8-17 DNAzyme modified with purine analogs in its catalytic core: The conservation of the five-membered moieties of purine residues

Article abstract of DOI:10.1016/j.bmcl.2012.05.044

8-17 DNAzyme is characterized by its recurrence in different in vitro selections and versatile cleavage sites, leading to extensive studies on its structural properties and applications. We evaluated the purine residues (A6, G7, G11, A12, G14, and A15) in the catalytic core of 8-17 DNAzyme of their five-membered ring moiety with purine analogs 1-5 to have an insight into the conservation of the residues at the level of functional groups. The 7-nitrogen atom in the AGC loop was demonstrated to be strictly conserved for the cleavage reaction. But such modifications exerted favorable effect at G11 of the base-pair stem and A12 in the single-strand loop, directing toward more efficient DNAzymes. Even the most conserved G14 could tolerate such modifications. These results demonstrated that chemical modification on the functional groups is a feasible approach to gain an insight into the structural requirement in the catalytic reaction of DNAzymes. It also provided modification sites for introduction of signaling molecules used for mechanistic and folding studies of 8-17 DNAzyme.

Full text of DOI:10.1016/j.bmcl.2012.05.044

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4238–4241
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Bioorganic & Medicinal Chemistry Letters
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8–17 DNAzyme modified with purine analogs in its catalytic core: The
conservation of the five-membered moieties of purine residues
Wei Rong a, Liang Xu b, Yang Liu b, Jianping Yu a, , Ying Zhou a, Keliang Liu b, Junlin He b,
⇑
⇑
a College of Life Science, Guizhou University, Guiyang 550025, China
b Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
a r t i c l e i n f o
a b s t r a c t
Article history:
8–17 DNAzyme is characterized by its recurrence in different in vitro selections and versatile cleavage
sites, leading to extensive studies on its structural properties and applications. We evaluated the purine
residues (A6, G7, G11, A12, G14, and A15) in the catalytic core of 8–17 DNAzyme of their five-membered
ring moiety with purine analogs 1–5 to have an insight into the conservation of the residues at the level
of functional groups. The 7-nitrogen atom in the AGC loop was demonstrated to be strictly conserved for
the cleavage reaction. But such modifications exerted favorable effect at G11 of the base-pair stem and
A12 in the single-strand loop, directing toward more efficient DNAzymes. Even the most conserved
G14 could tolerate such modifications. These results demonstrated that chemical modification on the
functional groups is a feasible approach to gain an insight into the structural requirement in the catalytic
reaction of DNAzymes. It also provided modification sites for introduction of signaling molecules used for
mechanistic and folding studies of 8–17 DNAzyme.
Received 10 April 2012
Revised 8 May 2012
Accepted 9 May 2012
Available online 17 May 2012
Keywords:
8–17 DNAzyme
Purine analogs
Catalytic cleavage
Chemical modification
7-Nitrogen atom
Ó 2012 Elsevier Ltd. All rights reserved.
Since the appearance of small artificial DNAzymes 10–23 and
8–17 and the development of their wide-broad applications, many
new DNAzyme entities have also been selected under specific con-
ditions.1–4 Interestingly, numerous 8–17 DNAzyme variants (e.g.,
17E and Mg5) have been selected independently from different
in vitro selection experiments.5–9 Their catalytic cleavage activity
and specific ion-dependence has led to a new class of metal biosen-
sors.10–12 Such recurrence of 8–17 motifs and versatile cleavage
sites13 initiated many studies on its catalytic mechanism and fold-
ing,14 either with residue replacement,15 spectroscopic meth-
ods,16,17 or contact photo-crosslinking experiments.18,19 In the
catalytic core, all of them share a highly conserved AGC triloop, a
Watson–Crick base-pair stem and the single-strand loop of four
or five residues (Fig. 1).13 Each residue was supposed to offer base
stacking and several functional groups to form a complex hydro-
gen-bond net responsible for the active conformation. Although
we could not yet conclude with a precise catalytic site or a role
of specific residues in the catalytic core of 8–17, extensive studies
are still being conducted to gain an insight about its structure and
mechanisms for a better design of metal biosensors and other
applications. Considering that the residues at each position in the
catalytic core is the result of repeated selection and the positive
role of base stacking, we started the chemical modification on spe-
cific functional groups around nucleobases in the catalytic core, in
3'-TCC TAG AT rG rA CCG AGG TA-5'
2.1
GGC TCC AT-3'
5'-AGG ATC TA T
A
3
G
14
C
C
C
G
A
G
G
8-17 DNAzyme
6
A
G
C
C
7
8
Figure 1. The 8–17 DNAzyme designed against
a DNA–RNA–DNA chimeric
substrate. The arrow showed the cleavage site between diribonucleotide rA-rG.
Bold letters indicated the most conserved purine residues.
order to have a better understanding of the conservation of nucle-
obases at the level of the functional groups and help the construc-
tion of more sensitive deoxyribozyme-based metal biosensors.
Of the three structural domains in the catalytic core of 8–17
DNAzyme, there are eight purine residues, including three most
conserved purine residues A6, G7, and G14, three guanine residues
(G5, G10, and G11) in the base-pair stem, and A12 and A15 in the
single-strand turn near the cleavage site. In this work, we present
the evaluation of the five-membered ring moiety of these adenine
and guanine residues except G5 and G10. The unnatural nucleoside
analogs 1–520–22 (Fig. 2) with no changes on the Watson–Crick
base-pairing functional groups were selected. By comparing with
parent 8–17 DNAzyme, the contribution of 7-N atom as well as
⇑
Corresponding authors. Tel.: +86 851 385 4677; fax: +86 851 385 6374 (J.Y.);
tel./fax: +86 10 6821 1656 (J.H.).
E-mail addresses: yujp666666@163.com (J. Yu), hejunlin@yahoo.com (J. He).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.044

W. Rong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4238–4241
4239
ment on the complexes between DNAzyme and its corresponding
full-DNA substrate 50-d(ATG GAG CCA GTA GAT CCT)-30 (D18, Ta-
ble 1) in the reaction buffer indicated that all the modifications
did not results in significant changes on thermal stabilities of the
complexes (Supplementary data). The DNAzyme–substrate com-
plex was formed in the reaction buffer. The cleavage activity of
8–17 and its analogs was determined under single-turnover condi-
tions in the presence of 20 mM Mg2+, with DNAzymes of 100-fold
more than the 32P-labeled substrate (Fig. 1).
O
N
O
N
NH2
HN
HN
N
N
N
N
N
N
H2N
HO
H2N
HO
N
O
O
O
HO
OH
OH
OH
1
2
3
O
N
NH2
In addition to its repeated appearance in 8–17 variants, AGC
loop has also been demonstrated to be highly conserved through
mutagenesis with canonical and non-canonical residues, their
functional groups, such as amino, keto, and 7-N atom are essential
for the catalytic activity. In our evaluation system for modified 8–
17, as shown in Table 2, the same phenomenon was also ob-
served.15 When the 7-N atom of G7 was deleted through the intro-
duction of compound 1 at G7 in 8-17DZ01, a decreased kobs was
obtained. Even a position shift of this nitrogen atom by compound
2 in 8-17DZ02 was not allowed, complete abolishment of cleavage
activity was observed. Similar effect was produced when the 7-N
atom of A6 was shifted to 8-position with compound 3 in 8-
17DZ03. These observations indicated that A6 and G7 were proba-
bly located very close to the catalytic center, all functional groups
around the two nucleobases might completely fold into the cata-
lytic conformation, either by forming hydrogen-bonding network
or positioning metal ions. In other words, the spacial distribution
of these functional groups has been best defined by A6 and G7 as
the repeated in vitro selection result. Further introduction of amino
group at this loop with compound 4 or 5 led to new interactions
accompanied by a complete loss of activity. This result further con-
firmed the absolute importance of the AGC loop in forming the cat-
alytic conformation at the level of functional groups.
NH2
NH2
HN
N
N
N
N
N
H2N
HO
N
O
O
HO
OH
OH
4
5
Figure 2. Purine analogs used for the modification on purine residues in the
catalytic core of 8–17 DNAzyme.
the five-membered ring moiety of purine residues was evaluated.
Their phosphoramidites were prepared as previously reported
(the preparation of phosphoramidite of 4 was presented in Supple-
mentary data). 8–17 DNAzyme was designed against the sequence
from the overlapping vpr and tat reading frames of HIV-1,23 and the
target sequence was synthesized as a chimeric DNA–RNA–DNA, in
which GA diribonucleotide was the cleavage site. 8–17 and its
modified analogs were synthesized with conventional solid-phase
synthesis and purified with denaturing PAGE (8% urea). All of them
were characterized by MALDI-TOF (Table 1). Thermal measure-
The three base-pair stem has been supposed to play a structural
role, offering stabilization for the catalytic conformation, based on
the fact that G–C and A–T base pairs are well-tolerated, even mis-
matched base pairs were found in 8–17 variants. Especially, this
stem could be replaced by T–Hg–T pair, with which mercuric ion
switches on the stem formation and subsequently the cleavage
reaction.24 Combining with fluorescent molecule labeling at the
recognition arms, such 8–17 variants have been used as the biosen-
sors for Hg2+. Based on these researches, the base pair in the stem
was hypothesized to be located inside the helix, and their func-
tional groups might not be involved in the catalytic conformation
directly. Indeed, removal of 7-N atom by compound 1 at G11 did
not produce significant effect on reaction rate (8-17DZ06,
kobs = 0.0077 0.0007 minꢀ1). However, when this 7-N was shifted
to 8-position by compound 2 at G11, about twofold increase in
reaction rate of 8-17DZ07 (kobs = 0.0131 0.0005 minꢀ1) was pro-
duced. This slight improvement indicated that the five-membered
Table 1
Characterization results of oligodeoxynucleotides by MALDI-TOF
a
DNAzymes
Sequences
MS (calcd)
9212.4
MS (found)
9212.1
8-17DZ00
agg atc taT CCG AGC CGG
ACG Agg ctc cat
agg atc taT CCG A1C CGG ACG
Agg ctc cat
agg atc taT CCG A2C CGG ACG
Agg ctc cat
agg atc taT CCG 3GC CGG ACG
Agg ctc cat
agg atc taT CCG A4C CGG ACG
Agg ctc cat
agg atc taT CCG 5GC CGG ACG
Agg ctc cat
agg atc taT CCG AGC CG1 ACG
Agg ctc cat
agg atc taT CCG AGC CG2 ACG
Agg ctc cat
agg atc taT CCG AGC CG4 ACG
Agg ctc cat
agg atc taT CCG AGC CGG 3CG
Agg ctc cat
agg atc taT CCG AGC CGG 5CG
Agg ctc cat
agg atc taT CCG AGC CGG AC1
Agg ctc cat
agg atc taT CCG AGC CGG AC2
Agg ctc cat
agg atc taT CCG AGC CGG AC4
Agg ctc cat
agg atc taT CCG AGC CGG
ACG 3gg ctc cat
agg atc taT CCG AGC CGG
ACG 5gg ctc cat
8-17DZ01
8-17DZ02
8-17DZ03
8-17DZ04
8-17DZ05
8-17DZ06
8-17DZ07
8-17DZ08
8-17DZ09
8-17DZ10
8-17DZ11
8-17DZ12
8-17DZ13
8-17DZ14
8-17DZ15
D18
9211.4
9212.4
9212.4
9269.2
9269.6
9211.4
9212.4
9269.6
9212.4
9269.6
9211.4
9212.4
9269.6
9212.4
9269.6
5521.0
9217.8
9212.1
9212.7
9266.7
9268.2
9215.8
9211.1
9268.7
9213.9
9268.4
9215.8
9213.1
9269.9
9216.5
9269.8
5520.9
Table 2
kobs of 8–17 DNAzyme and its modified analogs measured under single-turnover
conditionsa
No.
kobs (minꢀ1
)
No.
kobs (minꢀ1
)
8-17DZ00
8-17DZ01
8-17DZ02
8-17DZ03
8-17DZ04
8-17DZ05
8-17DZ06
8-17DZ07
0.0064 0.00055
8-17DZ08
8-17DZ09
8-17DZ10
8-17DZ11
8-17DZ12
8-17DZ13
8-17DZ14
8-17DZ15
0.0103 0.0010
0.0039 0.00036
0.0101 0.00105
0.0019 0.00017
0.0098 0.0013
0.0062 0.0005
0.0014 0.0001
<0.0001
0.0025 0.00006
NDb
ND
ND
ND
0.0077 0.0007
0.0131 0.0005
a
The cleavage reaction of DNAzyme (2 l
M) on the 32P-labeled substrate (20 nM)
atg gag cca gta gat cct
were conducted in the buffer (50 mM Tris–HCl, 20 mM Mg2+) at 37 °C (more details
a
The lowercase letters constitute both recognition arms, and the uppercase
letters represent the catalytic core of 8–17 DNAzyme.
see Supplementary data).
b
Not detected under present conditions.

4240
W. Rong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4238–4241
When G14 of 8–17 was further evaluated with compound 1 or 2, the
kobs of new DNAzymes 8-17DZ11 (kobs = 0.0019 0.00017 minꢀ1
)
and 8-17DZ12 (kobs = 0.0098 0.0013 minꢀ1) demonstrated that
7-N and 8-N atoms at G14 were helpful for the cleavage reaction.
Further perturbation with an extra amino group by compound 4
in this position could not offer more favorable effect on the reaction
(8-17DZ13, kobs = 0.0062 0.0005 minꢀ1). The cleavage pattern of
these three DNAzymes within 300 min was shown inFigure 3B. This
result told us that G14 is highly conserved as an integrate nucleo-
base, and its functional group distribution could be improved by
functional group modification.
For the adenine residues A12 and A15, mutations with com-
pound
3 resulted in loss of activity, as shown in Table 2
(kobs = 0.0039 0.00036 minꢀ1 for 8-17DZ09, and kobs = 0.0014
0.0001 minꢀ1 for 8-17DZ14, respectively), it indicated that their
7-N atom was involved in the catalytic reaction. More adverse effect
was observed for A15 when its 7-N was deleted. But the extra ami-
no group by compound 5 in these two positions resulted in opposite
effect. At A12, this extra amino group was beneficial for the reaction
(8-17DZ10, kobs = 0.0101 0.00105 minꢀ1), while it was deleterious
for A15 (8-17DZ15). The cleavage behavior of these DNAzymes
within 300 min was compared in Figure 3C.
Based on the residue replacement and modification of functional
groups on the catalytic core of 8–17 DNAzyme, we learnt that the
residue conservation is determined by both base stacking and the
interactions derived from functional groups. Chemical modifica-
tions on functional groups of specific nucleobases could give a more
detailed picture of the residue involvement in the cleavage reaction.
The two purine residues in the AGC loop was absolutely conserved
at the level of functional groups, while the five-membered moiety
of the conserved G14 could be modified with positive effect on
the cleavage behavior. Based on the effect of compound 4 at G11
and A12, these two positions were the possible sites for introduc-
tion of signaling molecules used in the research of mechanism
and folding of 8–17, through the 7-positioned amino group of com-
pound 4. By this way, favorable modifications could be selected on
the level of functional groups, either for more detailed understand-
ing of the structural requirement in the catalytic reaction of DNA-
zymes, or for more efficient versions of DNAzymes.
Acknowledgments
This research was supported by National Natural Science Foun-
dation of China 21072229 and Beijing Natural Science Foundation
7123223.
Figure 3. The effect of purine analogs incorporated into 8–17 DNAzyme on the
cleavage reactions: A, G11 replaced by 1 (8-17DZ06, d), 2 (8-17DZ07, N), 4 (8-
17DZ08, ꢀ), parent 8–17 (j); B, G14 replaced with 1 (8-17DZ11, s), 2 (8-17DZ12,
4), 4 (8-17DZ13, }); C, A12 replaced by 3 (8-17DZ09,q) and 5 (8-17DZ10,w), A15
replaced by 3 (8-17DZ14,5), and 5 (8-17DZ15,.), respectively.
Supplementary data
Supplementary data (the preparation of phosphoramidite of
compound 4 and Tm of the complexes between DNAzymes with
the corresponding full-DNA substrate) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2012.05.044.
moiety of G11 might be involved in the cleavage reaction by its 7-N
atom, and 8-N atom is a little more favorable for this positive effect
than 7-N atom. Further introduction of compound 4 remained al-
most the same rate increase. Their cleavage pattern within
300 min was shown in Figure 3A. The effect of compound 4 at
G11 and the extra amino group offers a potential spot for labeling
8–17 DNAzyme with signaling molecules in the studies of folding
of 8–17 DNAzyme.17
The third domain in 8–17 DNAzyme is the single-strand turn
close to the cleavage site, it differs from each other between its vari-
ants, ACGA in 8–17, TCGAA in 17E, and ACGAA in Mg5. C13G14 are
the most conserved residues, any substitution with other three
canonical residues led to deleterious effect on the cleavage activity.
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