Sequence-Specific Quantitation of Mutagenic DNA Damage via Polymerase Amplification with an Artificial Nucleotide

Article abstract of DOI:10.1021/jacs.9b11746

DNA mutations can result from replication errors due to different forms of DNA damage, including low-abundance DNA adducts induced by reactions with electrophiles. The lack of strategies to measure DNA adducts within genomic loci, however, limits our unde

Full text of DOI:10.1021/jacs.9b11746

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Article
Sequence-specific Quantitation of Mutagenic DNA damage
via Polymerase Amplification with an Artificial Nucleotide
Claudia M. N. Aloisi, Arman Nilforoushan, Nathalie Ziegler, and Shana J Sturla
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b11746 • Publication Date (Web): 20 Mar 2020
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Sequence-specific Quantitation of Mutagenic DNA damage via Poly-
merase Amplification with an Artificial Nucleotide
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Claudia M.N. Aloisi, Arman Nilforoushan, Nathalie Ziegler, and Shana J. Sturla*
Department of Health Sciences and Technology, ETH Zürich, Schmelzbergstrasse 9, 8092 Zurich, Switzerland.
Mutation spectra from colon cells of people with CRC resem-
bles the spectrum induced by the carboxymethylating agent po-
tassium diazoacetate (KDA).14 Spectra measured in the cancer-
relevant gene p53 of CRC tissues and in a KDA-treated p53
gene-containing plasmid were rich in C>T transition mutations,
potentially arising from the misincorporation of T opposite O6-
CMG during replication, as suggested by in vitro studies inter-
rogating the fidelity of replication past O6-CMG.15-17 When O6-
CMG was placed in a DNA template and replicated by trans-
lesion DNA synthesis (TLS) polymerases (Pols), it was found
that O6-CMG promotes the misincorporation of bases by Y- and
B-family TLS Pols.15 In another study, by transfecting a DNA
plasmid containing O6-CMG in human cells, it was concluded
that O6-CMG moderately blocks DNA replication and induces
mutations at substantial frequencies.16 While there is evidence
regarding the mutagenic potential of O6-CMG, there is however
a lack of data concerning the presence and accumulation of O6-
CMG in CRC hotspot regions of the genome.
To establish a cause-effect relationship between O6-CMG and
CRC, the detection and quantification of DNA damage in the
genome is critically needed. O6-CMG is typically detected by
P32 postlabeling,18 immunoblotting,19 affinity chromatog-
raphy,8 and mass spectrometry.20-22 The most sensitive O6-CMG
measurement was by ESI-MS3 with a LOQ of 73.4 amol,22 al-
lowing detection of 0.05 O6-CMG per 107 nucleotides in human
cells. However, for all approaches, only the total level of dam-
age can be determined and information on the genomic location
of O6-CMG is lost.
ABSTRACT
DNA mutations can result from replication errors due to differ-
ent forms of DNA damage, including low abundance DNA ad-
ducts induced by reactions with electrophiles. The lack of strat-
egies to measure DNA adducts within genomic loci, however,
limits our understanding of chemical mutagenesis. The use of
artificial nucleotides incorporated opposite DNA adducts by en-
gineered DNA polymerases offers a potential basis for site-spe-
cific detection of DNA adducts, but the availability of effective
artificial nucleotides that insert opposite DNA adducts is ex-
tremely limited, and furthermore, there has been no report of a
quantitative strategy for determining how much DNA alkyla-
tion occurs in a sequence of interest. In this work, we synthe-
sized an artificial nucleotide triphosphate that is selectively in-
serted opposite O6-carboxymethyl-guanine DNA by an engi-
neered polymerase and is required for DNA synthesis past the
adduct. We characterized the mechanism of this enzymatic pro-
cess and demonstrated that the artificial nucleotide is a marker
for the presence and location in the genome of O6-carboxyme-
thyl-guanine. Finally, we established a mass spectrometric
method for quantifying the incorporated artificial nucleotide
and obtained a linear relationship with the amount of O6-car-
boxymethyl-guanine in the target sequence. In this work, we
present a strategy to identify, locate and quantify a mutagenic
DNA adduct, advancing tools for linking DNA alkylation to
mutagenesis and for detecting DNA adducts in genes as poten-
tial diagnostic biomarkers for cancer prevention.
A nanopore-based O6-CMG sequencing approach has been re-
cently applied to study the behavior of O6-CMG during replica-
tion by Phi29 DNA Pol.23 In this system, the presence of the
adduct is associated with an alteration of current signal only for
pre-defined arrangements of 4-base contexts. Additionally, the
characteristic signal was obtained by using a low throughput
nanopore that is not commercially available.
INTRODUCTION
DNA integrity is continuously threatened by endogenous and
exogenous DNA-reactive chemicals. The resulting chemical
adducts to DNA can initiate adverse biological consequences
including cell death and mutation. O6-alkyl-guanines (O6-al-
kylGs) are mutagenic DNA adducts that have been linked to
carcinogenesis.1-4 They can form from anticancer drugs,5 anti-
biotics,6 and environmental exposures such as cigarette smoke
or red and processed meat consumption.7-11 In particular, O6-
carboxymethyl-guanine (O6-CMG; Fig. 1B) was significantly
higher in exfoliated colonocytes of people who eat meat vs. veg-
etarians,10 and has been detected upon in vitro gastrointestinal
digestion of meat,11 suggesting its formation as a molecular in-
itiating event in colorectal carcinogenesis (CRC) linked to meat
consumption.11-13
DNA adduct-directed artificial nucleotides have been devel-
oped as a basis for interrogating damaged DNA in duplex hy-
bridization or polymerase-mediated synthesis contexts.24 We
have reported a variety of nucleoside analogues that stabilize
damaged DNA vs. undamaged DNA.24-27 The heterocyclic im-
idic nucleoside analog derived from benzimidazolinone (Benzi)
and a derivative with an extended ρ-surface termed ExBenzi
(Fig. 1B) consist of a conjugated π-system, a hydrogen bond
accepting carbonyl group and an imino-based hydrogen bond
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donor. When placed opposite O6-alkylG adducts, the resulting
Page 2 of 14
duplexes are stabilized.27-31 When ExBenzi was included in
short oligonucleotides attached to gold nanoparticles, the pres-
ence of O6-alkylG adducts in target DNA strands disrupted na-
noparticle aggregation and induced a color change, indicating
the presence of the adduct.28 These hybridization probes pro-
vide an excellent quantitative read-out, however, do not involve
DNA amplification, and have so far been used to detect down
to 138 fmol of modified DNA in 6 pmol total DNA.
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Polymerase-mediated amplification has revolutionized the life
sciences, and the first report of its use to amplify a DNA adduct
involved the bypass-proficient Pol mutant KlenTaq M747K,30-
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and the heterocyclic imidic nucleotide triphosphate (TP)
Benzi. Benzi was selectively incorporated opposite O6-alkylG
and DNA complementary to the damaged templates could be
amplified by sequential repeats of primer extension reactions,
with the presence of Benzi marking the damage location. How-
ever, the approach lacked any quantitative analysis capacity
meaning that the amount of O6-alkylG in the DNA sample could
not be determined.
Figure 1 Single nucleotide incorporation opposite O6-
CMG (A) Scheme of KlenTaq M474K-mediated single nu-
cleotide-incorporation and DNA template sequence used in
this study (B) Single nucleotide-incorporation of T, Benzi
and ExBenzi opposite X = G or O6-CMG, catalyzed by
KlenTaq M474K, analyzed on denaturing polyacrylamide
gels and visualized by autoradiography (M = marker lane
with 23 nt primer).
To fulfil both the requirement of quantification of low-occur-
rence O6-CMG and the need to retain information regarding the
DNA sequence, we present herein a chemical-biochemical-an-
alytical combined strategy for the quantitative detection of O6-
CMG in a sequence-targeted manner. We synthesized (Scheme
S1) the triphosphate of the aromatic heterocyclic imide nucleo-
side ExBenzi and showed for the first time that it is specifically
incorporated opposite O6-CMG by an engineered Pol. We char-
acterized the incorporation rates of ExBenziTP vs. natural bases
opposite O6-CMG, and used molecular modelling based on the
KlenTaq M747K crystal structure to identify a structural basis
for ExBenzi selectivity. Finally, we developed a mass spectro-
metric method to quantify the incorporated ExBenzi nucleoside,
which reflects the amount of initial O6-CMG. With this combi-
nation of chemical probe, polymerase-mediated synthesis and
mass spectrometric analysis, we were able to detect and quan-
tify O6-CMG adduct in a specific DNA sequence context. With
the capacity to target any DNA sequence, this strategy is antic-
ipated to help elucidate how DNA damage in hotspot regions of
the genome impacts the mutagenesis and carcinogenesis pro-
cesses.
base most frequently incorporated opposite O6-CMG by Klen-
Taq M747K.30 The percentage of incorporated product was cal-
culated as the ratio of the amount of n+1 extension product to
the initial amount of primer (Fig. 1B). We found that opposite
O6-CMG, ExBenzi is more efficiently incorporated (59%) than
T (12%), and that it is incorporated at a higher level than the
previously reported Benzi (41%). Opposite a template contain-
ing G, there was almost no evidence for nucleotide incorpora-
tion (4% for both Benzi and ExBenzi vs. 12% for T).
To quantitatively assess the efficiencies of nucleotide incorpo-
ration and characterize how replication over O6-CMG by Klen-
Taq M747K depends on the presence of ExBenziTP, steady-
state kinetic analyses of these primer extension processes were
performed. Thus, the amount of incorporation of single nucleo-
tides was measured over time at increasing concentration of
each nucleotide (Fig. S2). Oligonucleotides resulting from sin-
gle incorporation of nucleotides were separated on polyacryla-
mide gel and visualized by autoradiography. The equilibrium
constant for binding affinity (KM) and the catalytic turnover
RESULTS AND DISCUSSION
Artificial nucleotide analogue is incorporated opposite O6-
CMG DNA adduct
Table 1 Steady-state kinetic parameters for nucleotide
incorporation by KlenTaq M747K DNA polymerase
Relativea
KM
kcat
[min-1
kcat/KM
[µM-1 min-1
X*
dNTP
[µM]
]
]
kcat/KM
To characterize the efficiency of incorporation of ExBenzi op-
posite O6-CMG, we performed primer extension experiments
using the engineered DNA Pol KlenTaq M747K, which has an
established capacity to bypass and incorporate chemically mod-
ified nucleotides. 30-32 Thus, a 5’-end radiolabeled 23-nucleotide
(nt) primer was annealed to a 28 nt template (SI, material and
methods) with either G or O6-CMG at nt position 24. The ability
of KlenTaq M747K to incorporate ExBenzi opposite G or O6-
CMG was tested by incubating the annealed primer-template
DNA with ExBenziTP and KlenTaq M747K at 55 °C for 10 min
(Fig. 1A). For comparison, the reaction was conducted with
other single nucleotides as controls, i.e. the previously reported
artificial nucleotide BenziTP and dTTP, which is the canonical
O6-CMG
C
T
689 ± 104
196 ± 29
4.1
3.8
0.006
0.019
0.04
0.13
0.44
Benzi
60 ± 14
3.7
0.062
ExBenzi
48 ± 7
1.5 ± 0.2
45 ± 7
6.8
26
0.142
17.5
1
G
C
T
123
0.34
2.2
0.048
Benzi
32 ± 6
39 ± 5
0.10
0.14
0.003
0.003
0.02
0.02
ExBenzi
a Relative kcat/KM equals catalytic efficiency (kcat/KM) relative to that of ExBenziTP
incorporation opposite O6-CMG
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opposite O6-CMG or G in DNA bound to KlenTaq M747K. Us-
(kcat) were derived for incorporation by KlenTaq M747K of
Benzi, ExBenzi, T or C, opposite G or O6-CMG (Table 1). For
ExBenzi incorporation, KM values for incorporation templated
by either damaged or undamaged DNA were very similar (48
and 39 µM, respectively). Interestingly, a higher binding affin-
ity to the enzyme was calculated for ExBenzi and Benzi com-
pared to T and to C (opposite O6-CMG). Indeed, the KM for in-
sertion of ExBenzi opposite O6-CMG was almost 4-fold lower
than the KM for insertion of T and 14-fold lower than the KM for
insertion of C (48, 196 and 689 µM respectively). Conversely,
kcat for the incorporation of ExBenzi opposite O6-CMG was al-
most 2-fold higher than for the incorporation of Benzi, T or C
in the same context. As a result, the catalytic efficiency (kcat/KM)
for the incorporation of ExBenzi opposite O6-CMG was 7- and
24-fold higher than for T or C, respectively, opposite O6-CMG
(Table 1). Remarkably, ExBenzi displayed the highest selectiv-
ity, almost 50-fold, for incorporation opposite damaged over
undamaged DNA (Table 1; kcat/KM = 0.142 vs. 0.003).
ing a molecular mechanics-based computational modeling ap-
proach, we built a model starting from the crystal structure of
KlenTaq M747K in a ternary complex with double-stranded
DNA and an incoming dCTP (PDB ID: 5O7T). We then re-
placed incoming dCTP with ExBenziTP, BenziTP or dTTP.
The template strand had either G (original structure) or O6-
CMG opposite the incoming base. Upon energy minimization
to identify the most stable and high-occupancy conformer of the
constructed DNA-enzyme complex (Fig. 2A), ExBenzi and O6-
CMG interacted by two hydrogen bonds (Fig. 2B, top): one be-
tween the N1 of O6-CMG and the –NH donor of ExBenzi (1.9
Å), and one between the NH2 donor of O6-CMG and the car-
bonyl group of ExBenzi (2.1 Å). These interactions are con-
sistent with previous models29, 31 and crystallographic analysis33
of a similar construct with Benzi. In contrast, the G:ExBenziTP
structure predicted only one hydrogen bond (2.0 Å) and a po-
tential steric clash between the –NH moiety on ExBenzi and the
–NH at the N1 position on O6-CMG (Fig. 2B, middle), expected
to hinder catalysis. As a result, the complex with O6-
CMG:ExBenziTP is predicted to be more stable (computed free
energy of -8.0 kcal/mol) than that of G:ExBenziTP (+32.0
kcal/mol). Furthermore, by overlapping the above computed
base pairs, we observed a different orientation for ExBenzi de-
pending on whether it is paired with O6-CMG or G (Fig. 2C).
When paired with G, the steric clash imposed a slight rotation
to ExBenzi that resulted in reduced planarity. The computation-
ally derived geometry and interactions of ExBenzi with O6-
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Structural basis for ExBenziTP selective incorporation op-
posite O6-CMG by KlenTaq M747K
We were interested to understand the physical basis for the
highly selective incorporation of ExBenzi opposite O6-CMG,
despite its large size, both when compared to other nucleotides
and in terms of template selectivity. Thus, we performed mo-
lecular modelling studies with ExBenziTP, BenziTP or dTTP
Figure 2 Molecular modelling of interactions in the active site of KlenTaq M747K (A) KlenTaq M747K (PDB ID: 5O7T)
was energy minimized with Molecular Operating Environment software. (B) In its active site (zoomed-in region), ExBenziTP
paired opposite O6-CMG (top) via two hydrogen bonds and opposite G (middle) via one hydrogen bond. dTTP paired opposite
O6-CMG (bottom) via two hydrogen bonds. (C) Overlay of base pairs O6-CMG:ExBenziTP and O6-CMG:TTP (similarly planarity,
bottom). Opposite G, ExBenzi is tilted (top). (D) Relative catalytic efficiency (Table 1) of incorporation of ExBenziTP opposite
G (blue bar), opposite O6-CMG (grey) and of TTP opposite O6-CMG (green). (E) 2D ligand-protein interactions are shown be-
tween KlenTaq M747K and ExBenziTP (top) and TTP (bottom)
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CMG vs. G help explain the experimentally derived catalytic ration experiments, except that the reaction mixture was supple-
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parameters (Table 1 and graph on Fig. 2D).
mented with all four natural dNTPs, with or without BenziTP
or ExBenziTP (Fig. 3A). Primer elongation products were ana-
lyzed on denaturing polyacrylamide gels and visualized by au-
toradiography. In the presence of all four natural dNTPs, repli-
cation was stalled at O6-CMG, resulting in little (17%) fully ex-
tended primer. However, when either BenziTP or ExBenziTP
was additionally added, O6-CMG was effectively bypassed
(44% and 63% full-length products, respectively). Finally, we
confirmed that replication of undamaged DNA (Fig. 3B, X=G)
in the presence of the four natural dNTPs occurs with full pri-
mer extension around 90% (band +5 and +6, Fig. 3B), regard-
less of the presence of BenziTP or ExBenziTP. Thus, BenziTP
and especially ExBenziTP promote full-length DNA synthesis
past O6-CMG, allowing replication of DNA containing O6-
CMG.
Having established ExBenzi as an effective complement to O6-
CMG, both when inserted and extended by KlenTaq M747K,
we tested whether a primer annealed to a template containing
O6-CMG could be amplified in the presence of ExBenzi. For
these studies, DNA primer and template were not pre-annealed:
steps of annealing, primer extension and duplex melting were
repeated to allow that the 28 nt DNA could template several
rounds of primer extension. To maximize selectivity and en-
zyme efficiency, we optimized amplification cycles (Fig. S4),
temperature, DNA and TP concentration (Fig. S5). Optimal
conditions consisted of 5 nM of 28 nt template, 300 nM of 23
nt primer, 10 µM of each dNTPs and of ExBenziTP, 50 nM
KlenTaq M747K (Fig. 4A). Alternating cycles (50) of DNA
melting (95 °C, 30 s), annealing (42 °C, 30 s) and extension
steps (55 °C, 30 s) were performed. Products were separated on
a polyacrylamide gel and visualized by fluorescence (Fig. 4B).
Under these conditions, a signal for the full-length product was
recorded only in the presence of ExBenziTP.
Having established a structural basis for template-selective in-
corporation of ExBenzi, we were interested to understand the
selective incorporation of ExBenziTP over TTP by KlenTaq
M747K. Thus, we modelled O6-CMG:TTP in the active site of
the enzyme and found a similar planarity (Fig. 2C) and hydro-
gen bond pattern (Fig. 2B, bottom) for O6-CMG:TTP and O6-
CMG:ExBenziTP, suggesting that in this instance interactions
between the paired bases do not determine the selectivity of
ExBenzi incorporation, or that differences in the interactions
are not evident in these models. We speculated that the interac-
tions of the incoming base, ExBenziTP vs. dTTP, with the en-
zyme might account for the highly favorable incorporation of
ExBenzi over T opposite O6-CMG, also based on the value of
KM calculated for ExBenziTP and dTTP incorporation, which
suggests a stronger binding affinity for ExBenziTP (Table 1).
Indeed, the energy calculated for the interaction of KlenTaq
M747K with ExBenziTP is -225.2 kcal/mol vs.-204.5 kcal/mol
with dTTP. Interactions between the triphosphate groups of the
incoming nucleotide and the enzyme made the largest contribu-
tion to this difference (Fig. 2E). For example, when Arg659,
predicted to interact strongly with the phosphate of ExBenziTP,
was mutated to Ala, the computed energy difference was +43
kcal/mol, whereas, when Phe667, predicted to interact with T
base, was mutated to Ala, there was not a significant impact on
computed energy (Fig. S3). The interaction with the enzyme
was predicted to be slightly more favorable for ExBenziTP
when compared to BenziTP as well (not shown), in agreement
with kinetic measurements (Table 1).
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The modeling studies performed herein suggest that a combina-
tion of interactions of the incoming nucleotide with the poly-
merase and between the bases of the nascent pair drives the se-
lective reaction. The interaction of an incoming base (e.g. TTP
and ExBenziTP) with the enzyme represents a first energetic
barrier in the incorporation process.34 We speculate that, once
in the active site, the interaction of the incoming TP with the
templating base promotes the rate-limiting conformational
changes of the enzyme required for insertion. Therefore, the sta-
bilization of the system caused by the lowest energy base pair
is hypothesized to be pivotal in driving the base insertion (e.g.
ExBenziTP opposite O6-CMG vs. G). Our findings provide a
structural and energetic basis for the rational development of
artificial nucleotides for DNA adduct detection, and imply that
the artificial nucleotides should be designed to compensate the
altered hydrogen bonding capacity of the DNA adduct and to
favorably interact with the Pol enzyme responsible for DNA
synthesis. Overall, modelling studies corroborated results of
primer extension (Fig. 1) and kinetics experiments (Table 1),
supporting the further investigation of ExBenzi as a marker for
O6-CMG.
Figure 3 Primer extension past O6-CMG (A) Polymer-
ase-mediated DNA synthesis (B) Full extension of DNA
primer complementary to undamaged (X = G) or damaged
(X = O6-CMG) DNA template, in the presence of all four
natural dNTPS (4), and of BenziTP (4 + BenziTP) or of
ExBenziTP (4 + ExBenziTP). Reactions were catalyzed by
KlenTaq M474K for 10 min, analyzed on denaturing poly-
acrylamide gels and visualized by autoradiography (M =
marker lane with 23 nt primer).
ExBenzi is required for efficient extension of a DNA primer
on a damaged DNA template
To test whether the polymerase can extend from ExBenzi once
it is incorporated opposite O6-CMG, we performed DNA primer
extension studies under conditions suitable for synthesis of a
full-length complement of the damaged DNA template. Reac-
tion conditions were the same as for single nucleotide incorpo-
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difficult to detect amounts. Thus, we developed a mass spectro-
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metric method for quantification of ExBenzi nucleoside from
the amplicons. Linear amplification reactions in the presence of
all four natural nucleotides and ExBenziTP were performed us-
ing either O6-CMG DNA or unmodified DNA as template, anal-
ogous to that described in the previous paragraph. Amplified
DNA was enzymatically hydrolyzed to nucleosides and purified
for mass spectrometric analysis.
ExBenzi nucleoside was quantified by LC-MS/MS in SRM
mode, by monitoring the transition m/z 301→185 (Fig. S7). To
correct for normal instrument variation and account for sample
loss during processing, we evaluated artificial nucleosides that
are structural analogues of ExBenzi, such as Benzi, the
perimidinone Per, the benzimidazole derivatives BIM and
ExBIM, as internal standards (IS) that could avoid the need for
synthesizing isotopically modified ExBenzi.28-31 ExBIM nucle-
oside was optimal in having similar chemical properties to
ExBenzi nucleoside, eluting at a similar retention time, and hav-
ing a unique mass transition of m/z 285→169. We optimized
the preparation of samples to achieve a similar recovery for
ExBenzi nucleoside and for the IS ExBIM nucleoside of 90%.
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Figure 4 Reiterated DNA primer extension past O6-CMG
(A) Scheme of cyclically repeated elongation past O6-CMG.
(B) Linear amplification of 6-FAM-labelled 23 nt primer
and O6-CMG 28 nt template in the presence or absence of
ExBenziTP. Reactions were analyzed on denaturing poly-
acrylamide gels and visualized by fluorescence (M1 =
marker lane with 23 nt primer; 4 = reaction with four natural
dNTPs; 4 + ExBenzi = reaction with natural dNTPs +
ExBenziTP; M2 = marker lane with 28 nt template, corre-
sponding to the extended primer).
To quantify ExBenzi nucleoside, calibration curves were pre-
pared in the presence of sample matrix, consisting of nucleo-
sides, enzyme and salts (1xKTQ, SI), as matrix effect was ob-
served for the ionization of both nucleosides. Standard samples
in matrix were prepared analogously to what described above
for the experimental samples. Briefly, linear amplification reac-
tions were run using unmodified DNA template under the usual
conditions but in the absence of ExBenzi. Samples were spiked
with internal standard ExBIM (10 nM) and with ExBenzi (5-
500 nM). By relating the signal of ExBenzi to that of ExBIM,
we could quantify ExBenzi nucleoside in a matrix with a LOQ
of 20 fmol (Fig. S8). Analysis of ExBenzi released from ampli-
cons that were templated with DNA containing O6-CMG
showed a linear increase of ExBenzi signal with increasing con-
centrations of O6-CMG in the template. ExBenzi could be de-
tected also when reactions were performed with unmodified
To assess whether the ExBenzi-promoted primer extension
from damaged DNA could be applied to longer DNAs, we per-
formed primer extension on a modified 4.7 kb plasmid. A 6-
FAM-labelled 11 nt primer complementary to a region of the
pEGFP-W plasmid preceding a G or O6-CMG position was
used (Fig. S6A). In the presence of all four natural dNTPs under
fixed conditions (55 °C 1 min-extension, 50 nM KlenTaq
M747K), the primer was extended up to around 200 bases when
annealed to the unmodified plasmid, but not the O6-CMG-
modified plasmid (Fig. S6B). When ExBenziTP also was
added, however, the primer was effectively extended past O6-
CMG (Fig. S6C), similar to what was observed with short oli-
gonucleotides (Fig. 4). Finally, longer products (>1 kb) could
be obtained without affecting the selectivity by lengthening the
elongation time to 4 min. These observations suggest the strat-
egies characterized with oligonucleotide substrates have rele-
vance for O6-CMG in longer DNA samples.
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5×103
4×103
3×103
2×103
1×103
0
These findings demonstrate that ExBenzi serves as a marker for
presence and location of O6-CMG. It is required as a partner for
replicating DNA past the adducts, up to around 200 bases. Fur-
thermore, the amount of primer extended with oligonucleotide
templates exceeded the initial amount of damaged template
DNA present by roughly 18-fold, thereby increasing the poten-
tial signal from the amount of damage to the amount of marker
probe. While these results support a biochemical basis for
boosting the DNA damage signal, analyzing incorporation of
ExBenziTP via gel assays limits the precision and sensitivity of
quantifying DNA adducts, and coupled higher performance an-
alytics strategies are needed.
0
50
100
150
200
O6-CMG (fmol)
Figure 5 Quantitation of ExBenzi in amplicons from
DNA containing O6-CMG. ExBenzi incorporated by
linearly-amplified primer reactions from DNA with var-
ying amounts of O6-CMG by mass spectrometry, based
on the calibration curve in Fig. S9. Data is average from
four independent replicates, each made of two technical
replicates. R2 = 0.95.
Quantification of ExBenzi nucleoside by mass spectrometry
is diagnostic of O6-CMG levels in DNA
Having optimized the artificial nucleotide-engineered polymer-
ase amplification strategy for O6-CMG in a DNA sequence, we
aimed to overcome limitations in quantifying O6-CMG at low,
5
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DNA, probably due to unspecific incorporation or non-covalent
Page 6 of 14
binding of ExBenzi, such as intercalation.35 We could reduce
the unspecific signal by disrupting such non-covalent interac-
tions by varying pH or salt concentration; however, these addi-
tional sample processing steps significantly reduced recovery
rates. Therefore, the background levels of ExBenzi in samples
arising from amplification of unmodified DNA was used for
background subtraction for the analysis. Thus, we found a linear
increase of ExBenzi with increasing initial O6-CMG levels in
the template (Fig. 5).
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CONCLUSION
Interest in understanding the genomic location of DNA adducts
is growing in medical sciences,41-45 due to causative links to mu-
tagenicity and carcinogenesis,46 as well as drug efficacy.47 The
O6-CMG DNA adduct focused on in this study is mutagenic and
hypothesized to be linked with CRC development.11-13 We de-
veloped a strategy combining sequence-specific extension, in-
crease in copy number of marker strands, and quantitation of a
unique synthetic nucleoside as a reporter of levels of O6-CMG
DNA adducts in a DNA oligonucleotide. The artificial synthetic
nucleotide ExBenzi is the most specifically and efficiently in-
corporated opposite O6-CMG reported to date. We elucidated a
structural basis for the high selectivity and specificity involving
complementarity of H bonds and planarity of the O6-
CMG:ExBenziTP pair in the KlenTaq M747K active site. Fi-
nally, we developed a mass spectrometric method for quantifi-
cation of ExBenzi nucleoside and showed that the amount of
ExBenzi incorporated during DNA synthesis correlates linearly
with the initial O6-CMG DNA, allowing therefore its quantita-
tion. The detection of O6-CMG with retention of DNA sequence
information presented in this study lays a foundation to address
relationships between DNA damage and mutations in hotspot
regions of the genome needed for predictive or pre-diagnostic
purposes.
9
The approach described here establishes for the first time a
chemical basis for amplification and quantitation of DNA dam-
age in a sequence-targeted manner. This approach is useful for
the study of isolated oligonucleotides or longer DNA, however,
the sensitivity is insufficient for sequence-specific adduct de-
tection of samples from cells. In cultured cells exposed to
azaserine (0-450 µM), O6-CMG levels were 0.3-9.1 lesions/107
nucleotides.22 The low occurrence of O6-CMG is even more rel-
evant in the case of sequence-specific detection; for example,
there are only 5 guanines per genome (i.e. 6.6 x 109 nucleotides)
belonging to codons 12 and 13 of the k-ras gene, which are
commonly mutated in CRC.36 With the combined strategy de-
scribed here, assuming all k-ras Gs are carboxymethylated, an
increase of sensitivity of a few orders of magnitude is needed.
Nonetheless, for cell-based applications, there are several as-
pects that can be developed in future studies to address this lim-
itation, including polymerase enzyme engineering/evolution,37-
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higher mass spectrometric sensitivity, and sample enrich-
ASSOCIATED CONTENT
Supplementary Information
ment.40
Bypass of O6-MeG occurs independent of ExBenzi
The Supporting Information is available free of charge
at https://pubs.acs.org/
In addition to the hurdle of extremely low abundance of DNA
adducts in cells, other adducts form, mostly O6-MeG, under re-
lated conditions.7-11 Since ExBenzi also can be incorporated op-
posite O6-MeG, and potentially unrelated adducts from differ-
ent sources, the presence of O6-MeG in biological samples by
the approach described here could lead to overestimation of O6-
CMG. Incorporation rates opposite O6-MeG are generally
higher than O6-CMG (6-fold increase for T and ExBenzi; Table
S1). Despite this, an integral selection feature of the O6-CMG-
targeting approach is that DNA synthesis past O6-CMG by
KlenTaq M7474K is low in the presence of natural dNTPs, re-
quiring ExBenzi for extension, whereas O6-MeG is readily ex-
tended in the absence of ExBenzi (Fig. S1B), due to the high
incorporation of T (Fig. S1A). Therefore, the combination of
ExBenziTP and KlenTaq M7474K is a poor indicator of O6-
MeG levels, and these observations are consistent with those
previously reported for Benzi.31 Nevertheless, further ap-
proaches may be envisioned, such as extending pre-annealed
primers first in the absence of ExBenziTP, resulting mainly in
the extension of primer annealed to O6-MeG, then adding
ExBenziTP to extend past O6-CMG targets (Fig. S1C). Future
work will be focused on characterizing chemical and biochem-
ical aspects that drive adduct-triphosphate interactions and on
targeting structural and enzymatic features to enable comple-
mentary strategies that address diverse adducts in heterogene-
ous biological samples.

Material and methods, characterization data for synthetic
triphosphate, supplementary figures S1-S8 and corre-
sponding references.
AUTHOR INFORMATION
Corresponding Author
*sturlas@ethz.ch
Acknowledgment
We are very grateful to Emma Sandell (ETH Zurich) for so
promptly and kindly sharing DNA plasmid.
Funding Sources
The research was supported by the Swiss National Science Foun-
dation (156280, 185020) and the European Research Council
(260341).
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