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Sensing DNA through DNA Charge Transport
Theodore J. Zwang
,
Edmund C. M. Tse
, and
Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
California 91125, United States
Abstract
DNA charge transport chemistry involves the migration of charge over long molecular distances
through the aromatic base pair stack within the DNA helix. This migration depends upon the
intimate coupling of bases stacked one with another, and hence any perturbation in that stacking,
through base modifications or protein binding, can be sensed electrically. In this review, we
describe the many ways DNA charge transport chemistry has been utilized to sense changes in
DNA, including the presence of lesions, mismatches, DNA-binding proteins, protein activity, and
even reactions under weak magnetic fields. Charge transport chemistry is remarkable in its ability
to sense the integrity of DNA.
Abstract
Here, we describe a variety of studies carried out in our laboratory probing the DNA duplex
and DNA-binding partners using DNA-mediated charge transport (DNA CT). Over the past
three decades, we have explored this chemistry and its application in sensing DNA.
1
5
Additionally, we have focused on how nature makes use of this chemistry for DNA sensing
and for long-range signaling across the nucleus of the cell.
6
,
7
Through a full range of strategies and platforms, we and others have characterized this
chemistry in detail. Two critical characteristics of this chemistry have been established.
DNA CT can occur over long molecular distances
. In fact, while ground state DNA CT has
been documented to occur over 34 nm,
8
the distance limit for DNA CT has yet to be
established. Many recent experiments suggest that DNA CT occurs over kilobase distances,
Present Address
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138
*
Corresponding Author
jkbarton@caltech.edu.
Notes
The authors declare no competing financial interest.
HHS Public Access
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ACS Chem Biol
. Author manuscript; available in PMC 2018 August 07.
Published in final edited form as:
ACS Chem Biol
. 2018 July 20; 13(7): 1799–1809. doi:10.1021/acschembio.8b00347.
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but DNA CT can occur over long distances only if the DNA duplex is well stacked.
Small
perturbations in DNA stacking perturb DNA CT
. Thus, base mismatches, lesions, and even
DNA-binding proteins that perturb the DNA base stack can be sensitively detected
electrochemically.
9
,
10
Using these parameters, DNA CT chemistry provides a powerful
means to sense DNA and the small and large molecules that interact with the DNA duplex.
1. PLATFORMS FOR MEASURING DNA CT
There are many different platforms that have been used to measure DNA-mediated charge
transport (DNA CT), illustrated in Figure 1. Early experiments testing DNA CT were
performed in solution and involved a photoexcited charge donor that transfers charge to an
acceptor through a DNA bridge.
11
A wide variety of donors and acceptors were used in
these experiments, ranging from transition metal complexes to purely organic moieties, base
analogs, and proteins.
12
14
More recent experiments have been conducted using electrodes,
typically gold or graphite, modified with a self-assembled monolayer of DNA.
15
18
Here,
DNA duplexes are linked to the surface using a covalent modification on the phosphate
backbone (alkane-thiols for gold or pyrene for graphite) that allows the duplexes to stand
upright,
17
facilitating interaction with DNA-binding molecules in solution. Redox
molecules, either noncovalently or covalently attached to the DNA, can then be reduced or
oxidized by applying a potential across the electrode surface.
3
The advantage to these electrochemical studies is that they allow measurements of ground
state CT, rather than CT through excited state photochemistry. Moreover, while the
chemistry is occurring on the electrode surface, in all respects it appears that the chemistry is
like that in solution; proteins bind to their specific cognate sites and carry out their various
enzymatic reactions with their specific nucleic acid substrates. One disadvantage is that the
rates of CT through the DNA duplex cannot be determined electrochemically, because for all
studies thus far conducted, even using DNA 100-mers, the rates of CT have been limited by
transport through the alkane linker.
19
It is this linker that keeps the duplex “upright.”
17
Measurements of base–base DNA CT in solution, using photoexcitation of 2-aminopurine,
nonetheless, show DNA CT to be on the picosecond time scale and gated by the motion of
the DNA bases.
20
24
Indeed, this chemistry provides a sensor also for the dynamics of DNA.
Other experimental setups have allowed for measurements of DNA conductivity. Conductive
atomic force microscopy has been used to create metal–DNA–metal junctions that can be
used as a circuit to measure the current–voltage characteristics of DNA.
25
The scanning
tunneling microscopy break junction technique measures the conductivity as the tip is
pushed toward and retracted away from the surface, apparently hybridizing and
dehybridizing the duplex.
26
The current is measured as a function of the distance of the tip
from the surface with the assumption that the stretch of separation where the current is
constant represents the conductivity of a DNA duplex bridge. Single DNA molecule circuits
have also been made that tether DNA between a nanotube gap and measure the change in
current that passes through the circuit; this experimental setup provides a measurement of
conductivity relative to that of the carbon nanotube.
27
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2. DNA CT CHARACTERISTICS FOR DNA SENSING
For all of these measurements, the “connection” to the DNA duplex is critical. For
experiments where DNA CT is monitored using small-molecule probes,
3
the careful
selection of the redox-active probe is essential. DNA-mediated charge transport occurs
efficiently only with redox-active molecules that couple effectively to the
π
-stack. Thus,
intercalators that stack well with the DNA duplex have been our most effective probes.
Intercalative redox probes such as methylene blue that are able to insert themselves into the
π
-stack undergo efficient DNA-mediated charge transport.
28
Other molecules, like the
positively charged ruthenium hexammine, associate electrostatically to the phosphate
backbone and are unable to access DNA CT.
29
In some cases, as with methylene blue,
different DNA binding modes are available. At low micromolar concentrations, methylene
blue primarily intercalates into DNA where it can undergo efficient DNA CT, but at higher
concentrations it can bind electrostatically where it cannot utilize DNA CT. Screening these
electrostatic interactions with increased salt concentrations promotes primarily intercalative
binding.
The importance of this coupling or connection to the DNA
π
-stack was highlighted in
comparing DNA CT in experiments monitoring photooxidation of guanine using two
fluorescent base analogs, 2-aminopurine, which stacks well with the duplex, versus etheno-
adenine, which does not.
13
The differences in CT rates and distance dependences were
remarkable. Even for electrochemistry experiments, probing the same DNA construct with
molecules that do and do not couple to the
π
-stack shows a significant difference in yield.
3
It
is also possible to use redox probes that selectively target mismatches or abasic sites and
stack within the open site, so that a DNA-mediated signal is found only if that mismatch or
abasic site is present. Critically, then, for all these experiments, DNA CT is only rapid and
over long-range if the connection is truly to the base pair stack.
4
It is also important when working with DNA to be aware that small changes in preparation
can have dramatic influences on the heterogeneity of samples and, therefore, the
reproducibility of experiments, especially when probing DNA that must be in the duplex
form and fully stacked. We have, for example, always utilized duplex DNA for initial
modifications of the electrode surface; single stranded DNA binds avidly to the gold surface
and cannot be easily displaced. Other concerns relate to the formation of DNA self-
assembled monolayers and the electrode attachments; these constructs may vary depending
on the chemistry used to attach the DNA, which include thiols for gold electrodes,
30
alkynes
for azide-terminated electrodes,
31
and pyrenes for graphite electrodes,
32
and can include
many less typical linkers depending on the desired surface attachment.
33
In all cases, it is
important that the linker allow the DNA to be positioned roughly perpendicular to the
electrode surface during experiments. The packing density of DNA is also rationally
controlled depending on the desired experiment, because, for example, proteins will not be
able to access and bind to DNA in a monolayer that is too densely packed.
18
Most
importantly, new electrochemical surfaces require characterization, particularly with respect
to quantitation of surface loading with the nucleic acid, and surface accessibility. Just as
packing densities may be too high for protein binding experiments, very low loading
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densities usually indicate surface contamination or DNA damage. For sensitive experiments
to detect DNA, the sensor needs to be chemically well-defined.
3
It is also essential to conduct experiments that verify that charge transport is DNA-mediated,
that the charge migrates through the DNA helix, for any new DNA sensor design. Ideally,
these experiments will disrupt DNA CT in a way that is recoverable or in a way that has
minimal other differences from the sensing experiments. One of the strongest confirmations
that charge transport occurs through DNA is by the inclusion of a single base mismatch or
abasic site that will disrupt the
π
-stacking. The main benefit of this method is that it changes
very little about the DNA structure that may influence other parts of the experiment but
should have a dramatic effect on charge transport that is mediated by the
π
-stack of the
duplex. Incorporation of a particularly ruinous mismatch, such as CC or CA, will result in a
significant decrease in the yield of DNA CT.
34
Guanine-containing mismatches tend to be
poor choices for this confirmation because they do not attenuate CT as dramatically. An
abasic site will have a more significant effect, but it is also a larger structural change to the
helix. If the experiments are run at relatively high temperatures, an abasic site is often a
better choice than a mismatch, because increasing the temperature decreases the attenuation
caused by a mismatch and potentiates the attenuation caused by an abasic site.
35
Larger
scale structural changes such as dehybridization or melting of the duplex may be used to
provide necessary confirmation of DNA CT in some context, especially when they are used
in the sensing experiment that is being established.
36
Careful use of multiple redox probes,
some that are able to undergo DNA CT, such as an intercalator, and others that are unable to
undergo DNA CT, for example ruthenium hexammine, can also be used to confirm a DNA-
mediated signal.
We developed a multiplexed chip that allows for measurements of four different DNA
monolayers on a single surface with 4-fold redundancy.
16
This multiplexing facilitates
carrying out the important controls in parallel. As an illustration of how this device may be
used, DNA-mediated CT to a covalently tethered Nile Blue redox probe was simultaneously
measured through four monolayers of DNA: 100 bp and 17 bp DNA duplexes with no
mismatch, and the same duplexes containing a single base mismatch.
8
The rate of electron
transport was calculated to be between 25 and 40 s
−1
for both duplex lengths, significantly
less than the 10
10
s
−1
rate of DNA CT found with picosecond spectroscopy,
22
,
37
because the
rate is limited by tunneling through the alkanethiol that tethers the DNA to the electrode.
19
Even so, substantial signal attenuation was observed for both duplex lengths upon
introduction of an intervening single base-pair mismatch in the DNA duplex. This set of
experiments demonstrates that the DNA-mediated CT is responsible for the redox signals
from Nile Blue on this multiplexed chip, even for a 100-mer, and also, remarkably, that such
an electrochemical biosensor utilizing DNA-modified electrodes can be used to identify a
single base error in a 100-mer.
It is more complicated to verify charge transport that is mediated by the DNA
π
-stack in
dried samples and DNA in other conditions that do not have known structures. Dehydration
or exposure to nonaqueous solvents can eliminate efficient DNA-mediated charge transport.
DNA is stabilized by a variety of hydrophobic and hydrophilic interactions; changing these
interactions can significantly change the resulting structure.
38
The precise structural changes
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caused by dehydration or exposure to most nonaqueous solvents are not very well
characterized, but it is clear that the equilibrium conformation of DNA is changed.
Some experiments conducted under nonaqueous conditions do show efficient DNA CT or
conductivity. Scanning tunneling microscope studies describing DNA conductivity are
generally performed under a vacuum to intentionally remove water that can make such
experiments difficult,
39
though some scanning tunneling microscope studies have been
conducted in humid environments with different results.
40
Other measurements, including
many conductive atomic force microscopy experiments, have shown varying degrees of
conductivity after rehydrating DNA that was deposited in a vacuum or washed with apolar
solvents.
25
27
Chemistry mediated by DNA CT in hydrated ionic liquids has also been
reported with careful consideration of the influence this environment has on the duplex
structure.
41
In these extreme conditions, the structure of DNA is unclear, so it is difficult to
make claims regarding DNA-mediated charge transport that are clearly deconvolved from
other aspects of the experiment. For example, ionic conduction through water may be what
is measured instead of DNA-mediated CT.
42
Thus, to properly understand the structure that
is being tested, it is essential to keep DNA hydrated with appropriate salt content during all
steps of preparation and experimentation, to characterize the DNA after procedures that may
change the structure, and to verify that the charge transport observed is mediated by DNA.
3. STACKING IS ESSENTIAL FOR DNA CT
The ability for DNA to mediate CT depends completely upon base stacking. It is thus not
surprising that some variations in DNA CT arise with the different DNA duplexes, the A, B,
and Z forms, all of which stack, albeit somewhat differently.
43
Using photoinduced CT
where rates can be measured, both the A and B forms of DNA show picosecond rates for the
DNA-mediated charge transport process.
44
Interestingly, in comparing the A and B forms,
using base–base CT, the A form shows rapid interstrand transport, because of interstrand
base overlap in the A form, whereas the B form, with no interstrand base overlap, shows
only rapid intrastrand CT. In electrochemical experiments, the A, B, and Z forms all display
long-range CT.
43
Here too, the intensity depends upon stacking and, particularly, the
coupling of the redox probe with the differing duplex conformations through stacking. The
A-form duplex shows the most intense DNA CT.
43
The B form follows next. The Z form,
which has the poorest
π
stacking of the duplex structures, exhibits significantly less efficient
yield of DNA CT to the intercalated redox probes; the peak current for B-form DNA is over
3 times larger than for Z-form DNA, and the total yield of DNA CT in a single potential
sweep differs by more than an order of magnitude; here, however, the different coupling of
the redox probe with each conformation needs to be taken into account. Indeed, the yield for
Z-form DNA is comparable to A- and B-form DNA using photooxidation assays and a
different redox probe.
45
Most importantly, single stranded DNA, if present in an unstacked
conformation which does not have an ordered
π
-stacked structure, does not facilitate
efficient charge transport. This phenomenon has been confirmed with electrochemical,
photooxidation, and direct conductivity studies.
22
,
27
,
43
It is important, however, to consider
the sequence for these experiments with single stranded DNA, since the extent of stacking
varies enormously depending upon the sequence.
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Perhaps the most unique characteristics of DNA CT is that even a small local disruption of
the
π
-stacking diminishes the efficiency of DNA CT. We have probed all of the mismatches
in DNA and, remarkably, all of the mismatches can be detected, even the GT mismatch,
which has a thermal stability equal to that of an AT base pair.
27
,
46
Interestingly, the presence
of mismatches lowers the rate and yield of DNA CT in a way that correlates with base pair
lifetime,
46
and this disruption occurs even though mismatched base pairs do not cause
significant structural changes.
47
Most remarkable is that the attenuating effects of
mismatches are evident independent of the sequence context.
48
These assays are not simply
measurements of thermal stabilization. Hence, DNA CT provides an exquisitely sensitive
and valuable assay for DNA mismatches, where different sequences may be tested under the
same experimental conditions.
Abasic sites and destabilizing lesions, such as 8-oxoguanine, also significantly diminish
DNA CT.
34
,
49
8-oxoG-A and 8-oxoG-C both destabilize the duplex structure, despite the
different locations of their modification, which is sufficient to attenuate DNA CT.
34
It is
because these base lesions are so easily detectable that the idea that nature might also use
this chemistry for detection inside the cell became reasonable to consider. Indeed, a whole
family of DNA repair enzymes has been found to contain [4Fe4S] clusters, redox cofactors
featured commonly in proteins, and many experiments we have carried out support the idea
that these repair proteins utilize DNA CT chemistry in their search for lesions within the
cell.
5
,
50
Significant kinks to DNA caused by protein binding, such as the TATA-binding
protein,
51
or chemical interactions with molecules such as cisplatin will also disrupt DNA
CT. Again, it is the stacking of bases that must be preserved for long-range CT, so that
anything that perturbs that base stacking turns off charge transport mediated by DNA.
It is worth noting that not all modifications to DNA structure diminish DNA CT. A
dephosphorylation of the backbone does not have a measurable effect on yield or efficiency,
52
nor does a full break in the DNA backbone, as long as base pair stacking is preserved.
52
,
53
Some changes in structure, such as methylation to generate 5-methylcytosine, do not
significantly influence DNA CT.
34
Also, proteins that do not interfere with the DNA
stacking upon DNA binding, as found with helix–turn–helix proteins
1
or even histones,
54
do
not interfere with DNA CT. We think of chromatin as packing up the DNA library to keep it
undamaged, yet long-range guanine oxidation can still occur in the nucleosome. For a
chromatin-bound DNA duplex, while the DNA duplex is wrapped gradually around the
histone core, the DNA base pairs are still well-stacked.
4. SENSING MISMATCHES/MUTATIONS AND DNA BASE LESIONS
The exquisite sensitivity of DNA-mediated charge transport to the structure of DNA allows
for it to be used to sense electrically phenomena that disrupt or alter the DNA duplex
structure. Depending on the design of the experiment and what is being tested, this
relationship can be utilized in a variety of ways to sense protein activity, the presence of
specific nucleotide sequences, or any changes in DNA structure.
Many different lesions and modifications can be detected electrochemically
via
their
disruption of DNA CT.
34
,
48
,
55
Damage products such as thymine dimers, O4-methyl-
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thymine, O6-methyl-guanine, 8-oxo-guanine, and 5-hydroxy-cytosine all disrupt DNA CT.
For the thymine dimer, it is clear that dimerization interrupts base stacking, but for the
various base lesions, it can be difficult to predict the degree to which they will disrupt DNA
CT, because it is not directly related to their influence on the thermodynamic stability of the
helix. More generally, structural modifications that disrupt the hydrogen bonding in Watson–
Crick base pairing will significantly disrupt DNA CT. Bulky modifications to bases or
changes to general conformation can have a large influence. Smaller changes, such as the
addition or subtraction of methyl groups that do not disrupt hydrogen bonding, do not appear
to have a large effect on DNA CT.
34
A chip-based technology utilizing DNA-functionalized electrodes was developed by the
Barton lab.
48
This chip-based technology uses DNA CT chemistry to probe the integrity of
double-stranded DNA sequences and detect single-base mismatches for early diagnosis of
genetic diseases.
48
This device uses an electrocatalytic cycle with [Fe(CN)
6
]
3−
and
methylene blue to amplify the difference in the yield of DNA CT for DNA with and without
a lesion (Figure 2). DNA CT to methylene blue occurs rapidly, and with reduced yield when
a lesion is present. [Fe(CN)
6
]
3−
regenerates the methylene blue, which allows it to be
rereduced
via
DNA CT. This redox cycling behavior amplifies any difference in CT yield
through DNA, making lesions even more apparent. This method has been applied to
distinguish commonly found DNA lesions and mutations from well-matched duplexes with
no damage. A two-electrode patterning and detection platform was further developed to
enhance spatial resolution of patterned DNA arrays and optimization of DNA lesion
detection through DNA-mediated CT with electrocatalysis.
31
This methodology enables
very sensitive discrimination.
Several other approaches exist for the detection of nucleic acids, some of which can take
advantage of DNA CT to signal substrate capture. These approaches tend to rely on
hybridization of either neutral peptide nucleic acid (PNA) or negatively charged DNA or
RNA to a target oligonucleotide, which have been reported to achieve sensitivities for their
target nucleic acid ranging from picomoles to zeptomoles (40 zmol in 4
μ
L samples).
56
59
Hybridization of a targeted nucleic acid with its complement on a sensor surface will restore
the DNA CT-capable duplex form, which upon addition of a redox active intercalator, will
result in different redox activity compared to the unhybridized DNA. The sensitivity of DNA
CT to mismatches can allow sensors to detect single nucleotide polymorphisms, with high
sensitivity, and use conditions that are independent of sequence, e.g., do not depend on
thermal melting.
5. ELECTRICALLY MONITORING PROTEIN BINDING AND ACTIVITY
The sensitivity of DNA charge transport to structural perturbations has allowed for unique
insight into the activity of many protein–DNA interactions. DNA-modified films used to
assay protein–DNA interactions are different from monolayers used to assay DNA lesions.
The first of these experiments involved the use of a low-density DNA monolayer containing
a covalently linked daunomycin probe near the duplex terminus and away from the protein
binding site.
1
The low density of the DNA film is essential for allowing access to the DNA-
binding proteins. Chronocoulometry on these DNA-modified surfaces in the presence and
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absence of different DNA-binding proteins found a significant difference in the yield of
charge transport to daunomycin, which was electrocatalytically regenerated by oxygen, that
was directly related to the way in which these proteins interacted with the DNA monolayers.
The methyltransferase
HHa1
catalyzes the methylation of cytosine in 5
-GCGC-3
by first
flipping a cytosine out from the duplex then inserting Gln 237 into the void left in the base
stack.
60
62
Incubating
HHa1
with the DNA monolayer without S-adenosylmethionine,
which is necessary for enzymatic activity, is enough to greatly diminish the yield of charge
transport (Figure 3).
63
65
DNA-binding by a mutant of
HHa1
, Q237W, that inserts an
aromatic Trp into the base-pair stack shows significantly less attenuation of charge transport;
here the Trp inserted in the stack serves to restore CT. When the film is incubated with a
protein that does not bind DNA, such as BSA, there is no change in current. Together, these
data show that the yield of DNA CT on this modified electrode depends upon the DNA
π
-
stack.
This DNA CT assay is sensitive to other types of DNA–protein interactions aside from base
flipping, so long as the protein perturbs the
π
-stacking of bases (Figure 3). The TATA-box
binding protein (TBP) does not flip bases, but rather kinks DNA ~90° upon binding to its
target site.
66
,
67
This interaction disrupts base stacking, but not base pairing, and is enough to
significantly diminish the yield of DNA CT.
1
,
51
As would be expected, more dramatic changes in DNA structure such as cutting with
restriction enzymes can also be monitored
via
this method. The binding of a restriction
enzyme, such as the endonuclease
PvuII
, which does not significantly perturb the DNA base
stack,
68
does not have a significant influence on the charge transport yield.
1
Upon
restriction, however, the daunomycin redox probe is released from the surface, which
significantly decreases the yield of DNA CT (Figure 3). Similar results have been observed
with many other restriction enzymes and redox probes.
1
,
8
,
69
,
70
Experiments with
Escherichia coli
photolyase show that DNA-modified films are also able
to monitor the activity of proteins that restore the
π
-stacked structure of DNA.
55
Cyclobutane pyrimidine dimers (CPD) are lesions which form as a result of a photoinduced
[2 + 2] cycloaddition between two adjacent pyrimidines on the same DNA strand. Upon
photolyase binding, the CPD is flipped out of the DNA helix into the protein’s active site,
where a reductive catalytic cycle is initiated upon blue light irradiation of a flavin cofactor
that repairs the CPD into individual pyrimidines. After repair, the monomer pyrimidines are
returned to the DNA, thus restoring its
π
-stacked structure.
71
73
Conveniently, DNA CT is
able to access the redox-active flavin, which allows for DNA duplex integrity to be observed
via
CT to the flavin, without need for an additional redox probe. Upon photoactivation of
photolyase bound to a DNA-modified electrode surface, an increase in DNA CT is observed,
indicating that this platform is electrochemically monitoring the repair of the CPD.
55
DNA-binding protein activity can also be used to sense reactions to modify DNA where the
modification itself does not affect DNA CT, as with base methylation. A recent DNA-based
biosensor was constructed to monitor the methylation of DNA by DNMT1, the human DNA
(cytosine-5)-methyltransferase, by using DNA CT and a methylation-specific restriction
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enzyme (Figure 4).
69
DNMT1 preferentially methylates hemimethylated DNA when it has
access to the cofactor
S
-adenosyl-
L
-methionine (SAM).
74
77
BssH
II is a restriction enzyme
that will cleave hemimethylated but not fully methylated DNA. Thus, if the DNA is
methylated by DNMT1, the duplex will be protected from restriction by
BssH
II and retain
high yield DNA CT. If there is no DNMT1 activity,
BssH
II will cleave the DNA and
decrease DNA CT yield. Here too, then, a sensitive probe for DNMTI activity can be
obtained.
6. TWO-ELECTRODE PLATFORM FOR SENSITIVE DETECTION
A goal with sensitive detection is to be able to monitor protein activity in cellular samples
without protein purification. Improvements in sensor design involving a two-electrode setup
allow for DNA CT to sense DNMT1 activity in crude lysate with minimal purification.
69
,
78
,
79
This bioanalytical platform utilizes two working-electrode arrays separated by a thin
layer of solution to detect biomolecules, nucleic acids, and DNA-binding proteins (Figure
4). The primary electrode is modified with a DNA monolayer, which has a constant applied
potential to reduce intercalated methylene blue. The methylene blue then diffuses in solution
and is oxidized by ferricyanide, thereby regenerating the methylene blue to be reduced
via
DNA CT in an electrocatalytic cycle. A second, reporting electrode is held at a potential to
oxidize the generated ferrocyanide, and the current at this electrode can be measured to
report the yield of DNA CT. This setup enables the reporting electrode to operate with
minimal background current, and the electrocatalysis increases the number of DNA CT
events that are possible, which together increase the sensitivity of this DNA CT sensing
platform.
A recent DNA-based biosensor for enzymatic activity of DNMT1 that has been linked to
tumorigenesis shows that this chemistry can be used to observe protein–DNA interactions
from biological samples with minimal purification.
78
,
79
Crude lysate from a colorectal
tumor and adjacent healthy tissue were incubated with a hemimethylated duplex DNA
substrate. Increased DNMT1 activity in the tumor samples further methylated the
hemimethylated substrate DNA and prevented the fully methylated form of the DNA from
being cut by subsequent exposure to restriction enzymes. The samples exposed to DNMT1
retain efficient DNA CT, and those not exposed to DNMT1 are cut by restriction enzymes
and have attenuated DNA CT, thus allowing DNA CT to be used as a sensor for aberrant
DNMT1 activity associated with colorectal tumors. These experiments were conducted with
pure DNMT1, DNMT1 added to cell lysate, colorectal tumor tissue, and healthy colorectal
tissue. The presence of cell lysate did not diminish the sensitivity of this assay to nanomolar
concentrations of DNMT1, indicating its potential for assaying biological samples with
minimal purification. Indeed, this assay was able to distinguish the increased DNMT1
activity in colorectal tumor tissue from adjacent healthy colorectal tissue without need for
purification.
It is noteworthy that what was key in these studies was the application of copper-activated
click chemistry to create an open monolayer. Activation occurred with control using the
second electrode. As a result, the DNA duplexes could be positioned with control, so that the
DNAs were not clumped together, permitting access of the many proteins in the cell lysate to
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the DNA. With this methodology and the two-electrode platform, cell samples could be
easily probed.
7. SENSING BY REDOX [4FE4S] CLUSTERS IN PROTEINS
Many DNA-processing enzymes have been shown to contain [4Fe4S] clusters that are
redox-active and able to be reduced and oxidized
via
DNA CT.
5
,
50
,
80
These proteins have a
wide variety of functions including those involved in base excision repair, nucleotide
excision repair, as well as helicases, DNA primase, and DNA polymerases.
81
87
Binding to
DNA shifts the redox potential of the [4Fe4S] clusters by about 200 mV and activates the
clusters toward oxidation, which allows the [4Fe4S]
2+/3+
redox couple to be close to +80
mV vs NHE, a potential accessible under physiological conditions.
80
,
88
Proteins with reduced and oxidized [4Fe4S] clusters have significant differences in affinity
for DNA.
89
Calculations based on the shift in redox potential caused by DNA binding
suggest that the affinity of proteins with an oxidized cluster is at least 2 orders of magnitude
stronger than proteins with a reduced cluster. Recently, experiments were conducted that
systematically varied the oxidation state of the [4Fe4S] cluster and measured how the redox
state of the metallocofactor influenced DNA binding affinity.
89
Electrophoretic mobility
shift assays, isothermal titration calorimetry, and microscale thermophoresis were used to
probe the nonspecific DNA binding of Endonuclease III, a base excision repair glycosylase
that repairs oxidized pyrimidines in
Escherichia coli
. The protein with the oxidized cluster
showed significantly stronger affinity for DNA. Microscale thermophoresis, which was able
to be performed under anaerobic conditions and best prevent extraneous oxidation, shows an
affinity for the oxidized state that is at least 550-fold greater than the protein with the
reduced cluster. Biophysical modeling suggests that this difference in affinity can be
explained primarily by changes in the electrostatic interactions between the cluster and the
DNA phosphate backbone without significant changes in the protein structure.
The difference in affinity of the different redox states of [4Fe4S] clusters combined with
DNA CT can be utilized in a strategy to rapidly detect and localize near DNA damage.
80
,
90
92
A basic model of genome scanning involving only facilitated diffusion and
instantaneous interrogation of the DNA integrity indicates that it is insufficient to probe the
entire
E. coli
genome within its doubling time. Intriguingly, DNA CT provides a means to
hasten this search. DNA CT between proteins bound to DNA occurs rapidly on the
picosecond time scale but only in cases where DNA
π
-stacking is unperturbed (Figure 5). In
situations where an intervening mismatch or other lesion disrupts the
π
-stack, DNA CT is
unable to occur efficiently between proteins. Since the reduction of oxidized proteins will
decrease their affinity for DNA and allow them to release and scan elsewhere, charge
transport can serve as an effective first step to aid protein binding where they are needed.
Altogether DNA CT decreases the amount of time for a repair protein to find its substrate
lesion and redistributes the repair protein in the vicinity of lesions. Proteins that have shown
the ability to participate in this redox-mediated damage search include DinG, MutY,
EndoIII, and XPD.
80
,
90
92
Proteins that contain [4Fe4S] clusters are able to communicate
with one another despite their origin or repair pathway, indicating the generality of this
mechanism for proteins to aid one another in their damage search.
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Mutations can be used to modify both the redox characteristics of proteins containing
[4Fe4S] clusters and their enzymatic activity. By using DNA-modified platforms,
electrochemical “sensing” of these proteins provides a means to probe those characteristics.
XPD, for example, is a 5
-3
helicase that is a key member in the nucleotide excision repair
process.
86
By utilizing a DNA-modified Au surface, wild-type XPD exhibits a redox wave
centered at −120 mV vs NHE.
93
The XPD L325V mutant displays an electrochemical signal
that is less than half that of WT XPD, suggesting that the mutant is deficient at performing
DNA CT and can therefore be identified using DNA-modified Au electrodes. Similar
inhibited CT behavior is also observed for the Y82A mutant of
E. coli
endonuclease III
(EndoIII), another [4Fe4S] cluster-containing DNA repair protein.
92
,
94
Other mutants of
EndoIII, including E200K, Y205H, K208E, and other DNA glycosylases, including WT
MutY and UDG,
18
display similar redox potentials, despite electrostatic perturbations in the
vicinity of the cluster, suggesting that binding to the DNA polyanion is the dominant
influence tuning the redox potential of the [4Fe4S].
This DNA electrochemistry can also be used to monitor the biochemical activity of [4Fe4S]
cluster-containing helicases, such as XPD and DinG. Upon the addition of ATP, the redox
signal corresponding to the [4Fe4S] cluster in DinG increases substantially in magnitude
(Figure 5).
91
Essentially, this electrochemical signal serves to “sense” enzymatic activity.
Presumably, this signal is associated with increased coupling of the cluster to the DNA on
reaction. Similar ATP-dependent electrochemical signaling was found in XPD.
93
Most recently, we were able to monitor a DNA-binding redox-switch in DNA primase.
95
In
eukaryotes, both DNA primase and DNA polymerase
α
contain [4Fe4S] clusters.
96
When
the protein domain, p58C, containing the cluster in primase was added to a DNA-modified
electrode, no signal was evident despite the fact that the domain was known to bind this
substrate as part of its activity. However, when the loosely associated domain was oxidized
electrochemically, a signal quickly emerged. Upon reduction, however, the signal was again
lost. In fact, these results pointed to primase utilizing a redox switch in its cluster for
substrate binding using DNA CT.
95
Here, primer initiation was proposed to be associated
with oxidation of the p58C cluster with electron transfer through DNA CT from polymerase
α
to primase, with rereduction, dissociation, and handoff once the primer DNA/RNA was
complete.
8. SENSING MAGNETIC FIELDS
Recent work has enabled the use of DNA CT in interesting ways to report changes in its
magnetic environment.
97
First work was conducted to establish chirality-induced spin
selectivity.
25
On the basis of that work, we utilized DNA CT in the presence of magnetic
fields to probe the ability of the DNA duplex to filter spin. Aqueous DNA monolayers were
formed on a ferromagnetic electrode substrate capped with a thin layer of gold. Magnetizing
the electrode generates a spin-polarized current when the applied potential is the negative of
the reduction potential of DNA-bound probe molecules. The sign of the polarization can be
switched by changing the direction of the applied magnetic field without influencing its
magnitude. When the redox probe is intercalated and undergoes DNA CT, a difference in
probe reduction yield is observed for the two magnetic field directions, indicating that one
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spin moves through the duplex with higher yield than the other (Figure 6). For a 60 bp B-
form duplex with covalently tethered Nile Blue, there was a 29% increase in methylene blue
reduction when the magnetic field was pointing up, which indicates that the duplex causes at
least a 55% spin polarization of electrons that are transported through. There is no magnetic
field effect found when DNA is not present, when using single stranded DNA, or when using
redox probes that are near a duplex but not undergoing DNA CT. Utilizing 5-methylcytosine
(
m
C) to create DNA oligomers, d(
m
CG)
n
, allows for a duplex to undergo a reversible B-to-Z
transition under conditions that allow for methylene blue to intercalate into both the B and Z
DNA. Remarkably, this switch in DNA helicity changes the magnetic field direction that
results in higher DNA CT yield. We find an upward magnetic field causing at least 36% spin
polarization for the right-handed B form, but the same duplex with the same magnetic field
has −19% spin polarization when switched to the left-handed Z form. Thus, spin transport
efficiency can be used to distinguish between B- and Z-form duplexes.
We also recently found that DNA CT can be used to sense the strength and direction of
magnetic fields when bound by magnetosensitive proteins.
70
We had earlier developed
electrochemical methods to monitor the repair of cyclobutane pyrimidine dimer (CPD)
lesions that disrupt DNA CT.
55
As proteins such as
E. coli
photolyase and a modified
Arabidopsis thaliana
cryptochrome I bind DNA on an electrode surface and repair the
pyrimidine dimer, high yield DNA CT is restored that allows efficient oxidation or reduction
of the redox-active flavin cofactor within the DNA-bound protein (Figure 7).
70
The repair of
these CPD lesions occurs
via
a reductive catalytic cycle upon irradiation of the flavin
cofactor with blue light. Intriguingly, this repair reaction is sensitive both to the magnetic
field strength and to magnetic field angle to which the photolyase and cryptochrome are
exposed, where the magnetic field generally dampens the restoration of high yield DNA CT.
The sensitivity of this repair reaction is exquisite, allowing for the detection of magnetic
fields as weak as 0.2 gauss, on the order of variations seen across the Earth. Increasing the
field strength eventually saturates this effect, with 30 gauss fields showing no difference
compared to 6000 gauss fields. The angle, but not the direction at which these fields is
applied relative to the electrode surface, determines the DNA CT dampening effect. The
largest dampening happens at fields that are perpendicular to the electrode surface, while the
weakest dampening occurs at fields that are parallel to the electrode surface. By removing
the applied magnetic field, the repair activity is restored and so is the yield of DNA CT to
the flavin cofactor. It is important to note that this magnetosensitivity relies on the uniform
orientation of the proteins on the electrode surface, as there is no observed
magnetosensitivity of CPD repair in solution.
70
Experiments with different DNA sequences and protein mutations were able to uncover the
mechanism by which the magnetic fields influence CPD repair. First, mutations in the active
site of
E. coli
photolyase were used to determine which part of the CPD repair pathway is
magnetosensitive. Mutations E274A and M345A near the CPD eliminated
magnetosensitivity, but N378C near the flavin retained magnetosensitivity, which suggested
that the magnetosensitivity arises from the dimer and not from the flavin. Next, uracil-
containing dimers were used to test this hypothesis, and it was found that U
U showed
diminished magnetosensitivity, while T
U and U
T both had no magnetosensitivity. These
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characteristics, together, suggest that the magnetosensitivity arises from a radical pair
involving the CPD. This chemistry harkens back to experiments conducted by Turro
et al
.
that showed how radical pair reactions can be controlled by weak magnetic fields.
98
These
data show how DNA CT can be used to sense magnetic fields, and it is intriguing to consider
whether nature takes advantage of this chemistry for
in vivo
magnetic sensing.
9. SUMMARY AND PROSPECTS
DNA-mediated charge transport is a fascinating phenomenon that relies on the
π
-stacked
structure present in some DNA conformations. Disrupting the
π
-stack inhibits efficient
charge transport, and recovering the
π
-stack can re-enable efficient charge transport. If
experiments are conducted thoughtfully, this exquisite sensitivity of DNA CT to structural
changes can be used confidently to sense a large variety of biological phenomena. To date,
there are numerous sensor designs that take advantage of this chemistry in order to detect
oligonucleotides, single nucleotide polymorphisms and lesions, protein binding, enzymatic
activity, and now DNA CT can even sense weak magnetic fields. Even nature appears to use
DNA CT in order to detect DNA damage and other structural changes, with implications that
it enables proteins to signal one another for efficient repair and coordination.
The future use of DNA CT for sensing phenomena is not limited to the types of experiments
described. Rather, the uses of DNA CT will continue to expand as more proteins that are
capable of modulating function using DNA CT are uncovered, such as recent discoveries
with primase and polymerase,
95
,
99
and as more is understood about the underlying
characteristics of DNA CT. Thus, as ever more intriguing uses and characteristics of DNA
CT are elucidated, the potential for DNA sensing will continue to grow.
ACKNOWLEDGMENTS
We are grateful to all our co-workers and collaborators for their efforts in developing new sensing technologies. We
also thank the NIH, GM61077, for financial support. E.C.M.T. acknowledges a Croucher Foundation Fellowship.
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Figure 1.
Platforms for the study of DNA-mediated charge transport (DNA CT). (a) DNA is
covalently tethered to an electrode surface with an intercalated redox probe. A cyclic
increasing or decreasing potential is applied that results in charge being transported through
the DNA either to or from the electrode, which can be measured as a change in the current
during a potential sweep. (b) DNA is covalently tethered between two electrodes. This type
of setup is used to measure the current between the two electrodes in conductive AFM and
STM break junction methods. (c) A ferromagnetic electrode influences the yield of charge
transport through DNA in different conformations, such as the Z-form shown above. (d)
Donor and
Acceptor molecules (ovals) are intercalated into a DNA duplex. Transition metal
complexes, Ru metallointercalators, Rh metalloinsertors, intercalating organic dyes, and
fluorescent base analogs are commonly used as donor and/or acceptor molecules.
Photoexcitation initiates charge transport through the DNA bridge and is measured using
spectroscopy or other means generally probing the donor or acceptor.
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