Design of hairpin ribozyme variants with improved activity
for poorly processed substrates
Irene Drude
1,
*, Anne Strahl
2
, Daniel Galla
2
, Oliver Mu
¨
ller
1,
 and Sabine Mu
¨
ller
2
1 Max Planck Institute for Molecular Physiology, Department I, Dortmund, Germany
2 Ernst-Moritz-Arndt Universita
¨
t Greifswald, Institut fu
¨
r Biochemie, Germany
Introduction
In recent years, a number of ribozymes, particularly
the rather small hammerhead and hairpin ribozymes,
have been designed for cleavage of therapeutically rele-
vant targets [1–5]. Cleavage occurs at conserved sites
that first need to be identified on the target; this is
followed by adapting the sequence of the ribozyme
substrate-binding domain to specifically recognize,
bind and cleave the chosen site on the target RNA.

¨
r Biochemie, Felix
Hausdorff Str. 4, 17487 Greifswald,
Germany
Fax: +49 (0) 3834 864471
Tel: +49 (0) 3834 8622842
E-mail:
*Present address
NOXXON Pharma AG, Max-Dohrn-Strasse
8-10, 10589 Berlin, Germany
Present address
University of Applied Sciences Kaiserslau-
tern, Campus Zweibru
¨
cken, Amerikastraße 1,
66428 Zweibru
¨
cken, Germany
(Received 26 August 2010, revised
1 December 2010, accepted 6 December
2010)
doi:10.1111/j.1742-4658.2010.07983.x
Application of ribozymes for knockdown of RNA targets requires the iden-
tification of suitable target sites according to the consensus sequence. For
the hairpin ribozyme, this was originally defined as Y
)2
N
)1
*G
+1

sites
were previously shown to be processed by the wild-type hairpin ribozyme.
However, our study demonstrates that, in the specific sequence context of
the substrate studied herein, compensatory base changes in the ribozyme
improve activity for cleavage (eight-fold) and ligation (100-fold). In partic-
ular, we show that A
+3
and A
+4
are well tolerated if compensatory muta-
tions are made at positions 6 and 7 of the ribozyme strand. Adenine at
position +4 is neutralized by G
6
fi U, owing to restoration of a Watson–
Crick base pair in helix 1. In this ribozyme–substrate complex, adenine at
position +3 is also tolerated, with a slightly decreased cleavage rate. Addi-
tional substitution of A
7
with uracil doubled the cleavage rate and restored
ligation, which was lost in variants with A
7
,C
7
and G
7
. The ability to
cleave, in conjunction with the inability to ligate RNA, makes these
ribozyme variants particularly suitable candidates for RNA destruction.
Abbreviations
CPG, controlled pore glass; dNTP, deoxynucleoside triphosphate; ds, double strand; EDTA, ethylene diamine tetraacetic acid; lcaa, long chain

bonds, noncanonical base pairs and a Watson–Crick
base pair between G
+1
in loop A and C
25
in loop B as
characteristic elements [18–21]. The consensus sequence
of the hairpin ribozyme, determined by site-directed
mutagenesis [22–27] and in vitro evolution methods
[25–30], defines the helical regions as being highly flexi-
ble in sequence, provided that complementarity is pre-
served. In contrast, base substitutions within the loops
strongly interfere with catalytic activity. Therefore,
suitable RNA substrates were originally supposed to
fulfill the following sequence requirements: reversible
cleavage occurs between the conserved G
+1
and N
)1
within the 5¢-Y
)
N
)1
*G
+1
U
+2
Y
+3
B

sequence YN*GUYB could be identified. Therefore,
we decided to search for a site that keeps at least some
of the required nucleotides intact, and to design hair-
pin ribozyme variants for cleavage at this specific site.
The major criterion for defining a suitable target site
was the presence of a G
+1
immediately at the cleavage
site, because substitution of G
+1
with any of the other
natural RNA bases has been shown to completely
abolish activity [26,28,33,34]. Although substitution of
G
+1
can be compensated for by corresponding substi-
tution of C
25
in loop B, regenerating the interdomain
Watson–Crick base pair, the catalytic activity of the
resulting double mutants was rather low [31]. There-
fore, we decided to retain the essential G
+1
, and chose
a site consisting of U
)2
G
)1
*G
+1

´
rez-Ruiz et al. [32]) also do not fully correspond to
the consensus sequence (Fig. 1A), and therefore
require additional investigation. In general, there are
two possible ways of adapting the ribozyme sequence
to a specific target sequence. Suitable ribozymes can be
developed by selection of active species from a random
library, or by rational design. For the hairpin ribo-
zyme, a number of crystal structures are available
[20,21,36–40]. Careful inspection of the crystal struc-
tures reveals that nucleobases at positions +3, +4
and )2 of the substrate strand interact only with nu-
cleobases in loop A of the ribozyme strand, without
I. Drude et al. AUG hairpin ribozyme
FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS 623
A C G G A
A
G
G A G -5
′
5′- G G G A G A
U G C C U U
N
G A
A
G C U C
G C
C G
U A
G C

A C G G A A
A
U G C C U U
N
G A
A
G C U C
G C
C G
U
G C
A
G
A
A
A
C
A
C
A
U
U
A
U
A
U
G
U G
A U
HP-CTNNB1 N7

3
′
A
U G
A U
HP-CTNNB1 N7
C
C
C
A
A
G
G
A
A
G
G
N
G C
C G
U
G C
A
G
A
A
A
C
A
C

C
U
U
C
G
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
U
U
U
U
U
U
G
C
C
C

C G
G
U
U
U
A
U
U
C
A
G
C
A
G C
A
C G
G
U
U
U
A
U
U
C
A
G
C
A
3′- C C C U C U
A

C
C
C
A
A
G
G
A
A
G
G
A
G
C
3′
A
C
C
C
A
A
G
G
A
A
G
G
A
G
C

G A
G A
A
A
U
U
G
G
+3
+3
+4
+4
B
C
–2
–2
Y
C
G
G
G
G
N
N
Hairpin ribozyme
consensus sequence
Hairpin ribozyme
wild-type sequence
CTNNB1 target sequence
Fig. 1. Hairpin ribozyme variants for knockdown of CTNNB1 mRNA. (A) Sequences of the wild-type hairpin ribozyme and the consensus

Watson–Crick base pair is required at this location.
Thus, we replaced G
6
with uracil in the CTNNB1 ribo-
zyme, assuming that the resulting A
+4
–U
6
base pair
would restore activity.
There are no literature data available on compensa-
tory mutations for substitutions at position C
+3
.
On the contrary, it has been shown that the single
substitution C
+3
fi A strongly decreases activity [26].
Careful inspection of crystal structures of the hairpin
ribozyme–substrate complex as four-way-junction
[20,21] and minimal junction-less [36–40] structures,
however, shows that C
+3
forms a noncanonical base
pair with the nucleobase at position 7 in the ribozyme
strand, which naturally is adenine. Therefore, we
investigated whether a single base substitution at posi-
tion 7 in the CTNNB1 ribozyme can compensate for
C
+3

cleav
R Á 3
0
P þ 5
0
P
Under these conditions, the time dependence of the
cleavage product concentration typically follows [42]:
½5
0
P¼½5
0
P
1
ð1 ÀðexpÞ
ðk
ðobs;cleavÞ
ÁtÞ
Þ
Hence, the kinetic parameters k
cleav
and K
m
can be
calculated from the observed cleavage rate k
obs,cleav
at
different ribozyme concentrations [R]
o
, using the

Ribozyme
S-CTNNB1-1 substrate S-CTNNB1-1 U
)2
C substrate
k
cleav
(min
)1
) K
m
(nM) Amplitude k
cleav
(min
)1
) K
m
(nM) Amplitude
Wild type 0.007 ± 0.0007 33 ± 3 0.34 ± 0.06 – – –
A7 0.025 ± 0.0007 20 ± 4 0.62 ± 0.02 0.17 ± 0.003 31 ± 3.1 0.6 ± 0.02
C7 0.026 ± 0.006 4.5 ± 2.5 0.64 ± 0.01 0.19 ± 0.003 8.8 ± 1.8 0.58 ± 0.008
G7 (0.005 ± 2) · 10
)5
34 ± 7 0.47 ± 0.02 0.05 ± 0.001 50 ± 6.2 0.33 ± 0.02
U7 0.057 ± 0.001 24 ± 3.6 0.86 ± 0.02 0.75 ± 0.02 29 ± 3.7 0.86 ± 0.02
I. Drude et al. AUG hairpin ribozyme
FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS 625
substrates, a stable ribozyme–substrate complex orga-
nized in a three-way junction results, with both liga-
tion fragments being tightly bound to the ribozyme, by
12 and 15 bp, respectively (Fig. 1C).

catalyzed the cleavage reaction with a k
cleav
of about
0.025 min
)1
, indicating an increase in the cleavage rate
of only three-fold to four-fold as compared with the
wild-type hairpin ribozyme. The G7 variant did not
show any improvement in activity. Only 30% cleavage
product could be detected after 4 h. With a k
cleav
of
0.005 min
)1
, it showed equally low activity as the wild-
type ribozyme for cleavage of the CTNNB1 substrate.
As mentioned above, the CTNNB1 substrate RNA
used has a uracil instead of a cytosine at position –2.
In order to evaluate the sole influence of base substitu-
tions in the ribozyme strand, a modified CTNNB1 sub-
strate was synthesized, carrying the consensus cytosine
at position –2, and the activities of the four variants
for this substrate were tested. C
)2
in the CTNNB1
substrate increased cleavage rate constants about
10-fold in each variant as compared with cleavage of
the U
)2
substrate (Fig. 3; Table 1). The U7 variant

0.02
0.03
0.04
0.05
0.06
k
obs
/min
–1
Ribozyme (nM)
0 50 100 150 200 250
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
A
B
Fraction of cleaved product
Reaction time (min)
Fig. 2. HP-CTNNB1 N7-mediated cleavage of S-CTNNB1-1 under
single turnover conditions. (A) Time course of reactions at 30-fold
excess of ribozyme over substrate. (B) Dependence of k
obs
values

substrate with a product yield similar to that of the
U
)2
substrate, although with clearly shorter reaction
times.
Intermolecular ligation kinetics
In order to fully characterize the designed hairpin ribo-
zyme variants, we also investigated the ligation behav-
ior. In-trans ligation kinetics were measured in reactions
with ribozyme, 3¢-cleavage product ⁄ ligation substrate,
termed S-CTNNB1-2, and 5¢-ligation substrate,
S-CTNNB1-3 or S-CTNNB1-3 U
)2
C, containing a 2¢,3¢-
cyclic phosphate terminus. Binding of ligation substrates
to the ribozyme resulted in the formation of a stable
ribozyme–substrate complex, forming a three-way-junc-
tion structure (Fig. 1C). Because of the stability of this
complex, ligation should be favored over cleavage,
although cleavage cannot be neglected. Therefore, the
determined ligation rate will reflect an approach to the
equilibrium between cleavage and ligation, provided
that cleavage is much faster than dissociation of the
ribozyme–product complex. It has to be taken into
account that the observed ligation rate will be the sum
of the cleavage and ligation rates. Kinetic parameters
were determined under single turnover conditions, with
increasing concentrations of ribozyme and S-CTNNB1-
3orS-CTNNB1-3 U
)2

Therefore, the observed ligation rate constant essen-
tially reflects the ligation step, as the reverse cleavage
2 min
10 min
30 min
1 h
2 h
4 h
6 h
8 h
2 min
10 min
30 min
1 h
2 h
4 h
6 h
8 h
2 min
10 min
30 min
1 h
2 h
4 h
6 h
8 h
2 min
10 min
30 min
1 h

a
0.01 ± 0.004 0.17 ± 0.01
A7 ND ND ND 1.3 ± 0.08 105 ± 18 0.29 ± 0.007
C7 ND ND ND 0.68 ± 0.05 123 ± 27 0.32 ± 0.01
G7 ND ND ND 0.26 ± 0.01 61 ± 9 0.18 ± 0.008
U7 1 ± 0.07 380 ± 39 0.34 ± 0.01 1.5 ± 0.08 168 ± 21 0.6 ± 0.03
a
k
obs,lig
at ribozyme saturation.
I. Drude et al. AUG hairpin ribozyme
FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS 627
reaction is negligible. The slightly higher K
m
value
(380 ± 39 nm) may be a result of inactive ribozymes
in the solution [45].
Next, ligation activities of the four variants were
investigated on CTNNB1 substrates with cytosine
instead of uracil at position )2 (S-CTNNB1-3U
)2
C).
As observed for the cleavage reaction, ligation was
also considerably improved by this substitution: rate
constants and product yields were increased for all
four variants (Table 2). All variants showed ligation
activity, with maximal product yields of 65% (U7),
30% (A7 and C7) and 20% (G7) (Fig. 6A). Although
the U7 and A7 variants catalyzed ligation with differ-
ent amplitudes, the rate constants were similar

0.4
0.5
k
obs
(min
–1
)
Ribozyme (nM)
0 50 100 150 200
250
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
A
B
Fraction of ligation product
Reaction time (min)
Fig. 5. HP-CTNNB1 U7-mediated ligation of S-CTNNB1-2 with S-
CTNNB1-3 under single turnover conditions. (A) Time course of the
reaction at 30-fold excess of ribozyme and S-CTNNB1-3 over S-
CTNNB1-2. (B) Dependence of k
obs
values on ribozyme concentra-
tion.

A
B
Fig. 6. Ligation of S-CTNNB1-2 with S-CTNNB1-3 U
)2
C catalyzed
by HP-CTNNB1 N7 under single turnover conditions. (A) Time
course of the reaction at 50-fold excess of ribozyme and
S-CTNNB1-3 U
)2
C over S-CTNNB1-2. (B) Dependence of k
obs
val-
ues on ribozyme concentration.
AUG hairpin ribozyme I. Drude et al.
628 FEBS Journal 278 (2011) 622–633 ª 2010 The Authors Journal compilation ª 2010 FEBS
time-resolved folding analysis of individual hairpin
ribozyme variants to look for differences in the folding
kinetics. For the purpose of the study presented here,
the ligation rate constant was assigned to the fast phase
of the reaction, being 0.01 ± 0.004 min
)1
for the wild
type, and 1.0 ± 0.07 min
)1
for ligation of S-CTNNB1-3
by the U7 variant and 1.5 ± 0.08 min
)1
for CTNNB1-3
U
)2

As known from the crystal structure, C
+3
in the sub-
strate interacts with A
7
in the ribozyme strand. There-
fore, we speculated whether substitution of A
7
would
compensate for C
+3
fi A. Four hairpin ribozymes car-
rying any of the four bases at position 7 have been
prepared (N7 ribozymes) and studied in cleavage and
ligation assays.
A wild-type hairpin ribozyme that recognizes the
CTNNB1 substrate showed 60-fold lower cleavage
activity and 100-fold lower ligation activity than with
the wild-type A*GUC substrate [35,41]. A
+4
in the
substrate is well tolerated if the ribozyme contains a
uracil at position 6, restoring a Watson–Crick base
pair. This result is somewhat surprising, as an A–U
base pair at this position never seemed to have
emerged from in vitro selection experiments [25–30],
such that the nucleobase at position +4 was included
in the consensus sequence as B = C, G or U, but not
A. A substrate with A
+3

These results expand hairpin ribozyme consensus
rules in different ways. On the basis of previous stud-
ies, it was concluded that the hairpin ribozyme accepts
any base except adenine at position +4 [25].
The results presented here indicate that A
+4
is toler-
ated without loss of activity, if the complementary
base is located at position 6 in the ribozyme strand,
allowing the essential Watson–Crick base pair to be
formed. Furthermore, as previously shown by Ander-
son et al. [26], the C
+3
fi A substitution almost com-
pletely abolished the cleavage activity of the wild-type
hairpin ribozyme. We did not observe such a strong
effect of C
+3
fi A on the ribozymes tested here, in
good agreement with the cleavage of G*GUA sub-
strates reported by Pe
´
rez-Ruiz et al. [32]. Apart from
the different substrate sequence, our A7 variant corre-
sponds to the sequence of the wild-type hairpin ribo-
zyme, with the only difference at position 6 being
uracil instead of guanine (Fig. 1). Apparently, the
G
6
fi U substitution, together with the altered sub-

and A
7
, in which, according to the crystal structure,
the excocyclic amino group of C
+3
donates a proton
to ring nitrogen N1 of A
7
[20,36–40]. In the A7 ribo-
zyme, the Watson–Crick base pair is formed between
A
+4
and U
6
followed by the noncanonical base pair
A
+3
–A
7
. Adenine provides a similar Watson–Crick
edge as cytosine, and the function of the exocyclic
amino group of C
+3
as a hydrogen donor can be
basically retained by adenine. The larger nucleobase
may be tolerated because of the different nature of the
neighboring Watson–Crick base pair, which is A–U
instead of G–C. An A–U base pair is less stable than
G–C, and thus might allow the neighboring A
+3

and pyrimidine
+3
–purine
7
(noncanonical) to purine
+4
–
pyrimidine
6
(Watson–Crick) and purine
+3
–pyrimidine
7
(noncanonical) in the U7 variant. This may be well
tolerated, owing to the ability of the modified base
pairs to provide the required base-pairing interactions
with similar spatial characteristics.
The most significant effect was observed upon
replacement of C
)2
with uracil. Both cleavage and liga-
tion activities suffered from this substitution, probably
because of the emerging wobble base pair between U
)2
and G
11
closing helix 2. This G–U wobble base pair,
located next to loop A, is presumably less capable of
stabilizing the required loop A conformation than the
regular Watson–Crick base pair that normally occurs

design study delivered functional ribozymes with less
time, material and costs. Moreover, our results demon-
strate that several changes in the substrate sequence can
be advantageous over just one base substitution, owing
to the cooperative effect of two or more base changes.
The discrepancy between cleavage and ligation activities
observed for the A7, C7 and G7 variants is a useful
property with regard to the use of ribozymes for mRNA
knockdown. Here, only cleavage is required, and liga-
tion activity is undesirable. Altogether, the results of our
study enlarge the window for application of tailor-made
ribozymes in molecular biology and medicine.
Experimental procedures
Substrate preparation
All substrates used for cleavage and ligation analysis were
chemically synthesized on a solid phase as described previ-
ously [48], with the use of phenoxyacetyl-protected phos-
phoamidites (ChemGenes, Wilmington MA, USA) and a
Gene Assembler Special synthesizer (Pharmacia, Freiburg,
Germany). For postsynthetic labeling of the 20mer cleavage
substrate and the 3¢-ligation substrate, 3¢-Amino Modifier
C-3 lcaa CPG (ChemGenes) was used as the solid phase.
Phenoxyacetyl and cyanoethyl protecting groups were
removed with a 1 : 1 mixture of 32% ammonia and 8 m
methylamine in ethanol at 65 °C for 30 min, followed by
lyophilization. Tert-butyldimethylsilyl protecting groups
were removed for 1.5 h at 55 °C in a 3 : 1 mixture of trieth-
ylamine trihydrofluoride and dimethylformamide, and the
reaction was stopped with 25% (v ⁄ v) water. RNA was pre-
cipitated with butanol and purified by PAGE on a 15 %

S-CTNNB1-3U
)2
C with 2¢,3¢-cyclic phosphate termini, RNA
substrate, DNAzyme and Tris (pH 7.5) were mixed to give a
final concentration of 2 lm RNA, 400 nm DNAzyme and
40 mm Tris, heated for 2 min at 95 °C, and incubated for
15 min at 37 °C. After addition of magnesium chloride to a
final concentration of 90 mm, the reaction was performed
for 2 h at 37 °C. RNA was purified on a 10% denaturating
polyacrylamide gel, eluted from the gel with 0.3 m sodium
acetate (pH 7.0) at 4 °C, and precipitated with ethanol.
Ribozyme synthesis
Hairpin ribozymes were transcribed in vitro from a dsDNA
template. The DNA template was obtained from a Klenow
polymerase-mediated fill-in reaction of two synthetic prim-
ers (5¢- CTG TAC TAA TAC GAC TCA CTA TAG GGA
GAT GCC TTN GAA
GCT CAG CTG AGA AAC ACG
AAT C-3¢ and 5¢-GCT CCT TCT CTG GGT AGC TGG
TAA TAT ACC GA A TGC GAA
GAT TCG TGT TTC
TCA GCT GAG C-3¢; biomers.net, Ulm, Germany) over-
lapping at their 3¢-ends by 22 nucleotides (underlined). Both
primers and 10 · KFI buffer (500 mm Tris, pH 7.6,
100 mm MgCl
2
and 500 mm NaCl) were mixed to give final
concentrations of 2 lm each primer and 1 · KFI buffer,
heated for 2 min at 90 °C, and incubated for 15 min at
37 °C. After addition of dNTPs (Fermentas, St Leon-Rot,

and incubated for a further 15 min at 37 °C. Reactions
were started by mixing substrate and ribozyme solutions.
At suitable time intervals, aliquots of 1 lL were taken, and
the reaction was immediately stopped by addition of 19 lL
of stop mix (7 m urea and 50 mm Na-EDTA). Samples
were stored on ice before analysis. All reactions were
repeated at least twice. Samples wer e analyzed on a 15% poly-
acrylamide gel with a DNA Sequencer Long ReadIR 4200
(LI-COR Bioscience Bad Homburg, Germany); data were
processed with gene imagir 4.05. The fraction of substrate
cleaved was plotted versus time, and fitted to the single
exponential equation
½3
0
P¼Að1 À e
Àkt
Þ
where [3¢P] is the product concentration, A is the ampli-
tude, k = k
obs,cleave
, and t is the time. Standard deviations
were less than 20% in each case. To determine the enzyme
specific constants, k
cleav
and K
m
, the obtained k
obs,cleav
values were plotted versus ribozyme concentration [R]
0

aliquots of 1.5 lL were taken and immediately added to
8.5 lL of stop mix, and samples were stored on ice until
analysis. Analysis of the ligation reaction was performed on
a DNA sequencer as described for cleavage reactions. The
fraction of ligation product was plotted versus time and fit-
ted to single-exponential or double-exponential equations.
The single-exponential equation was:
½P¼Að1 À e
Àk
obs;lig
Át
Þ
where [P] is the product concentration, A is the amplitude,
and t is the time. The double-exponential equation was:
½P¼A
0
þ A
1
ð1 À e
Àk
1
t
ÞþA
2
ð1 À e
Àk
2
t
Þ
where A

k
obs;lig
¼
k
app;lig
½R
0
K
m
þ½R
0
Acknowledgements
This work was supported by the Ju
¨
rgen Manchot
Foundation. A PhD scholarship of the Konrad-Adena-
uer Foundation to Irene Drude is gratefully acknowl-
edged.
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