Tài liệu Báo cáo khoa học: Efficient RNA ligation by reverse-joined hairpin ribozymes and engineering of twin ribozymes consisting of conventional and reverse-joined hairpin ribozyme units ppt

Efficient RNA ligation by reverse-joined hairpin ribozymes
and engineering of twin ribozymes consisting of
conventional and reverse-joined hairpin ribozyme units
Sergei A. Ivanov, Ste
´
phanie Vaule
´
on and Sabine Mu
¨
ller
Ruhr-Universita
¨
t Bochum, Bochum, Germany
In recent years RNA has become the focus of develop-
ment into new diagnostic and therapeutic schemes.
Antisense-RNA, ribozyme, aptamer and siRNA tech-
nologies have been developed and have found applica-
tion in molecular medicine [1–7]. Signalling aptamers
and aptazymes have been constructed that can sense a
number of molecules in real time and thus are valuable
diagnostic tools [8–10]. Furthermore, recently discov-
ered riboswitches that regulate gene expression in vivo
in response to specific metabolites [11–13] or tempera-
ture [14] may lead to new RNA-based therapeutic
strategies.
Elucidation of the molecular principles of RNA
functioning in a specific context has led to the engi-
neering of RNA molecules with new functions. Two
complementary strategies can be used in RNA engi-
neering: rational design and directed evolution.
Whereas directed molecular evolution relies on the cre-

ation of a repertoire of modified RNAs from which
beneficial variants are filtered, in a rational design
experiment, defined changes in the nucleotide sequence
and ⁄ or secondary structure of a specific RNA are
planned on the basis of a preconceived idea. This
requires detailed structural and mechanistic informa-
tion on the parent RNA. In cases where this informa-
tion is available, rational design has contributed to the
development of new functional RNA, for example, sig-
nalling aptamers and aptazymes [8–10].
Work in our laboratory has focused on the rational
design of functional RNA, in particular on the
development of hairpin-derived twin ribozymes for
site-specific alteration of RNA sequences, and fluores-
cent and affinity labelling of large RNA molecules
[15–18]. The hairpin ribozyme catalyses the reversible
site-specific cleavage of suitable RNA substrates, gen-
erating fragments with a 2¢,3¢-cyclic phosphate and,
respectively, a free 5¢-OH terminus [19,20]. In the
reverse reaction, the oxygen atom of the free 5¢-OH
group of one RNA fragment attacks the phosphorous
of the cyclic 2¢,3¢-phosphate group of another, result-
ing in ligation of the two fragments. In contrast to the
hammerhead ribozyme, the conformation of the hair-
pin ribozyme–substrate complex does not change signi-
ficantly upon cleavage: the two cleavage fragments
Keywords
rational design; RNA catalysis; RNA ligation;
sequence alteration; twin ribozyme
Correspondence

S. Mu
¨
ller, Ruhr-Universita
¨
t Bochum,
Fakulta
¨
t Chemie, Universita
¨
tsstrasse 150,
D-44780 Bochum, Germany
Fax: +49 234 321 4783
Tel: +49 234 322 7034
E-mail:
(Received 13 June 2005, accepted 15 July
2005)
doi:10.1111/j.1742-4658.2005.04865.x
In recent years major progress has been made in elucidating the mechanism
and structure of catalytic RNA molecules, and we are now beginning to
understand ribozymes well enough to turn them into useful tools. Work in
our laboratory has focused on the development of twin ribozymes for site-
specific RNA sequence alteration. To this end, we followed a strategy that
relies on the combination of two ribozyme units into one molecule (hence
dubbed twin ribozyme). Here, we present reverse-joined hairpin ribozymes
that are structurally optimized and which, in addition to cleavage, catalyse
efficient RNA ligation. The most efficient variant ligated its appropriate
RNA substrate with a single turnover rate constant of 1.1 min
)1
and a
final yield of 70%. We combined a reverse-joined hairpin ribozyme with a

conventional hairpin ribozyme to create a twin ribozyme that mediates the
insertion of four additional nucleotides into a predetermined position of a
substrate RNA, and thus mimics, at the RNA level, the repair of a short
deletion mutation; 17% of the initial substrate was converted to the inser-
tion product.
4464 FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS
remain oriented close to each other in the ribozyme–
product complex before dissociating to leave the free
ribozyme behind [21–23]. Therefore, in the hairpin
ribozyme reaction the entropic cost of ligation is rather
low and can be compensated for by the favourable
reaction enthalpy of formation of the 5¢,3¢-phospho-
diester bond via ring opening of the 2¢,3¢-cyclic phos-
phate [24]. This specific feature basically makes the
hairpin ribozyme a better ligase than it is an endonuc-
lease. However, dissociation of cleavage fragments
from the ribozyme has to be considered and thus the
ability to preferentially cleave or ligate a specific RNA
substrate strongly depends on the stability of the pro-
perly folded substrate–ribozyme complex. Strikingly,
the hairpin ribozyme is an efficient ligase if the ribo-
zyme substrate complex is folded into a stable secon-
dary and tertiary structure. By contrast, if secondary
and tertiary structure elements are less stable (yet sta-
ble enough to form a catalytically competent complex)
cleavage is favoured [25,26]. We exploited this specific
feature of the hairpin ribozyme in a scheme of site-
directed and patchwise exchange of RNA sequences
[16,17]. Combination of two hairpin ribozymes into
one molecule leads to twin ribozymes with two pro-

cessing sites at a suitable substrate RNA. Because of
the specific hairpin ribozyme cleavage–ligation charac-
teristics described above a fragment of residing
sequence is removed in the first part of the reaction
followed by binding of another separately added RNA
fragment in the gap left behind and its ligation to the
final product [16].
In addition to the conventional hairpin ribozyme,
we studied hairpin ribozymes with the two domains
(loop A and loop B) joined in reverse order [15,27,28]
(Fig. 1). To further complement our work on the
rational design of ribozymes for RNA sequence alter-
ation we were interested in using reverse-joined hairpin
ribozymes as building blocks for the construction of
twin ribozymes. In order to evaluate the structural
properties of reverse-joined hairpin ribozymes for func-
tional design, we first studied the cleavage and ligation
activity of reverse-joined hairpin ribozyme variants.
Based on the results, the most suitable ribozyme was
chosen for construction of a twin ribozyme. The ability
of this twin ribozyme to mediate site-specific alteration
of RNA sequence is demonstrated.
Results
RNA ligation by reverse-joined hairpin ribozymes
Reverse-joined hairpin ribozymes, first introduced by
Ohtsuka and colleagues [29,30], are derived from the
conventional hairpin ribozyme by dissecting the two
domains at the hinge between helix 2 and helix 3 and
rejoining helix 4 to helix 1 via a linker of six unpaired
residues [27] (Fig. 1). Linkers consisting of cytidine or

adenosine have been studied and it has been found
that a linker of six unpaired residues is suitable for
connecting the two domains [29,30]. Further extending
the linker length to 9, 12 or 18 residues increased the
cleavage rate by only a factor of two [29]. Therefore,
as well as to confine the conformational freedom of
the ribozyme structure, we initially used an A
6
-linker
for the design of reverse-joined hairpin ribozymes and
stabilized helix 3 by a UUCG tetraloop cap [15,27].
Fig. 1. (A) Schematic presentation of how reverse-joined hairpin ribozymes are derived from the conventional hairpin ribozyme. Loop A and
loop B domains of the conventional hairpin ribozyme are separated between helix 2 and helix 3. The loop B domain is turned through 180°
and helix 4 is rejoined with helix 1 via a single-stranded linker to generate a reverse-joined hairpin ribozyme. (B) Secondary structure of
reverse-joined hairpin ribozymes with substrates for ligation experiments. The circle indicates a 5¢-terminal fluorescein moiety used for detec-
tion. cp, 2¢,3¢ cyclic phosphate.
S. A. Ivanov et al. An engineered ribozyme for RNA sequence exchange
FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS 4465
Under appropriate conditions, reverse-joined hairpin
ribozymes with an A
6
-linker designed in our laborat-
ory efficiently catalysed RNA cleavage [27]. However,
ligation activity was rather poor; only about 1% of
cleavage products ⁄ ligation substrates were ligated
(S. A. Ivanov & S. Mu
¨
ller, unpublished results). Pre-
vious linker length variation experiments were carried
out to study the cleavage activity of reverse-joined

hairpin ribozymes without looking at ligation [29,30].
Therefore, to search for reverse-joined hairpin ribo-
zymes with improved ligation activity we reinvestigated
the influence of linker length using the constructs
shown in Fig. 1B. In order to favour ligation over
cleavage, helix 2 was extended to 16 bp (Fig. 1B). This
design should allow for a higher ligation yield due to
more stable binding of the 5¢-ligation fragment [25,26].
Furthermore, we used two different short substrates,
SG9 and SU9, for ligation. Whereas SU9 forms a 6 bp
duplex with the ribozyme, binding of SG9 generates
a duplex of only 5 bp due to the terminal G-A mis-
match. Thus, the nonpaired adenosine residue at the
hinge point can be integrated into the linker leading to
further enhancement of the degrees of conformational
freedom. We prepared reverse-joined hairpin ribo-
zymes with single-stranded linkers of 6, 7, 8 and 12
adenosine residues and compared their activity for
ligation of S17F5 cp with either SU9 or SG9 (Fig. 1B,
Table 1). This experimental design allowed us to look
at eight ribozyme–substrate complexes varying in the
length of the single-stranded linker and ⁄ or the length
of the duplex between the ribozyme and the 3¢-ligation
substrate. Ligation reactions were carried out under
conditions involving equimolar concentrations of ribo-
zyme and ligation fragments, as well as under single
turnover conditions (Table 1) in the presence of 10 mm
MgCl
2
and 2 mm spermine; the polyamine was previ-

ously found to be essential for efficient reverse-joined
hairpin ribozyme catalysis [27]. Initially, we used a
reaction temperature of 32 °C because this has been
shown to be optimal [29,30]. However, we observed
that ribozyme activity varied only slightly in measure-
ments at 32 and 37 °C. The construct HP–RJWTA7
showed the fastest reaction kinetics and gave the high-
est yields for ligation of the short fragment SG9 to
S17F5 cp under equimolar concentrations, as well as
under ribozyme saturation (Table 1). Thus, a linker
length of eight adenosine residues in combination with
a duplex of 5 bp between the 3¢-ligation substrate
and the ribozyme seems to be most favourable for
an efficient reaction among the studied species.
HP–RJWTA7 is also an efficient endonuclease; it
cleaves the substrate S40F3F5 with k
obs
¼ 0.4 min
)1
at
equimolar concentrations of ribozyme and substrate
(according to conditions used for sequence exchange
reaction, see below).
Design of a twin ribozyme and kinetic analysis
As a result of the studies described above, the opti-
mized structure of the reverse-joined ribozyme unit for
twin ribozyme design consists of two domains joined
by a single-stranded linker of eight adenosine residues
and binds the 3¢-terminal part of its substrate via a
duplex of 5 bp (Fig. 2A). The second part of the twin

ribozyme consists of a three-way junction hairpin ribo-
zyme as used in the twin ribozyme approach described
recently [16].
In order to learn about the activity of both ribozyme
units in the twin ribozyme, we determined the kinetic
parameters for cleavage as well as ligation at the two
sites in individual experiments (Fig. 3). Substrate
RNAs S40F5dA15 and S40F5dA31 were synthesized
to be cleaved at either of the specific sites. Cleavage at
the second site was abolished by replacing the attack-
ing 2¢-OH group with a hydrogen atom. Specifically,
either A15 or A31 was substituted by deoxyadenosine.
Cleavage reactions were carried out under single turn-
over conditions delivering the kinetic constants shown
Table 1. Ligation parameters of reverse-joined hairpin ribozymes with single stranded linkers of varying lengths.
Ribozyme HP-RJWTA6 HP-RJWTA7 HP-RJWTA8 HP-RJWTA12
3¢-substrate SU9 SG9 SU9 SG9 SU9 SG9 SU9 SG9
3¢-terminal base of substrate U G U G U G U G
5¢-terminal base of ribozyme A A A A A A A A
Linker length, n 6778891213
Duplex length, bp 6 5 6 5 6 5 6 5
k
lig
,min
)1
0.564 0.938 0.894 1.140 0.535 0.865 0.263 0.447
K
D
,nM 140.8 141.1 109.3 75.3 73.9 97.3 76.6 85.4
Ligation yield (%)

under equimolar conditions 35.2 43.7 35.0 43.3 31.5 43.0 21.7 33.1
under single turnover conditions 55.8 61.0 63.3 70.0 54.1 57.6 35.3 43.0
An engineered ribozyme for RNA sequence exchange S. A. Ivanov et al.
4466 FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS
A
B
Fig. 2. (A) Reaction scheme for HP–TWRJ-mediated fragment exchange. Substrate RNA S40F3F5 is annealed to HP–TWRJ (left) and cleaved
at two defined sites. The fragment extending between the two cleavage sites (16-mer, shown in red) is replaced on the ribozyme by the
oligonucleotide S20 cp (20-mer, shown in green) which subsequently becomes ligated to the flanking substrate fragments to form the
HP–TWRJ–product complex (right). Green circles indicate 5¢-and3¢-terminal fluorescein moieties used for detection. Reaction was carried
out in the absence and presence, respectively, of the oligonucleotide S6-anti, which is complementary to the six 5¢-terminal nucleotides of
HP–TWRJ (for details refer to main text). (B) Model duplexes used for determination of melting points. The sequence of the educt and prod-
uct duplex corresponds to the sequence of HP–TWRJ with initial substrate S40F3F5 and with product P44F3F5, respectively.
Fig. 3. Secondary structures of ribozyme substrate complexes used for measuring cleavage (A) or ligation (B) rates at either site. Cleavage
and ligation at the second site was abolished by replacing the essential adenosine in the substrate strand (A15 or A31) by deoxyadenosine.
Grey circles indicate 5¢- and 3¢-terminal fluorescein moieties used for detection.
S. A. Ivanov et al. An engineered ribozyme for RNA sequence exchange
FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS 4467
in Table 2. A similar setup was used to determine liga-
tion parameters: fragments with dA instead of A
(S35F5dA15cp and S29dA31) were used for ligation
with the corresponding substrate SG9 or S15F5 cp,
respectively. The results show that the twin ribozyme-
mediated cleavage, as well as ligation, proceeds with
similar activity at both individual sites; cleavage rates
are virtually identical, ligation rate constants vary by a
factor of only about 2.5.
Alteration of RNA sequence by the twin ribozyme
HP–TWRJ
The twin ribozyme HP–TWRJ was designed to pro-

mote the insertion of four specific nucleotides into a
predetermined site of an arbitrarily defined substrate
RNA as illustrated in Fig. 2A. In the ribozyme–
substrate complex, a stretch of four nucleotides in the
central part of the ribozyme strand (GAUU) bulges.
Cleavage at both predefined sites releases a 16-mer that
can easily dissociate from the ribozyme because of the
destabilizing bulge. The added RNA fragment S20 cp
contains the four additional nucleotides complement-
ary to the GAUU loop in the ribozyme strand. Hence,
binding of this oligonucleotide to the gap left by disso-
ciation of the 16-mer, converts the previously inter-
rupted duplex into a continuous one extended by 4 bp.
For thermodynamic characterization of the system, we
determined the melting points of model duplexes cor-
responding to the central part of the twin ribozyme–
substrate and ribozyme–product complexes (educt and
product duplex, Fig. 2B). There was a significant dif-
ference in the melting temperature of the two duplexes
(Table 3), supporting our strategy to drive the reaction
by a change in duplex stability. On the basis of the
melting temperatures obtained using varying duplex
concentrations, thermodynamic parameters were deter-
mined. The binding enthalpy for both duplexes varied
by 71.4 kcalÆmol
)1
and fragment exchange was associ-
ated with a favourable DG of )15.2 kcalÆmol
)1
(see

Experimental procedures and Table 3). This should
drive the reaction in the desired direction and favour
product formation.
The validity of the experimental design was checked
by analysing the time course of the reaction using a
fluorescence assay as described previously [15]. The
substrate RNA S40F3F5 was incubated with an equi-
molar amount of ribozyme at 37 °C. We used a reac-
tion temperature of 37 °C in order to compare the
results with our previous twin ribozyme studies [16].
The reaction was allowed to proceed for 30 min, after
which S20 cp was added in equimolar quantities to
ribozyme and initial substrate, and the reaction was
left to proceed at 37 °C for another 120 min. We ini-
tially chose this experimental design in order to be able
to observe the individual reaction steps. However, as
found later, the reaction occurs in a similar manner
when initial substrate, ribozyme and S20 cp are present
in the reaction mixture from the beginning (S. Vaule
´
on
&S.Mu
¨
ller, unpublished observations). Data for the
original setup are given in Fig. 4. After 30 min, char-
acteristic cleavage products were detected (Fig. 4B,
lane 2). Addition of the fragment S20 cp led to the for-
mation of new products detected as three additional
bands (lanes 3 and 4). These signals correspond to the
29- and 35-mer resulting from ligation of the 20-mer

to either the 9- or 15-mer cleavage product, and to the
desired product RNA (P44F3F5) resulting from liga-
tion of the 20-mer to both fragments as shown in
Fig. 4A. Continuing the reaction for 15 min after the
addition of S20 cp (total reaction time: 45 min) resul-
ted in conversion of 5% of starting material into the
44-mer product P44F3F5, whereas 13% of the sub-
strate S40F3F5 remained unprocessed (Fig. 4B, lane 3,
Fig. 4C). After another 105 min of incubation, further
enrichment of products involving ligation to the
20-mer fragment S20 cp was observed. The ratio of
product to initial substrate increased; 9% of substrate
RNA was converted to the 44-mer and 5% was left
unchanged (Fig. 4B, lane 4, Fig. 4C). These results
demonstrate favourable cleavage of the substrate fol-
lowed by dissociation of the 16-mer versus favourable
ligation of the 20-mer.
The yield of product RNA P44F3F5 could be fur-
ther increased by stabilization of the ribozyme active
Table 3. Thermodynamic parameters of model duplexes.
T
m
(°C)
a
DH
(kcalÆmol
)1
)
DS
(calÆK

)1
Æmol
)1
)
DG
37 °C
(kcalÆmol
)1
)
Educt duplex 38.1 )78.4 )223.0 )9.2
Product duplex 72.7 )149.8 )404.2 )24.4
a
At 500 nM oligonucleotide concentration.
Table 2. Kinetic parameters of HP–TWRJ catalyzed cleavage and
ligation reactions.
Substrates
k
react
,
(min
)1
)K(nM)
k
react
⁄ K,
(min
)1
lM
)1
)

Cleavage S40F5dA15 0.57 ± 0.01 16.1 ± 2.4 35.4
S40F5dA31 0.32 ± 0.02 12.0 ± 4.6 26.6
Ligation SG9 +
S35F5dA15cp
0.55 ± 0.03 22.9 ± 8.5 24.0
S29dA31 +
S15F5 cp
1.38 ± 0.09 34.8 ± 8.1 39.6
An engineered ribozyme for RNA sequence exchange S. A. Ivanov et al.
4468 FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS
structure. Theoretical analysis of HP–TWRJ folding
(using software rna structure 4.0) showed that
HP–TWRJ, in addition to the desired minimal energy
structure (DG ¼ –70.7 kcalÆmol
)1
), can fold into an
alternative nonfunctional structure with a virtually
identical Gibbs’ free energy (DG ¼ –70.5 kcalÆmol
)1
).
A short antisense oligonucleotide 3¢-CCCTCT-5¢,
complementary to the six 5¢-terminal nucleotides of
HP–TWRJ (Fig. 2A), assists proper folding; folding
analysis of this system revealed an energy difference
between both competing structures of 10 kcalÆmol
)1
in
favour of the desired functional structure. Carrying
out the sequence-exchange reaction described in the
presence of this antisense oligonucleotide increased the

yield of the final product to 17% (Fig. 4C).
To validate our data we repeated the reaction, using
for sequence exchange a 20-mer fragment internally
labelled with a Cy5 moiety [S20 cp(Cy5)] (Fig. 5). This
experimental setup allowed us to detect ligation pro-
ducts not only by fragment lengths analysis with an
ALF DNA sequencer (detection of fluorescein emis-
sion upon excitation at 488 nm), but also by virtue of
their unique fluorescence at 700 nm using a LI-COR
DNA sequencer (detection of Cy5 emission upon exci-
tation at 680 nm). As shown in Fig. 5A, there is a
clear conversion of the fast-running Cy5-labelled
20-mer into three slower running species corresponding
to ligation products of the Cy5-labelled 20-mer with
fluorescein-labelled 9-mer [29-mer (F3, Cy5)] and
15-mer [35-mer (F5, Cy5)], respectively, and to final
44-mer RNA product labelled with Cy5 and two fluo-
rescein moieties [44-mer (F3, F5, Cy5)].
In both experiments (compare Figs 4B and 5A)
there is a strong 35-mer signal resulting from ligation
of the 15-mer produced by cleavage in the first step of
the reaction to the added 20-mer fragment S20 cp.
This illustrates that ligation at the site of the conven-
tional hairpin ribozyme proceeds somewhat faster
(2.5-fold, Table 2) than ligation at the reverse-joined
ribozyme site.
Discussion
The twin ribozyme HP–TWRJ was designed to mediate
the specific exchange of two RNA fragments (Fig. 2).
A

B
C
Fig. 4. (A) Fragmentation and ligation scheme (compare with
Fig. 2). cp, 2¢,3¢-cyclic phosphate. (B) Monitoring the reaction. Lane
1, start of cleavage reaction. Lane 2, mixture after 30 min at 37 °C
(immediately before adding S20 cp). Lane 3, mixture 15 min after
addition of S20 cp (incubation at 37 °C continued, total reaction
time: 45 min). Lane 4, mixture after additional 105 min at 37 °C
(areas of peaks corresponding to S40F3F5 and P44F3F5 indicate
9% conversion to full length product and 5% remaining substrate).
For additional details see main text. Peak heights are standardized
by the data processing software, such that total peak integrals
of different lanes are not constant. (C) Time course of fragment
exchange reaction in the absence (solid lines) and presence
(dashed lines) of the antisense oligonucleotide S6-anti.
S. A. Ivanov et al. An engineered ribozyme for RNA sequence exchange
FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS 4469
During the process, 16 nucleotides of residing substrate
sequence are exchanged for 20 nucleotides, which are
added to the reaction mixture as a separate synthetic
RNA fragment. Recently, we communicated the devel-
opment of a twin ribozyme consisting of two hairpin
ribozymes connected in tandem that can catalyse the
same fragment-exchange reaction [16]. The twin ribo-
zyme described here was more challenging, because it
involves a reverse-joined hairpin ribozyme unit and
required more extensive design and evaluation.
Reverse-joined hairpin ribozymes were introduced
nearly 10 years ago [29,30]. Since then they have attrac-
ted little attention: there has been no follow-up demon-

strating the catalytic potential of these interesting
ribozyme structures beyond the work of the initial
developers and our laboratory. We studied the ligation
activity of reverse-joined hairpin ribozyme variants.
Interestingly, reverse-joined hairpin ribozymes act as
highly efficient ligases. The most active variant
HP–RJWTA7 ligated two substrates with 43% yield
when all reactants (ligation substrates and ribozyme)
were incubated at equimolar concentrations; under
single turnover conditions the yield increased to 70%
(Table 1). A conventional hairpin ribozyme variant
corresponding to the 3¢-terminal region of the twin
ribozyme HP–TWRJ delivered only 29% of ligated
product (compared with 43% for HP–RJWTA7, data
not shown). No high-resolution structure is available
for reverse-joined hairpin ribozymes. However, the
observed functionality implies that the active confor-
mation of reverse-joined hairpin ribozymes involves
a similar relative orientation of the two ribozyme
domains to that seen in the crystal structure of the con-
ventional hairpin ribozyme [21,31]. Variation in linker
length and ⁄ or the length of the duplexes flanking the
single-stranded linker will, therefore, influence the posi-
tions of the two loops in the folded structure. Our
results indicate that a single-stranded linker of eight
adenosine residues, as in HP–RJWTA7, and a 5 bp
duplex between the 3¢-ligation substrate and the ribo-
zyme allows proper folding of the complex into the act-
ive conformation with the required contacts between
loops A and B. Thus, to the best of our knowledge, this

is the first example of a reverse-joined hairpin ribozyme
that, under appropriate conditions, can ligate two suit-
able substrates with up to 70% yield.
AB
Fig. 5. (A) Monitoring the HP–TWRJ mediated fragment exchange involving a Cy5-labelled oligonucleotide S20 cp(Cy5). Lane 1, S20 cp(Cy5)
control. Lanes 2 and 3, incubation of S20 cp(Cy5) with HP–TWRJ in the absence of initial substrate under conditions of fragment exchange
reaction after 5 and 80 min. Lane 4, incubation of S20 cp(Cy5) with initial substrate S40F3F5 in the absence of HP–TWRJ under conditions
of fragment-exchange reaction. Lane 5, mixture after 30 min cleavage reaction at 37 °C (immediately before adding S20 cp(Cy5)). Lanes 6, 7
and 8, mixture 15, 120 and 180 min, respectively, after addition of S20 cp(Cy5) (incubation at 37 °C continued). Lane 9, Cy5 labelled 29-mer
as length control. Double bands result from an isomeric mixture (cis- ⁄ trans-isomers) of the Cy5 moiety used for labelling. (B) Schematic
presentation of HP–TWRJ mediated fragment exchange (compare with Fig. 2). Green circles indicate 5¢-and3¢-terminal fluorescein moieties;
the red circle indicates a Cy5 moiety used for detection.
An engineered ribozyme for RNA sequence exchange S. A. Ivanov et al.
4470 FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS
The reverse-joined hairpin ribozyme HP–RJWTA7
was combined with a three-way junction hairpin ribo-
zyme to generate the twin ribozyme HP–TWRJ. The
newly designed structure mediates the site-specific
exchange of two patches of RNA sequence with up to
17% yield, which is a remarkable improvement com-
pared with earlier versions of this twin ribozyme
[15,32]. The yield can possibly be increased further by
the additional destabilization of ribozyme–substrate
complexes in the region containing the sequence to be
exchanged (Fig. 2B, red). Although a bulge of four
nucleotides has been introduced to weaken binding of
this sequence, there is still a duplex of eight contiguous
base pairs hampering dissociation of the fragment to be
exchanged after cleavage. Further reduction of the
length of this duplex would facilitate dissociation.

However, this implies that the two ribozyme units of
the twin ribozyme are located closer together and this
may interfere with proper folding of the twin ribozyme
due to a sterical clash between both catalytic units. In
our previous studies, we observed that the sequence
and length of the helix between the two loops contain-
ing the cleavage ⁄ ligation site, as well the size of the
bulge, influence the exchange efficiency [16,32,33].
Therefore, the specific design of a custom-designed twin
ribozyme will strongly depend on the substrate to be
processed, such that variation of the distance between
the two sites seems reasonable only at this stage.
In summary, the demonstration of functionality of
HP–TWRJ provides proof of the principle that, in
addition to the conventional hairpin ribozyme, reverse-
joined hairpin ribozymes can also be used as building
blocks for the construction of twin ribozymes. Even
though HPTWRJ is not as efficient as its tandem-
configured relative [16], its successful construction and
the demonstration of its functionality supports the idea
of creating novel RNA catalysts by rational design.
Furthermore, reverse-joined hairpin ribozymes can act
on RNA substrates that are not readily accepted by
conventional hairpin ribozymes. For example, a hair-
pin ribozyme variant with the terminal base pair of the
ribozyme–substrate duplex at the hinge changed from
3
0
-CU-5
0

5
0
-GA-3
0
to
3
0
-GA-5
0
5
0
-CU-3
0
displayed 10-fold lower cleavage
activity compared with the wild-type ribozyme [15]. By
contrast, corresponding variants of the reverse-joined
hairpin ribozyme showed no significant difference in
cleavage behaviour. (C. Schmidt & S. Mu
¨
ller, unpub-
lished results). We attributed this result to enhanced
coaxial stacking of the two domains in the relevant
variant of the hinged conventional hairpin ribozyme
leading to enrichment of an extended inactive confor-
mation [34,35]. Because of the single-stranded linker,
joining the two domains in reverse-joined hairpin ribo-
zymes, coaxial stacking is less probable and therefore
not sensitive to the sequence at the hinge point.
A useful application for twin ribozymes is site-speci-
fic labelling or functionalization of transcripts in vitro

and possibly in vivo as we have recently demonstrated
with tandem configured twin ribozymes [18]. The twin
ribozyme accepts RNA fragments that are conjugated
with a dye (here Cy5). A number of other dyes and
modifications are incorporated equally well even in
rather long and structured RNA molecules [18]. Thus,
the twin-ribozyme strategy paves the way for site-speci-
fic labelling ⁄ modification of RNA molecules that are
too long for chemical synthesis. Furthermore, apart
from fragment-exchange reactions, simple clipping of a
fragment of desired length and sequence from a nat-
ural RNA may be a useful application. For example,
RNA fragments that involve modified nucleobases are
easily obtainable from naturally occurring RNA by the
use of twin ribozymes. Subsequently, these fragments
can be investigated using various analytical methods.
Another potential application is genotyping of single
nucleotide polymorphisms in human genomes [36].
Subsequent analysis of appropriate RNA fragments by
MS can reveal if a certain nucleotide in the target gene
has been altered [37,38]. Thus, the development and
application of twin ribozymes may lead to a number
of interesting strategies in molecular biology, genome
analysis and possibly molecular medicine. It is cer-
tainly advantageous having a number of twin-ribozyme
variants that can be adapted to and optimized for a
specific target. It has been shown previously that the
sequence of the substrate-binding domain of the
hairpin ribozyme can be adapted to cleave a desired
RNA substrate, just a few conserved nucleobases are

required [39]. The sequence requirements for conven-
tional and reverse-joined hairpin ribozyme substrates
are virtually the same [29,30]. However, the distinct
mode of joining the two ribozyme domains in both
variants leads to distinct acceptance of specific
sequences particularly in the region of the domain
hinge. The new designed twin ribozyme HP–TWRJ is
thus a valuable addition to the existing variants of a
tandem configured hairpin ribozyme.
Experimental procedures
Synthesis of ribozymes and substrates
Reverse-joined hairpin ribozymes HP–RJWTAn (n ¼ 6, 7,
8, 12) and the twin ribozyme HP–TWRJ were transcribed
in vitro from oligonucleotide templates using T7 RNA
S. A. Ivanov et al. An engineered ribozyme for RNA sequence exchange
FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS 4471
polymerase essentially as described previously [15]. Briefly,
double-stranded DNA templates were generated from two
synthetic primers (BioTez, Berlin, Germany) overlapping by
15 complementary bases (for generation of templates for
transcription of HP–RJWTAn) or 32 complementary bases
(for generation of the template for transcription of
HP–TWRJ). After primer annealing, DNA templates were
completed with DNA polymerase I, Klenow fragment exo
–
(Fermentas, St. Leon-Rot, Germany). Transcription was
carried out with T7 RNA polymerase at 37 °C in standard
transcription buffer (40 mm Tris ⁄ HCl pH 7.9, 6 mm MgCl
2
,

10 mm dithiothritol, 10 mm NaCl, 2 mm spermidine) over
3 h (HP–RJWTAn), or in Hepes buffer (20 mm Hepes
pH 8.0, 10 mm Mg(OAc)
2
,10mm NaOAc, 1 mm dithio-
threitol, 25 lgÆmL
)1
bovine serum albumin) for 4.5 h (HP–
TWRJ). Proteins were removed by phenol ⁄ chloroform
extraction and RNA was precipitated from ethanol. Ribo-
zymes were purified by denaturing gel electrophoresis (7 m
urea, acrylamide ⁄ bis-acrylamide 19 : 1) on a 10% poly-
acrylamide gel (HP–TWRJ) or a 20% polyacrylamide gel
(HP–RJWTAn). Product-containing bands were eluted with
2 m LiClO
4
overnight at room temperature and precipitated
from acetone.
Substrate oligoribonucleotides SU9, SG9 and S40F3F5
were synthesized using the phosphoramidite method on a
1 lmole scale using an automated DNA ⁄ RNA synthesizer
(Gene Assembler Special, Pharmacia Biotech, Freiburg,
Germany). 5¢-Fluorescein labelling was achieved by solid-
phase coupling of ‘fluoreprime’ phosphoramidite (Amer-
sham Biosciences, Freiburg, Germany). For labelling at the
3¢-end, controlled pore glass was used to which fluorescein
was attached via a thiourea functionality as a succinate
linkage (ChemGenes Corp., Wilmington, USA). It con-
tained a dimethoxytrityl-protected hydroxyl group which
after deprotection was used for chain elongation. All RNA

substrates were purified by electrophoresis on 15–20%
denaturing polyacrylamide gels, eluted with 2 m LiClO
4
and precipitated from acetone.
For internal Cy5 labelling of the 20-mer fragment
S20 cp(Cy5), a deoxythymidine carrying an amino function
(amino modifier C6dT phosphoramidite, ChemGenes
Corp.) was built into a 29-mer oligonucleotide during
chemical synthesis (5¢-GUCCAGAAA-NH
2
C6dT-CUCC-
CUCACAGUCCUCUUU-3¢). After standard deprotection
and gel purification the amino function was coupled with
Cy5-NHS ester (Amersham Biosciences). To this end,
30 nmol of the oligonucleotide were solved in 500 lL car-
bonate buffer (0.1 m pH 8.5) and mixed with 1 mg dye.
The reaction was allowed to proceed for 1 h at room tem-
perature in the dark with occasional shaking. The reaction
was stopped by ethanol precipitation. The side product
N-hydroxysuccinimid was removed by washing the resolved
precipitate over a NAP column; the labelled oligonucleotide
was separated from nonlabelled species by gel electrophor-
esis and then cleaved with a conventional hairpin ribozyme
HP–WTTL [27] to yield the 20-mer fragment S20 cp(Cy5)
with 2¢,3¢-cyclic phosphate. In the same way RNA frag-
ments S17F5 cp, and S20 cp containing a 2¢,3¢-cyclic phos-
phate group (cp) were obtained from cleavage of
appropriate chemically synthesized RNA molecules with
HP–WTTL [27]. Extinction coefficients of ribozymes and
substrates were calculated with oligoanalyzer 3.0 (http://

www.idtdna.com) taking into account the absorption of
fluorophores.
Ligation experiments
Individual ribozymes were mixed with the respective sub-
strate, SU9 or SG9 in Tris ⁄ HCl (pH 7.5) buffer, heated
at 90 °C for 1 min followed by incubation at 32 °C for
15 min. The second ligation substrate S17F5 cp was added
and the mixture was incubated at 32 °C for another
10 min. Reactions were started by the addition of MgCl
2
and spermine. The final volume of the reaction mixture was
10 lL, final concentrations were: 200 nm ribozyme, 20 or
200 nm SU9 and SG9, 200 nm S17F5 cp, 10 mm MgCl
2
,
2mm spermine, 15 mm Tris ⁄ HCl, pH 7.5. Ligation was
allowed to proceed at 32 °C for 30 min. Aliquots (2.5 lL)
were removed at suitable intervals and reactions were
quenched by addition to 3 lL stop-mix (10 mm EDTA in
90% formamide) followed by intensive vortexing, heating
at 90 °C for 1 min, and immediate cooling on ice. Each
experiment was repeated at least once. Samples were ana-
lysed by PAGE on 8% denaturing gels (7 m urea) using an
ALF DNA sequencer (Pharmacia Biotech) as described pre-
viously [15]. Data were processed and analysed using dna
fragment analyzer 1.2 Software (Pharmacia Biotech).
Kinetic constants of ligation reactions were determined
under single turnover conditions. Reactions were carried
out essentially as described above. However, owing to very
fast ligation the required volumes of MgCl

2
stock solution
as well as of spermine stock solution were placed on the
inner wall of the Eppendorf tube and reaction was started
by intensive vortexing. The final volume of the reaction
mixture was 15 lL; final concentrations were: 80–500 nm
ribozyme, 20 nm SU9 or SG9, respectively, 88–550 n m
S17F5 cp, 10 mm MgCl
2
,2mm spermine, 15 mm Tris ⁄ HCl,
pH 7.5. Reaction analysis was as described above. Appar-
ent first-order rate constants (k
obs
) were obtained from a
plot of product formation against time in the linear phase
of reaction. Rate and dissociation constants (k
lig
and K
D
)
were calculated from linear curve fitting using plots of k
obs
against k
obs
⁄ [E], where [E] ¼ ribozyme concentration
(Eaddie–Hofstee plot). The margin of error was < 10% in
all measurements.
Determination of kinetic parameters
Kinetic parameters of twin ribozyme cleavage reactions
using individual substrates S40F5dA15 and S40F5dA31

An engineered ribozyme for RNA sequence exchange S. A. Ivanov et al.
4472 FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS
were determined under single turnover conditions. Substrate
was mixed with ribozyme in Tris ⁄ HCl (pH 7.5) buffer, hea-
ted at 90 °C for 1 min and incubated at 37 °C for 15 min.
Reactions were started by addition of MgCl
2
and spermine,
portions of which were placed before on the Eppendorf
tube wall as described in ligation experiments. The final
volume of the reaction mixture was 15 lL; final concentra-
tions were: 40–200 nm ribozyme, 20 nm substrate, 10 mm
MgCl
2
,2mm spermine, 15 mm Tris ⁄ HCl, pH 7.5.
Kinetic parameters of twin ribozyme ligation reactions
were determined under single turnover conditions, using the
following protocol. Ribozyme was mixed with the respective
substrate, SU9 or SG9 in Tris ⁄ HCl (pH 7.5) buffer, heated
at 90 °C for 1 min followed by incubation at 37 °C for
15 min. The second ligation substrate S17F5 cp was added
and the mixture was incubated at 37 °C for another 10 min.
Reactions were started by addition of MgCl
2
and spermine.
The final volume of the reaction mixtures was 20 lL; final
concentrations were: 40–200 nm ribozyme, 20 nm short
substrate, 44–220 nm long substrate, 10 mm MgCl
2
,2mm

spermine, 15 mm Tris ⁄ HCl. Samples were processed as des-
cribed above; parameters of cleavage as well as of ligation
were determined from Eaddie–Hofstee plots.
Sequence exchange reaction
A mixture of ribozyme and substrate in Tris ⁄ HCl (pH 7.5)
buffer was heated at 90 °C for 1 min followed by incuba-
tion at 37 °C for 15 min. The cleavage reaction was started
by addition of MgCl
2
and spermine. The final volume of
the reaction mixtures was 20 lL; final concentrations were:
220 nm ribozyme, 220 nm substrate, 11 mm MgCl
2
, 2.2 mm
spermine, 16.5 mm Tris ⁄ HCl, pH 7.5.
After 30 min, an aliquot (2 lL) was removed from the
reaction mixture and substituted by an equal volume con-
taining the 20-mer fragment S20 cp (2 lL), such that ori-
ginal RNA fragments (ribozyme, initial substrate and
added 20-mer) had a final concentration of 200 nm. Aliqu-
ots (2.5 lL) were taken at suitable time intervals and added
to 3 lL of stop-mix on ice. Samples were analysed using an
ALF DNA sequencer, and data were processed using alf
fragment manager as described previously [15]. When
the reaction was carried out with the Cy5-labelled 20-mer
S20 cp(Cy5), samples were also analysed using a DNA
Sequencer 4200 (LI-COR Biosciences, Bad Homburg, Ger-
many) and the data were processed with gene imagir 4.05
(LI-COR).
Melting temperatures and thermodynamic

parameters
To generate duplexes individual amounts of oligonucleotides
(for sequence see Fig. 2B) were mixed in reaction buffer con-
taining 10 mm Tris ⁄ HCl, pH 7.5 and 10 mm MgCl
2
, heated
at 90 °C for 5 min followed by slow cooling to 20 °C.
Individual samples with duplex concentrations of 500 nm,
750 nm,1lm, 2.5 lm and 5 lm have been prepared.
Melting profiles were measured at 260 nm in 1 mL quartz
cells using a UV visible spectrometer CARY 1E (VARIAN
INC., Palo Alto, USA) equipped with a temperature control
device. Thermostat temperature was varied from 20 to
90 °C with a heating rate of 0.2 °CÆmin
)1
. To avoid solvent
evaporation 100 lL mineral oil was placed on the sample
surface. Melting points T
m
(°C) were defined as the maxima
of the first derivation. Individual values for DH and DS were
obtained by plotting R ln[C] against 1 ⁄ T
m
(K
)1
). The free
enthalpy of the exchange of the short fragment (Fig. 2, red)
for the longer fragment (Fig. 2, green) on the ribozyme at
298 K (DG) was defined as DG(298 K) ¼ DG
product duplex

(298
K) – DG
educt duplex
(298 K).
Acknowledgements
Financial support by DFG and Ju
¨
rgen Manchot
Foundation as well as a PhD studentship to SV by the
Ju
¨
rgen Manchot Foundation is gratefully acknow-
ledged.
References
1 Kurreck J (2003) Antisense technologies. Improvement
through novel chemical modifications. Eur J Biochem
270, 1628–1644.
2 Scanlon KJ (2004) Anti-genes: siRNA, ribozymes and
antisense. Curr Pharm Biotechnol 5, 415–420.
3 Long MB, Jones JP 3rd, Sullenger BA & Byun J (2003)
Ribozyme-mediated revision of RNA and DNA. J Clin
Invest 112, 312–318.
4 Puerta-Fernandez E, Romero-Lopez C, Barosso-del-
Jesus A & Berzal-Herranz A (2003) Ribozymes: recent
advances in the development of RNA tools. FEMS
Microbiol Rev 27, 75–97.
5 Rimmele M (2003) Nucleic acid aptamers as tools and
drugs: recent developments. Chembiochem 4, 963–971.
6 Alisky JM & Davidson BL (2004) Towards therapy
using RNA interference. Am J Pharmacogenom 4, 45–

51.
7 Berkhout A (2004) RNA interference as an antiviral
approach: targeting HIV-1. Curr Opin Mol Ther 6,
141–145.
8 Burgstaller P, Jenne A & Blind M (2002) Aptamers and
aptazymes: accelerating small molecule drug discovery.
Curr Opin Drug Discov Devel 5, 690–700.
9 Famulok M & Verma S (2002) In vivo-applied func-
tional RNAs as tools in proteomics and genomics
research. Trends Biotechnol 20, 462–466.
10 Silverman SK (2003) Rube Goldberg goes (ribo) nuc-
lear? Molecular switches and sensors made from RNA.
RNA 9, 377–383.
S. A. Ivanov et al. An engineered ribozyme for RNA sequence exchange
FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS 4473
11 Mandal M & Breaker RR (2004) Gene regulation by
riboswitches. Nat Rev Mol Cell Biol 5, 451–463.
12 Mandal M, Lee M, Barrick JE, Weinberg Z, Emilsson
GM, Ruzo WL & Breaker RR (2004) A glycine-depen-
dent riboswitch that uses cooperative binding to control
expression. Science 306, 275–279.
13 Brantl S (2004) Bacterial gene regulation: from tran-
scription attenuation to riboswitches and ribozymes.
Trends Microbiol 12, 473–475.
14 Chowdhury S, Ragaz C, Kreuger E & Narberhaus F
(2003) Temperature-controlled structural alterations of
an RNA thermometer. J Biol Chem 278, 47915–47921.
15 Schmidt C, Welz R & Mu
¨
ller S (2000) RNA double

cleavage by a hairpin-derived twin ribozyme. Nucleic
Acids Res 28, 886–894.
16 Welz R, Bossmann K, Klug C, Schmidt C, Fritz H-J &
Mu
¨
ller S (2003) Site-directed alteration of RNA
sequence mediated by a small engineered twin ribozyme.
Angew Chem Int Ed 42, 2424–2427.
17 Mu
¨
ller S (2003) Engineered ribozymes as molecular
tools for site-specific alteration of RNA sequence.
Chembiochem 4, 991–997.
18 Vaule
´
on S, Ivanov SA, Gwiazda S & Mu
¨
ller S (2005)
Site-specific fluorescent and affinity labeling of RNA
using a small engineered ribozyme. Chembiochem
in press.
19 Feldstein A, Buzayan JM & Bruening G (1989) Two
sequences participating in the autolytic processing of
satellite tobacco ringspot virus complementary RNA.
Gene 82, 53–61.
20 Hampel A & Tritz R (1989) RNA catalytic properties
of the minimum (-) sTRSV sequence. Biochemistry 28,
4929–4933.
21 Rupert PB, Massey AP, Sigurdsson ST & Ferre
´

-
D’Amare
´
AR (2002) Transition state stabilization by a
catalytic RNA. Science 298, 1421–1424.
22 Zhuang X, Kim H, Pereira MJ, Babcock HP, Walter
NG & Chu S (2002) Correlating structural dynamics
and function in single ribozyme molecules. Science 296,
1473–1476.
23 Nahas MK, Wilson TJ, Hohng S, Jarvie K, Lilley DMJ
& Ha T (2004) Observation of internal cleavage and
ligation reactions of a ribozyme. Nat Struct Mol Biol
11, 1107–1113.
24 Nesbitt SM, Erlacher HA & Fedor MJ (1999) The inter-
nal equilibrium of the hairpin ribozyme: temperature,
ion and pH effects. J Mol Biol 286, 1009–1024.
25 Fedor MJ (1999) Tertiary structure stabilization pro-
motes hairpin ribozyme ligation. Biochemistry 38,
11040–11050.
26 Fedor MJ (2000) Structure and function of the hairpin
ribozyme. J Mol Biol 297, 269–291.
27 Welz R, Schmidt C & Mu
¨
ller S (2001) Spermine sup-
ports catalysis of hairpin ribozyme variants to differing
extents. Biochem Biophys Res Commun 283, 648–654.
28 Ivanov SA, Volkov EM, Oretskaya TS & Mu
¨
ller S
(2004) Chemical synthesis of an artificially branched

hairpin ribozyme variant with RNA cleavage activity.
Tetrahedron 60, 9273–9281.
29 Komatsu Y, Kanzaki I, Koizumi M & Ohtsuka E
(1995) Modification of primary structures of hairpin
ribozymes for probing active conformations. J Mol Biol
252, 296–304.
30 Komatsu Y, Kanzaki I & Ohtsuka E (1996) Enhanced
folding of hairpin ribozymes with replaced domains.
Biochemistry 35, 9815–9820.
31 Rupert PB & Ferre
´
-D’Amare
´
AR (2001) Crystal struc-
ture of a hairpin ribozyme–inhibitor complex with
implications for catalysis. Nature 410, 780–786.
32 Mu
¨
ller S, Welz R, Ivanov S & Bossmann K (2004)
Twin ribozymes. In Highlights in Bioorganic Chemistry:
Methods and Application (Schmuck C & Wennemers H,
eds), pp. 404–419. Wiley-VCH, Weinheim.
33 Drude I (2005) Design und Synthese von Twinribozy-
men zur Reparatur von Insertions – und Punktmutatio-
nen. Diploma thesis, Ruhr-Universita
¨
t Bochum,
Fakulta
¨
t Chemie, Germany.

34 Esteban JA, Walter NG, Kotzorek G, Heckman JE &
Burke JM (1998) Structural basis for heterogenic
kinetics: reengineering the hairpin ribozyme. Proc Natl
Acad Sci USA 95, 6091–6096.
35 Murchie AIH, Thomson JB, Walter F & Lilley DMJ
(1998) Folding of the hairpin ribozyme in its natural
conformation achieves close physical proximity of the
loops. Mol Cell 1 , 873–881.
36 Kirk BW, Feinsod M, Favis R, Kliman RM & Barany
F (2002) Single nucleotide polymorphism seeking long
term association with complex disease. Nucleic Acids
Res 30, 3295–3311.
37 Lechner D, Lathrop GM & Gut IG (2002) Large-scale
genotyping by mass spectrometry: experience, advances
and obstacles. Curr Opin Chem Biol 6, 31–38.
38 Sauer S, Gelfand DH, Boussicault F, Bauer K, Reichert
F & Gut IG (2002) Facile method for automated geno-
typing of single nucleotide polymorphisms by mass
spectrometry. Nucleic Acids Res 30, 22.
39 Perez-Ruiz M, Barroso-delJesus A & Berzal-Herranz A
(1999) Specificity of the hairpin ribozyme. Sequence
requirements surrounding the cleavage site. J Biol Chem
274, 29376–29380.
An engineered ribozyme for RNA sequence exchange S. A. Ivanov et al.
4474 FEBS Journal 272 (2005) 4464–4474 ª 2005 FEBS