Tài liệu Báo cáo khoa học: An allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities doc

An allosteric DNAzyme with dual RNA-cleaving and
DNA-cleaving activities
Dazhi Jiang*, Jiacui Xu*, Yongjie Sheng, Yanhong Sun and Jin Zhang
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, China
Introduction
DNAzymes are efficient biological catalysts that
strengthen the catalytic power of nucleic acids [1,2]. To
date, a series of DNAzymes with RNA-cleaving (or
DNA-cleaving) activity have been obtained by in vitro
selection. Some investigations have focused on the
improvement of specific characteristics and functions
of these DNAzymes through rational design, including
the following: using oligo-DNAs [3,4] or different
wavelengths of light [5–8] as effectors to control the
catalytic activity of the DNAymes; engineering DNA-
zyme-based sensors for Mg
2+
[9,10], Cu
2+
[11], Hg
2+
[12,13], Pb
2+
[14], and UO
2
2+
[15]; and constructing
molecular logic gates and nanomotors [16–20]. How-
ever, engineering an allosteric DNAzyme with dual
RNA-cleaving and DNA-cleaving activities is very
challenging. To our knowledge, such a DNAzyme has

not been reported.
In this article, we report on a new catalytic activity
in a DNAzyme scaffold generated by rational recon-
struction, and the regulation of catalytic activity by a
conformational transition. We prepared a DNA-cleav-
ing DNAzyme, using a deoxyribonucleotide residue
grafting strategy, as a model system for designing a
bifunctional DNAzyme that undergoes the self-cleav-
age reaction, but also possesses the ability to catalyze
the cleavage of an RNA substrate (RS). An oligo-
RNA molecule played a double role as both the
substrate for the RNA-cleaving activity of the recon-
structed DNAzyme and as a ‘negative’ effector for
controlling the self-cleavage activity of the DNAzyme.
Keywords
activity; allosteric; DNAzyme; grafting;
regulation
Correspondence
J. Zhang, Key Laboratory for Molecular
Enzymology and Engineering of Ministry of
Education, Jilin University, Changchun,
130021 China
Fax: +86 431 88980440
Tel: +86 431 88980440
E-mail:
*These authors contributed equally to this
work
(Received 4 November 2009, revised 21
March 2010, accepted 1 April 2010)
doi:10.1111/j.1742-4658.2010.07669.x

A series of RNA-cleaving or DNA-cleaving DNAzymes have been
obtained by in vitro selection. However, engineering an allosteric
DNAzyme with dual RNA-cleaving and DNA-cleaving activities is very
challenging. We used an in vitro-selected pistol-like (PL) DNAzyme as a
DNA scaffold for designing a DNAzyme with dual catalytic activities. We
prepared the 46-nucleotide DNAzyme with DNA-cleaving activity
(PL DNAzyme), and then grafted the deoxyribonucleotide residues from
an 8–17 variant DNAzyme into the region of stem–loop I and the catalytic
core of the PL DNAzyme scaffold. This deoxyribonucleotide residue graft-
ing resulted in a DNAzyme with dual RNA-cleaving and DNA-cleaving
activities (DRc DNAzyme). Drc DNAzyme has properties different from
those of the original PL DNAzyme, including DNA cleavage sites and the
required metal ion concentration. Interestingly, the RNA substrate and
RNase A can act as effectors to mediate the DNA cleavage. Our results
show that RNA-cleaving and DNA-cleaving activities simultaneously coex-
ist in DRc DNAzyme, and the DNA cleavage activity can be reversibly
regulated by a conformational transition.
Abbreviations
DRc DNAzyme, DNA-cleaving and RNA-cleaving DNAzyme; PL, pistol-like; RS, RNA substrate.
FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS 2543
RNase was prepared as a ‘positive’ effector to reacti-
vate the DNAzyme via degradation of the ‘negative’
RNA.
Results and Discussion
Design of the DNA-cleaving and RNA-cleaving
DNAzyme (DRc DNAzyme)
We elected to use an in vitro-selected pistol-like (PL)
DNAzyme as a DNA scaffold for designing a DNA-
zyme with dual catalytic activities. The PL DNAzyme
(Fig. 1A) can efficiently catalyze Cu

2+
-dependent self-
cleavage, and is composed of a catalytic core spanning
nucleotides 27–46 and two base-paired structural ele-
ments (stems I and II) flanked by regions of ssDNA
[21]. The 5¢-arm of the enzyme binds the cleavable
sequence via Watson–Crick base pairs and the 3¢-arm
through formation of a DNA triplex.
The catalytic residues were derived from the 8–17
variant DNAzyme (Fig. 1B). The original 8–17 DNA-
zyme was isolated by in vitro evolution; this enzyme
can efficiently cleave RNA to provide 2¢,3¢-cyclic phos-
phate and the 5¢-hydroxyl termini of RNA fragments
[22]. In its catalytic core, the dinucleotides A6G7 of a
terminal AGC loop and C13G14 of a bulge loop are
essential, and serve as the key deoxyribonucleotide
residues involved in the cleavage of the RNA phospho-
diester bond [23–25]. Deoxyribonucleotides A12 and
A15 of a bulge loop are not conserved. A12 can be
changed to T12, and A15 can be changed to G15.
When 8–17 DNAzyme binds its substrate, a gÆT
wobble pair can be formed, and is considered to be
significant and crucial for the catalytic activity.
When the C deoxyribonucleotide is inserted in the
5¢-T28G29G30-3¢ sequence of PL DNAzyme, the
sequence is changed to 5¢-TCGG-3¢, which is the same
as the bulge sequence of 8–17 variant DNAzyme. The
stem and terminal loop (5¢-AGC-3¢) of 8–17 variant
DNAzyme replaces stem–loop I of the PL DNAzyme
scaffold, and the T deoxyribonucleotide of 8–17 vari-

ant DNAzyme is inserted between deoxyribonucleo-
tides 13 and 14 of the scaffold (Fig. 1C).
Characterization of DRc DNAzyme
Like the parent PL DNAzyme, DRc DNAzyme was
shown to catalyze self-cleavage in the presence of
aCu
2+
(Fig. 2A). Other metal ions, including
Mg
2+
,Ca
2+
,Mn
2+
,Co
2+
,Ni
2+
,Cd
2+
,Zn
2+
, and
Ba
2+
, failed to facilitate the cleavage activity. The
reconstructed DNAzyme was shown to use divalent
copper ions with high specificity, despite replacement
of the right domain of the DNAzyme scaffold. Incuba-
tion of DRc DNAzyme yielded two distinct DNA

cleavage products (P
a
and P
b
). To map the cleavage
site of the self-cleaving DNAzyme, we used denaturing
PAGE and ran the gel until the reaction products were
clearly separated (Fig. 2B). The P
a
and P
b
cleavage
fragments were produced upon DRc DNAzyme
scission at C14 and C24, respectively. The control,
PL DNAzyme, displayed four cleavage fragments
(P
a
,P
b1
,P
b2
, and P
b3
) which were cleaved at A14,
T28, G29, and G30, respectively.
To study the rate of DRc cleavage directly, the
DNAzyme was incubated at pH 7.0 and 23 °C. The
DRc DNAzyme exhibited a narrow functional range
for the concentration of Cu
2+

, with optimum activity
A
C
B
5′
5′
Fig. 1. Sequence and predicted secondary
structures of original DNAzymes and the
reconstructed DNAzyme. (A) Sequence and
secondary structure of a 46-nucleotide
self-cleaving PL DNAzyme. A triple helix
interaction (dots) occurs between the four
base pairs of stem II and four consecutive
pyrimidine residues near the 5¢-DNA. The
major site of DNA cleavage is indicated by
the black arrowhead. (B) Sequence and
secondary structure of 8–17 variant
DNAzyme. The capital letters represent
deoxyribonucleotides, and the small letters
represent ribonucleotides. (C) Sequence and
secondary structure of the reconstructed
DRc DNAzyme. The DRc DNAzyme can
form the DNA or RNA cleavage folded
motifs under different reaction conditions.
An RNA-cleaving and DNA-cleaving DNAzyme D. Jiang et al.
2544 FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS
being reached at 100 lm. The rate of DNA cleavage
was highly dependent on the concentration of Cu
2+
used in the reaction mixture. When the concentration

of Cu
2+
was higher or lower than 100 lm, the cleav-
age activity rapidly decreased (Fig. 3A). The original
PL DNAzyme showed a bell-shaped dependence on
Cu
2+
concentration from 10 mm to 1 nm, with cleav-
age essentially going to completion at 10 lm. DRc
DNAzyme and PL DNAzyme were incubated at 23 °C
in the presence of 100 lm Cu
2+
and buffers of varying
pH (5.0–8.5). The DRc DNAzyme cleavage product
increased to a maximum yield of 47% at pH 7.5
(Fig. 3B). A similar pattern was observed for PL
DNAzyme. To evaluate the effect of temperature on
DNA cleavage, we incubated DRc DNAzyme under
standard cleavage conditions while varying the temper-
ature. As compared with PL DNAzyme, DRc DNA-
zyme function seemed to have decreased sensitivity to
the reaction temperature. DRc DNAzyme exhibited a
broad functional range for temperature, with optimum
activity being reached at 23 °C (Fig. 3C).
After characterizing the DNA-cleaving activity of
DRc DNAzyme, we continued to study its RNA-
cleaving activity (Fig. 3D–F). Improved cleavage
activity has been observed upon replacement of Mg
2+
with Mn

2+
. To obtain RNA-cleaving rates over a
broad range of metal concentrations, cleavage reac-
tions in the presence of Mn
2+
(100–200 mm) were
performed at pH 7.5. The cleavage activity exhibited a
sharp metal concentration dependence, with maximal
activity at 1 mm (Fig. 3D). DRc DNAzyme was mod-
erately perturbed in its RNA-cleaving activity relative
to the 8–17 variant. Although the Cu
2+
and ascorbate
were important for the DNA-cleaving activity of DRc
DNAzyme, they did not support the RNA-cleaving
activity of DRc DNAzyme under our reaction
conditions (100 lm Cu
2+
,10lm ascorbate, 10 mm
Mn
2+
, and 50 mm Tris ⁄ HCl, pH 7.5).
To investigate the effect of pH on RNA cleavage,
the pH dependence of DRc DNAzyme was analyzed
between pH 4.92 and pH 9.18 in the presence of 1 mm
Mn
2+
(Fig. 3E), and was very similar to that of the
8–17 variant DNAzyme. It was not feasible to obtain
a quantitatively meaningful rate versus pH, because,

at high pH, Mn
2+
precipitation occurred. The RNA-
cleaving activity of DRc DNAzyme was assayed at
A
B
C
D
Fig. 2. The DNA-cleaving activity and cleavage sites of DRc DNAzyme. (A) 5¢-
32
P-labeled DRc DNAzyme was incubated in buffer A at 23 °C
with 10 l
M various divalent metal ions, which generated two labeled products (P
a
and P
b
) in the presence of Cu
2+
. Control reactions were
incubated in the absence of metal ions. Reaction products were separated by 20% denaturing PAGE and imaged by autoradiography. (B)
Trace amounts of 5¢-
32
P-labeled PL DNAzyme or DRc DNAzyme were incubated in buffer A, containing 10 lM CuCl
2
, 0.3 M NaCl, 10 lM
L
-ascorbate (except for PL–), and 30 mM Hepes at 23 °C. PL+ and PL) represent the presence and absence, respectively, of L-ascorbate in
the reaction. Lanes M I and M II were loaded with 5¢-
32
P-labeled synthetic DNAs of different lengths as indicated, each with a sequence that

corresponded to the respective 5¢-terminus of the substrate DNA. The letters indicate the 3¢-termini of these radiolabeled marker DNAs. (C,
D) Schemes for the substrate cleavage sites of PL DNAzyme and DRc DNAzyme, respectively. The arrowheads and asterisks denote the
positions of cleavage sites.
D. Jiang et al. An RNA-cleaving and DNA-cleaving DNAzyme
FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS 2545
different temperatures. The DNAzyme (Fig. 3F)
showed a linear temperature dependence between 25.8
and 53.7 °C.
When evaluating the optimal design for a DNAzyme
with respect to DNA-cleaving and RNA-cleaving
activities, we found that a number of nucleotides within
the catalytic core and substrate-binding arm of PL
DNAzymes were not highly conserved and could be
substituted by the structural domain derived from the
8–17 variant DNAzyme. We wanted to combine our
optimization of the arm design and modification of the
catalytic domain to yield an enhanced DNAzyme.
Unfortunately, other designed DNAzymes (DA1–DA3)
suggested that DNA-cleaving and RNA-cleaving activi-
ties could not coexist within a DNA motif, and the
double activities competed with each other. Because
DRc DNAzyme has comparatively high activities, we
focused substantial effort on its characterization. The
DRc DNAzyme with new catalytic activity can arise
from an existing DNAzyme scaffold, indicating that a
single DNA sequence can catalyze the two respective
reactions and assume either of two DNAzyme folds.
RNAzymes previously investigated have shown similar
properties [26,27]. The characterization data collected
(Fig. 3) provide a number of indications and constraints

for future modeling studies on the active structure and
evolution of the DNAzymes.
The regulating effects of RS and RNase on the
DNA cleavage of DRc DNAzyme
After characterizing the DNA-cleaving and RNA-
cleaving activities of DRc DNAzyme, we found that
an RS could act as an effector to control the DNA
cleavage of DRc DNAzyme (Fig. 4A). Regulation
proceeds via an effector-generated rearrangement of
the active site, where the DNA-cleaving active site
of DRc DNAzyme is hindered through the binding of
RS. The self-cleaving DRc DNAzyme was incubated
in reaction buffer B, containing 100 lm CuCl
2
, 0.3 m
NaCl, 10 lml-ascorbate, and 30 mm Hepes (pH 7.0)
at 23 °C. The self-cleavage of DRc DNAzyme was
A
BC
FED
Fig. 3. Characterization of the DNA-cleaving and RNA-cleaving reactions catalyzed by DRc DNAzyme. (A–C) Analyses of DNA cleavage at dif-
ferent Cu
2+
concentrations, pH values, and temperatures, respectively. All reactions were conducted using trace amounts of 5¢-
32
P-labeled
DRc DNAzyme. In (A), the CuCl
2
concentration was varied from 1 to 10 mM. The reactions were conducted at pH 7.0 (30 mM Hepes) and
23 °C with 0.3

M NaCl and 10 lML-ascorbate. In (B), the reactions were conducted under different pH conditions with 0.3 M NaCl, 10 lM
L
-ascorbate, and 10 lM CuCl
2
, and were incubated at 23 °C. In (C), the effect of reaction temperature on DRc DNAzyme function was
assessed with cleavage assays conducted as described in (A), except that 10 l
M CuCl
2
was present and the temperature was varied from
12 to 40.7 °C. (D–F) Analyses of RNA cleavage at with different Mn
2+
concentration, reaction pH values, and temperatures, respectively. All
reactions were conducted using 20 n
M DNAzyme and 2 nM 5¢-
32
P-labeled RS. In (D), the MnCl
2
concentration was varied from 0.1 to
200 m
M. The reactions were conducted at 37 °C and pH 7.5 (50 mM Tris ⁄ HCl). In (E), reactions were conducted under different pH condi-
tions with 10 m
M Mn
2+
, and were incubated at 37 °C. In (F), the reaction temperature was varied from 25.8 to 53.7 °C. The reactions were
conducted at pH 7.5 (50 m
M Tris ⁄ HCl) with 10 mM Mn
2+
.
An RNA-cleaving and DNA-cleaving DNAzyme D. Jiang et al.
2546 FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS

measured in the presence of various concentration of
RS regulator, and the percentage cleavage versus RS
concentration yielded a sigmoidal curve (Fig. 4B). The
presence of a 12-nucleotide RNA regulator that is
complementary to the RS recognition domain dramati-
cally decreased the rate of DNA self-cleavage. We
systematically varied the length of the RS to determine
the effects on regulation of the DNA self-cleaving
activity. Enlarging the RS to 18 bp essentially abol-
ished the catalytic activity, as expected. This might be
attributed to steric interference in the DNA-cleaving
catalytic domain of DRc DNAzyme. Reducing the RS
to 6 bp increased the rate of DNA cleavage, which
approached the self-cleaving rate of DRc DNAzyme in
the absence of RS. RSs with greater lengths were more
efficient at decreasing the DNA self-cleavage of DRc
DNAzyme.
Here, the RS actually acted as a ‘negative’ effector
to regulate the DNA-cleaving activity of DRc DNA-
zyme. We were interested in constructing a reversible
control for catalytic activity. RNase is a type of nucle-
ase that catalyzes the degradation of RNA into smaller
components. Bishop and Klavins reported that
RNase H had been used to reverse binding in a deoxy-
ribozyme nanomotor [20]. In our study, RNase A and
RNase H were selected as ‘positive’ effectors to elimi-
nate the RS regulation. As shown in Fig. 4C, RNase A
was more efficient than RNase H in triggering the
DNA-cleaving activity of DRc DNAzyme. Under the
conditions of the DNA-cleaving regulated system,

most RSs exist in a single-stranded state, and a few
RSs can form a DNAÆRNA duplex with the RNA
substrate recognition domain of DRc DNAzyme.
RNase A cleaved ssRNA and RNase H specifically
A
CB
Fig. 4. The regulating effects of RS and RNase on DNA cleavage. (A) Scheme for reversible modulation of DNA self-cleavage. (B) The RS
acted as a ‘negative’ effector to decrease the DNA cleavage. The data fit the Boltzmann equation and the curve is sigmoidal. (C) RNase A or
RNase H acted as ‘positive’ effectors to renew the DNA cleavage.
D. Jiang et al. An RNA-cleaving and DNA-cleaving DNAzyme
FEBS Journal 277 (2010) 2543–2549 ª 2010 The Authors Journal compilation ª 2010 FEBS 2547
hydrolyzed RNA in a DNAÆRNA duplex. We specu-
lated that this difference between RNase A and
RNase H might lead to different efficiencies in
‘positive’ regulation. When compared with RNase H,
RNase A was better suited as a ‘positive’ effector.
We used a simple and invasive method to reversibly
modulate the DNA cleavage rate of DRc DNAzyme
with RNA substrate and RNase A. Such regulatory
biocatalyst systems offer several advantages, including
the ease of RNA substrate and DNAzyme synthesis
without any chemical modification, and convenient use
of RNase A as a common bioreagent, an attractive
property for DNAzymes that have various applications.
In conclusion, we have described a key residue graft-
ing strategy for generating RNA-cleaving activity in a
self-cleaving DNAzyme. The DNAzyme with DNA-
cleaving and RNA-cleaving activities was constructed
by incorporating the catalytic domain of 8–17 variant
DNAzyme into the right domain of the secondary

structure of PL DNAzyme, demonstrating that dual
activities can coexist in a small DNA scaffold. Using
the RNA substrate and RNase A, we constructed a
simple conformational switch to control the DNA-
cleaving activity of DRc DNAzyme. The generation of
a new active site within a DNAzyme scaffold and reg-
ulation of the catalytic activity provide further insights
into the engineering of DNAzymes.
Experimental procedures
Materials
The DNA sequences (PL DNAzyme, 5¢-GAATTCTAATAC
GACTCAGAATGAGTCTGGGCCTCTTTTTAAGAAC-3¢;
8–17 variant DNAzyme, 5¢-AATACTCCGAGCCGGTCG
GGCCTC-3¢; DRc DNAzyme, 5¢-GAATTCTAATACTCC
GAGCCGGTCGGGCCTCTTTTTAAGAAC-3¢) were pre-
pared by automated synthesis, and purified by 16%
denaturing PAGE (Sangon, Shanghai, China). The RS
(5¢-gaggcagguauu-3¢) was also prepared by automated syn-
thesis and purified by HPLC (TaKaRa, Dalian, China).
[
32
P]ATP[cP] was purchased from Furui. T4 polynucleotide
kinase was purchased from TaKaRa. RNase A and RNase
H were purchased from MBI. All chemical reagents were
purchased from BBI.
Activity assays for the DNAzyme
To assess the cleavage activity of the DNAzyme, radiola-
beled RNA or DNA were first generated by enzymatically
tagging the 5¢-termini of synthetic RSs or self-cleaving
DNAzymes. The reaction mixture contained 10 mm MgCl

2
,
5mm dithiothreitol, 2 lm RS or self-cleaving DNAzyme,
0.4 lm [
32
P]ATP[cP] ( 20 lCi; 1 Ci = 37 GBq), and
0.5 lL of T4 PNK (10 UÆlL
)1
), and the mixture was incu-
bated at 37 °C for 1 h.
Prior to the self-cleavage activity assays, the DNAzyme
was first denatured by heating to 90 °C for 2 min, and then
incubated at 0 °C for 5 min. Trace amounts of 5¢-
32
P-labeled
DNAzyme were incubated in reaction buffer A, containing
10 lm CuCl
2
, 0.3 m NaCl, 10 lml-ascorbate and 30 mm
Hepes (pH 7.0) at 23 °C. To assess the RNA-cleaving activ-
ity of the DNAzyme, cleavage reactions were performed by
combining 20 nm DNAzyme and 2 nm 5¢-
32
P-labeled RS in
the presence of 10 mm Mn
2+
in 50 mm Tris ⁄ HCl (pH 7.5).
Mixtures were incubated at 37 °C for 30 min. The reaction
was terminated after a designated period of time by the
addition of stop solution containing 60 mm EDTA, 8 m urea,

0.02% (w ⁄ v) xylene cyanol, and 0.02% (w ⁄ v) bromophenyl
blue solution. Cleavage products were separated by 20%
denaturing PAGE and visualized by autoradiography.
Regulation of the DNA cleavage of the DNAzyme
For ‘negative’ regulation assays, reactions were initiated by
the addition of a mixture of 1 nm 5¢-
32
P-labeled DNAzyme,
reaction buffer B (100 lm CuCl
2
, 0.3 m NaCl, 10 lm
l-ascorbate, 30 mm Hepes, pH 7.0) and varying concentra-
tions (0–100 nm) of RS (as a ‘negative’ effector) at 23 °C.
For ‘positive’ regulation assays, reactions were con-
ducted at 23 °C for a designated period of time, using 1 nm
5¢-
32
P-labeled DNAzyme, 100 nm RS, reaction buffer B,
and 10 ng of RNase A (or 5 U of RNase H). Cleavage
products were separated by denaturing PAGE and imaged
by autoradiography.
Acknowledgement
This work was supported by grants from the National
Natural Science Foundation of China (General Pro-
gram No. 30770479).
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