of 17
Electrochemical Assay for the Signal-on Detection of Human
DNA Methyltransferase Activity
Natalie B. Muren
and
Jacqueline K. Barton
*
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
California 91125, USA
Abstract
Strategies to detect human DNA methyltransferases are needed, given that aberrant methylation by
these enzymes is associated with cancer initiation and progression. Here we describe a non-
radioactive, antibody-free, electrochemical assay in which methyltransferase activity on DNA-
modified electrodes confers protection from restriction for signal-on detection. We implement this
assay with a multiplexed chip platform and show robust detection of both bacterial (
SssI
) and
human (Dnmt1) methyltransferase activity. Essential to work with human methyltransferases, our
unique assay design allows activity measurements on both unmethylated and hemimethylated
DNA substrates. We validate this assay by comparison with a conventional radioactive method.
The advantages of electrochemistry over radioactivity and fluorescence make this assay an
accessible and promising new approach for the sensitive, label-free detection of human
methyltransferase activity.
INTRODUCTION
In mammals, DNA methylation is the most prominent form of epigenetic gene regulation
and is a critical long-term gene silencing mechanism.
1,2
This covalent addition of a methyl
group to the carbon-5 position of cytosine at predominantly 5
-CG-3
sites is catalyzed by
DNA methyltransferases, which use the cofactor S-adenosyl-L-methionine (SAM) as a
methyl donor. DNA methylation is central to many normal cellular processes including
development, X chromosome inactivation, gene regulation, and transposon silencing, among
others.
1,2
However, aberrant DNA methylation has been associated with multiple disease
states including developmental abnormalities such as ICF (immunodeficiency, centromere
instability, and facial abnormalities) syndrome and Rett syndrome,
3,4
autoimmune diseases
such as lupus,
5
and many types of cancer.
6–8
The link between abnormal DNA methylation and cancer has recently become an area of
intense, widespread research, and both excessive methylation (hypermethylation) and
deficient methylation (hypomethylation) have been identified in diverse tumor types.
7,9
While hypermethylation can contribute to oncogenesis by the silencing of tumor suppressor
genes,
8
hypomethylation may activate oncogenes or latent retrotransposons, or cause
chromosome instability.
7
In many cases, these harmful methylation states have been linked
to the abnormal expression and activity of DNA methyltransferases.
8,10–13
Mammalian DNA methyltransferases include Dnmt1, Dnmt3a, and Dnmt3b and while all
three catalyze the same reaction, they play different roles in establishing methylation
patterns in the genome. Dnmt1 transmits methylation patterns across cell divisions by
completing methylation on newly replicated strands at 5
-CG-3
sites that carry methylation
*
Corresponding Author: jkbarton@caltech.edu.
NIH Public Access
Author Manuscript
J Am Chem Soc
. Author manuscript; available in PMC 2014 November 06.
Published in final edited form as:
J Am Chem Soc
. 2013 November 6; 135(44): 16632–16640. doi:10.1021/ja4085918.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
on the template strand alone.
1
Thus Dnmt1 is characterized as a maintenance
methyltransferase and displays a significant preference for hemimethylated DNA
substrates.
14
Dnmt3a and Dnmt3b, in contrast, are characterized as
de novo
methyltransferases because of their activity at unmethylated 5
-CG-3
sites, primarily during
embryogenesis when new methylation patterns must be set.
1,15
These inherently different
activities contribute to the complex roles of methyltransferases that are now being elucidated
in a growing number of cancers.
Understanding how methyltransferase activity contributes to cancer initiation and
progression is a very attractive goal; abnormalilties in methyltransferase activity usually
occur far before other signs of malignancy and could thus be used for early cancer
detection.
7,8
Additionally, identification of cancers with a certain methylation phenotype
(hypermethylation or hypomethylation) can help specify an effective course of treatment.
7,16
Clearly, the expansion of this new field, including the study of methyltransferase activity in
cancer cells, the characterization of anti-methylation drugs, and the screening of patients for
early cancer diagnosis, requires effective and accessible assays to measure methyltransferase
activity.
While radioactive labeling with [methyl-
3
H]-SAM is the current standard for assaying
methyltransferase activity,
14,17
the desire to avoid radioactive reagents has motivated the
development of diverse alternatives including PCR-based bisulfite conversion,
18
HPLC,
19
and fluorescence and colorimetric assays.
20,21
Additionally, assays have been developed that
utilize digestion of DNA by methylation-sensitive restriction enzymes. In these assays,
methylation of a specific DNA sequence confers protection from digestion by the
corresponding restriction enzyme, and results are typically visualized by fluorescence.
22–24
Alhough non-radioactive, these methods still carry significant drawbacks including time-
consuming sample preparation and data analysis, bulky detection equipment, expensive
antibodies and fluorescently labeled substrates, and inflexible detection schemes that are not
compatible with human methyltransferases.
Electrochemical strategies overcome many of these drawbacks, providing non-radioactive,
low-cost, portable sensors that have high potential for use in clinical settings.
25,26
Despite
these advantages, relatively little work has been aimed at the electrochemical detection of
methyltransferase activity. Reported electrochemical strategies include the direct oxidation
of individual DNA bases to detect 5-methylcytosine
27
and several methods that use
methylation-sensitive restriction enzymes. These include restriction-based signal modulation
with DNA-functionalized gold nanoparticles,
28
restriction-facilitated binding of redox-active
moieties such as carbon nantubes,
29
probe-modified DNA,
30
and redox-active enzymes,
31
and DNA monolayers with methylation-sensitive restriction sites that bear either
electrochemical
32–35
or photoelectrochemical
36
reporters. Though diverse, these strategies
are limited in that they are either demonstrated with synthetic 5-methylcytosine alone and
not enzymatic methylation or they are only applicable to the detection of bacterial
methyltransferase activity. As human methyltransferase activity is sensitive to the
methylation state of the DNA substrate (unmethylated or hemimethylated), assays for the
study of human methyltransferases must allow for the use of both substrates.
Here we describe a new electrochemical assay in which either an unmethylated or
hemimethylated DNA substrate may be used for the sensitive detection of both bacterial and
human methyltransferase activity. In this assay, multiplexed, DNA-modified electrodes
bearing covalent redox probes
26,37
are combined with a methylation-sensitive restriction
enzyme to convert methylation into an electrical signal (Figure 1). Such DNA-modified
electrodes have been used previously to detect protein binding
38
and restriction
activity,
37–39
and the electrochemistry of the covalent methylene blue redox probe employed
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here has been thoroughly characterized.
40
Importantly, our multiplexed chip platform
(Figure 1, inset) allows for the direct comparison of up to four types of DNA substrates and
four methyltransferase conditions side by side on the same surface.
37
For this assay,
electrodes are modified with DNA that contains the human methylation site (5
-CG-3
)
within the recognition site of a methylation-sensitive restriction enzyme. Upon treatment of
the electrodes with active methyltransferases, these sites become methylated, thereby
protecting the DNA from restriction during subsequent restriction enzyme treatment. With
the DNA intact, the redox signal from the probe is retained (signal-on). In the absence of
active methyltransferases, the DNA remains unmethylated and is readily cut, causing the
near complete disappearance of the redox signal (signal-off).
Importantly, this work addresses the special requirements and challenges associated with
human methyltransferase detection. First, DNA substrate versatility is critical as the primary
human methyltransferase, Dnmt1, has a strong preference for hemimethylated 5
-
m
CG-3
sites. To meet this requirement, we demonstrate this assay with both the
BstU
I and
BssH
II
restriction endonucleases (recognition sites of 5
-CGCG - 3
and 5
-GCGCGC-3
,
respectively). While
BstU
I does not support the use of a hemimethylated substrate because
hemimethylation of its recognition site alone blocks restriction,
BssH
II allows for the use of
both unmethylated (5
-GCGCGC-3
) and hemimethylated (5
-G
m
CGCGC-3
) DNA because
both substrates are readily cut if not further methylated. Second, work with human
methyltransferases involves the exposure of electrode surfaces to greater amounts of protein
material due to the larger size and lower activity of these proteins, as compared to bacterial
methyltransferases. To overcome the obstructive effects of high protein content on
electrochemical signals, we introduce a simple and effective protease treatment step. With
these important adaptations we are able, uniquely, to detect human methyltransferase
activity by an electrochemical method. We demonstrate this assay for the sensitive detection
of bacterial
SssI
and human Dnmt1 methyltransferase activity with a multiplexed, low cost
format that may easily be applied to high throughput studies or utilized in research and
clinical laboratories.
MATERIALS AND METHODS
Materials
All standard and modified phosphoramidites were purchased from Glen Research. Modified
methylene blue dye for coupling was synthesized as described previously.
40
S-adenosyl-L-
methionine (SAM) and lambda DNA were purchased from New England Biolabs. Tritiated
SAM (
32
H-SAM) was purchased from Perkin Elmer. All other chemicals for the preparation
of protein buffers and DNA-modified electrodes, and for use in
32
H-SAM experiments were
purchased from Sigma-Aldrich and used as received. Multiplexed chips were fabricated at
Caltech as described previously.
37
Protein Preparation
All proteins were purchased from commercial sources.
Sss
I methyltransferase, BSA, and the
restriction endonucleases
BstU
I,
BssH
II, and
Rsa
I were purchased from New England
Biolabs and used as received unless otherwise indicated. Protease from
Streptomyces griseus
was purchased as a dry powder from Sigma-Aldrich and stored as a 250
μ
M solution in 40%
glycerol in phosphate buffer without NaCl (5 mM phosphate, pH 7) at −20°C. Human
Dnmt1 was purchased from BPS Bioscience. Buffer exchange by size exclusion spin column
(10 kDa cutoff, Amicon) was performed on Dnmt1 and
BssH
II prior to electrochemistry
experiments to remove dithiothreitol (DTT), which disrupts DNA-modified electrodes upon
heating. The exchange was performed according to manufacturer instructions at 4°C. Dnmt1
was exchanged into Dnmt1 activity buffer (50 mM Tris-HCl, 1 mM EDTA, 5% Glycerol,
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pH 7.8), while
BssH
II was exchanged into a methylation/restriction (M/R) buffer (10 mM
tris-HCl, 50 mM NaCl, 10 mM MgCl
2
, pH 7.9)
DNA Sequences
For the detection of methyltransferase activity with
BstU
I, electrodes were modified with the
sequence 5
-HS- (CH
2
)
6
-GACTGA
GTAC
T
CGCGACTGA -3
with an unmethylated
methylene blue-modified complement. The
BstU
I restriction site (5
-
CGCG - 3
) is
underlined. As a control, this sequence also contains the
Rsa
I restriction site (5
-
GTAC
-3
)
which is italicized. For experiments with synthetically methylated DNA, the
BstU
I
restriction site was fully methylated on both strands (5
-
m
CG
m
CG -3
).
For the detection of methyltransferase activity with
BssH
II, electrodes were modified with
the sequence 5
-HS- (CH
2
)
6
- GACTGAGTACT
GCGCGCACTGA -3
with an
unmethylated methylene blue-modified complement. The unmethylated
BssH
II restriction
site (5
-
GCGCGC -3
) is underlined. DNA was also prepared with a hemimethylated
BssH
II
restriction site (5
-
G
m
CGCGC -3
).
DNA Synthesis
All DNA was synthesized with an Applied Biosystems 3400 DNA synthesizer. Thiolated
strands were prepared with a C6-S-S phosphoramidite at the 5
terminus. Strands containing
methylated cytosine were synthesized with a 5-methyl dC-CE phosphoramidite. DNA for
methylene blue coupling was prepared with an amino-C6-dT phosphoramidite at the 5
terminus. All DNA was purified by reverse-phase HPLC with a polymeric PLRP-S column
(Agilent) and characterized by mass spectrometry. For methylene blue-modified DNA,
coupling was carried out in solution as described previously.
40
Briefly, HPLC-purified DNA
was suspended in 0.1 M NaHCO
3
and combined with an equimolar amount of modified
methylene blue dye in DMSO. The mixture was allowed to shake overnight at room
temperature. The coupled DNA was then purified by Nap-5 size exclusion column (GE
Healthcare) before further purification by HPLC. For thiolated DNA, the disulfide was
reduced to the free thiol with 100 mM DTT in 100 mM tris-HCl buffer, pH 8.3 at room
temperature for 45 minutes, and then purified by Nap-5 column and HPLC. To prepare
duplexes, all DNA stocks were desalted, resuspended in phosphate buffer (5 mM phosphate,
50 mM NaCl, pH 7), and quantified by UV/Vis absorption at 260 nm. Equimolar amounts
(50
μ
M) of complementary strands were combined and thermally annealed.
Multiplexed Chip Preparation and Assembly
Prior to application of DNA solutions, chips were cleaned with acetone and isopropanol,
dried, and further cleaned by UV ozone. The chips were then assembled with a rubber
gasket and clamp, and a solution of 25
μ
M DNA in phosphate buffer was immediately
applied (20
μ
L DNA per quadrant). DNA film assembly was allowed to proceed overnight
at room temperature in a humid environment.
Electrochemistry
All electrochemistry was carried out with a standard potentiostat and multiplexer console
(CH Instruments). A three-electrode system was employed including a Pt wire auxiliary
electrode and Ag/AgCl reference electrode (Cypress Systems). DNA-modified chips were
first backfilled with 1 mM mercaptohexanol in phosphate buffer with 5% glycerol for 45
minutes at room temperature. For all electrochemistry, cyclic voltammetry (CV) scans were
performed at a 100 mV/s scan rate over a potential window of 0 mV to −500 mV. Scans of
the 16 electrodes on a chip were performed sequentially. Following each treatment step,
chips were rinsed and scanned with 200
μ
L of the specified buffer in a common well. Signal
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size was measured as the CV cathodic peak area. The reported variation in the data
represents the standard deviation across all electrodes measured for a given condition.
For the detection of
Sss
I activity, scans were performed in M/R buffer. Individual quadrants
of chips were treated with
Sss
I in M/R buffer with 160
μ
M SAM. A reaction volume of 10
μ
L was used for each quadrant. The
Sss
I solution was allowed to incubate on the chips at
room temperature for 2 hours. Chips were then rinsed and treated with a solution of 10
μ
g/
mL lambda DNA in M/R buffer for 45 minutes at room temperature. Following this, chips
were rinsed with 500 mM NaCl in M/R buffer to ensure complete
Sss
I dissociation. Next,
chips were treated with 1,000 units/mL of
BstU
I in M/R buffer at room temperature for 1
hour. As a control, chips were then treated with 1,000 units/mL
Rsa
I in M/R buffer at room
temperature for 30 minutes.
A similar procedure was followed for the detection of Dnmt1. As Dnmt1 activity buffer
lacks ionic and charged components (such as NaCl, MgCl
2
, and spermidine which inhibit
Dnmt1 activity),
41
it is not optimal for DNA electrochemistry. Thus, after Dnmt1
methylation was allowed to proceed in Dnmt1 activity buffer, an optimized scanning buffer
(5 mM phosphate, 50 mM NaCl, 4 mM MgCl
2
, 4 mM spermidine, 50
μ
M EDTA, 10%
glycerol, pH 7) was used for all electrochemical scans. Dnmt1 with 100
μ
g/mL BSA and
160
μ
M SAM were applied to individual chip quadrants and chips were incubated at 37°C
for 2 hours in a humidified container. Chips were then treated with 1
μ
M protease in
phosphate buffer for 1 hour at 37°C. After thorough rinsing, chips were treated with 1,500
units/mL of
BssH
II in M/R buffer at 37°C for 1 hour. For measurements of
Sss
I activity on
the
BssH
II 22-mer, scans were also performed in optimized scanning buffer and protease
treatment was used. Including the methyltransferase and restriction enzyme incubations, the
total assay time for
Sss
I or Dnmt1 is about 5 hours.
32
H-SAM Methyltransferase Activity Assay
Methyltransferase activity was also determined by a conventional
32
H-SAM activity assay,
based partially on previously published procedures.
14,17
Briefly, 20
μ
L reactions were
prepared with 20
μ
M DNA, 0.5
μ
Ci
32
H-SAM (~3
μ
M), and the methyltransferase sample in
appropriate activity buffer. For reactions with Dnmt1, 100
μ
g/mL BSA was included. For
the DNA substrate, the same
BssH
II unmethylated and hemimethylated 22-mer sequence
used for electrochemistry was employed, but without any probe or thiol modifications. For
each experiment, positive (15 nM
Sss
I) and negative (no protein) controls were included.
The reactions were mixed thoroughly and incubated at 37°C for 2 hours. The reactions were
then stopped with 30
μ
L of a 10 % TCA solution, spotted onto DE81 filter paper
(Whatman), and allowed to air dry for 15 minutes. The filter papers were then soaked
separately in 10 mL of 50 mM Na
2
HPO
4
for 15 minutes, and then rinsed with 50 mM
Na
2
HPO
4
and 95% cold ethanol. The filter papers were then dried before measurement by
liquid scintillation counting.
RESULTS AND DISCUSSION
Electrochemical Detection of SAM-dependent
SssI
Methyltransferase Activity
We first established this assay for the detection of
SssI
methyltransferase, the bacterial
analog to human methyltransferases, which also binds the site 5
-CG-3
and methylates the
C-5 position of the target cytosine.
42
To confirm that electrochemical signal protection is
due exclusively to DNA methylation, the dependence of
SssI
-mediated protection on the
essential SAM cofactor was evaluated. A 20-mer DNA duplex with a covalent methylene
blue redox probe at one end was designed with a centrally located
BstU
I restriction site (5
-
CGCG - 3
) and an adjacent
Rsa
I restriction site (5
-GTAC - 3
). Importantly, while
SssI
can
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methylate the
BstU
I site, it does not recognize the
Rsa
I site. Multiplexed chips were
modified in three quadrants with the unmethylated
BstU
I 20-mer and treated side by side
with 15 nM
Sss
I with SAM, 15 nM
Sss
I alone, or SAM alone. As a positive control, the
fourth quadrant was modified with the synthetically methylated
BstU
I 20-mer and left
untreated (Figure 2).
Initially, after
SssI
treatment of the electrodes, large electrochemical signals are observed
from the methylene blue-modified DNA film. Following
BstU
I treatment, electrodes treated
with
Sss
I and SAM show near complete signal protection (98% ± 2%), while those treated
with
Sss
I alone or SAM alone show the same, minimal signal protection (12% ± 1% and
13% ± 1%, respectively). Thus, the signal-on response of this assay depends on the presence
of the methyl donor, SAM. Electrodes modified with the synthetically methylated
BstU
I 20-
mer show complete signal protection (no measurable signal decrease).
To further rule out other possible modes of DNA protection, such as
SssI
-DNA binding or
nonspecific
SssI
aggregation that physically blocks the
BstU
I restriction site, the electrodes
were then treated with
Rsa
I, a restriction enzyme that is not inhibited by 5
-CG-3
methylation. With this treatment, the remaining redox signals for all DNA types are reduced
to the same, near complete level of attenuation (4% ± 1% signal remaining for all). As the
Rsa
I recognition site is located well within the binding footprint of
Sss
I,
43
DNA binding or
aggregation by
Sss
I that prevents access of
BstU
I to the DNA, would also prevent the access
of
Rsa
I. These results indicate that the observed DNA protection is due specifically to DNA
methylation catalyzed by
Sss
I.
Concentration Dependence of
Sss
I Methyltransferase Activity
The concentration range over which
Sss
I methyltransferase activity is detected with this
assay was then evaluated. Multiplexed chips modified with the unmethylated
BstU
I 20-mer
were treated with a range of
Sss
I concentrations (0–16 nM) including up to four different
concentrations on the same chip (Figure 3, inset). The percent signal protected from
BstU
I
restriction at each
Sss
I concentration, compiled from 4 chips with 4–8 electrodes measured
at each concentration, was used to make an activity curve (Figure 3). Near complete signal
protection is observed with
Sss
I concentrations of 8 nM and higher (96 % ± 3% at 8 nM;
99% ± 1% at 16 nM). Between the narrow
Sss
I concentration range of 4 nM and 2 nM, there
is a sharp loss of signal protection (91% ± 5% at 4 nM; 21% ± 5% at 2 nM). Below 2 nM
Sss
I, signal protection is not distinguished from the baseline signal that remains when
electrodes are left untreated (7% ± 1%). Thus the limit of detection for
Sss
I is 2 nM.
A Hemimethylated DNA Substrate and Protease Treatment for Human Dnmt1 Detection
To accommodate the most prominent human methyltransferase, Dnmt1, which shows strong
preferential activity at hemimethylated 5
-
m
CG-3
sites, DNA substrates with a
hemimethylated
BssH
II restriction site (5
-G
m
CGCGC-3
) were utilized. Importantly,
BssH
II requires full methylation of either 5
-CG-3
site within its recognition sequence to
prevent DNA restriction. Given that Dnmt1 is larger than
SssI
(molecular weights of 185 kD
and 42 kD, respectively) as well as less active, studies with Dnmt1 require the exposure of
electrodes to solutions with greater amounts of total protein material. Upon addition of 100
nM Dnmt1 to DNA-modified electrodes, substantial broadening of the redox peak is
observed along with some signal loss (Figure 4). These effects severely interfere with
activity comparisons across a wide Dnmt1 concentration range. Unlike work with
Sss
I,
where binding can be reversed by competitor DNA and buffer washes, these treatments are
not effective for Dnmt1.
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To address this issue, a protease treatment step was introduced to remove bound
methyltransferases; following methyltransferase treatment, prior to
BssH
II treatment, chips
were treated with a 1
μ
M solution of protease from
Streptomyces griseus
. Electrochemical
scans of a chip with methyltransferase-treated and untreated quadrants at these sequential
steps are shown in Figure 4. After the first 37°C heated incubation, signals from untreated
electrodes show a small decrease in peak height, without peak broadening, that typically
occurs the first time a chip is heated (77% ± 1% of the original peak height). In contrast,
signals from electrodes treated with 100 nM Dnmt1 show a much larger decrease in peak
height (46% ± 3% of the original peak height) with substantial peak broadening. After
protease treatment, however, the relative peak heights of treated and untreated electrodes are
equalized (74% ± 2% and 73% ± 1% of the original peak heights, respectively) and peak
broadening of the Dnmt1-treated electrodes is reversed.
From this equal starting point, methylation can then be visualized by treatment with
BssH
II;
signals from electrodes treated with 100 nM Dnmt1 are largely protected (89% ± 3%) while
minimal signal remains for untreated electrodes (5% ± 1%). Although proteases are
routinely used to remove interfering proteins in nucleic acid isolation and
in situ
hybridization (ISH) protocols, protease treatment has not yet been described as a strategy to
enhance electrochemical biosensing on DNA-modified surfaces. Demonstrated here,
protease treatment removes bound proteins, restores signal sharpness, and equalizes
electrode signals such that Dnmt1 activity may be accurately quantified and compared over
a wide range of methyltransferase concentrations.
Substrate Preference of Human Dnmt1
To measure the substrate preference and cofactor dependence of Dnmt1 electrochemically,
multiplexed chips were modified in two quadrants with the hemimethylated
BssH
II 22-mer
and in two quadrants with the unmethylated
BssH
II 22-mer. One quadrant of each DNA
substrate was then treated with 50 nM Dnmt1 with SAM, while the other quadrant of each
DNA substrate was treated with 50 nM Dnmt1 alone (Figure 5a, top row). As a comparison,
chips were assembled and treated similarly with
Sss
I (30 nM), which is a
de novo
methyltransferase and shows no preference for hemimethylated or unmethylated 5
-CG-3
sites (Figure 5a, bottom row).
44
For electrodes modified with the hemimethylated
BssH
II
22-mer and treated with Dnmt1 and SAM, substantial signal protection is observed (80% ±
2%). The same treatment shows minimal protection of the unmethylated
BssH
II 22-mer
(18% ± 2%). In contrast, electrodes treated with 30 nM
Sss
I and SAM show the same high
level of signal protection regardless of whether the hemimethylated or unmethylated
BssH
II
22-mer is the substrate (91% ± 2% and 92% ± 2%, respectively). These quantified data are
shown in Figure 5b, left column. For electrodes treated with Dnmt1 without SAM, a near
complete lack of signal protection is observed for both the hemimethylated and
unmethylated
BssH
II 22-mers (5% ± 1% and 4% ± 1%, respectively). Hemimethylated and
unmethylated electrodes treated with
Sss
I without SAM also show the same, minimal level
of signal protection (5% ± 1% for both). These important, SAM-free controls further
confirm that methylation is the mode of signal protection and demonstrate that both the
hemimethylated and unmethylated
BssH
II 22-mers are cut equally by
BssH
II when not
protected by methylation.
These electrochemical activity and substrate preference results for Dnmt1 and
Sss
I were
further validated by conventional
32
H-SAM Assay. For these experiments, the same
hemimethylated and unmethylated
BssH
II 22-mers, without redox probe or thiol
modifications, were employed. Mirroring the electrochemical result, Dnmt1 shows
substantially more activity on the hemimethylated
BssH
II 22-mer, while
Sss
I shows the
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same activity on both the unmethylated and hemimethylated 22-mers (Figure 5b, right
column).
Concentration Dependence of Dnmt1 Methyltransferase Activity
With protease treatment to establish a uniform initial point of comparison, the concentration-
dependent response of this assay for Dnmt1 activity was next evaluated. Multiplexed chips
modified with the hemimethylated
BssH
II 22-mer were treated with a range of Dnmt1
concentrations (0 – 100 nM), followed by protease and
BssH
II treatments (Figure 6, inset).
An activity curve was derived by compiling data from 4 chips with 4–12 electrodes
measured at each concentration (Figure 6). Near complete signal protection is achieved by
Dnmt1 concentrations of 50 nM and higher (83% ± 2% at 50 nM; 89% ± 3% at 100 nM).
The dynamic range of Dnmt1 is much broader than that of
Sss
I, spanning 5–50 nM. A
detection limit of 5 nM Dnmt1 is the concentration below which signal protection is no
longer distinguished from the signal that remains on untreated electrodes (11% ± 1% at 5
nM; 6% ± 1% for untreated electrodes).
Comparison of Assay Responses by
SssI
and Dnmt1
Results from this electrochemical assay reflect the clear differences in activity between
bacterial
Sss
I and human Dnmt1, as well as their distinct roles as
de novo
and maintenance
methyltransferases, respectively. Side by side electrochemical analysis of activity on
unmethylated and hemimethylated DNA substrates shows the hemimethylated substrate
preference of Dnmt1 and the absence of a preference for
Sss
I, a result mirrored in
32
H-SAM
experiments. Additionally for this assay, while
Sss
I exhibits a sharp, switch-like, signal
protection response with a narrow dynamic range of 2–8 nM, the dynamic range for Dnmt1
activity spans a much broader 5–50 nM. For
Sss
I, both the limit of detection and full
dynamic range of the assay fall below its solution dissociation constant (
K
d
) of 11 nM.
45
This indicates that methylation-induced signal protection is not limited by substrate access
and affinity; once the concentration of
Sss
I reaches a minimal level that allows appreciable
binding, the DNA film is efficiently methylated and protected. In contrast, the
K
d
of 23 nM
for Dnmt1 falls in the middle of the broad dynamic range measured by this assay.
46
The contrasting dynamic ranges exhibited by
Sss
I and Dnmt1 clearly reflect inherent
differences in activity (as expected, parallel
32
H-SAM experiments verify that the tested
Sss
I
is significantly more active than the tested Dnmt1), but may also reflect more subtle
differences that are exaggerated by this surface platform. The broader dynamic range of
Dnmt1 suggests that the DNA film is a substantially more difficult substrate matrix for
Dnmt1 than it is for
Sss
I. At nearly 4.5-fold larger than
Sss
I, the size of Dnmt1 may be a
limiting factor for methylation in the confined surface environment. Not only is the larger
size likely a greater hindrance to turnover rates at the surface, Dnmt1 may also not be
physically able to access all of the DNA sites in the film that
Sss
I is able to access. The
greater maximum signal-on magnitude achieved by
Sss
I over Dnmt1 provides additional
support that DNA access is more limited for Dnmt1. Notably, access to the DNA film is not
a significant issue for the restriction enzymes used in this assay; not only are
BstU
I and
BssH
II comparable in size to
Sss
I, but individual DNA duplexes are made more and more
accessible as progressive restriction of the DNA film occurs.
We can take clues from these divergent responses by
Sss
I and Dnmt1 to further refine the
DNA-modified electrode platform that is used to carry out this assay. For assays that require
binding of a target molecule to a nucleic acid probe on a surface, the physical accessibility
of the nucleic acid probe is a critical factor that can dictate both the sensitivity and dynamic
range of a biosensor platform.
47
Notably, previous studies have shown that for self-
assembled monolayers like those used in this work, thiolated DNA duplexes form films of
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heterogenenous density on gold surfaces.
48
Thus for this assay, different DNA film
morphologies (e.g. fully accessible duplexes spaced evenly across the surface or dense
island patches of DNA for which only outer duplexes are accessible to protein activity), may
present more or less challenging landscapes for a given methyltransferase to access and fully
protect from restriction. The film density and morphology of DNA-modified electrodes must
be a central point of consideration for modulating the sensitivity and dynamic range of this
assay for different applications.
Comparison to Current Approaches for the Detection of Methyltransferase Activity
As compared to other approaches in the literature to measure methyltransferase activity, a
notable advantage of this assay is the small sample volume (10
μ
L) required for analysis.
For colorimetric and fluorescence methods in solution, sample volumes typically range from
at least 20 – 150
μ
L.
21–24,49,50
Furthermore, the sample volume of our assay is only
restricted by our current electrochemical platform design and thus hardly at a lower limit.
Unlike other assays that are constrained by the configuration and sensitivity of
spectrophotometers and fluorescence readers, the flexibility of electrochemical platforms
inherently supports miniaturization. Still, with our current configuration,
Sss
I activity can be
observed by as little as 20 fmoles (0.8 ng) of protein while Dnmt1 activity can be observed
by as little as 50 fmoles (9 ng) of protein.
Several electrochemical assays have been described for the detection of
Sss
I activity based
on methylation protection of DNA films from cutting by the
Hpa
II restriction enzyme.
32–35
Our assay advances beyond these previous electrochemical approaches in that it allows
measurements on both hemimethylated and unmethylated DNA substrates. Thus, our assay
uniquely permits the detection of human methyltransferase activity by an electrochemical
method.
Currently, commercially available ELISA-like kits are the main alternative to radioactive
methods for the detection of human methyltransferase activity in both purified samples and
crude cell lysates. The sensitivity and dynamic range of our electrochemical assay for
Dnmt1 detection is comparable to these commercially available kits,
49,50
but the cost and
complexity of necessary reagents and equipment for our approach is substantially less.
These commercial assays require primary and secondary antibodies along with colorimetric
reagents or fluorescent labels and an absorbance or fluorescence microplate reader.
49,50
In
contrast for our assay, multiplexed chips and DNA substrates may be inexpensively
fabricated in bulk and the
BssH
II restriction enzyme is available cheaply from commercial
sources. Furthermore, the advantages of electrochemical instrumentation for biosensing are
well established; potentiostats are relatively simple, inexpensive devices that require
minimal maintenance and can be made portable, thereby making them accessible to a wide
range of research and clinical settings.
25,26
Beyond cost and ease of implementation, an
additional advantage of our assay is that the activity of a sample on hemimethylated and
unmethylated DNA substrates may be measured separately and compared on the same
device. This capacity to assess the maintenance vs.
de novo
methyltransferase activity of a
sample is critical for studies involving human methyltransferases because of the profound
impact of these specific roles on cancer processes.
CONCLUSIONS
Described here is a multiplexed, signal-on, electrochemical assay for the sensitive detection
of bacterial and human methyltransferase activity. This non-radioactive, antibody-free assay
is robust and generally requires less sample volume than other methods. The electrochemical
basis of this assay provides numerous advantages, including minimal, portable equipment,
inexpensive reagents, and a format that may be easily adapted to even more effective
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electrode architectures. The unique capability of this assay to provide a side-by-side report
of activity on unmethylated and hemimethylated DNA substrates is a key feature for studies
with human methyltransferases. As we continue to discover the epigenetic basis of
cancerous transformation, the demand for effective and inexpensive strategies to detect
human methyltransferase activity will continue to grow. Widely accessible approaches, like
the electrochemical assay presented here, will be necessary to meet this demand for cancer
detection and treatment.
Acknowledgments
This work was supported by NIH GM61077. We also thank the Parsons Foundation for a fellowship to NBM. We
thank C. Pheeney and P. Bartels for assistance with multiplexed chip fabrication. Multiplexed chip fabrication was
completed in the Kavli Nanoscience Institute at Caltech.
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