DNA-nanoparticle micelles as supramolecular fluorogenic substrates enabling catalytic signal amplification and detection by DNAzyme probes

Article abstract of DOI:10.1039/c0cc02291h

Catalytic DNA molecules have tremendous potential in propagating detection events via nucleic acid sequence selective signal amplification. However, they suffer from product inhibition limiting their widespread utility. Herein, this limitation is overcome utilizing a novel fluorogenic substrate design consisting of cooperatively assembled DNA-nanoparticle micelles.

Full text of DOI:10.1039/c0cc02291h

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DNA–nanoparticle micelles as supramolecular fluorogenic substrates
enabling catalytic signal amplification and detection by DNAzyme
probeswz
Miao-Ping Chien, Matthew P. Thompson and Nathan C. Gianneschi*
Received 1st July 2010, Accepted 12th August 2010
DOI: 10.1039/c0cc02291h
Catalytic DNA molecules have tremendous potential in
propagating detection events via nucleic acid sequence selective
signal amplification. However, they suffer from product inhibition
limiting their widespread utility. Herein, this limitation is
overcome utilizing a novel fluorogenic substrate design consisting
of cooperatively assembled DNA–nanoparticle micelles.
To enzymatically amplify a signal one requires a triggering
mechanism that connects the detection event to the catalytic
signal transduction process. This is the cornerstone of
biologically regulated catalytic systems for signal transduction
in living systems. Several effective enzymatic systems have
been reported that use various types of DNA sequence and
structure selective processes for detecting DNA via catalytic
1
–19
turnover for signal amplification.
several efforts to harness catalytic single-stranded DNA
These have included
(
ssDNA) sequences as phosphodiesterases with ribonuclease
2
0–23
activity (DNAzymes
) in a variety of assay formats usually
aimed at activating fluorogenic nucleic acid substrates via
2
4–32
strand cleavage reactions (Fig. 1).
DNAzymes offer a
unique opportunity for detection and signal amplification
because unlike proteinaceous enzymatic endonucleases,
DNAzymes are not restricted to a given sequence but rather
are synthesized in the laboratory to desired specifications
making them selective for any given sequence. In this respect,
DNAzymes are unique and potentially powerful tools in
biodiagnostics because they have tailorable sequence selectivity
and are potentially catalytic. Herein, their utility as selective,
triggerable catalysts for signal amplification by true turnover
is demonstrated in the context of a DNA detection protocol
Fig. 1 The design of supramolecular substrates capable of multiple
turnovers compared to single-stranded DNA (ssDNA) substrates.
Active DNAzymes recognize fluorogenic substrates and catalyze
strand cleavage at RNA bases. (a) ssDNA substrates are typically
modified with fluorophore and quencher pairs for activation via
sequence selective cleavage. (b) The DNA–nanoparticle micelles
formed via the assembly of DNA–brush copolymer surfactants.
Dye-labelled DNA strands within the particles are recognized and
cleaved by a DNAzyme enabling detection of fluorescence.
3
3
enabled by supramolecular fluorogenic substrates. Given the
product having the same DNA sequence and one must
balance turnover of cleaved substrate with binding energy as
with any system designed with substrate selectivity and
turnover in mind. We reasoned that if one could generate
a cooperatively assembled supramolecular fluorogenic substrate
then DNAzyme catalytic activity would be enhanced by virtue
of increasing effective DNA substrate concentration upon
numerous modes of selective recognition open to ssDNA (e.g.
3
4–36
18,37
hybridization,
aptamer-based recognition,
enzymatic
3
8
39
substrates, thermal responsiveness ) it is expected that
harnessing DNAzymes in catalytic detection protocols will
have broad utility.
Currently, a major limitation of DNAzymes in signal
amplification and detection via catalysis is that they are
product inhibited and are therefore typically limited to a single
turnover or less. This limitation is caused by substrate and
1
7,40–43
DNAzyme recognition of the probe.
A DNA–brush
copolymer capable of assembling into an aggregate structure
was found to be capable of achieving this goal (Fig. 1).
The key benefit to this assembly over material simply
decorated with DNA is that in this system the substrate is
within the internal structure increasing the density of the
probe. We reasoned that a micellar aggregate consisting
Department of Chemistry & Biochemistry, University of California,
San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
E-mail: ngianneschi@ucsd.edu
w This article is part of the ‘Emerging Investigators’ themed issue for
ChemComm.
z Electronic supplementary information (ESI) available: Experimental
details including synthesis and characterization. See DOI: 10.1039/
c0cc02291h
of a material formed from the DNA substrate itself
would allow sequence selective turnover not accessible to
single-stranded DNA fluorogenic substrates such as molecular
3
4,36
beacons.
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 167–169 167

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fluorogenic substrate particle is greatly enhanced compared
to the single-stranded fluorescent DNA substrate (F-ssDNA)
(
Fig. 2). The multiple turnovers observed for the DNAzyme
on the particle compared with the F-ssDNA is presumably due
to the high effective concentration of substrate in the particle
shell enabling enhanced, efficient shell strand truncation.
Importantly, the ordinarily product inhibited DNAzyme is
capable of multiple turnovers and complete shell degradation
occurs within 15 minutes following addition to a solution of
the particles (complete turnover was confirmed via calibration
curve, see ESIz). This is in comparison to the low turnover
observed for F-ssDNA substrate over the same time scale.
To demonstrate the utility of this substrate design in signal
2
6,28
amplification via DNAzyme-mediated sensing,
designed an assay (Fig. 3) composed of a DNAzyme
we
(
(
DNAzyme-1), its inhibitor (DNA-Inh), a target sequence
DNA-T; sequence from HIV-1 gag/pol gene) and a supra-
molecular substrate particle, labelled P-1 in Fig. 3, encoded
with single-stranded substrate (sequence = DNA-1). First,
DNAzyme-1 was mixed for 30 min with DNA-Inh, designed
to hybridize via the recognition sequences in the DNAzyme
and block the active site. This inhibited DNAzyme complex
was then mixed with varying concentrations of DNA-T and
incubated for 30 min. Once added, DNA-T rapidly invades
into the inhibited DNAzyme duplex (DNAzyme-1ꢂDNA-Inh),
releasing active DNAzyme-1 and forming the new, longer
duplex DNA-TꢂDNA-Inh. Substrate particles (P-1) were
Fig. 2 Structure of supramolecular substrate micelles and turnover
of supramolecular fluorogenic substrate particle vs. single-stranded
fluorescent substrate (F-ssDNA). TEM data indicate 20 nm particles
assembled from the DNA–brush copolymer. PEG = polyethylene
glycol incorporated via phosphoramidite chemistry, see ESIz for
structural details. Conditions: particle DNA (1 mM), F-ssDNA
(1 mM), DNAzyme (5 nM). Buffer: Tris (20 mM, pH 7.4), MgCl
2
(
50 mM), room temp. DNAzyme utilized in these studies has sequence;
0
DNAzyme-1: 5 -GGAGAGAGATCCGAGCCGGTCGAAGGGT-
0
GCGA-3 .
The DNAzyme catalyst consists of a conserved DNA
0
0
sequence forming a catalytic domain with 5 - and 3 -ends as
2
2
recognition sequences. Therefore, a DNA substrate with a
single RNA base (rA) as the site of cleavage was chosen for
incorporation in the DNA–brush copolymer architecture used
to form micellar aggregates (Fig. 2). The DNA substrate was
designed to incorporate two sequences complementary to the
recognition portions of the DNAzyme on either side of the
RNA-base cut-site as well as incorporating a fluorescein
0
moiety on the 3 -termini of the strand to allow for detection
and analysis of the cleavage reaction by fluorescence
spectroscopy. The polymers utilized in these systems were
formed with low polydispersity and well-defined block
structure amenable to post-polymerization modification with
0
the 5 -amino modified oligonucleotide (see ESIz). The resulting
DNA–brush copolymer was dialyzed in buffered water (Tris,
Fig. 3 Selective and sensitive DNAzyme catalyzed DNA–shell
truncation. Inhibited DNAzyme-1 (duplex: DNAzyme-1ꢂDNA-Inh)
is treated with target strand (DNA-T) over a range of concentrations
causing displacement of DNAzyme-1 and formation of a new duplex,
DNA-TꢂDNA-Inh. Activated DNAzyme-1 binds to and cleaves the
particle shell releasing fluorescein-labelled ssDNA. (a) DNAzyme-1
triggering by target DNA (DNA-T). (b) Selectivity of DNAzyme
catalyzed reactions; fluorescence measurements taken 5 minutes
following addition of respective DNAzymes. Columns left to right:
2
0 mM, pH 7.4) to obtain spherical DNA nanoparticles
approximately 20 nm in diameter as shown by transmission
electron microscopy (TEM, Fig. 2), dynamic light scattering
3
DLS) and atomic force microscopy (AFM). Initially,
3
(
ꢀ1
particles were mixed with DNAzymes in 10 000 g mol
molecular weight cut off (MWCO) centrifuge tubes. Cleavage
of the DNA shell at the RNA base (rA) cut-site releases a
fluorescent 10 base ssDNA product into solution from the
particle surface resulting in a shell containing a truncated
(1) signal from P-1 without DNAzyme. (2) P-1 mixed with DNAzyme-1.
(
3) P-1 mixed with DNAzyme-2. (4) P-2 (from copolymer with
9
3
-base long sequence. By centrifuging at 14 000 ꢁ g for
sequence, DNA-2) mixed with DNAzyme-2. (5) F-ssDNA mixed with
0 seconds at given time points, the ssDNA product was
DNAzyme-1. Conditions: particle DNA (1 mM), DNAzyme (5 nM).
separated from particles as it passed through the MWCO filter
for analysis by fluorescence. The identity of the product was
confirmed by MALDI mass spectrometry (see ESIz).
The DNAzyme catalytic efficiency in the supramolecular
2
DNA-Inh (5 nM). Buffer: Tris (20 mM, pH 7.4), MgCl (50 mM),
0
room temp. DNAzyme-2: 5 -AACACACACTCCGAGCCGGTC-
0
0
GAAAGCTTTCTGAT-3 . DNA-2: 5 -ATCAGAAAGCTrAGGTG-
0
TGTGTT-3 -PEG-Fluorescein.
1
68 Chem. Commun., 2011, 47, 167–169
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added to the solution of activated DNAzyme-1 causing
particle shell recognition and cleavage. Product strands were
again separated by filtration from particles and analyzed by
fluorescence. The observed pM sensitivities (Fig. 3a) for the
detection and signal amplification of ssDNA confirm the
turnover of substrate on the particle by DNAzyme-1.
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In addition, this process is sequence selective, as evidenced
by the selective inhibition and triggering of DNAzyme-1 and
the observation that the non-complementary DNAzyme-2 has
no effect on P-1 (Fig. 3b). In turn, DNAzyme-2 catalyzes shell
degradation when mixed with particles containing the
complementary sequence, DNA-2 (P-2). The above studies
illustrate two key features of this system: (1) particles are
sensitive to low concentrations of catalytically active
DNAzyme. (2) The particles are susceptible to degradation
in a sequence selective and concentration dependent fashion.
In conclusion, the present study has introduced a novel
approach to substrate design enabling a DNAzyme to be
utilized in a truly catalysis-based amplification and detection
assay. This affords a route toward DNAzyme detection assays
of various types where the myriad ways of recognizing ssDNA
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