nihms91231.pdf

DNA-Mediated Electrochemistry

Alon A. Gorodetsky

Marisa C. Buzzeo

, and

Jacqueline K. Barton

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,

California 91125

E-mail:

Alon A. GorodetskyMarisa C. BuzzeoJacqueline K. Barton

[jkbarton@caltech.edu]

Abstract

The base pair stack of DNA has been demonstrated as a medium for long range charge transport

chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to

structural perturbations in the base pair stack as occur with lesions, single base mismatches, and

protein binding. We have exploited this sensitivity for the development of reliable electrochemical

assays based on DNA charge transport at self-assembled DNA monolayers. Here we discuss the

characteristic features, applications, and advantages of DNA-mediated electrochemistry.

1. Introduction

Since the elucidation of the double helical structure of DNA, scientists have been fascinated

by the possibility that the stacked aromatic base pairs of the duplex may promote charge

transport (CT) over significant distances (1,2). Consequently, the nature of the conductive

properties of duplex DNA has attracted substantial interest (3–7). Over the past two decades,

a wide-ranging collection of experiments has both revealed the fundamental details of DNA-

mediated CT and illustrated its potential for sensing applications.

Initial solution experiments featured photoinduced DNA-mediated CT between well defined

donor and acceptor sites (

8,9

). While long-range CT has been shown to yield oxidative damage

in DNA up to 200 Å away from the bound oxidant (10,11), DNA CT has also been found to

be exquisitely sensitive to the integrity of the base pair stack (

9,12

) and to the coupling of the

donors and acceptors with the DNA (13). Indeed, this sensitivity has prompted both the

consideration of biological roles for DNA CT (14) and the construction of electrochemical

DNA-based sensors for mutations, base lesions, and protein-binding (15).

Electrochemical techniques provide a particularly convenient means for the study of

heterogeneous electron transfer at solid surfaces (16). Typically, redox-active molecules are

modified with thiol-terminated alkyl chains and self assembled as well ordered monolayers on

metal surfaces (17). Under an applied potential, electrons or holes are then transferred to

pendant, redox-active head groups with the rates and yield of charge transfer (as measured

electrochemically) providing information on the structure of the thiol terminated linker. In our

laboratory, we have extended this methodology to the study and application of DNA-modified

surfaces where charge transfer is

mediated

by the DNA monolayers, which are self-assembled

onto conductive substrates.

Before embarking on a general discussion of the application of this methodology, it is important

to have a clear understanding of the architecture of DNA-modified surfaces. Independent of

the material employed, duplex DNA is modified at the 5’ end of a single strand with a linker,

Correspondence to: Jacqueline K. Barton,

jkbarton@caltech.edu

NIH Public Access

Author Manuscript

Bioconjug Chem

. Author manuscript; available in PMC 2009 December 1.

Published in final edited form as:

Bioconjug Chem

. 2008 December ; 19(12): 2285–2296. doi:10.1021/bc8003149.

NIH-PA Author Manuscript

which allows it to interact with the electrode surface (Figure 1). In the case of gold, this tether

is an alkane thiol; for graphitic surfaces, it is pyrene. Electrodes are then incubated with

modified duplexes and an organized monolayer is allowed to self-assemble. A redox-active

molecule is introduced either by exposure to a solution or through covalent attachment to the

5’ terminus of the single strand lacking the linker. Subsequent addition of an inert backfilling

agent, such as mercaptohexanol, promotes the removal of non-specifically adsorbed duplexes

and single strands.

We have extensively characterized these DNA monolayers on both gold and graphite via

electrochemical and physical techniques (15). Structural characterization has confirmed that

the DNA duplexes within a DNA monolayer adopt an upright orientation, at an angle relative

to the surface. The uniformity and reliability of this structure enables the duplexes to serve as

an extension of the active electrode surface, thus allowing DNA-bound, redox-active molecules

to be reduced via the

-stack. Importantly, fabrication of the surface with rigid, structurally

defined DNA duplexes, rather than with single stranded DNAs that can stick to the surface in

ill-defined conformations, provides an enormous level of control and reliability in the

construction of the monolayers. Moreover, since the electrochemistry of the DNA probe is

remarkably sensitive to the integrity of the base pair stack, single base mismatches and lesions,

located between the surface and the probe dramatically attenuate the electrochemical yield of

CT. These findings have been extended to general assays for the study of DNA/RNA hybrids

and RNA transcripts. Furthermore, we have been able to detect a wide variety of DNA-binding

proteins based on their association and electronic communication with the base pair stack.

These data have underscored the strength and versatility of this methodology.

It is noteworthy that the charge transport properties of DNA monolayers are not simply of

utility in the context of electrochemistry. The base pair stack of each individual duplex serves

as a conduit for charge transfer, as recently confirmed by single molecule experiments within

our laboratory (18). In fact, when considered in the context of an electrical circuit, the DNA

duplexes and bound redox-active probes act as transducer elements with extraordinary gain;

biochemical binding events such as hybridization are sensitively transduced into electrical

signals. This emphasizes the role of DNA as a remarkably unique medium that provides

unprecedented opportunities for studies and applications of long-range CT.

Finally, this review is by no means exhaustive but rather seeks to provide an overview of DNA-

mediated electrochemical studies undertaken within our group. Here, we present our

explorations of features characteristic of DNA-mediated CT that have been critical for

applications of DNA monolayers as biosensors. In fact, several other reviews have detailed the

use of electrochemical DNA sensors (19–21), and where appropriate, examples from other

authors are presented.

2. Surface Characterization

For proper interpretation of experimental observations, extensive physical characterization of

these self-assembled DNA monolayers is crucial. A gamut of experimental techniques can be

utilized for investigating the structure and characteristics of DNA monolayers including (but

not limited to) radioactive labeling (22–25), fluorescence self-interference (26–28), scanning

tunneling microscopy (29–31), atomic force microscopy (23,25,32–36), scanning

electrochemical microscopy (37–39), and surface plasmon resonance (40,41). When dense

DNA monolayers are desired, the monolayers are assembled in the presence of Mg

to allow

for close packing of the duplexes (schematically illustrated in Figure 1). Our group, in

particular, has focused on surface characterization via radioactive labeling and scanning probe

techniques. These investigations have indicated that DNA monolayers adopt an upright

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orientation and have predictable surface coverages, thereby allowing us to develop a detailed

picture of DNA monolayer morphology.

Quantification of Surface Coverage Via Radioactive Labeling

Radioactive labeling of the duplexes provides a reliable measure of surface coverage for DNA

monolayers. In a typical radioactive labeling experiment, the 5’ end of the DNA

complementary to the thiolated strand is labeled with polynucleotide kinase, and the amount

of DNA is quantified directly via scintillation counting after self-assembly. On gold and

graphite, these measurements indicated surface coverages of ~40 pmol/cm

for well packed

DNA monolayers self assembled in the presence of Mg

(22,25

) and coverages of ~12 pmol/

for loosely-packed DNA monolayers in the absence of Mg

(23). Interestingly, the well

packed monolayer coverages obtained via radioactive labeling correspond to only ~75–85 %

of the theoretical coverages of ~60 pmol/cm

with DNA helices packed perpendicular to the

surface. This fact strongly hinted that the DNA is oriented at an angle with respect to the surface.

The reliability and sensitivity of radioactive labeling relative to electrochemical assays cannot

be understated. For example, monolayer formation can be

qualitatively

monitored via

attenuation of an anionic probe such as ferri/ferrocyanide, Fe(CN)

3-/4-

(22) In addition, a

cationic probe such as ruthenium hexammine can be utilized to quantify DNA coverages semi-

quantitatively (42,43

), but this common electrochemical assay of surface coverage can lead to

errors of up to 65 % within each experiment (44). Accordingly, in our laboratory, radioactive

labeling studies have proven to be particularly illuminating.

Scanning Tunneling Microscopy of DNA Monolayers

While radioactive labeling affords a quantitative measurement of the overall surface coverage,

the electronic structure of local areas of a DNA monolayer can be probed via scanning tunneling

microscopy (STM), providing atomic level information (29). Indeed, STM allows for the

investigation of the electronic properties of a DNA as a function of duplex orientation.

Therefore, the images obtained yield information on not only the morphology but also the local

density of states of the sample. At negative potentials, where the DNA adopts an upright

orientation, agglomerates of duplexes are visible because the tip can electronically access the

oriented DNA in an effective metal-molecule-metal junction. However, at positive potentials,

the DNA is encouraged to lie down and the DNA film is effectively not visible to the STM tip.

These data indicated that the DNA duplexes comprising a DNA monolayer aggregate in small

hexagonal groups of ~10 nm diameter. Furthermore, the inclusion of a mismatch within the

monolayer dramatically attenuates communication between the tip and the surface,

demonstrating that the STM is accessing the local density of states of the full DNA duplex.

Atomic Force Microscopy of DNA Monolayers

The morphology of self-assembled DNA monolayers can be further explored at the nanometer

scale with atomic force microscopy (AFM). These investigations have indicated that 5’ tethered

films are essentially smooth and featureless within the resolution of the AFM tip with the DNA

dispersed in a uniform manner (32). AFM measurements also provide important information

about the morphology and depth of the DNA monolayer at various substrate biases. By applying

a large downward force to the monolayer with the AFM tip, a bare spot can be uncovered on

the DNA-modified surface. Height contrast measurements between this bare spot and the

surrounding DNA monolayer indicate a film depth which ranges from 4.2 to 4.6 nm for 15 mer

duplexes at open circuit on both gold and graphite. In fact, the height of these films can be

reversibly modulated from 2 nm (the diameter of the duplex) at positive potentials to 5.5 nm

(the full length of the duplex when normal to the surface) at negative potentials (Figure 2).

These data, in conjunction with the above experiments, indicate that the duplexes adopt an

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upright orientation at negative potentials and are oriented at a ~45 ° angle with respect to the

surface in the absence of an applied bias 25,32).

In a complementary study, we also investigated alternative linkages of the thiol to the DNA.

Interestingly, 3’ rather than 5’ grafted DNA monolayers did not adopt an upright orientation

relative to the gold surface, as confirmed by their corresponding film depth of only ~2 nm.

Since vertical orientation of the monolayer is absolutely essential for studies based on DNA-

mediated CT, this work underscored the importance of considering linker placement when

designing self-assembled DNA monolayers (33).

Several other AFM investigations of DNA monolayers are particularly noteworthy, especially

when considering the electrochemical monitoring of DNA with larger macromolecules, such

as proteins. Loosely packed DNA monolayers assembled in the absence of Mg

are necessary

for improved accessibility of proteins, but such monolayers have a distinct “island”

morphology since the DNA has room to lie flat on the gold surface in the absence of an applied

potential (23). However, monolayers backfilled with a short chain alkanethiol such as

mercaptohexanol were demonstrated to adopt an upright orientation relative to the gold surface

even at open circuit (

35,36

). Furthermore, these studies showed that even loosely packed films

formed from longer 24 mer and 30 mer duplexes adopt an upright orientation. This well defined

morphology of “deeper” monolayers is a crucial design point to consider in the sequence-

specific detection of DNA binding proteins with extensive binding footprints.

3. DNA-mediated Electrochemistry of Small Molecules Bound to DNA

We have explored numerous redox-active probes in our investigations of DNA CT, and at first

glance, many molecules appear adequate for these studies. The large collection of experiments

conducted in our laboratory over the past decade, however, has taught us essential lessons

regarding the selection of an appropriate redox probe. Three criteria are paramount: the species

must (i) be electronically well coupled to the base pair stack, (ii) be reliably linked to the duplex

and (iii) undergo stable reduction or oxidation within a potential window that does not

compromise the integrity of the DNA monolayer. Furthermore, in addition to affording insight

into the optimum probe design, our systematic studies clearly indicate that no one probe is

ideally suited for every experiment. Instead, different markers offer distinct advantages and

must be individually evaluated for specific applications.

The behavior of the redox probes employed in our studies do, however, display some common

features when monitored electrochemically. When bound to DNA, small molecules appear to

act as surface-bound species as indicated by a linear relationship between the peak current and

scan rate. This relationship is not strictly linear for DNA-bound redox proteins, where the

proteins are not situated in one fixed position or conformation and thus exhibit a slight diffusive

component. Nonetheless, covalently probes bound to DNA typically do not display a square-

root-dependence of the peak current on the scan rate that is indicative of free diffusion. In

addition, DNA binding typically shifts the redox potential of the probe by 20–50 mV from

direct reduction at the bare surface and the voltammetry adopts a skewed shape relative to a

classically adsorbed couple; the reductive and oxidative peaks are separated slightly due to the

intervening DNA/alkanethiol spacer. Electrochemical reversibility and stability are also

specific to each probe, but they can be interrogated simply by repetitive cycling over the

potential window of interest. Taken together, all of these features allow us to distinguish DNA-

mediated processes from ones involving direct electrochemistry.

Early work in our laboratory employed probes that were not covalently attached to DNA but

instead interacted with the base pair stack electrostatically or via intercalation (22). Although

the majority of our studies have involved the intercalator methylene blue, we have also studied

the interaction between DNA and anionic ferricyanide, cationic ruthenium hexammine, the

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tris-heteroleptic intercalator Ir(bpy)(phen)(phi)

, and the anti-tumor agent daunomycin

(Figure 3). While these non-covalent markers have afforded a significant foundation for the

study of DNA-mediated CT, they are intrinsically limited in the mechanistic detail and

experimental control they can provide. As a result, more recent efforts have largely focused

on developing covalently bound probes, specifically those that are electronically well coupled

to the base pair stack. This latter family began with investigations of cross-linked daunomycin,

and has been more recently expanded to include other covalently attached molecules such as

anthraquinone and phenoxazine derivatives (Figure 3).

Electrochemistry of Small Molecules Non-covalently Bound to DNA

In our earliest electrochemical experiments, methylene blue (MB), a well studied aromatic

intercalator that undergoes a 2e

−

/1H

reduction in aqueous systems, was employed as a reporter

of DNA-mediated processes (

22). (Figure 4) shows a schematic illustration of the reduction of

MB at a DNA monolayer; a reversible redox couple is seen with a midpoint potential centered

−

10 to

−

50 mV vs. NHE, shifted by ~30 mV from bare gold. The measured peak currents

scale linearly with scan rate, indicating that the redox molecule is surface-bound, and a

combination of electrochemical and spectroscopic measurements give a saturation value of 1.4

(2) molecules per duplex at high MB concentrations. This latter finding suggests that, for well

packed monolayers, MB is constrained to the top of the film. Any observed reduction, therefore,

can be attributed to a DNA-mediated process, as the surface is inaccessible to the molecule.

To characterize this redox process in greater detail, a number of systematic studies were

conducted (44–49

. Initially, the behavior of the intercalator MB was compared to the groove-

binder ruthenium hexammine (47). At high salt concentrations, where interaction with the

charged duplex is inhibited, fewer molecules are reduced, thus illustrating the importance of

efficient and tight binding to the monolayer for charge transfer. The reduction of these two

probes was also monitored on surfaces that had been passivated with electropolymerized 2-

naphthol. If the DNA were truly serving as a medium for charge transport, passivation would

not affect the process being measured electrochemically. The reduction of ruthenium

hexammine, which is thought to proceed via facilitated diffusion along the duplex, decreased

by 70 % while reduction of MB was relatively unaffected. Although this study does not

elucidate the mechanism of MB reduction, it supports the notion of a DNA-mediated process.

To further prove that the reduction of distally bound probes proceeds in a DNA-mediated

fashion, we explored the electrochemistry of small molecules at DNA monolayers containing

single base mismatches positioned between the electrode surface and the probe (45). It was

theorized that if the DNA base pairs combined to form a continuous,

-stacked conduit for

charge transfer, that the disruption of a single base pair, as in a mismatch, could interrupt the

CT pathway. The presence of a single base mismatch causes no global change in the duplex

structure, and yet, the inclusion of an intervening CA mismatch proved to have a dramatic

effect on the efficiency of charge transfer to DM. These results are in excellent agreement with

STM studies of matched and mismatched duplexes and with studies of photoinduced DNA CT

in solution (9)

(vide supra)

. Interestingly, not only could several mismatches be detected but

the DNA-mediated chemistry was also found to be independent of sequence context.

Furthermore, this exquisite sensitivity to base pair stacking has since been observed for

numerous intercalating probes including MB and Ir(bpy)(phen)(phi)

but not for the groove-

binding ruthenium hexammine. Thus the observed electrochemistry is DNA-mediated and a

general characteristic of the DNA, independent of the redox probe.

To increase the sensitivity of mismatch detection and further improve discrimination, MB was

coupled to freely diffusing Fe(CN)

in an electrocatalytic cycle (Figure 5). In this catalytic

process, MB undergoes a two e

−

reduction via the base pair stack, and upon reduction to

leucomethylene blue, loses some affinity for DNA, thus dissociating from the duplex (

45,46).

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In turn, the cycle continues when leucomethylene blue is reoxidized by Fe(CN)

in solution,

and binds again to DNA for another cycle of DNA-mediated reduction. It should be noted that

Fe(CN)

cannot be reduced directly at the negatively charged monolayer owing to repulsion

from the polyanionic DNA monolayer. The entire process is effectively governed by the on/

off kinetics of the methylene blue/leucomethylene blue redox couple, as confirmed by rotating

disk electrode experiments (

48). In fact, during the course of a single voltammogram, the DNA

is repeatedly sampled, amplifying the absolute signal of MB and increasing the signal

attenuation associated with a mismatch.

The detection of all naturally-occurring mismatches and nearly all common lesions was

achieved with the MB/Fe(CN)

electrocatalytic couple via cyclic voltammetry and

chronocoulometry (Figure 6). In addition, low detection limits were achieved in a chip-based

format, with mismatch discrimination possible at electrodes as small as 30

∝

m in diameter

(46). It should also be noted that the relatively stable GA mismatch could

not

be detected

without the aid of the described catalytic cycle, emphasizing the utility of electrocatalysis as

an electrochemical tool. Furthermore, even base lesions that cause minor thermodynamic

destabilization to the duplex could be detected with electrocatalysis: a systematic investigation

revealed that the efficiency of CT was dramatically decreased even by subtle base

modifications that altered base structure, steric bulk, or hydrogen-bonding (49). Perhaps not

surprisingly, modifications with methyl groups that do not participate in hydrogen-bonding

had little to no effect on the DNA-mediated electrochemistry. The sensitive detection of all

single base mismatches independent of sequence context is remarkable and has proven to be

valuable in the application of DNA electrochemistry for the diagnosis of single nucleotide

polymorphisms.

MB electrochemistry was also extensively explored through self-assembled DNA monolayers

on highly ordered pyrolytic graphite (HOPG), onto which the DNA is anchored to the surface

via a pyrene moiety (25). As a surface, HOPG provides an attractive alternative since its

accessible potential window is no longer limited by the oxidative reduction or desorption of

thiols. The behavior of MB on graphite is quite similar to that on gold. Evidence that the charge

transfer is DNA-mediated is provided by the facile detection of the CA and GT mismatches.

The wider potential window afforded by HOPG allowed for the study of the voltammetry of

Ru(bpy)

dppz

and Os(phen)2dppz

. As expected, the electrochemistry of both intercalators

was found to be DNA-mediated.

Electrochemistry of Small Molecules Covalently Bound to DNA

To facilitate the systematic investigation of charge transduction of DNA, it became necessary

to employ redox-active probes that are covalently conjugated to the base pair stack. Our initial

efforts focused on covalently attached daunomycin (DM), which can be site-specifically cross-

linked to the exocyclic amine of guanine, forming a covalent bond, as shown in (Figure 7)

(50–52). Most importantly, this allows for controlled placement of the probe within the

sequence. Since functionalization with DM occurs after the synthesis of the DNA, it does,

however, necessitate that all guanines other than the one to be linked are replaced by inosine,

a close base analogue. For the purpose of electrochemical measurements, this substitution has

no influence on the results, but this requirement does have to be considered when developing

sensing applications. Importantly, covalent attachment of this probe for DNA electrochemistry

has made possible several seminal conclusions about DNA-mediated CT involving

ground-

state

reactants.

In initial experiments, the separation between the gold surface and DM adduct was varied from

15 Å to 45 Å, and no effect on the yield or rate of electron transfer was observed (50). The

inclusion of a mismatch in the intervening DNA, however, completely shuts off the

electrochemistry of DM, conclusively demonstrating that CT is DNA-mediated. Subsequently,

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with DM held in a fixed position in the duplex, the length of the thiolated alkyl tether was

varied to probe the effect of tether length on the rate of charge transfer (51). While the yield

of CT remained the same regardless of chain length (the number of intervening methylene units

was increased from 4 to 9), significantly, the rate of electron transfer decreased with increased

tether length with a

of 1.0 per –CH

–unit, as expected for the variation in coupling with

bonded systems. Therefore, CT through the alkyl tether, not through 30 Å of

-stacked DNA,

was the rate-limiting step of the DNA-mediated reduction. Taken together, these findings

illustrate that the DNA-DM construct behaves as a single redox-active entity with dramatically

different rates of CT through the stacked DNA and the a-bonded alkyl chain.

In a later study, DM was employed to prove that DNA-mediated electrochemistry occurs via

the base pair stack and not the sugar-phosphate backbone (52). With two DM moieties

crosslinked to guanine residues, voltammograms of duplexes containing a nick in the backbone,

a nick and a mismatch, a nick on both strands, and no modifications were compared. Nicks in

the phosphate backbone did not attenuate CT, but, consistent with previous results, mismatches

significantly attenuate the electrochemistry of DM. Interestingly, a later study on HOPG

showed that the electrochemistry of thiols incorporated within the sugar-phosphate backbone

was DNA-mediated but proceeded at low yield (53). Therefore, while DNA electrochemistry

proceeds through the base pair stack rather than through the sugar-phosphate backbone, it can

promote reactions on the DNA backbone.

While DM afforded several key findings for our applications, it does possess some inherent

limitations. As mentioned above, all guanine residues, other than the one to be linked, have to

be replaced by inosines, limiting its applicability in biosensors. Moreover, the stability of the

linkage between DM and DNA is heat-sensitive. As a result, we have been focused on

developing a new generation of probes that can be covalently attached to DNA with synthetic

flexibility, are stable, and are electronically well coupled to the base pair stack.

Through a variety of experiments conducted both in our laboratory and others, it has become

apparent that efficient coupling of the probe to the base stack is critical for efficient DNA CT.

Recently, the groups of Saito and Gooding introduced thymines modified with anthraquinone

as effective, covalently attached probes of charge transfer at DNA modified surfaces (

54–57).

Using this technology, Gooding and coworkers demonstrated the application of these modified

probes to the detection of primer extension reactions (54,55), and Saito and coworkers

demonstrated the photostimulated detection of mismatches with applications to genotyping of

single nucleotide polymorphisms (

56,57

). However, the low current densities obtained in these

studies led us to conduct a comparative study of anthraquinone linked to DNA through

conjugated and saturated tethers (58). Importantly, we found efficient redox chemistry and

mismatch sensitivity for anthraquinone only when linked through an alkyne and not when

tethered by an alkyl chain (Figure 8). Thus not only the choice of probe but also how the probe

is coupled to the base pair stack are key to effective DNA-mediated CT.

More recently, we have investigated two phenoxazine-based probes, Redmond Red and Nile

Blue, as covalent reporters of DNA CT (Figure 3). Redmond Red is commercially available,

affording facile incorporation into DNA with no sequence restrictions (

59), and Nile Blue can

be attached in high yield to DNA by reacting its exocylic amine with a carboxy-NHS-ester-

thymine (

60). These probes can be incorporated via standard phosphoramidite chemistry, have

virtually no sequence restrictions, are stable to light/heat, and support DNA-mediated

electrochemistry. Accordingly, they are preferable to DM for routine use and high throughput

experiments; both of these probes have already been applied for electrochemical monitoring

of protein/DNA interactions (

vide infra

). Furthermore, Nile Blue has proven effective for

detection of hybridization and has displayed electrocatalytic activity (unpublished results).

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Redmond Red, moreover, has proven to be a particularly sensitive reporter of abasic sites in

DNA. The development of these probes holds great promise for future experiments.

4. Electrochemical Detection of Hybridization and Transcription Products

The studies described thus far report of findings achieved with pre-hybridized duplexes on

gold or graphite; the monolayers were formed exclusively via self-assembly of duplex DNA

prior to electrochemical interrogation. It is of tremendous importance, from both a fundamental

and an applications perspective, to demonstrate similar behavior of DNA-mediated

electrochemistry when duplexes are instead formed

in situ

. This approach allows for repeated,

internally normalized investigations of a single surface, thereby laying the groundwork for the

detection of lesions, mutations, and mismatches in chip-based genetic analysis.

Initial promising results have led current efforts to focus on the design and development of an

electrochemical assay for the products of transcription. Consequently, we first explored DNA-

mediated CT in DNA/RNA hybrids. Given the known correlation between several RNA

sequences and the development of disease, the ability to simply and quantitatively detect the

concentration of particular RNA sequences could ultimately be exploited for both disease

diagnosis and chemotherapeutic design.

Electrochemical Detection of Mismatches In Situ

A comparative

in situ

experiment on modified gold surfaces allowed for a CA mismatch to be

reversibly detected via DNA-mediated CT (45). DNA was immobilized on two separate

electrodes, one bearing duplexes with well matched complements, the second containing

duplexes with a single base mismatch. Following exposure to a solution of DM, the electrodes

exhibited the behavior expected for their respective complements, namely the signals were

much larger for the well matched duplex. The duplexes were then dehybridized by heat

denaturation and re-incubated with the opposite complement; the resulting well matched

duplex exhibited an increased signal while the response from the mismatched duplex displayed

significant attenuation. Importantly, the signals observed for single-stranded DNA were

qualitatively different from those observed for either mismatched or well matched DNA,

confirming duplex formation.

By utilizing the MB/Fe(CN)

electrocatalytic couple, highly sensitive detection limits for

hybridization have been achieved (46). Monolayers containing well matched DNA were first

self-assembled at gold microelectrodes manufactured in a chip-based format and investigated

with chronocoulometry. The DNA was subsequently rehybridized with 100 pM of mismatched

complement before chronocoulometry was performed again (Figure 9). Dramatic signal

attenuation was observed

in situ

in the presence of even a single mismatch. Controls performed

with single stranded DNA again yielded voltammetry which was broad, weak, irreproducible,

and qualitatively different, again confirming duplex formation.

In addition to cyclic voltammetry and chronocoloumetry, an alternative electrochemical

technique, electrochemical impedance spectroscopy, can also report on duplex formation

situ

. Differences in the surface coverage are reflected in the electron-transfer resistance,

(

), as determined by this measurement. Using Fe(CN)

as an electrochemical reporter,

electrode surfaces were taken through several dehybridization/rehybridization cycles (using

simple heat denaturation and room-temperature annealing), where the impedance at the surface

was monitored at each step. The sensitivity of this technique allowed for excellent

discrimination between dehybridized and rehybridized surfaces, allowing for label-free

detection of duplex formation (44).

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Electrochemistry Through DNA/RNA Hybrids

As a complement to the hybridization studies described above, our laboratory has also

investigated DNA-mediated charge transport through DNA/RNA hybrids directly. It is known

that these naturally-occurring intermediates adopt a conformation that more closely resembles

A-form DNA, whereby the helix is significantly wider and more compact than canonical B-

form. While these structural differences could potentially interfere with a charge transport

pathway down the helix, electrochemical studies within our laboratory have demonstrated that

DNA/RNA hybrids are indeed capable of conducting charge in a manner similar to DNA/DNA

duplexes (

61,62

). Non-covalent MB reduced via a DNA/RNA hybrid exhibits nearly identical

voltammetric behavior to that observed with DNA. Moreover, electrocatalytically coupling

MB with Fe(CN)

affords discrimination of all possible mismatches in DNA/RNA hybrids,

as was monitored by chronocoulometry. These studies reveal that DNA-mediated CT can serve

as an effective methodology for the detection of transcription products. Current efforts are now

focused on the development of a sensitive and quantitative electrochemical hybridization assay

for mRNA using covalently attached anthraquinone.

5. Electrochemical Monitoring of Protein/DNA Interactions

Self-assembled DNA monolayers also provide particularly convenient platforms for electrical

monitoring of protein/DNA interactions. These interactions play crucial roles in many cellular

processes such as transcription, repair, and replication. In particular, the association of

transcription factors with DNA is an important area for exploration in proteomics and

genomics; it is these interactions that control the developmental and regulatory responses of

the cell, often in a complicated fashion. Therefore, the development of convenient and

inexpensive methodologies for monitoring protein-DNA interactions remains of critical

importance.

In a typical experiment, a loosely packed DNA monolayer is self-assembled with or without

a covalently appended probe and the surface is backfilled (Figure 10). Subsequently,

voltammetry of the DNA-modified electrode is recorded before and after addition of protein.

Detection of protein binding can be achieved in two ways: 1) proteins that perturb the base pair

stack will attenuate the yield of DNA CT to a distally-bound electroactive probe and 2) proteins

featuring a redox-active cofactor such as an iron-sulfur cluster will cause the appearance of a

new DNA-dependent electrochemical signal. These experiments have particularly benefited

from the development of covalently attached probes which have allowed for conclusive

quantification of the yield of DNA CT before and after protein binding. Furthermore, such

electrochemical assays are general and based on exquisitely sensitive DNA-mediated

electrochemistry, so a wide range of protein/DNA interactions and binding motifs can be

sequence-specifically interrogated in real time.

Detection of Transcription Factors

Our early studies at DNA-DM monolayers demonstrated that we could easily detect TATA-

binding protein (TBP), a ubiquitous transcription factor that bends duplex DNA by ~90°

(23). The addition of TBP to a DNA-modified surface results in distortion of the DNA and

lowers the yield of DNA CT (Figure 10). Later, we extended this methodology to the detection

of TBP at DNA-modified microelectrodes (

60). At DNA monolayers modified with Nile Blue,

TBP could be readily detected at either the macro-or microscale. However, microelectrodes

allowed for the rapid detection of nanomolar concentrations of this transcription factor: 300

nM TBP could be reversibly detected with total signal loss in less than 30 seconds. Furthermore,

30 nM concentrations of TBP were detected even in the presence of micromolar amounts of

bovine serum albumin, EndonucleaseIII, or Bam HI methyltransferase. These data hinted at

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the potential of assays based on DNA CT for the high throughput, multiplexed electrical

monitoring of numerous DNA binding proteins on a single chip.

Detection of Base Flipping Enzymes

Methyltransferases are base-flipping enzymes that catalyze the transfer of a methyl group from

S-adenosyl-methionine to adenine or cytosine within a DNA duplex. Establishing base flipping

by a protein was historically quite difficult, usually requiring a crystal structure, but the

electrical detection of base flipping using DNA electrochemistry has been found to be a

sensitive, rapid, and attractive method for this characterization.

While methyl substitution does not appreciably perturb the base pair stack, methyltransferases,

in carrying out the base flip, significantly attenuate DNA-mediated CT. Based upon this

attenuation, we have detected base flipping in binding both uracil DNA glycosylase and

HhaI

methyltransferase to their cognate sequences (23). At a surface featuring a DNA-DM

monolayer, for example, the addition of

M.HhaI

disrupts the integrity of the base pair stack

with the concomitant decrease in the DM redox signal. Based upon the crystal structure of the

protein/DNA complex, it is apparent that upon base flipping the internal cytosine of the

recognition sequence 5’-GCGC-3’,

M.HhaI

also intercalates Gln 237, a non-aromatic residue

into the base pair stack, filling the space occupied by the flipped out cytosine, and interrupting

the

-stacking within the duplex. Accordingly, electrochemistry experiments were conducted

with a Q237W mutant enzyme. In this case, we expected that the mutant protein would insert

the aromatic tryptophan residue into the

-stack upon base flipping, so little signal loss would

be found; indeed, little attenuation in DM signal upon binding the mutant protein was observed.

Moreover, to establish that the lack of attenuation was the result of restoration of the

-stack

and not simply poor DNA binding by the mutant, we also examined binding of both wild type

and mutant

M.HhaI

to a DNA-DM monolayer containing an abasic site at the position of what

would be the flipped out cytosine. On this DNA-DM monolayer, the DM redox signal is only

weakly detected both in the absence of protein and in the presence of wild type

M.HhaI,

owing

to the poor stacking of the duplex with an intervening abasic site. Nonetheless, in the presence

of the Q237W mutant, the DM redox signal is restored. This enhanced signal for DM on binding

the mutant reflects insertion of the tryptophan from the mutant protein within the base pair

stack, so as to restore proper stacking in the DNA-DM duplex. Hence DNA-binding proteins

are seen to modulate DNA CT both positively and negatively, depending upon how they affect

the conformation of the DNA.

Electrical Monitoring of Proteins in Real Time

Another advantage of monitoring protein binding to DNA electrically comes from the fact that

this technique allows the sensitive detection of events in real time. This was illustrated first by

showing double-stranded cleavage on a DNA-DM monolayer using the

Pvu

II restriction

enzyme (23). In the presence of magnesium ion,

Pvu

II cleaves DNA sequence-specifically.

Addition of

Pvu

II to a DNA monolayer containing the PvuII recognition sequence with DM

attached to the top of the film but without magnesium causes little perturbation in the DM

redox signal. However, the addition of magnesium ion is seen to trigger loss of the DM signal;

as the protein cleaves the DNA, the fragment containing the redox probe is released into

solution. Interestingly, the kinetics of this signal loss parallels closely that seen for the protein

restricting DNA in solution followed by DNA cleavage analysis using gel electrophoresis. The

advantage of the electrochemistry technique, however, is clear: the restriction reaction can be

monitored in real time.

Monitoring the repair of thymine dimers in DNA by photolyase provides another illustration

of how protein/DNA reactions may be monitored sensitively and in real time using DNA

electrochemistry (63

). Photolyase is a repair enzyme from

E. coli

containing a flavin cofactor

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