Báo cáo khoa học: Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10–23 potx

Mapping of the functional phosphate groups in the
catalytic core of deoxyribozyme 10–23
Barbara Nawrot, Kinga Widera, Marzena Wojcik*, Beata Rebowska, Genowefa Nowak and
Wojciech J. Stec
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences, Lodz, Poland
The RNA-cleaving DNA enzymes, like most ribozymes,
require a divalent metal cation for their cleavage activity
[1]. Among metal ion-dependent DNA enzymes,
deoxyribozyme 10–23, first selected and characterized
by Santoro & Joyce [1,2], has been examined most
extensively both in vitro and in vivo [3–5]. This enzyme
consists of a 15-nucleotide conserved catalytic core and
variable substrate recognition arms (Fig. 1A). Cleavage
of an RNA substrate is highly sequence-specific, and
occurs between the bulged 5¢-purine and paired 3¢-pyr-
imidine nucleosides, resulting in the formation of the
two products, a 5¢-terminal product with a 2¢,3¢-cyclic
phosphate, and a 3¢-terminal product containing an OH
group at its 5¢-end. The enzyme preferentially uses Mg
2+
for its activity, although other divalent metal ions are
accepted as cofactors [1,2,6]. To date, the structure of
the substrate–deoxyribozyme 10–23 active complex
remains unknown [7,8], and the mechanistic details of
the catalytic reaction are not fully understood. There-
fore, much effort has been devoted to determine the role
of individual nucleotides in the 10–23 catalytic core, as
well as their relative importance [9–13]. Despite numer-
ous studies performed on a mutant deoxyribozyme 10–
23 containing chemical modifications inserted into the
catalytic core, the role of particular phosphates within

this domain has not been investigated in detail. We have
studied this issue by systematic modification of each
phosphate of the core with phosphorothioate (PS) ana-
logs, in which one of the two nonbridging oxygen atoms
of the phosphate group was replaced with a sulfur atom.
Keywords
catalysis; deoxyribozyme; phosphorothioate;
rescue effect; thio effect
Correspondence
B. Nawrot, Department of Bioorganic
Chemistry, Centre of Molecular and
Macromolecular Studies of the Polish
Academy of Sciences, Sienkiewicza 112,
90-363 Lodz, Poland
Fax: +48 42 6815483
Tel: +48 42 6816970
E-mail:
*Present address
Medical University of Lodz, Department of
Structural Biology, Zeligowskiego, Poland
(Received 4 October 2006, revised 29
November 2006, accepted 18 December
2006)
doi:10.1111/j.1742-4658.2007.05655.x
The RNA phosphodiester bond cleavage activity of a series of 16 thio-de-
oxyribozymes 10–23, containing a P-stereorandom single phosphorothioate
linkage in predetermined positions of the catalytic core from P1 to P16,
was evaluated under single-turnover conditions in the presence of either
3mm Mg
2+

or 3 mm Mn
2+
. A metal-specificity switch approach permitted
the identification of nonbridging phosphate oxygens (proR
P
or proS
P
)
located at seven positions of the core (P2, P4 and P9–13) involved in direct
coordination with a divalent metal ion(s). By contrast, phosphorothioates
at positions P3, P6, P7 and P14–16 displayed no functional relevance in the
deoxyribozyme-mediated catalysis. Interestingly, phosphorothioate modifi-
cations at positions P1 or P8 enhanced the catalytic efficiency of the
enzyme. Among the tested deoxyribozymes, thio-substitution at position P5
had the largest deleterious effect on the catalytic rate in the presence of
Mg
2+
, and this was reversed in the presence of Mn
2+
. Further experiments
with thio-deoxyribozymes of stereodefined P-chirality suggested direct
involvement of both oxygens of the P5 phosphate and the proR
P
oxygen at
P9 in the metal ion coordination. In addition, it was found that the oxygen
atom at C6 of G
6
contributes to metal ion binding and that this interaction
is essential for 10–23 deoxyribozyme catalytic activity.
Abbreviations

AP, 2-aminopurine; DNAzyme, RNA-cleaving deoxyribozyme; PS, phosphorothioate; s
6
G, 6-thioguanosine.
1062
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Journal compilation ª 2007 FEBS
The PS modification represents the most conservative
elemental replacement for the phosphate, although the
sulfur atom is slightly larger than the oxygen atom, and
the P–S bond is 0.3 A
˚
longer than the P–O bond [14].
Oligonucleotides possessing stereodefined PS internucle-
otide linkages have been found useful for clarifying the
function of proR
P
and proS
P
positions at the scissile site
of oligonucleotide substrates. The ribozyme-assisted
cleavage reactions were conducted in the presence of
divalent metal cations with different affinities for oxygen
and sulfur [15–20]. According to the HSAB (Hard and
Soft, Acid and Base) rule [21], a reduction in the clea-
vage rate of a ‘soft’ thio-substituted substrate should be
observed in the presence of ‘hard’ Mg
2+
cation (the thio
effect), and restoration of a normal cleavage rate of a
sulfur-containing substrate should occur in the presence

of thiophilic cations such as Mn
2+
,Zn
2+
,orCd
2+
,in
increasing order (the rescue effect). Analysis of these
types of interaction led to a better understanding of the
mechanistic aspects of the action of naturally occurring
catalytic ribozymes: group I and II introns [22–25], the
RNA subunit of RNase P [26,27], and the hammerhead
ribozymes [18,28–30]. The successful application of P-
chiral phosphorothioates in those mechanistic studies
prompted us to establish the role of phosphate groups in
the catalytic core of deoxyribozyme 10–23. First, we
introduced a P-stereorandom single PS linkage in prede-
termined positions of the catalytic core in 16 thio-deoxy-
ribozymes 10–23 (P1–P16; Table 1, entries 2–17), and
conducted metal-specificity switch experiments with
Mg
2+
and thiophilic Mn
2+
. These experiments showed
that catalytically important phosphate groups were
positioned within the catalytic domain of the enzyme.
The role of the particular oxygen atoms of the selected
phosphate groups is also discussed. Moreover, we ana-
lyzed the function of the oxygen moiety at C6 of nucleo-

side G
6
positioned within the catalytic loop, by either its
removal [substitution with 2-aminopurine (AP) nucleo-
side] or its replacement with a sulfur atom by using the
6-thioguanosine (s
6
G) mutant enzyme. Kinetic measure-
ments of these deoxyribozyme variants, along with data
obtained by Zaborowska et al. [11], proved the import-
ance of the oxygen of the carbonyl group at G
6
for the
catalytic activity of deoxyribozyme 10–23.
Results and Discussion
The influence of PS modification on the catalytic
activity of deoxyribozyme 10–23
The functional role of the individual phosphate groups
in the catalytic core of deoxyribozyme 10–23 was
examined by determination of the thio effect and the
Mn
2+
-dependent rescue effect of thio-substituted de-
oxyribozymes bearing a single PS linkage from P1 to
P16, where the P1 phosphate is a 5¢-phosphate of nuc-
leotide 1 (G
1
) (Table 1, entries 2–17). The PS deoxyri-
bozymes were synthesized by automated solid-phase
synthesis, in which one of the iodine oxidation steps

was replaced by sulfurization [31]. Each oligomer was
an R
P
and S
P
(c. 1 : 1) diastereomeric mixture (Fig. 1).
The activity of thio-substituted deoxyribozymes was
tested against a short target substrate homosequential
with mRNA of aspartyl protease Asp2 (BACE1, acces-
sion number AF190725, between nucleotides 1801 and
1817) (Fig. 1). It has already been demonstrated that
deoxyribozyme 10–23 accepts not only short RNA
substrates but also modified substrates containing a
DNA backbone with RNA nucleotides (5¢-purine and
3¢-pyrimidine ribonucleotides) positioned at the scissile
bond of the target oligonucleotide [32–34]. We pre-
pared a 17-nucleotide chimeric DNAÆRNA substrate
with the sequence 5¢-d(ACAGATGA)GUd(CAACC-
CT)-3¢, which was easier to synthesize and chemically
more stable than an RNA oligonucleotide.
All kinetic experiments were performed at a satur-
ating concentration of the unmodified deoxyribozyme
1 or thio-deoxyribozymes 2–17 (10 lm) with
32
P-labe-
led substrate (0.1 lm) in the presence of 3 mm MgCl
2
.
The cleavage product (9-mer) and the substrate were
quantified by autoradiography following electrophor-

A
B
O
B
O
P
O
S
O
B
O
O
B
O
P
S
O
O
B
O
S
P
R
P
Fig. 1. (A) The structure of deoxyribozyme 10–23. The target sub-
strate is a chimeric DNAÆRNA oligonucleotide homosequential to
the mRNA of BACE1 (nucleotides 1801–1817). Substrate–enzyme
binding occurs via the Watson–Crick mode of base-pairing. The
arrow indicates the cleavage site. The positions of the phosphate
groups of the catalytic core are numbered from P1 to P16. (B) P-

chiral PS internucleotide bonds in PS DNA of S
P
-sense and R
P
-
sense of chirality, respectively.
B. Nawrot et al. Metal-binding site in deoxyribozyme 10–23
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Journal compilation ª 2007 FEBS
1063
esis in 20% polyacrylamide gels. The observed rate
constants (k
obs
) were calculated according to the equa-
tion given in Experimental procedures, and compared
with the rate constant of the unmodified deoxyribo-
zyme (k
rel
).
The data presented in Table 1 and Fig. 2A indicate
that thio-substitutions at phosphates P2, P4 and P9–13
lowered the k
rel
values by c. 50%. The replacement of
3mm Mg
2+
with 3 mm Mn
2+
resulted in a restoration
of the activity to the level of the k

Mn
obs
of the unmodified
enzyme (Table 1, Fig. 2B), implying a possible coordi-
nation of the metal cation to one of the two (the
proR
P
or the proS
P
) oxygen atoms. However, it should
be noted that the k
obs
values were c. 30-fold higher in
the presence of Mn
2+
compared with Mg
2+
(Table 1).
A similar high rate of the cleavage reaction in the pres-
ence of Mn
2+
was reported previously [1,35]. As pro-
posed by Breaker et al. [35], it is possible that the
higher activity of deoxyribozyme 10–23 in the presence
of Mn
2+
may result from the fact that Mn
2+
,asa
stronger Lewis acid, participates more effectively in

catalysis steps such as the acceleration of the ribose
2¢-hydroxyl group deprotonation, stabilization of a
negative charge that may develop on the nonbridging
oxygen in a transition state, and ⁄ or stabilization of the
negative charge on the oxygen atom of the 5¢-leaving
group.
Among the tested modified enzymes, the biggest thio
effect (a 16-fold reduction in the cleavage activity;
Table 1, Fig. 2A) was found for the PS enzyme modi-
fied at position P5. The reduction was much bigger
than the two-fold reduction expected if only one of the
diastereomers coordinated the metal ion, suggesting
that the sulfur atoms in both the proR
P
and proS
P
positions hindered direct contact with metal ions.
Interestingly, this PS enzyme regained its activity in
the presence of Mn
2+
, with the k
Mn
obs
value being 176-
fold higher than the k
Mg
obs
value. This value, however,
was still c. 3-fold lower than that measured for the
unmodified reference at the same conditions (Table 1).

It seems that the slightly lower reaction rate of this PS
enzyme in the presence of Mn
2+
might be attributed to
Table 1. Single-turnover rate constants of the cleavage reactions catalyzed by unsubstituted and thio-substituted deoxyribozyme 10–23. NA,
value not available.
Entry
DNAzyme
abbreviation ⁄ PS
position
5¢fi3¢
sequence of
the catalytic core
a
k
Mg
obs
(min
)1
)
b
k
Mg
rel
c
Thio effect k
Mn
obs
(min
)1

)
d
k
Mn
rel
e
k
Mn
obs
⁄ k
Mg
obs
(rescue effect)
1 d(AGGCTAGCTACAACGAT) 0.27 ± 0.028 1 1.0 8.00 ± 0.42 1 30
2 P1 d(A
PS
GGCTAGCTACAACGAT) 0.85 ± 0.042 3.10 0.3 0.36 ± 0.031
f
3.0
f
NA
3 P2 d(AG
PS
GCTAGCTACAACGAT) 0.15 ± 0.018 0.56 1.8 8.10 ± 0.57 1.01 54
4 P3 d(AGG
PS
CTAGCTACAACGAT) 0.24 ± 0.0078 0.89 1.1 8.00 ± 1.10 1.00 33
5 P4 d(AGGC
PS
TAGCTACAACGAT) 0.16 ± 0.0071 0.59 1.7 8.60 ± 1.40 1.08 54

6 P5 d(AGGCT
PS
AGCTACAACGAT) 0.017 ± 0.0014 0.06 16 3.00 ± 0.071 0.38 176
7 P6 d(AGGCTA
PS
GCTACAACGAT) 0.21 ± 0.0071 0.78 1.3 8.20 ± 1.10 1.03 39
8 P7 d(AGGCTAG
PS
CTACAACGAT) 0.24 ± 0.070 0.89 1.1 8.70 ± 0.071 1.10 36
9 P8 d(AGGCTAGC
PS
TACAACGAT) 0.44 ± 0.014 1.60 0.6 0.16 ± 0.038
f
1.30
f
NA
10 P9 d(AGGCTAGCT
PS
ACAACGAT) 0.14 ± 0.014 0.52 1.9 8.40 ± 0.64 1.05 60
11 P10 d(AGGCTAGCTA
PS
CAACGAT) 0.14 ± 0.028 0.52 1.9 9.30 ± 0.71 1.20 66
12 P11 d(AGGCTAGCTAC
PS
AACGAT) 0.15 ± 0.018 0.56 1.8 8.20 ± 1.10 1.03 55
13 P12 d(AGGCTAGCTACA
PS
ACGAT) 0.15 ± 0.014 0.56 1.8 10.00 ± 1.70 1.30 67
14 P13 d(AGGCTAGCTACAA
PS

CGAT) 0.14 ± 0.024 0.52 1.9 9.20 ± 0.71 1.20 66
15 P14 d(AGGCTAGCTACAAC
PS
GAT) 0.28 ± 0.014 0.96 1.0 9.30 ± 0.64 1.20 33
16 P15 d(AGGCTAGCTACAACG
PS
AT) 0.38 ± 0.035 1.40 0.7 9.40 ± 0.71 1.20 25
17 P16 d(AGGCTAGCTACAACGA
PS
T) 0.24 ± 0.030 0.89 1.1 7.40 ± 0.78 0.93 31
18 P1 ⁄ P8 d(A
PS
GGCTAGC
PS
TACAACGAT) 0.76 ± 0.080 2.80 0.78 ± 0.048
f
6.5
f
NA
a
The sequences of PS deoxyribozymes 10–23 containing a single PS linkage of stereorandom P-configuration (equal amounts of R
P
and
S
P
diastereomers) in the selected positions of the catalytic core marked from P1 (phosphate bond between A
0
and G
1
) to P16 (phosphate

bond between A
15
and T
16
).
b, d
RNA cleavage reactions were performed in 20 mM Tris ⁄ HCl (pH 7.5), containing 100 mM NaCl,
b
3mM
Mg
2+
or
d
3mM Mn
2+
under single-turnover conditions with 0.1 lM 5¢-end
32
P-labeled substrate and 10 lM deoxyribozyme, at 37 °C.
c
k
Mg
rel
¼ the ratio of the k
obs
values of modified deoxyribozyme to unmodified deoxyribozyme, in the presence of Mg
2+
.
e
k
Mn

rel
¼ the ratio
of the k
obs
values of modified deoxyribozyme to unmodified deoxyribozyme, in the presence of Mn
2+
.
f
Reactions were performed in
20 m
M Tris ⁄ HCl (pH 7.5), containing 100 mM NaCl and 0.06 mM Mn
2+
under single-turnover conditions with 0.1 lM 5¢-end
32
P-labeled sub-
strate and 10 l
M deoxyribozyme, at 37 °C. Values of k
obs
for unsubstituted and thio-substituted deoxyribozyme reactions represent mean
values of four independent experiments, and errors indicate deviations between individual experiments. The obtained data were normal-
ized to a k
obs
of 0.12 ± 0.014 min
)1
for reaction of the unmodified deoxyribozyme in 0.06 mM Mn
2+
.
Metal-binding site in deoxyribozyme 10–23 B. Nawrot et al.
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FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences

Journal compilation ª 2007 FEBS
the bulky sulfur atom, which could influence the geom-
etry of metal ion interactions, or the geometry of the
catalytic conformation of the core. However, this par-
tial inability of thiophilic metal ions to fully rescue
catalysis does not eliminate the possibility that this
modified phosphate is in direct contact with a catalyti-
cally important cation [36].
Other modified deoxyribozymes, containing thio-
substitutions at positions P3, P6, P7, P14, P15 and
P16, retained catalytic activity comparable with that of
the unmodified enzyme in the presence of Mg
2+
and
Mn
2+
. These data constitute strong evidence against
direct coordination of a metal cation to both the
proR
P
and proS
P
phosphate oxygen atoms at these
positions during catalysis. Also, it is possible that a
sulfur atom in these positions does not alter the struc-
ture of the catalytically active core of deoxyribozyme.
This observation suggests the possibility of using parti-
ally modified PS analogs of deoxyribozymes to
improve their stability against intracellular endonuc-
leases in cellular systems.

The catalytic activity of double PS-substituted
deoxyribozyme 10–23
PS modification at positions P1 or P8, surprisingly,
accelerated the cleavage rates (3-fold and 1.6-fold,
respectively) in the presence of Mg
2+
as well as Mn
2+
(Table 1, Fig. 3). The k
obs
and k
rel
values for these
enzymes were calculated from the reactions performed
in 3 mm Mg
2+
or 0.06 mm Mn
2+
. The concentration of
Mn
2+
was reduced 50-fold, because reactions per-
formed in the presence of 3 mm Mn
2+
reached comple-
tion in less than 5 s, making kinetic analysis
impossible. Whereas the P8 substitution had only a
minor effect both in the presence of Mg
2+
and in the

presence of Mn
2+
, causing a 30–60% increase in k
rel
,
the effect of the double substitution P1 ⁄ P8 was strik-
ingly different, depending on the metal ion present.
There was no increase of the enzyme efficiency in the
presence of Mg
2+
, compared to P1 substitution itself,
but in the presence of Mn
2+
the k
rel
for the P1 ⁄ P8
enzyme was over two-fold higher than the k
rel
for the
P1 enzyme and 6.5-fold higher than that for the
unmodified reference (Table 1, Fig. 3). For the P1 ⁄ P8
PS congener, the k
Mg
obs
and k
Mn
obs
values were nearly iden-
tical, despite a 50-fold difference in the concentration
of metal ions present in the catalysis reaction, and the

k
Mn
obs
value for this mutant enzyme was three-fold
higher than the k
Mn
obs
value for the unmodified reference.
The obtained data demonstrate that the P1 ⁄ P8
A
unmodified
P2
P3
P4
P5
P6
P7
P9
P10
P11
P12
P13
P14
P15
P16
unmodified
P2
P3
P4
P5

P6
P7
P9
P10
P11
P12
P13
P14
P15
P16
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
k
rel
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
k
rel

B
Fig. 2. Comparison of the relative rates of cleavage (k
rel
) of thio-
substituted deoxyribozymes 10–23 in the presence of 3 m
M MgCl
2
(A) and 3 mM MnCl
2
(B).
0
1
2
3
4
5
6
7
8
unmodified Mg
2+
unmodified Mn
2+
P1 Mg
2+
P1 Mn
2+
P8 Mg
2+
P8 Mn

2+
P1/P8 Mg
2+
P1/P8 Mn
2+
k
rel
Fig. 3. Comparison of the relative rates of cleavage (k
rel
) of thio-
substituted deoxyribozymes 10–23 in the presence of 3 m
M MgCl
2
(white bars) and 0.06 mM MnCl
2
(gray bars).
B. Nawrot et al. Metal-binding site in deoxyribozyme 10–23
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Journal compilation ª 2007 FEBS
1065
PS-mutant enzyme is c. 50-fold more active in the
presence of Mn
2+
than in the presence of Mg
2+
, and
its k
obs
value in the presence of 3 mm Mn
2+

could
reach a value of c. 40 min
)1
. This is a four times
higher value than the highest one so far reported in
the literature for catalytic nucleic acids [35]. Moreover,
this double PS congener is c. 150-fold more active in
the presence of Mn
2+
than the unmodified reference in
the presence of Mg
2+
. A possible explanation for these
results is that both pairs of oxygen atoms at the P1
and P8 phosphates do not directly interact with metal
ions, and such a double PS modification, together with
the presence of Mn
2+
, facilitates a catalytically favora-
ble conformation of the 10–23 core. Moreover, one
cannot exclude the possibility that the 10–23 enzyme
operates with two metal ions interacting with different
sets of residues.
Our finding that the introduction of a PS bond at
the P1 site of deoxyribozyme 10–23 causes about
three-fold stimulation of the cleavage rate, irrespective
of the metal ion used, demonstrates that chemical
modifications of the deoxyribozyme backbone can be
used to improve both its stability and its catalytic effi-
ciency in cellular experiments.

Effect of P-chirality on the catalytic activity
of deoxyribozyme 10–23
In order to obtain a deeper insight into the functional
role of the oxygen atoms of the P5 phosphate group in
the catalytic core of deoxyribozyme 10–23, we pre-
pared two PS deoxyribozymes with stereodefined R
P
-
PS or S
P
-PS linkages at that position and measured
the rate of RNA cleavage under analogous conditions
in the presence of 3 mm Mg
2+
(Fig. 4). We found that
R
P
-PS and S
P
-PS substitutions at position P5 reduced
k
Mg
rel
by a factor of 34 and 21, respectively (Table 2,
Fig. 5A). As k
obs
values were measured at a saturating
concentration of the PS enzymes, their lowered activity
could not be attributed to decreased substrate binding,
thus implying that sulfur substitution disrupted specific

Mg
2+
interactions with nonbridging phosphate oxy-
gens. In 3 mm Mn
2+
buffer, the R
P
-PS and S
P
-PS
deoxyribozyme P5-mediated cleavage activity was
significantly enhanced (73-fold and 108-fold increase
of k
obs
values, respectively; Table 2, Fig. 5B). The
observed thio effect and rescue effect values for partic-
ular P-chiral diastereomers slightly differed from those
determined for the diastereomeric mixture of this PS
enzyme, and these differences may result from experi-
mental errors. The remarkable increase of the catalytic
rate for the reactions carried out in the presence of
Mg
2+
and each of the P-chiral diastereomeric deoxyri-
bozymes suggests that Mn
2+
can stimulate 10–23
enzyme activity in a way that depends on the simulta-
neous metal ion interactions with both nonbridging
oxygens at position P5. Thus, earlier suggestions are

fully confirmed by our findings [13].
Other stereodefined PS deoxyribozymes with a PS
bond at position P9 (prepared synthetically by using
the same pair of diastereomeric T
PS
A phosphoramidite
monomers), as well as those modified at positions P3
and P7, were evaluated. The latter two pairs of diaster-
0 5 10 15 20
0
20
40
60
80
100
Time [min]
Time [min]
Degradation of substrate [%]
0 50 100 150 200 250
0
25
50
75
100
Degradation of substrate [%]
0 4 10 20 30 45 60 90 120 150 180 210 240 min
0 0.16 0.5 1.0 2.0 4.0 6.0 8.0 10 20 30 min
A
B
C

D
Fig. 4. Comparison of the Mg
2+
-dependent activity of the unmodi-
fied deoxyribozyme 10–23 with that of thio-substituted deoxyribo-
zyme R
P
-P5 in the presence of 3 mM MgCl
2
. Time course of
cleavage reaction of a chimeric DNAÆRNA oligonucleotide by the
unmodified (A, B) and R
P
-P5 (C, D) deoxyribozymes.
Metal-binding site in deoxyribozyme 10–23 B. Nawrot et al.
1066
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Journal compilation ª 2007 FEBS
eomeric deoxyribozymes were prepared from diastereo-
meric R
P
dimers and S
P
dimers, G
PS
C [37]. We chose
this sequence following Taira and coworkers’ sugges-
tion that the proR
P
phosphate at position P7 (between

G
6
and C
7
) might be important for the catalytic activ-
ity of deoxyribozyme 10–23 (unpublished results). The
stereodefined deoxyribozymes (R
P
and S
P
at positions
P3, P7 and P9) were characterized in cleavage reac-
tions similar to those described above. The kinetic
parameters calculated for these reactions are listed in
Table 2 and shown in Fig. 5. Interestingly, in these
experiments we found higher thio effects and rescue
effects (10 and 115, respectively) for the R
P
-PS deoxy-
ribozyme P9, and a lack of these effects for its S
P
counterpart, which implies direct involvement in the
metal ion coordination of the proR
P
, but not proS
P
,
oxygen at position P9. The k
rel
values for nonbridging

phosphate oxygens at positions P3 and P7 reach sim-
ilar values, indicating a lack of direct coordination of
a metal cation to the proR
P
and proS
P
oxygen atoms
at these positions. These findings further confirm our
previous data obtained for the mixtures of diastereo-
mers of PS deoxyribozymes.
We compared our data with those published for
hammerhead ribozymes containing site-specific PS
modifications at either the proR
P
or proS
P
positions
[17]. Single-turnover relative rates of RNA cleavage,
determined at 10 mm Mg
2+
, were reduced three-fold
for R
P
-PS isomers at positions A
13
and A
14
, and S
P
-PS

isomers at positions A
6
and U
16.1
, 10-fold for the R
P
isomer at position A
9
, and 1000-fold for the R
P
isomer
at position U
1.1
, relative to the reactions performed by
the hammerhead enzyme. In the analogous reactions
performed in the presence of 10 mm Mn
2+
, k
rel
values
for R
P
-PS isomers at positions A
9
and U
1.1
increased
two-fold and 10-fold, respectively [17]. Thus, the thio
and rescue effect values observed in our studies for PS
deoxyribozymes were much stronger than those

observed for hammerhead constructs, except for the
k
rel
value determined in the presence of Mg
2+
for the
R
P
-PS isomer at position U
1.1
of the hammerhead
ribozyme.
Mutational analysis of nucleoside in position 6
of the catalytic core
We were interested in whether there are any other lig-
ands in the 10–23 catalytic core that might be directly
involved in stabilization of the catalytically active
architecture of the deoxyribozyme. As has already
been proven, the hammerhead ribozyme metal-binding
site utilizes both nonbridging oxygen atoms of the A
9
phosphate as well as nitrogen N7 of the subsequent
guanosine unit G
10.1
[38]. We were interested in deter-
mining whether the nucleotide residue following the A
5
unit in deoxyribozyme 10–23 plays any role in cata-
lysis. Although the exact metal-binding site of deoxyri-
bozyme 10–23 is not yet known, it has already been

suggested by Kurreck and coworkers that A
5
and G
6
residues within the catalytic core could be directly
involved in metal ion binding [11,13]. To characterize
the functional role of the oxygen moiety at C6 of G
6
,
we replaced this guanosine with its analogs, s
6
G and
AP nucleoside (Fig. 6), creating two analogs of the
DNA enzyme, s
6
G-zyme and AP-zyme, respectively
(Table 3). The k
rel
values observed for these enzymes
Table 2. Single-turnover rate constants for stereodefined thio-deoxyribozyme-mediated reactions in the presence of Mg
2+
and Mn
2+
.
Entry
DNAzyme
abbreviation ⁄ PS
position
a
k

Mg
obs
(min
)1
)
b
k
Mg
rel
d
Thio effect k
Mn
obs
(min
)1
)
c
k
Mn
rel
e
k
Mn
obs
⁄ k
Mg
obs
f
(rescue effect)
1 Unmodified 0.27 ± 0.028 1 1 8.0 ± 0.42 1 30

4a R
P
-P3 0.30 ± 0.015 1.1 0.90 9.1 ± 0.57 1.1 30
4b S
P
-P3 0.33 ± 0.028 1.2 0.83 7.5 ± 0.35 0.94 23
6a R
P
-P5 0.0077 ± 00078 0.029 34 0.56 ± 0.042 0.070 73
6b S
P
-P5 0.013 ± 0.0014 0.048 21 1.4 ± 0.14 0.18 108
8a R
P
-P7 0.29 ± 0.003 1.1 0.91 9.4 ± 0.28 1.2 32
8b S
P
-P7 0.28 ± 0.028 1.04 0.96 8.6 ± 0.50 1.1 31
10a R
P
-P9 0.026 ± 0.0007 0.096 10 3.0 ± 0.21 0.38 115
10b S
P
-P9 0.12 ± 0.005 0.44 2.3 0.98 ± 0.021 0.12 8.2
a
R
P
and S
P
are absolute configurations at the P-chiral center at a given PS linkage.

b, c
All RNA cleavage reactions were performed in
20 m
M Tris ⁄ HCl (pH 7.5), containing 100 mM NaCl and
b
3mM Mg
2+
or
c
3mM Mn
2+
under single-turnover conditions with 0.1 lM 5¢-end
32
P-
labeled substrate and 10 l
M deoxyribozyme, at 37 °C. Values of k
obs
for nonsubstituted and thio-substituted deoxyribozyme reactions repre-
sent mean values of four independent experiments, and errors indicate deviations between individual experiments.
d
k
Mg
rel
ratio of the k
obs
values for the modified and unmodified deoxyribozymes in the presence of Mg
2+
.
e
k

Mn
rel
ratio of the k
obs
values for the modified and unmodi-
fied deoxyribozymes in the presence of Mn
2+
.
f
The values of the rescue effect were calculated from k
Mn
obs
⁄ k
Mg
obs
.
B. Nawrot et al. Metal-binding site in deoxyribozyme 10–23
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Journal compilation ª 2007 FEBS
1067
(shown in Table 3) demonstrate that the stimulation of
the catalytic activity in the presence of Mn
2+
was sim-
ilar for unmodified and s
6
G-substituted enzymes. The
observed thio effect is about 20, and the rescue effect
is 28, implying that the oxygen atom of the carbonyl
moiety serves as a metal ion ligand. In contrast to the

inosine substitution [11], exchange of the G
6
base with
AP nucleoside resulted in complete loss of catalytic
activity, independent of the metal ion (no substrate
cleavage over 8 h; Table 3). These findings clearly indi-
cate that the oxygen at C6 is essential for the catalytic
activity of deoxyribozyme 10–23, whereas the exo
amino group of G
6
is not of functional importance.
In addition, we extended our mutational analysis to
the nucleoside at position 6 by the replacement of G
6
with a 7-deaza-dG unit. This substitution resulted in a
104-fold loss of activity of the DN
7
-zyme in the pres-
ence of Mg
2+
, suggesting that the N7 nitrogen partici-
pates in the formation of a functionally important
intramolecular hydrogen bond within the deoxyribo-
zyme 10–23 catalytic core. The k
obs
for this enzyme
increased by almost three orders of magnitude upon
addition of Mn
2+
, and was about 30-fold greater than

that for the unmodified reference (Table 3). We do not
offer any rational explanation for the nature of the
extremely high k
Mn
obs
⁄ k
Mg
obs
value. One can only speculate
that this  1000 rescue value for the 7-deazaguanosine-
modified enzyme may result from conformational rear-
rangement of this modified 10–23 core in the presence
of the soft metal ion, involving hydrogen bond pat-
terns within the catalytic loop.
Implications and Conclusions
The present results support the idea that phosphate
oxygens of the catalytic core of deoxyribozyme 10–23
participate in stabilization of the catalytically active
conformation. Using sulfur-modified deoxyribozymes,
we identified phosphate groups important for catalysis.
We found that the metal-binding site of deoxyribo-
zyme 10–23 involves both nonbridging oxygens of the
P5 phosphate of adenosine at position 5, and the oxy-
gen atom of the 6-carbonyl group of the subsequent
nucleoside (G
6
). Our model of the metal-binding site
in the catalytic core of deoxyribozyme 10–23 includes
the interactions of divalent cations with both the pro-
R

P
and proS
P
oxygens of P5, and an interaction with
the oxygen ligand at C6 of the subsequent guanosine
nucleotide (Fig. 7). One can argue that in this model
the distances between the oxygen ligands of P5 and the
oxygen of G
6
are too large to be spanned by a single
metal ion. However, it is possible that the architecture
of the active conformation of the catalytic core allows
for such interactions, or that more than one metal ion
is involved in catalysis. Contributions of other ligands
cannot be excluded, and the first candidate is the
proR
P
oxygen of phosphate P9, between the T
8
and A
9
nucleosides (Table 2). It is also possible that other
functional groups of the catalytic core serve as metal
ion ligands, because, as we have already suggested,
there are at least seven more nonbridging phosphate
oxygens, at positions P2, P4, P9, P10, P11, P12 and
P13, which exhibit remarkable thio and rescue effects.
Besides the oxygen ligands of the internucleotide
bonds, some other functional groups, as indicated in
other studies [11], may form intraloop hydrogen bonds

or coordinate to metal ion(s) directly or by water
bridges.
In conclusion, the reported data, along with results
obtained by systematic site-directed PS substitutions,
enabled the proposal of a model for the metal-binding
site in the catalytic core of deoxyribozyme 10–23. In
P3
P
R
P3
P
S
P5
P
R
P5
P
S
P7
P
R
P
7
P
S
P9
P
R
P
9

P
S
P
3
P
R
P
3
P
S
P
5
P
R
P
5
P
S
P
7
P
R
P
7
P
S
P
9
P
R

P
9
P
S
unmodified
unmodified
0.00
0.25
0.50
0.75
1.00
1.25
0.00
0.25
0.50
0.75
1.00
1.25
1.50A
B
k
rel
k
rel
Fig. 5. Comparison of the relative rates of cleavage (k
rel
) of PS-ster-
eodefined thio-deoxyribozymes 10–23 in the presence of 3 m
M
MgCl

2
(A) and 3 mM MnCl
2
(B).
Metal-binding site in deoxyribozyme 10–23 B. Nawrot et al.
1068
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Journal compilation ª 2007 FEBS
this model, the plausible ligands for metal coordina-
tion are the proR
P
and proS
P
oxygen atoms of the P5
phosphate, and the proR
P
oxygen at position P9, as
well as the carbonyl oxygen of the guanosine unit at
position 6 of the 10–23 catalytic core. In addition, sev-
eral other phosphate oxygens and nucleobase func-
tional groups can serve as metal-binding ligands
and ⁄ or hydrogen bond acceptors within the catalytic
core, but no detailed information is yet available.
Therefore, further experiments are required to identify
possible metal-binding ligands and to study the struc-
ture of deoxyribozyme 10–23 at the atomic level, either
by molecular modeling or by solution of the crystal
structure.
In addition, our observations that nonbridged oxy-
gens at phosphates at positions P3, P6, P7, P14 and

P15 could be replaced by a sulfur without substantial
loss of activity, and that the introduction of the PS
bond at the P1 and P8 sites stimulated catalytic activ-
ity, provided us wi th a starting point for the creation
of variants of deoxyribozyme 10–23 with not only
improved catalytic effectiveness but also better stability
against cellular endonucleases (such studies are pres-
ently being carried out in our laboratory).
Fig. 6. Chemical structures of the various nucleotide analogs employed in the current study.
Table 3. Single-turnover kinetics of cleavage reaction mediated by the deoxyribozyme 10–23 modified at position 6 of the catalytic core. ND,
not determined.
Deoxyribozyme Substitution k
Mg
obs
(min
)1
)
a
k
Mg
rel
c
k
Mn
obs
(min
)1
)
b
k

Mn
rel
d
k
Mn
obs
⁄ k
Mg
obs
e
Unmodified (WT) None 0.27 ± 0.028 1 8.0 ± 0.42 1 30
G
6
fi adenosine
f
ND – – – –
G
6
fi inosine
f
As WT – – – –
s
6
G-zyme G
6
fi 6-thio-dG 0.013 ± 0.0028 0.048 0.37 ± 0.035 0.046 28
AP-zyme G
6
fi 2-aminopurine nucleoside ND – ND – –
DN

7
-zyme G
6
fi 7-deaza-dG 0.0026 ± 0.00028 0.0096 2.6 ± 0.28 0.33 1000
a, b
All RNA cleavage reactions were performed in 20 mM Tris ⁄ HCl (pH 7.5), containing 100 mM NaCl and
a
3mM Mg
2+
or
b
3mM Mn
2+
,
under single-turnover conditions with 0.1 l
M 5¢-end
32
P-labeled substrate and 10 lM deoxyribozyme, at 37 °C. Values of k
obs
for unmodified
and mutated deoxyribozyme reactions represent mean values of three independent experiments, and errors indicate deviations between
individual experiments.
c
k
Mg
rel
¼ ratio of the k
obs
values of modified deoxyribozyme to unmodified deoxyribozyme, in the presence of Mg
2+

.
d
k
Mn
rel
¼ ratio of the k
obs
values of modified deoxyribozyme to unmodified deoxyribozyme, in the presence of Mn
2+
.
e
The values of the res-
cue effect were calculated from k
Mn
obs
⁄ k
Mg
obs
f
.
B. Nawrot et al. Metal-binding site in deoxyribozyme 10–23
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Journal compilation ª 2007 FEBS
1069
Experimental procedures
Deoxyribozymes and substrate
The unmodified deoxyribozyme and its substrate oligonu-
cleotide (Fig. 1) were synthesized using an ABI 394 DNA
synthesizer (Applied Biosystems, Inc., Foster City, CA) and
commercially available phosphoramidite monomers (Glen

Research, Sterling, VA). Base-modified deoxyribozymes
(AP-zyme and DN
7
-zyme) were synthesized routinely using
commercially available monomers (2-aminopurine 2¢-deoxy-
ribonucleoside and 7-deaza-2¢-deoxyguanosine phosphoram-
idite monomers; Glen Research). The s
6
G-zyme was
synthesized routinely using protected 6-thio-2¢-deoxyguano-
sine phosphoramidite prepared according to the published
procedure [39] and commercially available UltraMILD
phosphoramidites (Glen Research). The deprotection step
was performed as described previously [39]. Oligomers were
purified by RP-HPLC (ODS Hypersil column, Alltech
Associates, Inc., Deerfield, IL) followed by preparative elec-
trophoresis in a 20% polyacrylamide gel containing 7 m
urea. PS-stereodefined oligonucleotides were synthesized by
incorporation of PS dinucleoside building blocks into the
oligonucleotide chain according to our recently described
procedure [37]. The structure and purity of the PS oligonu-
cleotides were confirmed by MALDI-TOF MS and RP-
HPLC, as well as by PAGE. The absolute configuration at
the chiral phosphorus center was assigned enzymatically
with stereospecific nP1 (Sigma-Aldrich, St Louis, MO) and
svPDE (Boehringer Mannheim, Germany) nucleases.
Oligonucleotide labeling
The substrate oligonucleotide of an RNAÆDNA chimeric
sequence (Fig. 1) was 5¢-labeled with [c-
32

P]ATP and
T4 polynucleotide kinase (Amersham, Little Chalfont,
UK). A mixture containing 10 mm Tris ⁄ HCl (pH 8.5),
10 mm MgCl
2
,7mm 2-mercaptoethanol, 30 lm (0.1 A
260
unit) oligonucleotide, 1 lL (10 lCi) of [c-
32
P]ATP and T4
polynucleotide kinase (6 units) was incubated for 30 min at
37 °C, and then heat denatured and stored at ) 20 °C.
Enzymatic assay
The substrate cleavage reactions were performed under
single-turnover conditions with the DNA enzyme in 100-
fold excess over the substrate. The 5¢-labeled substrate
(0.1 lm) was incubated with deoxyribozyme (10 lm)in
20 mm Tris ⁄ HCl (pH 7.5) containing 100 mm NaCl, and
3mm MgCl
2
or 3 mm MnCl
2
,at37°C. After various
time intervals, 10 lL aliquots were withdrawn, and the
cleavage reaction was stopped by addition of 50 mm
EDTA and by cooling on ice. Before electrophoresis, 8 lL
of formamide containing 0.03% bromophenol blue and
0.03% xylene cyanol was added to each sample, and the
cleavage products were separated from noncleaved sub-
strate by electrophoresis in 20% polyacrylamide gel under

denaturing conditions. The amount of product was deter-
mined by autoradiography with PhosphorImager (Molecu-
lar Dynamics, Sunnyvale, CA), and the observed rate
constants (k
obs
) were calculated from a pseudo-first-order
reaction equation, Y ¼ [EP] [1 ) exp(– k
obs
t)], where Y is
the percentage of the cleaved product at time t, and EP is
the endpoint, showing the percentage of cleaved product
at the plateau of reaction. Reactions were carried out near
to completion. Endpoints between 80% and 90% were
used in kinetic analyses. In all cases, good fits to the
appropriate kinetic model were obtained, with R
2
> 0.96.
The k
obs
values for cleavage of the substrate by modified
deoxyribozymes represent mean values of at least three
independent experiments, and errors indicate deviations
between individual experiments. The error bars in Figs 2,
3 and 4 were calculated in the following manner. The rel-
ative k-values (k
rel
) were calculated as a ratio of the k
obs
values for the modified and unmodified enzyme. The
upper limits for k

rel
were calculated as a ratio of
(k
obs
M+SD
M
) ⁄ (k
obs
U ) SD
U
), where k
obs
M and
SD
M
, and k
obs
U and SD
U
, are the mean reaction rates
and SD errors for the modified and unmodified enzymes,
respectively. Similarly, lower limits for k
rel
were calculated
from the equation (k
obs
M ) SD
M
) ⁄ (k
obs

U+SD
U
).
To ensure that the substrate was completely saturated
by the deoxyribozyme, the rate constants at concentrations
of the deoxyribozyme increasing from 1 to 30 lm were
measured (data not shown). The rate of cleavage was inde-
pendent of the concentration of the deoxyribozyme above
10 lm, indicating that the chemical step within the deoxy-
ribozyme-assisted substrate cleavage was a rate-limiting
step.
The ‘thio effect’ was calculated as a ratio of k
Mg
obs
of
the reference unmodified enzyme to k
Mg
obs
of the particular
N
N
N
NH
O
NH
2
O
P
O
O

O
O
O
O
P
O
O
A
O
O
O
T
O
Mg
2+
O
O
T
O
O
P
O
O O
A
O
?
?
pro-R
P
P5

P9
G
6
A
5
pro-R
P
pro-S
P
T
4
A
9
T
8
Fig. 7. Model for the metal-binding site in the catalytic core of
deoxyribozyme 10–23. No clear evidence is given concerning whe-
ther these coordinations are to the same or different magnesium
ions.
Metal-binding site in deoxyribozyme 10–23 B. Nawrot et al.
1070
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Journal compilation ª 2007 FEBS
modified enzyme, and the ‘rescue effect’ was calculated as a
ratio of k
Mn
obs
to k
Mg
obs

of modified enzyme.
Acknowledgements
The authors thank Professor J. Connolly of Glasgow
University for critical reading of the manuscript and
valuable suggestions. This work was supported by
the Ministry of Science and Higher Education
(Poland) through the Centre of Molecular and
Macromolecular Studies, Polish Academy of Sciences,
under Decision 70⁄ E-63 ⁄ SN-014 ⁄ 2006 and ICGEB
project CRP ⁄ POL04-01.
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