Redmond Red, a fluoropore containing a redox-active phenoxazine core, has been explored as a new electrochemical probe for the detection of abasic sites in double-stranded DNA. The electrochemical behavior of Redmond Red-modified DNA at gold surfaces exhibits stable, quasi-reversible voltammetry with a midpoint potential centered around −50 mV versus NHE. Importantly, with Redmond Red positioned opposite an abasic site within the DNA duplex, the electrochemical response is significantly enhanced compared to Redmond Red positioned across from a base. Redmond Red, reporting only if well-stacked in the duplex, represents a sensitive probe to detect abasic sites electrochemically in a DNA-mediated reaction

Barton, Jacqueline K.

Buzzeo, Marisa C.

English

Caltech Authors - Main

Redmond Red as a Redox Probe for the DNA-mediated Detection
of Abasic Sites
Marisa C. Buzzeo and Jacqueline K. Barton*
Division of Chemistry and Chemical Engineering California Institute of Technology, Pasadena,
California 91125
Abstract
Redmond Red, a fluoropore containing a redox active phenoxazine core, has been explored as a new
electrochemical probe for the detection of abasic sites in double stranded DNA. The electrochemical
behavior of Redmond Red-modified DNA at gold surfaces exhibits stable, quasi-reversible
voltammetry with a midpoint potential centered around -50 mV versus NHE. Importantly, with
Redmond Red positioned opposite an abasic site within the DNA duplex, the electrochemical
response is significantly enhanced compared to Redmond Red positioned across from a base.
Redmond Red, reporting only if well stacked in the duplex, represents a sensitive probe to detect
abasic sites electrochemically in a DNA-mediated reaction.
Since its discovery, the ability of DNA to conduct charge has been exploited in a variety of
arenas, including time resolved spectroscopy (1-3), conductivity measurements (4-7), and the
development of biosensors (8). Our group, in particular, has explored the versatility of this
intrinsic property of DNA by performing extensive electrochemical studies on surfaces
modified with DNA duplexes (9-15). Typically in such systems, thiol-modified
oligonucleotides are tethered to a gold electrode at the 5′ terminus by a Au-S bond and further
modified at the opposite end with a redox probe, thus allowing the duplexes to act as an
extension of the conducting medium. The reduction of the attached probe, as observed
voltammetrically, serves as an indicator of the transport of charge via the DNA duplex.
Importantly, it has been shown that DNA-mediated charge transport is remarkably sensitive
to the integrity of the base stack; upon introduction of a single base mismatch located between
the electrode and the redox probe, measured currents are dramatically diminished (9-10). This
behavior not only provides conclusive evidence that the observed electrochemical reactions
are indeed DNA-mediated but also allows naturally occurring perturbations, which may only
subtly interrupt π-stack overlap, to be detected (11-15).
Among these possibly destabilizing defects are abasic sites, which appear naturally and
frequently among DNA sequences as the result of hydrolytic cleavage of the glycosidic bond
(16-23).Specific enzymes are responsible for correcting these damaged sites prior to
transcription and replication. When this repair pathway is delinquent, however, the persistence
of these defects can impart deleterious effects on genetic encoding, leading to the development
of cancer, among other diseases (24-26). Because they represent a potential threat, the reliable
and specific detection of abasic sites is critical, although still very much under exploration.
Development of an electrochemical system to detect these unwanted sites specifically could
E-mail: jkbarton@caltech.edu
*Author to whom correspondence should be addressed
Supporting Information Available: Synthetic details, electrochemical experimental conditions, and characterization data (HPLC, MS,
UV-vis, Tm) of modified DNA. This material is available free of charge via the Internet at http://pubs.acs.org/BC.
NIH Public Access
Author Manuscript
Bioconjug Chem. Author manuscript; available in PMC 2009 November 19.
Published in final edited form as:
Bioconjug Chem. 2008 November 19; 19(11): 2110–2112. doi:10.1021/bc800339y.
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contribute to the understanding of their occurrence and repair. Here, we employ Redmond Red
(RR), obtained as a commercially available phosphoramidite and used commonly as a
fluorophore, as a new redox probe for the detection of abasic sites. We find that the width of
the covalently tethered phenoxazine derivative is particularly well accommodated when
positioned opposite an abasic site within a DNA duplex, thus facilitating efficient DNA-
mediated reduction as a “turn on” probe for their presence (Figure 1).
Alkane thiol-modified DNA strands were synthesized as described previously (14,15) with
Redmond Red internal to the sequence and hybridized to a complement containing an abasic
site in the opposite position. All syntheses were performed using standard phosphoramidite
chemistry. Redmond Red-modified strands were cleaved from the solid support resin using
0.05 M K2CO3 in MeOH at ambient temperature for 12 – 17 h. Complementary strands were
cleaved using concentrated NH4OH at 60 °C for 8 – 12 h. Samples were purified twice by
reversed-phase HPLC and characterized by MALDI -TOF mass spectrometry and UV-visible
spectroscopy (see Supporting Information). For those oligonucleotides containing an abasic
site, the commercially available tetrahydrofuranyl analogue was used (27); the naturally
occurring, hemiacetal abasic residue is unstable and reactive on the time scale of DNA
synthesis.
Freshly etched gold macroelectrodes were incubated with 50 μM modified duplex DNA
overnight at 5 °C in the presence of 100 mM MgCl2 to encourage formation of densely packed
films. Electrochemical measurements were performed at ambient temperature in phosphate
buffer (5 mM sodium phosphate, 50 mM NaCl, pH 7) using a CH Instruments 760B
potentiostat. A saturated calomel electrode served as a reference electrode, and a platinum wire
as a counter. Square wave voltammograms were recorded at a frequency of 15 Hz and an
amplitude of 0.025 V. Under such conditions, Redmond Red-modified DNA (RRDNA)
displays stable, quasi-reversible voltammetry with a midpoint potential centered around -50
mV versus NHE. The measured peak currents scale linearly with scan rate, indicating that RR
behaves as a surface-bound species, as anticipated, and is not under diffusional control.
To test if the observed voltammetric response is in fact dependent on the presence of an abasic
site, the same thiolated RRDNA was then hybridized to complementary sequences containing
a guanine (G) or thymine (T) opposite the probe, and the resulting voltammetry recorded (see
Figure 2 for sequences). The observed reductive signal is severely affected. Figure 3 shows a
comparison of typical square wave voltammograms obtained for both assemblies, and Figure
4 presents a comparison of the average peak areas measured for the different sequences. More
than a two-fold increase is consistently observed when RR is situated opposite an abasic site
versus an intact DNA strand. It therefore becomes apparent that inclusion of an opposing base
precludes well stacked insertion of RR into the duplex, thus preventing efficient reduction
through the π-stack. Conversely, when an abasic residue is located on the complementary
strand, the phenoxazine derivative, itself having a similar dimension to a base pair, is now able
to insert into the duplex and become available for DNA-mediated reduction. The enhancement
of signal seen for the well stacked Redmond Red is consistent with earlier results that have
shown the redox signal to reflect how well the probe is coupled into the base stack (14,28).
This discrimination of abasic sites indicates that the DNA-mediated reduction of RR can
provide a convenient means by which to detect the abasic defect.
We also investigated whether or not RR would respond voltammetrically to a base deletion. A
complementary strand was synthesized in which the position opposite the RR was occupied
not by an abasic site, but instead by the base that corresponds to the next position on the RR
strand; in short, no complementary base was assigned to RR (Figure 2). Under these conditions,
we considered that the RR may be pinched out of the sequence, allowing the bases above the
probe to fully hybridize and thus inhibiting reduction of the phenoxazine moiety. For these
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experiments, the length of the oligonucleotide was extended by six bases in order to prevent
fraying above the probe site while keeping the position of Redmond Red relative to the
electrode surface constant. As observed by both cyclic and square wave voltammetry, duplexes
containing the deleted site do indeed consistently yield diminished currents, and the magnitude
of the effect (300 % increase for an abasic site) is in line with those results observed when a
G or T is situated opposite RR (Figure 4). This indicates that the probe is, in fact, excluded
from the base stack in order to allow for the remaining bases to hybridize and is therefore not
available for reduction in a DNA-mediated fashion.
Early work first demonstrated the sensitivity of electrochemical techniques to the presence of
abasic sites resulting from depurination in chromosomal DNA: differential poloragraphic
responses were observed for non-denatured DNA versus the apurinic form (29-31). More
recently, studies have reported the detection of these damaged sites specifically in synthetic
oligonucleotides using chronopotentiometric and square wave voltammetric techniques
(32-35). These systems, however, possess intrinsic limitations that prevent them from serving
as practical diagnostics. In the first example, the target DNA must be consumed via acid-
induced oligonucleotide digestion (32), while the other detection schemes are specific to
thymine-related mutations (33-35). Here, the redox probe is covalently attached to the DNA,
the target oligonucleotide remains intact, and the electrochemical measurement is fast, simple,
and selective. Moreover, electrocatalytic coupling may provide enhanced signal
discrimination, thus affording the increased sensitivity needed on the genomic scale (11,12).
The studies presented here once again highlight how sensitive DNA-mediated charge transport
chemistry can be in reporting on DNA structure. Earlier we used DNA-mediated
electrochemistry to characterize perturbations in the intervening DNA structures using a well
stacked probe, but here how well the probe is itself stacked serves as a specific reporter of local
conformation. The selective enhancement found with RR opposing an abasic residue confirms
that to detect efficient reduction of the probe at a distance from the electrode, the reduction
must be DNA-mediated. The dependence of probe detection on the spatial constraints of the
duplex underscores the general sensitivity of DNA-mediated charge transport chemistry to
local DNA conformation.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
We are grateful to the National Institutes of Health (Grant GM 61077) for their financial support.
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Figure 1.
Structure of Redmond Red (RR), an electroactive phenoxazine derivative covalently tethered
to DNA, along with a schematic representation of the basis of the abasic site probe. With
Redmond Red placed opposite an abasic residue (left), DNA-mediated reduction can proceed,
but if opposite a base (right), RR is excluded from the duplex and DNA-mediated reduction
cannot proceed. Note that the three-ring phenoxazine core is comparable in size to a base pair.
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Figure 2.
Sequences employed to determine the sensitivity of the DNA-mediated reduction of Redmond
Red to the presence of the opposing base. Shown are the thiol-modified strands, along with the
variable complementary strands, where “RR” indicates a Redmond Red moiety, and “__” an
abasic site.
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Figure 3.
Electrochemical reduction at a RRDNA-modified electrode, observed by square wave
voltammetry, when RR is positioned opposite an abasic site (left) or a guanine residue (right);
the reductive signal is significantly attenuated in the latter case. Peak areas were determined
by integrating above the baseline of the voltammograms, thus accounting for differences in the
background responses.
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Figure 4.
Bar chart representation of the differences observed in peak areas for the reduction of Redmond
Red when positioned opposite an abasic residue, a base, or a deletion site within a DNA duplex.
Peak areas, determined from square wave voltammetry, have all been normalized to the area
of the gold surface as determined by sulfuric acid etches performed immediately prior to
measurement. Error bars shown represent one standard deviation of the measurement.
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Redmond Red as a Redox Probe for the DNA-Mediated Detection of Abasic Sites

Abasic DNA structure, reactivity, and recognition.

Abasic sites in DNA: repair and biological consequences in Saccharomyces cereVisiae.

and de los

Apurinic sites as mutagenic intermediates.

Changes in DNA properties due to treatment with the pesticides malathion and DDVP.

Charge separation in DNA via consecutive adenine hopping.

Conductivity of a single DNA duplex bridging a carbon nanotube gap.

Coupling into the base pair stack is necessary for DNAmediated electrochemistry.

DNA binding shifts the redox potential of the transcription factor SoxR.

DNA glycosylases in the base excision repair of DNA.

Electrical detection of TATA binding protein at DNA-modiﬁed microelectrodes.

Electrochemical detection at low temperature for a speciﬁc nucleobase of target nucleic acids by an abasic site-containing DNA binding ligand.

Electrochemical detection of abasic site-containing DNA.

Electrochemical detection of lesions in DNA.

Electrochemical DNA sensors.

Electrochemical SNPs detection using an abasic sitecontaining DNA on a gold electrode.

Femtosecond dynamics of DNAmediated electron transfer.

Hole mobility in DNA A tracts.

In situ electrochemical distance tunneling spectroscopy of ds-DNA molecules.

Inﬂuence of abasic and anucleosidic sites on the stability, conformation, and melting behavior of a DNA duplex: correlations of thermodynamic and structural data.

Instability and decay of the primary structure of DNA.

Intercalative stacking: a critical feature of DNA charge-transport electrochemistry.

Long range electron transfer through DNA ﬁlms.

Mutation detection by electrocatalysis at DNA-modiﬁed electrodes.

Origin of endogenous DNA abasic sites in Saccharomyces cereVisiae.

Oscillographic polarography of highly polymerized deoxyribonucleic acid.

Recent progress in the biology, chemistry, and structural biology of DNA glycosylases.

Single-base mismatch detection based on charge transduction through DNA.

Single-strand interruptions in replicating chromosomes cause double-strand breaks.

Solution structure of duplex DNA containing an extrahelical abasic site analog determined by NMR spectroscopy and molecular dynamics.

Study of single-nucleotide polymorphisms by means of electrical conductance measurements.

The stalling of transcription at abasic sites is highly mutagenic.

Thermodynamic consequences of an abasic lesion in duplex DNA are strongly dependent on base sequence.

Use of an abasic site-containing DNA for electrochemical SNP detection.

Variable-temperature measurements of the single-molecule conductance of double stranded DNA.

Voltammetry of native double-stranded, denatured, and degraded DNAs.

https://authors.library.caltech.edu/12558/5/nihms-91115.pdf

Redmond Red as a Redox Probe for the DNA-Mediated Detection of Abasic Sites

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