nihms97829.pdf

Electrical Detection of TATA Binding Protein at DNA-Modified

Microelectrodes

Alon A. Gorodetsky

Ali Ebrahim

, and

Jacqueline K. Barton

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

California 91125

Sensitive methods to detect the specific binding of proteins to DNA are essential in monitoring

pathways for gene expression and in the development of new diagnostics of disease states.

There are several high-throughput, array-based methodologies currently available for

monitoring transcription factor binding to DNA including immunoprecipitation of reversibly

crosslinked chromatin/protein complexes,

capture of fluorescent antibodies by epitope-tagged

DNA-binding proteins,

and amplification of sequences methylated by enzyme/DNA adenine-

methyltransferase fusions.

However, these methods rely on fluorescence detection and require

DNA amplification and/or protein labeling.

Electrochemical assays based on DNA-mediated charge transport at microelectrodes offer an

alternative approach.

DNA electrochemistry has been useful in assays to detect mutations and

lesions

–

as well as for the detection of protein binding at self-assembled DNA monolayers.

–

Moreover, DNA electrochemistry provides the opportunity for highly sensitive detection

in a multiplexed format.

However, there are few reports of the electrical detection of protein

binding utilizing DNA monolayers at small scales.

Using DNA-modified

macroelectrodes

we have previously detected micromolar concentrations of TATA-binding protein (TBP),

ubiquitous transcription factor that severely bends the DNA duplex upon binding.

Here, we

describe the rapid and specific detection of nanomolar concentrations of TBP at

microelectrodes

modified with DNA containing a new, easily prepared redox probe.

The strategy utilized for detection is schematically illustrated in Figure 1. Two complementary

17-mer strands incorporating the 5

′

-TATAAAG-3

′

TBP binding site were prepared. The 5

′

end

of one strand was modified with an alkane thiol, and the terminal T at the 5

′

end of the

complementary strand was modified with Nile Blue (NB), our redox-active probe.

The

thiol- and NB-modified strands were thermally annealed in equimolar amounts to form duplex

DNA. Loosely packed DNA films were then self-assembled for 24–48 h on gold electrodes

(Bioanalytical Systems) using procedures described.

Binding of TBP to its target site, as

found earlier,

kinks the DNA duplex and perturbs the base pair stack, attenuating the DNA-

mediated reduction of the redox probe (Figure 1).

Cyclic voltammetry (CV) and square wave voltammetry (SWV) of both a macroelectrode and

a microelectrode modified with NB–DNA duplexes are shown in Figure 2. The CV at the

macroelectrode is reversible with a midpoint potential of

−

220 mV (±50) versus Ag/AgCl.

For macroelectrodes, the cathodic and anodic waves are symmetric (though not overlapping),

E-mail: jkbarton@caltech.edu.

Supporting Information Available:

Synthesis and characterization of the Nile Blue/DNA conjugate. Details of the KCl wash showing

reversible TBP binding. Voltammograms after addition of lower concentrations of TBP to DNA-modified microelectrodes.

Voltammograms revealing the effect of oxygen and alternative gold surfaces. Details of protein preparation and quantification. This

material is available free of charge via the Internet at http://pubs.acs.org.

NIH Public Access

Author Manuscript

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 21.

Published in final edited form as:

J Am Chem Soc

. 2008 March 12; 130(10): 2924–2925. doi:10.1021/ja7106756.

NIH-PA Author Manuscript

and a plot of peak current as a function of scan rate is linear as expected for a surface-bound

species.

For microelectrodes with critical dimensions

≤

m, the CV differs in shape and

the midpoint potential appears to be systematically shifted by

∼

100 mV.

–

It is also notable

that the background capacitance is significantly smaller at the microelectrode relative to the

signal size.

Upon addition of 300 nM TBP, reduction of DNA-bound NB is dramatically attenuated at both

the macro- and microscales (Figure 2).

For macroelectrodes, addition of the protein causes

a signal loss of 60% with the signal slowly decreasing over a period of 15 min. For

microelectrodes, 100% signal loss occurs in less than 30 s. The consistently greater signal loss

and appreciably faster equilibration on the microelectrode may reflect better access of the

protein to the NB-modified DNA on the smaller surface. It is also noteworthy that binding of

TBP, in kinking the DNA,

may bring NB in closer proximity to the surface, yet a

decrease

is observed in the NB signal. This observation is fully consistent not with contact of NB to the

surface but instead a DNA-mediated reduction that is inhibited by bending the intervening

DNA.

TBP binding and the resulting signal attenuation are fully reversible on a microelectrode

(Supporting Information). A microelectrode incubated with TBP can be washed in 1 M KCl,

promoting protein dissociation

and rapid recovery of the NB signal. Electrodes can be taken

through this cycle up to three times with no loss of response. In addition, detection of low

concentrations of TBP is easily achieved (Supporting Information). At 30 and 15 nM protein

concentrations, complete NB signal loss is observed. At 3 nM protein, the NB signal is partially

attenuated, as expected based upon the affinity of the protein for its target sequence.

The detection event is furthermore specific to TBP (Figure 3). Bovine serum albumin (BSA),

a protein that does not bind DNA, has little effect on DNA-mediated electrochemistry or on

signal attenuation associated with TBP binding.

Micromolar Endonuclease III (EndoIII),

a glycosylase that targets damaged pyrimidines (not unlike the NB-modified thymine), also

has little effect on the DNA-mediated NB signal.

Finally, the addition of BamHI

methyltransferase (BAM), a base-flipping methylase that targets 5

′

-GGATCC-3

′

yields

little change in the voltammetry or the signal decrease associated with TBP binding. Although

EndoIII and BAM likely bind nonspecifically to the DNA film at these concentrations, they

do not inhibit specific binding by TBP. These results are therefore consistent with observing

a sequence-specific TBP binding event.

We have thus demonstrated the electrochemical detection of nanomolar TBP binding to DNA

in the presence of other proteins with a rapid and sensitive electrochemical assay. This

methodology is based upon an interruption of DNA-mediated electrochemistry associated with

protein binding and therefore may be generally applied in detecting other proteins that perturb

the DNA base stack. Our ability to detect protein binding to DNA reliably at small scales

provides a foundation for rapid, electrical assays of DNA-binding proteins on a single chip.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We are grateful to the NIH (GM61077) for their financial support.

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′

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22. We have found that the midpoint potential of NB–DNA monolayers on macroelectrodes vary

depending upon the gold surface utilized, leading to an uncertainty of

∼

50 mV in the potentials,

although they have a higher certainty within a given surface. Moreover, there is a systematic negative

shift in potential of

∼

100 mV on the microelectrodes versus the macroelectrodes. Comparable shifts

have been seen by others (see ref 18 b,c) and may reflect the different geometries of DNA monolayers

on the different surfaces. See Supporting Information for further details.

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24. At small scales, we observe a partially sigmoidal shape for the voltammetry of Nile Blue–DNA films.

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as reported in Ju H, Shen C. Electroanalysis 2001;13:789.. See Supporting Information.

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. Author manuscript; available in PMC 2009 September 21.

NIH-PA Author Manuscript

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Figure 1.

Schematic illustration of addition of TBP to a DNA monolayer modified with Nile Blue (NB),

followed by DNA kinking and attenuation of DNA-mediated electrochemistry.

Gorodetsky et al.

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Figure 2.

CV (top) and SWV (bottom) of NB–DNA at macroelectrodes of 2 mm diameter (left) and

microelectrodes of 25

m diameter (right). Voltammetry without TBP (blue) and with 300 nM

TBP (red) is shown at pH 7 in a degassed phosphate buffer containing 5 mM NaP

, 50 mM

NaCl, 4 mM MgCl

, 4 mM spermidine, 50

M EDTA, 10% glycerol. After addition of protein,

solutions were briefly stirred by a gentle argon purge. The sequence is thiol-5

′

GAGA

TATAAAG

CACGCA-3

′

plus NB-modified complement. The potentials are reported

versus Ag/AgCl with a CV scan rate of 50 mV/s and an SWV frequency of 15 Hz.

Gorodetsky et al.

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Figure 3.

CV of three NB–DNA modified microelectrodes (10

m) before and after addition of proteins.

CVs before protein addition are in blue. From left to right, the black CVs represent addition

of BSA (2

M); BSA (1

M) + EndoIII (1

M); and BSA (1

M) + BAM (1

M). CVs in red

reflect the subsequent addition of 30 nM TBP to each electrode. The data have been smoothed

for clarity. The sequence is thiol-5

′

-GAGA

TATAAAG

CACGCA-3

′

plus NB-modified

complement, and the potentials are reported versus Ag/AgCl with a CV scan rate of 50 mV/s.

Gorodetsky et al.

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J Am Chem Soc

. Author manuscript; available in PMC 2009 September 21.

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