Modular assembly of a Pd catalyst within a DNA scaffold for the amplified colorimetric and fluorimetric detection of nucleic acids

Article abstract of DOI:10.1002/anie.201206006

Phosphine ligands were covalently tethered to short oligonucleotide strands such that active palladium complexes formed in the presence of a specific nucleic acid target. This catalytic center on the double-stranded DNA then converted water-soluble iodo-BODIPY dyes, present in excess, into highly emissive deiodinated reporters (see picture). This approach is suitable for the rapid colorimetric or fluorimetric detection of nucleic acid targets with a lower detection limit of just 10pM. Copyright

Full text of DOI:10.1002/anie.201206006

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DOI: 10.1002/anie.201206006
DNA Sensing
Modular Assembly of a Pd Catalyst within a DNA Scaffold for the
Amplified Colorimetric and Fluorimetric Detection of Nucleic Acids**
Deepak K. Prusty, Minseok Kwak, Jur Wildeman, and Andreas Herrmann*
Catalytic signal amplification is a powerful tool for the
detection of chemical and biological analytes. In chemistry it
has been employed in sensing various toxic metal ions (e.g.
Pd2+, Pb2+, Cu2+, and Hg2+)[1] as well as small molecules such
as carbon monoxide[2] and thiols.[3] In a biological context,
catalytic reactions have enabled the highly sensitive detection
of scarce analytes. They have been widely utilized, for
instance, in the detection and assay of proteins,[4] antibodies,[5]
and nucleic acids.[6]
In the case of nucleic acids, DNA-templated catalytic
processes in particular have been successful. Fluorogenic
transformations of this type have been exploited for the
amplified detection of deoxyribonucleotides (ODNs) both in
homogeneous solutions[7] and in living cells.[8] In this
approach, DNA probes are labeled with poorly fluorescent
precursors and assembled with target nucleic acids into
catalytic hybrids. These then chemically convert the precur-
sors into fluorescent reporters[9] (e.g. through the Staudinger
reaction,[10] transthioesterification,[11] and aminolysis).[12] The
turnover rate and detection signal can be further improved by
repeated thermal cycling. Besides variation of temperature,
another external stimulus for signal amplification is light.
Photochemical reactions have been employed to trigger the
photocatalytic formation of singlet oxygen for the generation
of fluorescent reporters.[13] While such systems have resulted
in the amplified detection of DNA, RNA, and peptide nucleic
acids (PNAs), the approach remains limited by hurdles such
as the covalent attachment of profluorescent molecules or
photosensitizers to probe ODNs and the need for external
stimuli to achieve multiple turnovers.
DNA phosphorylation,[15] and the formation of nucleopeptide
linkages.[16] The same structural properties allow ODNs to
serve as effective scaffolds for complex formation with
transition metals to form hybrid catalytic systems; Michael
addition,[17] Friedel–Craft alkylation,[18] ester hydrolysis,[19]
and an enantioselective Diels–Alder reaction[20] have been
demonstrated using this concept.
Here we apply the concept of DNA-directed transition-
metal catalysts in an entirely new strategy for catalytic signal
amplification. We use ligand-labeled probe strands to form
a palladium complex in the presence of a specific target DNA
sequence. The catalytic center formed on the double-stranded
(ds) DNA then efficiently converts water-soluble profluor-
escent iodo dyes, present in excess, into highly emissive
deiodinated reporters. Additionally, since neither the precur-
sor nor the reporter is covalently attached to DNA, no
external trigger is required to amplify the fluorescence signal:
deiodination of the precursor upon diffusion to the catalytic
site is sufficient. This simple DNA-directed catalyst assembly
opens the possibility of generating multiple fluorescence
signals per hybridization event.
Boron dipyrromethane (BODIPY) derivatives were
selected as chromophores because of their 1) high fluores-
A potential solution to these drawbacks is grounded in the
use of DNA as a structural component, rather than an analyte,
in catalytic systems. ODNs on their own are versatile
components for catalysis, able to adopt complex three-
dimensional structures to catalyze DNA/RNA ligation,[14]
[*] D. K. Prusty, M. Kwak, J. Wildeman, Prof. Dr. A. Herrmann
University of Groningen, Zernike Institute for Advanced Materials
Department of Polymer Chemistry
Nijenborgh 4, 9747 AG Groningen (Netherlands)
E-mail: a.herrmann@rug.nl
[**] The authors acknowledge financial support from the EU (ERC
starting grant NUCLEOPOLY and collaborative project ECCell), the
Netherlands Organization for Scientific Research (NWO-Vici), and
the Zernike Institute for Advanced Materials. The help of Andrew
Musser and Alessio Marcozzi is appreciated very much by the
authors.
Figure 1. Chemical structures of the water-soluble BODIPY chromo-
phores and their fluorescence spectra. a) Pd-catalyzed dehalogenation
of profluorescent mono-/diiodinated precursors (1 and 2) to give the
fluorescent deiodinated reporters (3 and 4). b) Fluorescence spectra of
the mono (solid line) and diiodo (dashed line) precursors (left) and
the corresponding reporters (right). Numbers in brackets indicate the
FQY (Ffl) of each compound, as determined against a cresyl violet
reference in methanol. See p. 5 in the Supporting Information for
additional photophysical properties. TPPTS=sodium triphenylphos-
phinetrisulfonate.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201206006.
Re-use of this article is permitted in accordance with the Terms and
Conditions set out at http://angewandte.org/open.
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cence quantum yield (FQY), 2) high extinction coefficient,
and 3) photostability.[21] We investigated mono- and diiodi-
nated BODIPY precursor chromophores (1 and 2, respec-
tively; see Figure 1a). To ensure water solubility, which is
essential for applications in biological systems, a precursor
was modified with four triethylene glycol chains. Both
profluorescent compounds 1 and 2 proved highly soluble in
aqueous media (> 10 mgmLÀ1) and were synthesized in 10
and 30% overall yield, respectively (see pp. 2–3 in the
Supporting Information for synthetic details and structural
characterization). In both compounds, iodine atoms were
incorporated at the C2 and/or C6 positions of the BODIPY
core, to favor intersystem crossing to the triplet manifold.[22]
The photophysical properties of the precursors and their
deiodinated products (3 and 4) were initially investigated to
confirm their suitability as substrates for fluorogenic reactions
in water. Compounds 1 and 2 were individually subjected to
palladium-catalyzed deiodination by dissolution in sodium
acetate (NaOAc) buffer (0.5m, pH 5.0) in the presence of
a water-soluble Pd catalyst (Na2PdCl4·TPPTS; see pp. 2–3 in
the Supporting Information for details). The UV/Vis absorp-
tion and fluorescence spectra of the products (3 and 4)
obtained after 4 h of shaking at room temperature differed
markedly from those of the corresponding precursors. As
anticipated, the absorption maxima of the dehalogenated
products were strongly blue-shifted (Figure 1b; Figures S1
and S2). Indeed, the color change of the aqueous solution
from red to yellow upon deiodination was evident to the
naked eye, which implies the possibility for colorimetric
detection (similar to Figure 2c). The fluorescence emission
maxima of 3 and 4 also exhibited a blue shift, with
fluorescence intensities increasing 35- and 80-fold, respec-
tively, relative to the profluorescent substrates. The significant
difference in the fluorescence intensity increase was antici-
pated in light of the higher background intensity of monoiodo
precursor 1, which we attribute to its having fewer heavy
atoms than the diiodo precursor 2. Moreover, the fluores-
cence quantum yields (Ffl) of fluorophore 3 (Ffl3 = 0.68) and 4
(Ffl4 = 0.81) were 22 and 40 times greater than those of their
precursors (Table S1). At this stage, it is important to note
that the fluorogenic reaction does not proceed in the absence
of any of three reaction components: phosphine ligands and
Pd, which form the catalytic complex, and iodo-BODIPY.
The suitability of this fluorogenic deiodination reaction
for DNA detection was tested with a DNA-templated catalyst
(Figure 2a). Triphenylphosphine ligands were individually
coupled through amide bonds to the 5’-end of probe L and 3’-
end of probe R (see Scheme S4 and p. 7 in the Supporting
Information for details on the purification and character-
ization). After HPLC purification, the phosphine-labeled
probes were annealed with the target strand (or template, T)
in a hybridization buffer (see p. 8 in the Supporting Informa-
tion). Subsequent addition of Na2PdCl4 with the reducing
agent NaBH4 resulted in an active catalytic complex owing to
the close proximity of the ligands attached to L and R. Low
probe concentrations (ꢀ 1 mm) were chosen so that Pd
complex formation would only occur through hybridization
to the template. It should be noted that in initial experiments
we investigated different templates T with various nucleotide
Figure 2. DNA-directed assembly of the catalyst and subsequent
generation of the fluorophore. a) Pd-catalyzed detection of the target
sequence (T, 30-mer) mediated by the complexation of phosphine-
modified ODN probes L and R (15-mer and 14-mer, respectively) with
Pd to form the Cat. site. b,c) Photographs of reaction mixtures
(precursor 2, L, R, Na2PdCl4, and NaBH4 in NaOAc buffer) with (right)
and without (left) T (1 nm) taken under UV (365 nm) exposure (b) and
ambient light (c). In the presence of the template, conversion of 2 to 4
resulted in a clear color and fluorescence emission.
gaps (0–4 nt) between the two annealing sites of L and R.
When the two probes were separated by 0, 1, and 2 nt an
active catalyst was formed; at greater distances (3 and 4 nt)
dehalogenation did not occur (data not shown). Since the
highest activity was achieved with a single nucleotide gap (see
Figure 2a), this hybrid catalyst in combination with profluor-
escent BODIPYs was used to detect the presence of a target
DNA sequence, which we investigated in terms of reaction
kinetics, scope, and detection limit.
The DNA-directed catalyst for the fluorogenic reaction
was tested at a range of concentrations of the target strand T
(from 1 nm to 1 fM) and iodo-BODIPY 1 or 2, with fixed
concentrations of the probes R and L, and catalyst Na2PdCl4,
and NaBH4 (see p. 8 in the Supporting Information). The
reactions were performed in NaOAc buffer at pH 5 under
inert conditions. It is noteworthy that the reaction required an
acidic pH probably to prevent complexation of Pd with the
nucleobases. After gentle mixing for 4 h, the reaction
mixtures exhibited the expected intense fluorescence result-
ing from multiple turnovers of the deiodination reaction
(Figure 2b). As negative controls, all reactions were also
performed without the template or catalyst, or with a template
containing a single-base mismatch (T-sbm). When the fully
complementary template was used, 90% of the fluorescence
maximum was reached after 4 min for a template concen-
tration of 1 nm, and saturation was achieved within 10 min
(Figure 3a, curve 1). This rate of reaction is twice that of
a DNA-templated Heck reaction used to deiodinate an
analogous BODIPY-DNA conjugate.[22a] A possible explan-
ation for this improvement is that the dehalogenation
reaction entails fewer intermediates than the Heck cross-
coupling.[23] It should be also noted that at lower template
concentrations (100 pm and 10 pm) the reaction is so rapid
(Figure 3a, curves 2 and 3) that detection assays could be
performed within only several minutes. In contrast, the use of
T-sbm in identical conditions slowed the reaction dramati-
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Figure 3. Fluorimetric determination of the reaction kinetics and the limit of detection of the catalytic conversion. a) Evolution of fluorescence
intensity over time at target concentrations (CT) of 1 nm to 10pm (curves 1–3), in the presence of T-sbm with catalyst (curve 4), in the presence of
T without catalyst (curve 5), and without T (curve 6) for the conversion of 1 into 3. b) Comparison of the reaction kinetics for the conversion of
1 into 3 under an argon atmosphere (curve 1) and under ambient air (curve 2) in the presence of CT =1 nm and 10 nm monoiodo substrate.
c) Fluorescence intensities corresponding to the the conversion of 1 into 3 for CT =1, 10, 100, 500, and 1000 fm (squares) and without template
(circles) at a fixed concentration of the monoiodo substrate of 500 fm. Magnified sub-picomolar range of the graph (inset). All fluorogenic
dehalogenations were monitored at 510 nm (lex =500 nm).
cally, reducing the initial increase in intensity by 65-fold
(Figure 3a, curve 4). Comparing this finding to an analogous
However, the amplified conversion to reporter dyes in our
system and the high extinction coefficient of BODIPY
analogues made such simple and immediate visual detection
possible without any additional instruments.
À
study using the Pd-catalyzed C C cross-coupling (55-fold
slowing),[22a] we see a greater degree of sequence selectivity
using the DNA hybrid catalyst.
When fluorescence rather than the relatively small
absorption shift is used as the sensing parameter, the
detection limit improves by up to 5 orders of magnitude.
This limit of detection was defined as the target concentration
at which the fluorescence signal could be distinguished from
that of the corresponding negative control without the
template, namely CT = 100 fm with diiodo-BODIPY 2 and
10 fm using monoiodo-BODIPY 1 as the chromophore
precursors (see p. 11 in the Supporting Information). The
difference in the detection limits for the two compounds is
a simple consequence of number of iodine atoms in the
precursors. Below a target ODN concentration of 100 fm, the
catalytic complex can still quite efficiently remove single
iodine atoms from the abundant profluorescent substrates,
yielding a strong signal for the conversion of 1 to 3. However,
precursor 2 must be deiodinated twice to produce a significant
fluorescence signal, and at such low concentrations of the
catalytic complex the predominant product is monohalogen-
ated 5 (Scheme S3), which has a low FQY (Ffl5 = 0.03) and
exhibits a much smaller blue shift in both fluorescence and
absorption (Figure S3). The different conversion capabilities
of the mono and diiodo precursors at low target concen-
trations were investigated further in terms of the quantitative
threshold for complete fluorogenic conversion as a function
of CT (see p. 12 in the Supporting Information). The lower
conversion efficiency for diiodo precursor 2 (Figure 4) at all
tested target concentrations is consistent with a single deiodi-
nation event per diiodo molecule. This result merits further
investigation to better understand catalytic conversions at low
concentrations. Single-molecule spectroscopy may be a suit-
able tool to study the efficient and sensitive fluorogenic
catalytic conversion presented here in greater depth, as using
DNA in our catalytic model offers several advantages: facile
functionalization, immobilization, and sequence-specific
assemblies.
In further experiments we investigated the scope and
utility of the DNA-mediated Pd-catalyzed dehalogenation
reaction. Since an inert gas atmosphere complicates the
applicability of an analytical assay we performed the catalysis
in the presence of air. As can be seen from Figure 3b, the
presence of oxygen affects the catalysis; however, the initial
rates for the two reactions are very similar, indicating that the
transformation still proceeds at a fast rate even in oxygenated
environments. In the next step, we performed the catalytic
dehalogenation assay in more complex environments. We
studied the performance of the catalyst in the presence of
crude cell extract and proteins (bovine serum albumine
(BSA) and DNA polymerase) (see p. 10 in the Supporting
Information). The results indicate that the catalyst remains
active in crude cell extract and in the presence of the
polymerase; however, it loses its activity when BSA is present
(Figure S6). It is not surprising that no activity was detected
for the sample containing BSA. This protein, which contains
35 cysteines, was applied in twofold excess relative to Pd and
it is well known that thiols are toxic for palladium catalysts.
From these measurements it can be concluded that the DNA-
mediated Pd catalyst system is robust enough for practical
sensing applications.
In terms of sensitivity, we investigated both colorimetric
and fluorimetric limits of detection. A series of reaction
mixtures with differing target concentrations and a fixed
concentration of 2 (0.1 mm) was incubated for 4 h, and a clear
transition from red to yellow could be observed with the
naked eye for target concentrations down to 1 nm (Figure 2c).
This visual ODN detection limit in our DNA-templated
system is somewhat limited compared to that of other
systems, such as plasmonic enhancement of inorganic par-
ticles[24] and the DNA-photograph technique,[25] because the
observation here depends solely upon the absorption shift of
the iodinated versus the deiodinated BODIPY from lmax
533 to 500 nm (Figure S1a in the Supporting Information)
=
In summary, we have developed a novel and modular
DNA–transition-metal hybrid catalyst based on the powerful
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Figure 4. Equivalents of di-/monoiodo (black/gray) dye molecules
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Received: July 26, 2012
Published online: October 17, 2012
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Keywords: bodipy · DNA catalyst · DNA sensing ·
fluorogenic reaction · signal amplification
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