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 Te
Trang 1catalytic 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 Mg2+
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: bnawrot@bio.cbmm.lodz.pl
*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
3 mm Mg2+or 3 mm Mn2+ A metal-specificity switch approach permitted the identification of nonbridging phosphate oxygens (proRP or proSP) 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
Mg2+, and this was reversed in the presence of Mn2+ Further experiments with thio-deoxyribozymes of stereodefined P-chirality suggested direct involvement of both oxygens of the P5 phosphate and the proRPoxygen at P9 in the metal ion coordination In addition, it was found that the oxygen atom at C6 of G6contributes 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.
Trang 2The 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 proRPand proSPpositions 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’ Mg2+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 Mn2+, Zn2+, or Cd2+, 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
Mg2+and thiophilic Mn2+ 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 G6positioned 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 (s6G) 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 G6for 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
Mn2+-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 (G1) (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 RPand SP (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 MgCl2 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
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.
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Trang 3esis in 20% polyacrylamide gels The observed rate
constants (kobs) were calculated according to the
equa-tion given in Experimental procedures, and compared
with the rate constant of the unmodified
deoxyribo-zyme (krel)
The data presented in Table 1 and Fig 2A indicate
that thio-substitutions at phosphates P2, P4 and P9–13
lowered the krel values by c 50% The replacement of
3 mm Mg2+with 3 mm Mn2+resulted in a restoration
of the activity to the level of the kMnobs of the unmodified
enzyme (Table 1, Fig 2B), implying a possible
coordi-nation of the metal cation to one of the two (the
proRP or the proSP) oxygen atoms However, it should
be noted that the kobs values were c 30-fold higher in
the presence of Mn2+compared with Mg2+(Table 1)
A similar high rate of the cleavage reaction in the
pres-ence of Mn2+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 Mn2+ may result from the fact that Mn2+, as a
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 proRP and proSP
positions hindered direct contact with metal ions Interestingly, this PS enzyme regained its activity in the presence of Mn2+, with the kMnobs value being 176-fold higher than the kMgobs 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 Mn2+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¢ fi 3¢
sequence of the catalytic core a kMgobs(min)1) b kMgrelc Thio effect kMnobs(min)1) d kMnrele
k Mn obs ⁄ kMgobs (rescue effect)
18 P1 ⁄ P8 d(APSGGCTAGCPSTACAACGAT) 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 RPand
SPdiastereomers) in the selected positions of the catalytic core marked from P1 (phosphate bond between A0and G1) to P16 (phosphate bond between A 15 and T 16 ).b, dRNA cleavage reactions were performed in 20 m M Tris ⁄ HCl (pH 7.5), containing 100 m M NaCl,b3 m M
Mg 2+ or d 3 m M Mn 2+ under single-turnover conditions with 0.1 l M 5¢-end 32 P-labeled substrate and 10 l M deoxyribozyme, at 37 C.
c kMgrel ¼ the ratio of the k obs values of modified deoxyribozyme to unmodified deoxyribozyme, in the presence of Mg 2+ e kMnrel ¼ the ratio
of the k obs values of modified deoxyribozyme to unmodified deoxyribozyme, in the presence of Mn2+. fReactions were performed in
20 m M Tris ⁄ HCl (pH 7.5), containing 100 m M NaCl and 0.06 m M Mn 2+ under single-turnover conditions with 0.1 l M 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 kobsof 0.12 ± 0.014 min)1for reaction of the unmodified deoxyribozyme in 0.06 m M Mn 2+
Trang 4the bulky sulfur atom, which could influence the
geom-etry of metal ion interactions, or the geomgeom-etry 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 Mg2+ and
Mn2+ These data constitute strong evidence against
direct coordination of a metal cation to both the
proRP and proSP 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 Mg2+as well as Mn2+ (Table 1, Fig 3) The kobs and krel values for these enzymes were calculated from the reactions performed
in 3 mm Mg2+or 0.06 mm Mn2+ The concentration of
Mn2+ was reduced 50-fold, because reactions per-formed in the presence of 3 mm Mn2+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 Mg2+and in the presence of Mn2+, causing a 30–60% increase in krel, 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 Mg2+, compared to P1 substitution itself, but in the presence of Mn2+ the krel for the P1⁄ P8 enzyme was over two-fold higher than the krelfor 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 kMgobsand kMnobs values were nearly iden-tical, despite a 50-fold difference in the concentration
of metal ions present in the catalysis reaction, and the
kMnobs value for this mutant enzyme was three-fold higher than the kMnobs 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 P11P12P13 P14P15P16
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
krel
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
krel
B
Fig 2 Comparison of the relative rates of cleavage (krel) of
thio-substituted deoxyribozymes 10–23 in the presence of 3 m M MgCl 2
(A) and 3 m M MnCl2(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+
krel
Fig 3 Comparison of the relative rates of cleavage (k rel ) of thio-substituted deoxyribozymes 10–23 in the presence of 3 m M MgCl2 (white bars) and 0.06 m M MnCl2(gray bars).
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Trang 5PS-mutant enzyme is c 50-fold more active in the
presence of Mn2+than in the presence of Mg2+, and
its kobs value in the presence of 3 mm Mn2+ 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 Mn2+than the unmodified reference in
the presence of Mg2+ 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 Mn2+, 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 RP
-PS or SP-PS linkages at that position and measured
the rate of RNA cleavage under analogous conditions
in the presence of 3 mm Mg2+(Fig 4) We found that
RP-PS and SP-PS substitutions at position P5 reduced
kMgrel by a factor of 34 and 21, respectively (Table 2,
Fig 5A) As kobsvalues 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
Mg2+ interactions with nonbridging phosphate
oxy-gens In 3 mm Mn2+ buffer, the RP-PS and SP-PS
deoxyribozyme P5-mediated cleavage activity was
significantly enhanced (73-fold and 108-fold increase
of kobs 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
Mg2+and each of the P-chiral diastereomeric
deoxyri-bozymes suggests that Mn2+ 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 TPSA phosphoramidite monomers), as well as those modified at positions P3 and P7, were evaluated The latter two pairs of
0 20 40 60 80 100
Time [min]
Time [min]
0 25 50 75 100
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 RP-P5 in the presence of 3 m M MgCl2 Time course of cleavage reaction of a chimeric DNAÆRNA oligonucleotide by the unmodified (A, B) and R P -P5 (C, D) deoxyribozymes.
Trang 6eomeric deoxyribozymes were prepared from
diastereo-meric RP dimers and SP dimers, GPSC [37] We chose
this sequence following Taira and coworkers’
sugges-tion that the proRP phosphate at position P7 (between
G6 and C7) might be important for the catalytic
activ-ity of deoxyribozyme 10–23 (unpublished results) The
stereodefined deoxyribozymes (RP and SP 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 RP-PS
deoxy-ribozyme P9, and a lack of these effects for its SP
counterpart, which implies direct involvement in the
metal ion coordination of the proRP, but not proSP,
oxygen at position P9 The krelvalues 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 proRP and proSP 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 proRP or proSP positions
[17] Single-turnover relative rates of RNA cleavage,
determined at 10 mm Mg2+, were reduced three-fold
for RP-PS isomers at positions A13and A14, and SP-PS
isomers at positions A6 and U16.1, 10-fold for the RP
isomer at position A9, and 1000-fold for the RPisomer
at position U1.1, relative to the reactions performed by
the hammerhead enzyme In the analogous reactions
performed in the presence of 10 mm Mn2+, krelvalues for RP-PS isomers at positions A9 and U1.1 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
krel value determined in the presence of Mg2+for the
RP-PS isomer at position U1.1 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 A9 phosphate as well as nitrogen N7 of the subsequent guanosine unit G10.1[38] We were interested in deter-mining whether the nucleotide residue following the A5 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 A5 and G6 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 G6,
we replaced this guanosine with its analogs, s6G and
AP nucleoside (Fig 6), creating two analogs of the DNA enzyme, s6G-zyme and AP-zyme, respectively (Table 3) The krel 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 kMgobs(min)1) b kMgreld Thio effect k Mn
obs (min)1) c k Mn
rel e
kMnobs⁄ k Mg obs f
(rescue effect)
a RPand SP 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 m M NaCl and b 3 m M Mg 2+ or c 3 m M Mn 2+ under single-turnover conditions with 0.1 l M 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 kMgrel ratio of the kobs values for the modified and unmodified deoxyribozymes in the presence of Mg 2+ e kMnrel ratio of the kobsvalues for the modified and unmodi-fied deoxyribozymes in the presence of Mn2+.fThe values of the rescue effect were calculated from kMnobs⁄ k Mg
obs
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Trang 7(shown in Table 3) demonstrate that the stimulation of
the catalytic activity in the presence of Mn2+was
sim-ilar for unmodified and s6G-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 G6 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 G6is not of functional importance
In addition, we extended our mutational analysis to
the nucleoside at position 6 by the replacement of G6
with a 7-deaza-dG unit This substitution resulted in a
104-fold loss of activity of the DN7-zyme in the
pres-ence of Mg2+, 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 kobs for this enzyme increased by almost three orders of magnitude upon addition of Mn2+, 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 kMnobs⁄ kMgobs 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 (G6) 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-RP and proSP 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 G6 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 proRPoxygen of phosphate P9, between the T8and A9 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
P5 P
P
P S
P R
P
5 P R
P
7 P R
P
9 P R
P S
un m
odif
ied
un m
odif
ied
0.00
0.25
0.50
0.75
1.00
1.25
0.00
0.25
0.50
0.75
1.00
1.25
1.50
A
B
krel
krel
Fig 5 Comparison of the relative rates of cleavage (krel) of
PS-ster-eodefined thio-deoxyribozymes 10–23 in the presence of 3 m M
MgCl 2 (A) and 3 m M MnCl 2 (B).
Trang 8this model, the plausible ligands for metal
coordina-tion are the proRP and proSP oxygen atoms of the P5
phosphate, and the proRP 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 with 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 kMgobs(min)1) a kMgrelc kMnobs(min)1) b kMnreld kMnobs⁄ kMgobse
a, b All RNA cleavage reactions were performed in 20 m M Tris ⁄ HCl (pH 7.5), containing 100 m M NaCl and a 3 m M Mg 2+ or b 3 m M Mn 2+ , under single-turnover conditions with 0.1 l M 5¢-end 32
P-labeled substrate and 10 l M 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 kMgrel ¼ ratio of the k obs values of modified deoxyribozyme to unmodified deoxyribozyme, in the presence of Mg 2+
d
kMnrel ¼ ratio of the k obs values of modified deoxyribozyme to unmodified deoxyribozyme, in the presence of Mn2+.eThe values of the res-cue effect were calculated from k Mn
obs ⁄ k Mg obs
f
FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences
Trang 9Experimental 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 DN7-zyme) were synthesized routinely using
commercially available monomers (2-aminopurine
2¢-deoxy-ribonucleoside and 7-deaza-2¢-deoxyguanosine
phosphoram-idite monomers; Glen Research) The s6G-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-32P]ATP and
T4 polynucleotide kinase (Amersham, Little Chalfont,
UK) A mixture containing 10 mm Tris⁄ HCl (pH 8.5),
10 mm MgCl2, 7 mm 2-mercaptoethanol, 30 lm (0.1 A260 unit) oligonucleotide, 1 lL (10 lCi) of [c-32P]ATP and T4 polynucleotide kinase (6 units) was incubated for 30 min at
37C, 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
3 mm MgCl2 or 3 mm MnCl2, at 37C 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 (kobs) were calculated from a pseudo-first-order reaction equation, Y¼ [EP] [1) exp(– kobst)], 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 R2> 0.96 The kobs 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 (krel) were calculated as a ratio of the kobs values for the modified and unmodified enzyme The upper limits for krel were calculated as a ratio of (kobsM + SDM)⁄ (kobsU) SDU), where kobsM and
SDM, and kobsU and SDU, are the mean reaction rates and SD errors for the modified and unmodified enzymes, respectively Similarly, lower limits for krelwere calculated from the equation (kobsM) SDM)⁄ (kobsU + SDU)
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 kMgobs of the reference unmodified enzyme to kMgobs of the particular
N
NH O
NH2 O
P
O
O O
O O
O
P
O
O A
O
O
O T
O
O T O O P
O
?
?
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.
Trang 10modified enzyme, and the ‘rescue effect’ was calculated as a
ratio of kMnobsto kMgobsof 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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FEBS Journal 274 (2007) 1062–1072 ª 2007 Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences