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

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

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

, 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

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,

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

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

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

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,

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