Harnessing nature's insights: Synthetic small molecules with peroxidase-mimicking DNAzyme properties

Article abstract of DOI:10.1002/chem.201101337

Substitute features: The demonstration that small DNAs can acquire enzymatic properties (DNAzyme activity) is appealing since it makes protein-based biotechnologies simpler. Herein, two further simplifications of this process are proposed: the substitution of DNAs for small molecules, called DOTA-templated synthetic G-quartets (DOTASQ), and of hemin for the more practically convenient iron(III) porphyrin (FeTPPS; see scheme). Copyright

Full text of DOI:10.1002/chem.201101337

COMMUNICATION
DOI: 10.1002/chem.201101337
Harnessing Natureꢀs Insights: Synthetic Small Molecules with Peroxidase-
Mimicking DNAzyme Properties
[a]
Loic Stefan, Hai-Jun Xu, Claude P. Gros, Franck Denat, and David Monchaud*
By the end of the 1990s, Dipankar Sen and co-workers
found that small, and G-rich, DNAs and RNAs could ac-
quire protein-like catalytic properties, and particularly
H O -mediated peroxidase activity, upon interaction with
hemin) for two synthetic small molecules (DOTASQ and
FeTPPS, vide infra) and on their evaluation as an original
and fully synthetic DNAzyme catalytic system.
On the basis of inspiring studies from Sherman and co-
workers, who reported on the design of small molecule-
based template-assembled synthetic G-quartets (so-called
2
2
hemin, the cofactor of many oxidative enzymes (including
[1]
cytochrome P450 or horseradish peroxidase). This proper-
ty, already known as ribozyme for RNA, was consequently
termed DNAzyme for DNA; this activity originates in the
ability of the studied G-rich oligonucleotides to fold into a
peculiar tridimensional G-quadruplex architecture. This
structure, which results from an intramolecular folding of
the oligonucleotides, is promoted by the coplanar associa-
tion of four guanine residues through a Hoogsteen-type H-
bond pattern, and is further stabilised by the presence of
[13]
TASQ), we recently developed a novel series of TASQ in
which the template is the water-soluble DOTA moiety
(1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic
[14]
acid).
Resulting compounds have consequently been
named DOTASQ (DOTA-templated synthetic G-quartet,
Figure 1B). We demonstrated by NMR spectroscopy studies
that the DOTASQ is able to adopt a closed conformation
(i.e., a stable intramolecular G-quartet fold; Figure 1B,
right) in a physiologically relevant environment (i.e., buf-
fered solution at pH 7.2, with high ionic strength, e.g.,
+
+
[2]
biologically relevant cations (Na , K ). The resulting
DNA/RNA architectures are comprised of stacked G-quar-
tets, and thus offer accessible G-quartets as binding pockets
for hemin.
[14]
>10 mm KCl). We also interpret the ability of DOTASQ
to interact efficiently with quadruplex DNA (whatever the
nature of the sequence it folds from: whether it is the short
Upon interaction with the G-quartet, hemin is indeed ac-
tivated for both its reaction with H O and the breakdown
[2]
sequences from human telomeres or the promoter region
2
2
[15]
of the resulting hemin–H O complex in the presence of the
of oncogenes, like c-myc or c-kit) as a solid, yet indirect,
proof of the existence of the closed conformation in solu-
tion, the recognition efficiency presumably originating in a
nature-inspired association between two G-quartets, one
2
2
substrate to be oxidised (Figure 1A). Even if this approach
has its trade-offs (particularly the low catalytic efficiency of
the DNA system as compared to native enzymes), it is indis-
putable that the use of small DNA or RNA molecules as
biocatalysts appears appealing since oligonucleotides are
more practically convenient than proteins and offer brand-
[14]
being native (G-quadruplex) the other artificial (ligand).
We thus believe that DOTASQ can be interesting biocata-
lyst-mimicking small molecules. Indeed, the intramolecular
G-quartet fold could offer a binding site to hemin (the catal-
ysis principle is schematically depicted in Figure 1C), and
therefore, could pave the way for a reduction in the com-
plexity of the DNAzyme system (neither DNA syntheses
nor purification/folding steps are required).
[3]
new possibilities. However, to make this process still more
appealing and to expand its scope, which includes detection
[
4]
[5]
[6]
of various cations, proteins, enzymatic activity, DNA/
[7]
[8]
RNA analytes, nucleotide modifications, and even cancer
[9]
[10]
cells as a whole, some biophysical
or histochemical
[11]
[12]
assays, as well as DNA logic gates, it is of high interest
to develop a DNAzyme system smaller, cheaper, and above
all, more readily available on a larger scale than DNA or
RNA. To this end, we report herein on the substitution of
the two “natural” DNAzyme components (DNA and
Toward this goal, we firstly evaluated the ability of
DOTASQ to interact with hemin by UV/Vis titrations as al-
[1]
ready reported by Sen et al. We performed these experi-
ments in a Caco.KTD buffer (10 mm lithium cacodylate
buffer, pH 7.2, 10 mm KCl/90 mm LiCl, 0.05% Triton X-100,
0
.1% DMSO). The choice of these particular conditions is
justified by the fact that all our previous investigations with
[
a] L. Stefan, Dr. H.-J. Xu, Prof. C. P. Gros, Prof. F. Denat,
Dr. D. Monchaud
Institut de Chimie Molꢀculaire de l’Universitꢀ de Bourgogne
DOTASQ were performed in Caco.K buffer (10 mm lithium
[14]
cacodylate buffer, pH 7.2, 10 mm KCl/90 mm LiCl),
and
(
ICMUB)
that the poor solubility of the hemin requires the addition of
low percentages of both nonionic detergent (Triton X-100)
CNRS UMR5260, 9, Avenue Alain Savary, 21000 Dijon (France)
Fax : (+33)380-396-117
E-mail: david.monchaud@u-bourgogne.fr
[1]
and highly solubilising organic solvent (DMSO). As shown
in Figure 2A, the addition of increasing amounts of
DOTASQ-C5 (n=5, Figure 1B) results in large red-shifted
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101337.
Chem. Eur. J. 2011, 17, 10857 – 10862
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10857

hyperchromism that indicates
a
DOTASQ-mediated disaggregation of
[1]
hemin. This demonstrates a good in-
teraction between DOTASQ and
hemin (quantified through the appar-
ent affinity constant K =135 nm, see
d
the Supporting Information for de-
tailed K calculations).
d
Similar experiments were carried
out with DOTASQ-C1 (n=1, Fig-
ure 1B), and the observed interaction
lies in the same nanomolar range
(
K =170 nm). For the sake of compar-
d
ison, a similar titration was performed
with a G-quadruplex DNA formed
from the human telomeric sequence
5
’
(
the 22-nucleotide sequence AG3-
3’
AHCTUNGRTNNEG(U T AG ) known as 22AG). We se-
2 3 3
lected this quadruplex primarily be-
[
2]
cause of its biological relevance,
+
even if its K -promoted “antiparallel”
quadruplex structure (Figure 1A)
makes it a more modest DNAzyme
[10b,16]
catalyst
than “parallel” quadru-
[17]
plexes, like c-myc.
We focused on
the 22AG-quadruplex also because we
initially aimed at using DNAzyme for
telomerase activity assays, as pio-
[6a–c]
neered by Willner and co-workers.
In our hands, 22AG interacts strongly
with hemin (K =235 nm) in an effi-
d
ciency range comparable to that of
DOTASQ. This is somewhat surpris-
ing since it implies that the surround-
ing elements present in the quadru-
plex architecture (loops, grooves) do
not drastically influence the hemin ap-
parent affinity. This might originate in
the fact that hemin is negatively
charged at pH 7, and thus insensitive
to the negatively charged quadruplex
loops and/or grooves (with phosphate
groups).
We therefore decided to evaluate
the ability of DOTASQ to catalyse
Figure 1. A) Schematic representation of the
DNAzyme activity promoted by the G-quad-
ruplex 22AG. B) Chemical formulae of the
DOTASQ (DOTASQ-C1: n=1, DOTASQ-
C5: n=5) and schematic representation of its
“
open” and “closed” conformations. H-bonds
are represented as dotted lines. The 3D struc-
tures were obtained with CAChe Worksystem
Pro Version 7.5 (ꢁ 2000–2006 Fujitsu, Ltd.).
C) Schematic representation of the DNAzyme
activity promoted by a DOTASQ. D) Chemi-
cal formulae of ABTS, hemin and oxidised
hemin.
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Chem. Eur. J. 2011, 17, 10857 – 10862

DNAzyme Mimicking Synthetic Small Molecules
COMMUNICATION
the hemin-mediated oxidation of ABTS (2,2’-azinobis(3-eth-
ylbenzothiazoline)-6-sulfonic acid) by H O following a pre-
2
2
viously established protocol (2 mm ABTS, 1 mm hemin in Ca-
co.KTD buffer; the reaction was initiated by the addition of
[18]
0
.6 mm H O ). As depicted in Figure 2B, the DNAzyme
2 2
process promoted by DOTASQ-C5 works effectively, in a
dose-response manner since the efficiency of the catalysis
(
monitored by the appearance of a UV/Vis signal at 420 nm,
characteristic of the oxidation product of ABTS) is in direct
correlation with the concentration of DOTASQ-C5 (the
higher the DOTASQ-C5 loading, up to 50 mm, the better the
catalysis). To further validate this proof-of-concept, we first-
ly confirmed that this activity is related to the putative intra-
molecular G-quartet folding by repeating experiments with
a DOTASQ derivative in which all guanines were O-pro-
tected (by a benzylic group) and thus incapable of self-as-
sembly in G-quartet (Prot.DOTASQ-C5).
As depicted in Figure 2C, Prot.DOTASQ-C5 was ineffec-
tual in promoting ABTS oxidation, thereby supporting that
the G-quartet folding is a key requisite for DNAzyme activi-
ty. We thus decided to compare this activity to the one re-
[16]
ported for 22AG. As depicted in Figure 2C, G-quadruplex
DNA is far more active than DOTASQ since an interesting
activity is obtained with only 2 mm 22AG. For the sake of
comparison, we quantified the catalytic efficiencies of this
process: given that these experiments were carried out
under saturating substrate conditions (2 mm ABTS vs. 1 mm
hemin) catalytic rates can be quantified as k , which repre-
cat
sents the number of ABTS molecules that are oxidised by a
single molecule of catalyst in a given unit of time (see the
Supporting Information for detailed kcat calculations). The
kcat values calculated for 22AG and DOTASQ-C5, 9.71 and
À1
0
.36 h , respectively, clearly highlight the better performan-
ces of the quadruplex DNA.
These results are interesting since we demonstrated
before (by UV/Vis titrations) that hemin interacts with
2
2AG and DOTASQ-C5 with a comparable apparent affini-
ty. This highlights the that the surrounding elements present
in the 22AG architecture (loops, grooves) define a binding
pocket that is optimised for DNAzyme activity whilst it
does not enhance hemin-apparent affinity, probably by pro-
viding a better protection for hemin against oxidative degra-
[1]
dation as initially postulated by Sen and co-workers on the
[19]
basis of the pioneering works of Brown and Jones. Finally,
DOTASQ-C1 was also evaluated as catalyst. As depicted in
Figure 2C, it is less active than DOTASQ-C5 (calculated
Figure 2. A) UV/Vis titration of hemin (1 mm) with increasing amounts of
DOTASQ-C5 (from 0 to 5 mm, in the direction of the arrow) in Ca-
co.KTD buffer, pH 7.2, at 258C. B) Dose-response curves for the DNA-
+
zyme activity of DOTASQ-C5, quantified by the absorbance of ABTSC
(
signal at 420 nm) as a function of time. Experiments were carried out
with hemin (1 mm), H (0.6 mm), ABTS (2 mm) and DOTASQ-C5
from 0 to 50 mm) in Caco.KTD buffer, pH 7.2 at 258C. C) Comparison of
DNAzyme activities. Experiments were carried out with hemin (1 mm),
(0.6 mm), ABTS (2 mm) in the absence of DNA or ligand (no ma-
2
O
2
(
2 2
H O
trices) or with 22AG (2 mm), DOTASQ (50 mm, viz. DOTASQ-C5, Prot.-
DOTASQ-C5 and DOTASQ-C1) in Caco.KTD buffer, pH 7.2 at 258C.
D) Structures of DOTASQ-C1, DOTASQ-C5 and Prot.DOTASQ-C5.
Chem. Eur. J. 2011, 17, 10857 – 10862
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10859

D. Monchaud et al.
À1
kcat =0.29 h ), which could originate from a less stable quar-
hemin has poor practical convenience because of its very
low solubility both in water (or buffers, which imposes the
equally inconvenient use of detergents, like Triton X-100)
and in DMSO, which is problematic for the preparation of
mother solutions even at 1 mm. Replacing polyanionic
DNAs by a cationic small molecule offers the possibility of
tet fold (due to its shorter guanine arms) given their compa-
rable solubility in the experimental conditions (see the Sup-
porting Information). To further confirm these results, we
decided to perform similar experiments with another colori-
metric substrate. On the basis of the work recently reported
[20]
[21,22]
by Dong et al.,
we selected 3,3’,5,5’-tetramethylbenzi-
substituting hemin for a highly water soluble iron
phyrin.
ACHTUNGTRENUNNG( III) por-
dine sulfate (TMB). The oxidation of TMB is a two-step
procedure, with a coloured intermediate that elicits UV/Vis
maxima at 370 and 652 nm and a final product characterised
by an absorbance maximum at 450 nm (Figure 3A). As de-
picted in Figure 3B, DOTASQ-C5 catalyses TMB oxidation
kcat =0.02 vs. 0.27 h for 22AG); this confirms that the
DOTASQ–hemin system operates in a substrate-independ-
ent manner.
We thus turned our attention on the last “natural” compo-
nent of the DNAzyme process, that is, the hemin. This bis-
We selected iron AHCUTNGRNENUG( III) meso-tetra(4-sulfonatophenyl)por-
phyrin chloride (FeTPPS) since this porphyrin is highly
water soluble (stock solutions in water are routinely pre-
pared at 10 mm), water stable (see the Supporting Informa-
tion), resistant to highly oxidative conditions and known to
perform H O -mediated oxidations for more than two de-
À1
(
2
2
[26]
cades. For being an ideal candidate, the selected porphy-
rin must be ineffectual in promoting the oxidation of the
chromophoric substrate alone, and must be activated upon
interaction with DOTASQ (like hemin). In that sense,
FeTPPS is particularly interesting since it forms a m-oxo-
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
anionic iron
ACHTUNGTRENNUNG( III) porphyrin is the natural co-catalyst of the
[1]
native peroxidase. Hemin is quite unique since it is one of
the sparse examples (with NMM and Cu-APC) of an
anionic small molecule that displays affinity for quadruplex
DNA. This explains why the replacement of hemin by an-
other iron(III) porphyrin (or other) candidate has not been
investigated. This is, however, critically important, since
[23]
[24]
[27]
dimer in solution at physiological pH that is known to be
[26b]
catalytically inactive.
We verified this by UV/Vis titra-
[25]
tions, since both forms display characteristic Soret bands (at
394 and 410 nm for the monomeric aquo complex and the
ACHTUNGTRENNUNG
[27]
dimer, respectively). In our conditions (Caco.K buffer, see
the Supporting Information), FeTPPS indeed adopts a UV/
Vis signature characteristic of the dimeric, and so catalyti-
cally inactive, form.
The interaction between DOTASQ and FeTPPS was sub-
sequently investigated again by UV/Vis titrations. As depict-
ed in Figure 4A, the addition of increasing amounts of
DOTASQ-C5 deeply modified the FeTPPS UV/Vis signa-
ture, and both the bathochromic (4 nm; Figure 4B) and hy-
pochromic (~20%) shifts are reminiscent of what has been
[28]
[29,30]
observed with cyclodextrins
or BSA,
two molecules
that have been reported to favour the monomeric form of
FeTPPS (bound FeTPPS is characterised by a maximum at
[27a]
4
14 nm, similar to the monomeric hydroxo complex).
The presence of a clean isosbestic point (at 424 nm; Fig-
ure 4A, dotted arrow) clearly indicates a transition between
two well-defined species upon addition of DOTASQ,
namely, free and bound FeTPPS. Similar titration performed
with 22AG clearly demonstrates the complete lack of quad-
ruplex–FeTPPS interaction (see the Supporting Informa-
tion). With these results in hand, we decided to study the
ability of DOTASQ–FeTPPS complexes to catalyse the oxi-
dation of ABTS, under simplified conditions since Caco.K
buffer was used instead of Caco.KTD. As depicted in Fig-
ure 4C, FeTPPS is inactive for promoting the oxidation of
ABTS both alone (in direct line with its dimeric state) and
in the presence of 22AG-quadruplex (in direct line with the
lack of quadruplex–FeTPPS interaction). However, upon in-
teraction with DOTASQ-C5 the resulting DOTASQ–
FeTPPS complex acquires an interesting catalytic efficiency
Figure 3. A) Schematic representation of the enzymatic (enz.) two-step
oxidation of TMB (see ref. [21]). B) Comparison of DNAzyme activities.
À1
(
kcat =0.36 h ; see the Supporting Information), which is
[31]
similar to that of DOTASQ-C5–hemin (vide supra).
Experiments carried out with hemin (1 mm), TMB (0.5 mm),
2 2
H O
These results clearly demonstrate that a nature-inspired pro-
cess, like DNAzyme activity, can be successfully turned into
(
(
1.5 mm) in the absence of DNA or ligand (no matrices), or with 22AG
2 mm) and DOTASQ-C5 (50 mm) in Caco.KTD buffer, pH 7.2 at 258C.
10860
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ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 10857 – 10862

DNAzyme Mimicking Synthetic Small Molecules
COMMUNICATION
molecules, by virtue of their special supramolecular proper-
ties. These two simplifications (quadruplex DNA for pro-
tein, then G-quartet for quadruplex DNA) have their trade-
offs, notably a gain of practical convenience on the one
hand and a decreased catalytic efficiency on the other. How-
ever, even if the herein reported results are admittedly
modest as compared to the quadruplex–hemin system, they
are, nevertheless, highly interesting since they validate the
proof-of-concept that small molecules, and particularly an
isolated G-quartet devoid of surrounding elements, can ac-
quire DNAzyme activity. Additionally, the use of DOTASQ
enables the substitution of hemin for more practically con-
venient FeTPPS, which together greatly contribute to a
simplification of the DNAzyme process that is now readily
amenable to larger scales. With such a simplification, we are
now working on the development of more active TASQ-
zyme catalysts through the synthesis of DOTASQ analogues
that will display more stable G-quartet folds (like “two-
quartet-layer” analogues) and we hope to report on these
results soon.
Acknowledgements
The authors deeply acknowledge J.-L. Mergny and L. Lacroix for fruitful
ꢂ
discussions, CheMatech company for helpful assistance for DOTASQ
synthesis, F. Cuenot for his help in writing funding proposals, G. Bordeau
for his valuable bibliographic assistance, the CNRS, the Conseil Rꢀgional
de Bourgogne and the Universitꢀ de Bourgogne (through the 3MIM inte-
grated project and FABER grant 2009-149E-149CE) and the Agence Na-
tionale de la Recherche ((ANR-10-JCJC-0709)) for funding.
Keywords: biomimetics · bioorganic chemistry · catalysis ·
DNAzymes · hemin
[
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[
Chem. Eur. J. 2011, 17, 10857 – 10862
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www.chemeurj.org
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Supporting Information). Its catalytic activity was subsequently in-
vestigated and we found that BSA indeed catalyses the FeTPPS-
mediated oxidation of ABTS, but modestly (ꢀ7.5-fold less actively)
as compared to the corresponding BSA–hemin system. So, as depict-
ed in the Supporting Information, both BSA and DOTASQ interact
with the two prophyrins, but while only hemin can be considered as
a valuable cofactor for BSA, both hemin and FeTPPS act as equally
efficient cofactors for DOTASQ.
[31] The DOTASQ–FeTPPS system was not evaluated for the catalysis
of the oxidation of TMB, because the UV/Vis properties of FeTPPS
impeded the proper evaluation of the appearance of the 370, 450
and 652 nm signals.
[
[
[
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[
[
9
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[
20] Terbium complexes of DOTASQ are better quadruplex ligands than
DOTASQ (see ref. [14]) but their high sensitivity toward the DNA-
zyme experimental conditions have precluded their evaluation as
catalysts.
Received: May 2, 2011
Published online: August 29, 2011
10862
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Chem. Eur. J. 2011, 17, 10857 – 10862