the 5'-cleaved fragments were selected
for adenine-specific religation with the 3'-cleaved fragments; after
positive selection for adenine-specific RNA cleavage, purified
5'-cleaved fragments were incubated with a large excess of 3'-cleaved
fragments in cleavage buffer. Unligated RNAs were recovered and
incubated again with a large excess of 3'-cleaved fragments in the
presence of adenine. Religated ribozymes were recovered and amplified.
During each selection round, the randomized RNA pool was dissolved in
the cleavage buffer, heated to 65 °C for 3 min, and cooled slowly
(3 °C/min) to 23 °C (denaturation and renaturation steps). The
solution was then incubated at room temperature, and the cleavage
reaction was stopped by adding 1 volume of loading solution (30 mM EDTA, 80% formamide). The RNA concentration was 50 µM in the first generation (G0), and 20-70
µM in the other selection rounds. The reaction times for
negative selection were 2-50 h for G0-G9 and 2-4 h for G9-G11, and
the negative selections in G0-G8 were repeated. The uncleaved RNAs
recovered from the second negative selection were dissolved in cleavage
buffer, denatured and renatured, and then incubated with 3 volumes of
cleavage buffer supersaturated in adenine. Reaction times for positive
selection were 3-20 h for G0-G5 and 10-40 min for G6-G11. After the
loading solution had been added to stop the reaction, the cleaved
products were purified by denaturing PAGE, ethanol-precipitated (52.5 µg/ml of glycogen was added as carrier), and resuspended in distilled water.
The pooled RNA was reverse transcribed using 20 units/µl Moloney
murine leukemia virus reverse transcriptase (MMLV-RT,
Invitrogen) with 20 µM reverse primer P2 at
37 °C for 1 h. The cDNA was amplified by PCR with 1 µM each primer (P1 and P2). The thermal cycle program was
94 °C for 30 s, 56 °C for 30 s, and 72 °C for
60 s; the cycle was repeated 16 times. After ethanol precipitation
and resuspension in water, the newly selected DNA library served to
produce the RNA pool for the next selection round by in
vitro transcription.
During the last two selection rounds, two additional steps following
positive selection were performed to isolate ribozymes able to religate
in the presence of adenine. First, adenine-cleaved RNA was dissolved in
cleavage buffer in the presence of a 10-fold excess of ligation
substrate (LS). LS is a 15-nucleotide-long DNA/RNA mixed
oligonucleotide (Genset) in which the sequence corresponds to the
3'-cleaved product (Fig. 1). Its 5'-end is composed of 9 ribonucleotides, and its 3'-terminus is composed of 6 deoxyribonucleotides because 2'-OH are not necessary in this region.
Cleaved RNA and LS were heated to 65 °C for 3 min and cooled slowly
(3 °C/min) to 8 °C (denaturation and renaturation steps in
cleavage buffer prior to ligation). The mixture was incubated at
8 °C for 2 h. The ligation reaction was stopped, and unligated
fragments were purified by denaturing PAGE. Finally, the unligated RNAs
were again dissolved in cleavage buffer in the presence of a 10-fold excess of LS and then denatured and renatured prior to ligation. Three
volumes of a 8 °C cleavage buffer supersaturated with adenine were
added, and the solution was incubated at 8 °C for 2 h. The ligated products purified by denaturing PAGE were used for reverse transcription as described above.
After each cleavage or ligation reaction, aliquots (2 µl containing
0.2-1 µg of RNA) were analyzed by denaturing 10% PAGE and ethidium
bromide staining. RNA fragments were revealed by UV trans-illumination
and scanned. The light intensities of the fragments were quantified
using an NIH Image analyzer. It was thus possible to monitor the
evolution of cleavage or ligation efficiencies in the presence or
absence of adenine during the selection process.
Cloning--
Ribozymes positively selected at G11 were cloned
(TOPO TA cloning kit, Invitrogen) after conversion of RNA to
double-stranded DNA by RT-PCR. Plasmids were prepared from isolated
clones (Plasmix minipreps, Talent), and the cloned DNAs were
sequenced. After plasmid purification and in vitro
transcription of the PCR-amplified inserted fragments, individual RNAs
were prepared for further studies.
Cleavage Reactions of Individual RNA
Molecules--
Double-stranded DNA templates were prepared from
individual plasmids by PCR using primers P1 and P2. The RNAs were
transcribed from the DNA templates and purified as described above.
Individual RNAs (4 µM) were dissolved in cleavage buffer
and subjected to the denaturation and renaturation steps. The solutions
were then incubated with 3 volumes of cleavage buffer alone (negative
controls) or with 3 volumes of adenine containing cleavage
buffer (2.7 mM adenine). Aliquots were removed from the
mixtures at various times and added to 1 volume of loading solution.
After overnight incubation with adenine or 3 days of incubation in
cleavage buffer alone, the reactions were analyzed by denaturing PAGE
and ethidium bromide staining. The observed rate constant values
(kobs) were calculated using the equation
S/(S + L) = a(1
exp(
kobst)) (30), where S is the
concentration of the cleaved product, L the concentration of the
precursor, t is time, and a is the percentage of
cleaved product versus total RNA at equilibrium.
Ligation Reactions of Individual RNA Molecules--
Individual
RNAs were subjected to overnight adenine-assisted self-cleavage, and
the cleaved fragments were purified as described above. Individually
cleaved RNAs (4 µM) were dissolved in cleavage buffer
with 72 µM LS, denatured, and renatured prior to
ligation. The solutions were then incubated with 3 volumes of cleavage
buffer either alone (negative controls) or supplemented with 2.7 mM adenine. Reactions were analyzed, and the
kobs values were calculated, except that S was
taken as the concentration of the ligation product.
Adenine Dependence of Cis-cleavage Reactions--
Individual
RNAs (4 µM) were dissolved in cleavage buffer. After
denaturation and renaturation, 8 aliquots were prepared and incubated
with 3 volumes of varying concentrations of adenine in cleavage buffer
(0.027 to 20 mM). Aliquots were removed after 30, 60, 90, and 120 min, the cleavage reactions were analyzed, and the
kobs were plotted as a function of adenine concentration.
Magnesium Dependence of Cis-cleavage Reactions--
Individual
RNAs (4 µM) were dissolved in cleavage buffer with
MgCl2 concentrations ranging from 0.1 µM to
10 mM. After denaturation and renaturation, the solutions
were incubated with 3 volumes of adenine (30 mM) in
cleavage buffer with the corresponding MgCl2 concentrations. Aliquots were removed at various times, the cleavage reactions were analyzed, and the kobs values
were plotted as a function of MgCl2 concentration.
Cis-cleavage Reactions with Small Molecules--
Individual RNAs
were dissolved in cleavage buffer and subjected to the denaturation and
renaturation steps. Aliquots were incubated with 3 volumes of 3-5
different concentrations of small molecules in cleavage buffer (from
0.1 mM to 150 mM depending on the solubility of
the compound). The molecules tested for cis-cleavage were
1-methyladenine, 3-methyladenine, 6-methyladenine, purine, xanthine,
hypoxanthine, uracil, and imidazole. Aliquots were removed at various
times and analyzed as described above. The kobs
values were plotted as a function of small molecules concentrations.
Studies of the pH Dependence of Cis-cleavage
Reactions--
Individual RNAs were dissolved in cleavage buffers with
pH values ranging from 4 to 10 prior to the denaturation and
renaturation steps. The buffers used were 40 mM sodium
acetate for pH 4 and 5, 40 mM HEPES for pH values from 6 to
8.2, and 40 mM glycine for pH 8.6, 9, and 10. After
denaturation and renaturation, the solutions were incubated with 3 volumes of 32 mM adenine dissolved in the cleavage buffers
with the respective pH values. Aliquots were removed at various times,
and the cleavage reactions were analyzed as described above. The
experiment was repeated using 32 mM imidazole instead of
adenine and were also performed without cofactor. The
kobs values for adenine-assisted cleavage,
imidazole-assisted cleavage, and RNA cleavage without cofactor were
plotted as a function of pH.
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RESULTS |
In Vitro Selection--
In vitro selection began with a
pool of 85-nucleotide-long hairpin ribozymes containing 20 degenerate
nucleotides. The randomized positions were all located in loop A (4 randomized nucleotides) and in loop B (16 randomized nucleotides).
Degeneracy was introduced in these positions because loops A and B
contain the nucleotides required for catalytic activity in the
wild-type ribozyme (Fig. 1). Our
selection procedure was designed to select inactive ribozymes in which
catalytic activity could be rescued by the addition of free exogenous
adenine.

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Fig. 1.
Wild-type hairpin ribozyme and randomized
starting construct sequences. A, minimal wild-type
self-cleaving hairpin ribozyme. The cleavage site is indicated with an
arrowhead. B, randomized construct used
for in vitro selection. 20 degenerate nucleotides are
located within loops A and B at the positions required for catalysis.
3'- and 5'-extensions are added for hybridization with replication
primers.
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The initial RNA population was composed of ~1015
molecules and expected to express 1012 different ribozyme
variants. After transcription from the DNA pool, two successive
negative selections were carried out by incubating the transcripts in
the absence of free adenine. The uncleaved RNAs were isolated by
denaturing PAGE and incubated in the presence of free adenine. The
70-nucleotide-long 5'-cleaved products were purified by PAGE. The
selected molecules were converted to DNA and amplified by RT-PCR. The
alternation of the negative and positive selection steps was repeated
for 12 rounds. The progress of in vitro selection is shown
in Fig. 2.

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Fig. 2.
Progress of in vitro
selection. Self-cleavage or self-ligation percentages
during each round of selection. Adenine-assisted cleavage
percents are represented by black bars, and adenine-assisted
ligation percents are represented by white bars. RNA
self-cleavage and self-ligation percentages without adenine are
represented by gray bars. The + and symbols indicate
the presence or absence, respectively, of adenine during the incubation
of RNA. Incubation times are also indicated at the bottom of
the figure.
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During the first four selection rounds, the amount of cleaved RNA was
too small to be detected after negative or positive selection. RNA
cleavage became detectable as of the fifth selection round (G4), but
further selection rounds were necessary to eliminate adenine-independent self-cleaving ribozymes from the population. Adenine-cleaved hairpin ribozymes became predominant in the population at G8. Selection for adenine-assisted self-cleavage was continued to
G11. From G9 to G11, the second negative selection step was omitted
because most of the adenine-independent self-cleaving ribozymes were
removed in G8. In addition, during G10 and G11, adenine-cleaved
ribozymes also served to select for adenine-assisted religation. After
positive selection and purification of the active molecules, the RNA
was incubated with a 10-fold molar excess of LS without free adenine.
Unligated fragments were purified by PAGE and incubated again with a
10-fold excess of LS in the presence of free adenine (incubation times
are shown in Fig. 2). Religated molecules were purified by PAGE and
converted to DNA by RT-PCR. During G10, self-cleavage of the negative
and positive selections were 1 and 10%, respectively, and the
self-ligation product after negative and positive selections for
religation were undetectable. During G11, self-cleavage of the negative
and positive selections were 1 and 30%, respectively, and
self-ligation of the negative and positive selections for religation
were 1.5 and 6%, respectively.
Sequence of Clones--
The RNA pool selected for adenine-assisted
cleavage and religation during G11 was converted to DNA by RT-PCR and
cloned. Two different sequences were obtained after sequencing of 14 clones: ADHR1 and ADHR2 (adenine-dependent
hairpin ribozyme). The sequences and secondary
structures are shown in Fig.
3A.
The ADHR1 aptamer displays four mutations with respect to the wild-type
ribozyme (U20, A36, G38, and C39 instead of A20, G36, A38, and U39 in
the wild-type RNA) and the ADHR2 aptamer displays 6 mutations with respect to the wild-type ribozyme (U20, C24, C26, C37, U38 and G39
instead of A20, A24, A26, U37, A38, and U39 in the wild-type RNA). Both
aptamers share a common mutation, U20 in place of A20 in the wild-type
RNA. As expected, the guanosine located downstream of the cleavage site
(G+1), which is not in the initial randomized zone, is conserved. C25
and U42, which were included in the randomized zone, are
conserved. These 3 nucleotides are believed to be the major
determinants for the stability of the tertiary structure of the
wild-type ribozyme by creating H-bond networks that stabilize the
ribozyme in its docked conformation (31, 32). It is thus very likely
that ADHR1 and ADHR2 share the same global docked and active
conformation as the wild-type hairpin ribozyme. In contrast, several
critical nucleotides of the catalytic site of the wild-type ribozyme
were replaced: G36 and A38 were changed to A36 and G38 in ADHR1; A24,
A26, and A38 were changed to C24, C26, and U38 in ADHR2. There must be
more significant differences in the conformation at the active site of
ADHR1 and ADHR2 compared with the wild-type ribozyme because these
nucleotides are located in the vicinity of the scissile phosphate
(33).


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Fig. 3.
Analysis of ADHR1 and ADHR2 self-cleavage
and self-ligation activities. A, secondary
structures of aptamers. The cleavage site is indicated with an
arrowhead. Nucleotides that were changed with respect to the
wild-type ribozyme are indicated with gray circles.
Respective kobs and end point values in the
presence or absence of adenine are shown under each aptamer secondary
structure; + or symbols indicate that reactions were performed
with or without adenine, respectively. B, cleavage and
ligation kinetics analyzed by 10% denaturing PAGE and ethidium bromide
staining. Ade(+) and Ade( ) kinetics were performed with or without
adenine, respectively. Prec, full-length RNA precursor;
5'-Clv, 5' cleaved product; 5'-Prec, 5'
cleaved product precursor for the ligation reaction; Lig,
ligation product. C, ADHR1 and ADHR2 self-cleavage and
self-ligation kinetics plots. Clv ade(+) and Clv
ade( ) indicate that cleavage kinetics were performed with or
without adenine, respectively. Lig ade(+) and Lig
ade( ) indicate that ligation kinetics were performed with or
without adenine, respectively.
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Analysis of Self-cleavage and Self-ligation
Activities--
Cis-cleavage and cis-ligation activities of both sets
of clones were assayed by measuring the kobs in
the presence or absence of adenine (Fig. 3). In the experiments carried
out without adenine, the rates were too low to reach the end points for
which values are needed to calculate the kobs.
In effect, after 3 days of incubation of the ribozymes in the absence
of adenine, only 2-4% cleavage or ligation was reached. Thus, the
kobs values were calculated on the basis of the
initial rates of reaction and estimated to be ~10-00-fold higher
than the uncatalyzed reaction under the same condition. During
cis-cleavage and cis-ligation, each clone has very little activity in
the cleavage buffer alone but becomes active with 2.7 mM
adenine (values are shown in Fig. 3A). The kobs values are in the same order of magnitude
for both aptamers. ADHR1 has slightly higher values than ADHR2, and
under these experimental conditions the two ribozymes are only
10-30-fold less active than the wild-type ribozyme. These experiments
demonstrate that both mutated hairpin ribozyme aptamers are inactive
alone but are activated by adenine. Adenine enhances the aptamer
cleavage and ligation activities more than 300-fold.
Adenine and MgCl2 Concentration Effects on Cis-cleavage
Activity--
The kobs values for self-cleavage
were measured for both adenine-dependent ribozymes as a
function of adenine concentration (Fig.
4A). The apparent dissociation
constant (Kd) values of ADHR1 and ADHR2 for adenine
are 7.6 and 2.6 mM, respectively, and the
kobs reached at saturating adenine concentration
are 0.04 and 0.011 min
1, respectively. The affinity of
ADHR2 for adenine is higher than that of ADHR1, but ADHR1 is more
effective at saturating adenine concentration.

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Fig. 4.
Effects of adenine and Mg2+
on ADHR1 and ADHR2 catalysis. A, plots of the
observed rate constants versus adenine concentration.
Apparent dissociation constants for adenine
(Kdapp(ade)) and maximal
kobs values of each aptamers are shown below the
graph. B, plots of the observed rate constants
versus Mg2+ concentration.
Kdapp for Mg2+ and maximal
kobs values of each aptamer are shown below
the graph. The concentration of adenine used in this experiment was
30 mM.
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The kobs were also measured as a function of
MgCl2 concentration (Fig. 4B). As expected,
millimolar concentrations of Mg2+ are required for
catalysis. The apparent Kd values of ADHR1 and ADHR2
for Mg2+ are 1 and 4 mM, respectively, and
kobs reached at saturating MgCl2
concentrations are 0.04 and 0.017 min
1, respectively.
ADHR2 needs more Mg2+ than ADHR1 to be fully active, and
ADHR1 can reach higher activity at saturating Mg2+
concentration. In addition, previous studies (34) with comparable wild-type constructs reported 3 mM Mg2+ for
50% cleavage kobs, which thus are similar to
ADHR1 and ADHR2.
Cis-cleavage Activity with Small Molecules--
The
kobs values were measured for both
adenine-dependent ribozymes in the presence of various
concentrations of small molecules (Fig.
5). Both aptamers remain inactive in the
presence of 3-methyladenine, xanthine, and uracil. This finding
indicates that the N-3 and the C-2 positions of adenine are
required for recognition or activation. Low cleavage rates were
observed in the presence of hypoxanthine with ADHR1 and ADHR2.
Hypoxanthine differs from adenine by substitution of the 6-amino group
with a carbonyl group. The Kd values for
hypoxanthine are similar to those of adenine. Hypoxanthine thus likely
binds both aptamers in the same way as adenine, without inducing the
same catalytic effects. Chemical substitutions at position 6 of adenine
rather decrease or disrupt catalysis than binding. In effect, ADHR1
retains low activity in the presence of purine and 6-methyladenine.
Their Kd values confirm that the 6-amino group of
adenine is not involved in ADHR1 recognition. Its absence in purine
induces a 40-fold decrease in activity as compared with adenine, but
its methylation in 6-methyladenine has no significant effect on
activity. Position 6 of adenine is probably involved in hydrogen bond
formation required for catalysis. The activation of ADHR1 cleavage
induced by imidazole is more striking because, although its affinity is
lower compared with adenine, its catalytic efficiency is close to that
of adenine (Fig. 5). The imidazole moiety alone is therefore able to
induce nearly the same catalytic effect as adenine, even in
the absence of its 6-amino group. The imidazole moiety of adenine is
thus crucial for catalysis by the ADHR1 ribozyme. In contrast, ADHR2 is
capable only of low level self-cleavage in the presence of imidazole.
No activity was detected for ADHR2 in the presence of either
6-methyladenine or purine. Compared with the hypoxanthine data, this
would mean that the 6-amino group of adenine is able to induce
catalysis but not in the same way as ADHR1. Its absence or methylation
completely abolishes activity, and its replacement by a carbonyl group
yields a 25-fold lower activity with respect to adenine. ADHR2 is
active with 1-methyladenine, which has an affinity slightly higher than
that of adenine. Substitution at position 1 with a methyl group yields
nearly the same catalytic efficiency as adenine. As opposed to ADHR1,
which remains inactive with 1-methyladenine, the N-1 atom of
adenine is not involved in either ADHR2 recognition or cleavage
induction. Taken together, the data related to small molecules indicate
that adenine recognition and/or catalysis induction are not achieved in
the same manner for ADHR1 and ADHR2.

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Fig. 5.
Effect of adenine analogs on ADHR1 and ADHR2
catalysis. A, plots of the observed rate constants
versus cofactor concentration. Cofactors used for ADHR1 and
ADHR2 self-cleavage are indicated near the corresponding curves.
Ade, adenine; 1MA, 1-methyladenine;
6MA, 6-methyladenine; Hyp, hypoxanthine;
Imi, imidazole; Pur, purine. B,
cofactor affinities and catalytic effects. The maximal
kobs and apparent Kd of ADHR1
and ADHR2 for each cofactor are shown. The values are deducted from the
curves presented in A. N.D. indicates that the
Kd and kobs values were not
detected because activity was too low in the presence of the
cofactor.
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Effects of pH on Cis-cleavage Activity in the Presence of Adenine
and Imidazole--
The kobs values at pH
ranging from 4 to 10 were measured for both aptamers in the absence or
presence of cofactor (32 mM adenine or 32 mM
imidazole. Plots of kobs as a function of pH for
both aptamers are shown in Fig. 6. In the
presence of adenine, the kobs values of ADHR1
are very low, between pH 4 and 6. They increase as of pH 6 and reach a
plateau between pH 8.6 and 10. The ADHR1 imidazole pH profile is
roughly similar to that of the adenine pH profile; the
kobs values in the presence of imidazole are
higher at low pH but are two times lower at the plateau than in the
presence of adenine. Self-cleavage of ADHR1 in buffer alone yields a
flat pH profile. The kobs is not measurable at
low pH, and self-cleavage becomes detectable at pH 7.5 and increases
slightly with pH. At pH 10, kobs values are
55-fold lower than in the presence of adenine and 25-fold lower than in
the presence of imidazole). In the presence of adenine, ADHR2
self-cleavage is very low between pH 4 and 5. It increases as of pH 6 to reach a plateau at pH 8.2. The ADHR2 imidazole pH profile is as flat
as the ADHR2 pH profile without cofactor. These results suggest that
deprotonation events are rate-limiting for adenine-assisted catalysis
by both aptamers.

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Fig. 6.
Effect of pH on ADHR1 and ADHR2 catalysis.
A, plots of the observed rate constants for ADHR1
self-cleavage versus pH in the presence of 32 mM
adenine or 32 mM imidazole or in the cleavage buffer alone.
B, plots of the observed rate constants for ADHR2
self-cleavage versus pH in the presence of 32 mM
adenine or 32 mM imidazole or in the cleavage buffer alone.
The identity of the cofactor used is indicated near the corresponding
curve. Ade, adenine; Imi, imidazole;
N.C., no cofactor.
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DISCUSSION |
The successful selection procedure allowed us to discover new
hairpin ribozymes that are strongly dependent on adenine for their
reversible self-cleavage reaction. Screening of 14 selected clones yields two different aptamer sequences. ADHR1 is largely prevalent over ADHR2 in the final selected population (13:1). This can
be explained by the cleavage and ligation kinetics studies. First,
ADHR1 has slightly higher kobs values. Its
relative ratio to ADHR2 probably increases after short-time positive
selection steps. In addition, two positive selections for religation
were performed during the last two selection rounds (Fig. 2). For
ADHR1, both the rate and equilibrium constants for ligation are higher than for ADHR2. Hence, it is easy to understand that ADHR1 predominates over ADHR2 after 12 selection rounds for adenine-assisted cleavage and
after two rounds of selection for adenine-assisted religation. This can
now be understood in terms of selection conditions. Indeed, ADHR2
requires more Mg2+ than ADHR1 does to be fully active.
During in vitro selection the Mg2+ concentration
was 6 mM, which is not optimal for ADHR2 catalysis.
The analysis of cis-cleavage and cis-ligation kinetics indicates that
the self-cleavage reaction in the presence of adenine is reversible.
This confirms that adenine is part of the catalyst and not of the
reactants. If adenine were acting as a reactant (for example in direct
nucleophilic attack of the scissile phosphate), it would have been
trapped either in the 3'- or 5'-side products. In the first case,
religation of the 5'-side product with fresh LS (carrying a 5'-OH end)
would not have succeeded either in the presence or absence of adenine.
In the second case, the 5'-side product would not have religated or
would have been able to religate either in the presence or absence of
free adenine. ADHR1 and ADHR2 thus catalyze the same kind of reaction
as the wild-type hairpin ribozyme. Hydrolysis of the RNA backbone
proceeds via the nucleophilic attack of the scissile phosphate by the
adjacent 2'-oxygen, and the release products are 2'-3' cyclic phosphate
and 5'-OH termini.
The investigations of the effects of various adenine analogs and the
studies of the effects of pH were undertaken to determine the level of
implication of adenine in catalytic self-cleavage of ADHR1 and ADHR2.
The results of these studies can be explained in several ways. First,
it is possible that both aptamers are subjected to a pH-triggered
conformational change enhancing catalysis. This remains unlikely,
because the pH profile of wild-type ribozyme catalysis is flat between
pH 5 and 9 (35). ADHRs 1 and 2 are believed to share a tertiary
structure close to that of wild-type, and such a
pH-dependent conformational switch observed in both aptamers but absent from the wild-type does not seem very likely. The
more probable explanation would be that both aptamers use catalytic
strategies in which a deprotonation event (absent or not detectable in
the wild-type RNA) is rate-limiting in the chemical cleavage mechanism
(acid/base catalysis). Proton transfer would thus be the limiting step
in catalysis, and the protons could come from ribozyme chemical groups
with shifted pKa values or from exogenous
adenine used as cofactor for cleavage catalysis. Indeed, the use of
catalytic Mg2+-complexed hydroxide ions cannot formally be
excluded, but this last hypothesis remains very unlikely because the
wild-type ribozyme does not use Mg2+ in catalysis (36).
Indeed, Mg2+ dependence of both aptamers is similar to
wild-type Mg2+ requirements. It is thus probable that ADHR1
and ADHR2 require Mg2+ only for folding and not for
catalysis per se. The involvement of exogenous adenine in
direct catalysis is probably different for ADHR1 and ADHR2.
ADHR1 is able to react with a wider variety of adenine analogs than
ADHR2. Among these analogs, imidazole (pKa
value, 7) yields comparable catalytic efficiency as adenine. It is thus
probable that the imidazole moiety of adenine is involved directly in
the catalytic step. The N-9 atom of adenine has a
pKa value of 9.8, which can be shifted toward
neutrality as a result of the interaction with ADHR1 RNA. The N-1 atom
(pKa value, 4.1), in which methylation abolishes
activity, is also a candidate for acid/base catalysis, but it is not
present in imidazole. Models in which N-9- or N-1-deprotonated adenine
directly abstract the proton of the 2'-attacking nucleophile oxygen to
enhance the catalytic rate (general base mechanism) or chemical
mechanisms in which N-9- or N-1-protonated adenine releases its proton
to the 5'-oxo leaving groups (general acid mechanism) are thus
consistent with our data.
The involvement of adenine in ADHR2 catalysis seems to be different.
The imidazole part of adenine seems to be less involved than in ADHR1
catalysis. Methylation of the N-1 atom has no effect on catalysis.
Mechanisms involving the adenine N-1 atom with a neutral
pKa available for the abstraction of the 2'
attacking hydroxide proton or for proton release to the 5'-oxo leaving
group can thus be excluded for purposes of ADHR2 catalysis.
Adenine-assisted ADHR2 self-cleavage seems to be more sensitive than
ADHR1 to chemical substitutions at position 6 of adenine. The 6-amino
group may thus play a more important role in catalysis by ADHR2 than by ADHR1. We propose two additional chemical explanations. In the first
case, ADHR2-bound adenine would simply help to position the RNA
reacting groups and would not act directly on catalysis. In the second
case, exogenous adenine would act as wild-type ribozyme adenosine 38 by
stabilizing the transition state with hydrogen bonds involving its
6-amino group (37). In these two models, the pH profiles of the ADHR2
adenine-assisted self-cleavage would be due to protonation state
changes in RNA catalytic groups.
ADHR1 and ADHR2 aptamers do not use the same catalytic strategies as
the wild-type hairpin ribozyme, which is believed to use a transition
state stabilization strategy. We have thus selected new hairpin
ribozyme variants that are dependent strictly on the presence of
exogenous adenine for catalysis. These new
adenine-dependent ribozymes act with different catalytic
strategies with respect to wild-type ribozyme, probably having
different ways of handling exogenous adenine as cofactor for catalysis.
These aptamers are of great interest considering the prebiotic RNA
world hypothesis. Adenine is itself a prebiotic analog of histidine and
could have been used by ribozymes of the RNA world in the same way as
histidine is used by present day enzymes. Our work thus supports the
view that the use of small exogenous cofactors by ribozymes of the RNA
world could have constituted an easy way of expanding its catalytic and
metabolic repertoires.