In-Gene Quantification of O6-Methylguanine with Elongated Nucleoside Analogues on Gold Nanoprobes

Article abstract of DOI:10.1021/jacs.6b03599

Exposure of DNA to chemicals can result in the formation of DNA adducts, a molecular initiating event in genotoxin-induced carcinogenesis. O⁶-Methylguanine (O⁶-MeG) is a highly mutagenic DNA adduct that forms in human genomic DNA upon reaction with methylating agents of dietary, environmental, or endogenous origin. In this work, we report the design and synthesis of novel non-natural nucleoside analogues 1′-β-[1-naphtho[2,3-d]imidazol-2(3H)-one)]-2′-deoxy-d-ribofuranose and 1′-β-[1-naphtho[2,3-d]imidazole]-2′-deoxy-d-ribofuranose and their use for quantifying O⁶-MeG within mutational hotspots of the human KRAS gene. The novel nucleoside analogues were incorporated into oligonucleotides conjugated to gold nanoparticles to comprise a DNA hybridization probe system for detecting O⁶-MeG in a sequence-specific manner on the basis of colorimetric readout of the nanoparticles. The concept described herein is unique in utilizing new nucleoside analogues with elongated hydrophobic surfaces to successfully measure in-gene abundance of O⁶-MeG in mixtures with competing unmodified DNA.

Full text of DOI:10.1021/jacs.6b03599

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Article
6
In-gene Quantification of O-Methylguanine with
Elongated Nucleoside Analogues on Gold Nanoprobes
Ioannis A. Trantakis, Arman Nilforoushan, Heidi A. Dahlmann, Celine K Stäuble, and Shana J Sturla
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b03599 • Publication Date (Web): 17 Jun 2016
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In-gene Quantification of O -Methylguanine with Elongated
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Nucleoside Analogues on Gold Nanoprobes
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Ioannis A. Trantakis*, Arman Nilforoushan, Heidi A. Dahlmann, Celine K. Stäuble, and Shana J.
Sturla*
Department of Health Sciences and Technology, ETH Zürich, Schmelzbergstrasse 9, 8092 Zurich, Switzerland
ABSTRACT: Exposure of DNA to chemicals can result in the formation of DNA adducts, a molecular initiating event in
6
6
genotoxin-induced carcinogenesis. O -Methylguanine (O -MeG) is a highly mutagenic DNA adduct that forms in human
genomic DNA upon reaction with methylating agents of dietary, environmental, or endogenous origin. In this work, we
report the design and synthesis of novel non-natural nucleoside analogues 1’-β-[1-naphtho[2,3-d]imidazol-2(3H)-one)]-2’-
6
deoxy-D-ribofuranose and (1’-β-[1-naphtho[2,3-d]imidazole]-2’-deoxy-D-ribofuranose and their use for quantifying O -
MeG within mutational hotspots of the human KRAS gene. The novel nucleoside analogues were incorporated into
oligonucleotides conjugated to gold nanoparticles to comprise a DNA hybridization probe system for detecting O -MeG
6
in a sequence-specific manner on the basis of colorimetric readout of the nanoparticles. The concept described herein is
unique in utilizing new nucleoside analogues with elongated hydrophobic surfaces to successfully measure in-gene
6
abundance of O -MeG in mixtures with competing unmodified DNA.
1
3
INTRODUCTION
DNA is exposed to chemical and environmental
in DNA, further supports the etiologic involvement of
6
O -MeG in colon and lung carcinogenesis, and
underscores the value of detecting its presence in tumor-
suppressor or tumor-promoter gene sequences as a
potential prognostic marker for cancer risk.
insults such as ultraviolet radiation, reactive oxygen
species, and alkylating agents. Endogenous and
exogenous methylating agents react with and modify
7
DNA nucleobases, producing primarily N -methylguanine
Assessing the accumulation of adducts such as
7
6
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(
N -MeG) and O -methylguanine (O -MeG). These
O -MeG in DNA is a considerable challenge owing to
adducts are present in DNA isolated from blood cells and
their extremely low occurrence, which is on the order of
only tens to thousands per genome.
quantified by P-postlabeling,
1
6
14-17
6
various human tissues. O -MeG is formed in DNA by SN -
O -MeG has been
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32
15,17
14,18,19
type methylating agents, represented by N-nitroso
immunoassay
and
20
compounds including known human carcinogens present
mass spectrometry,
completely digested prior to analysis. Therefore it is not
possible to assess O -MeG abundance in particular
wherein the DNA must be
3
in the environment, diet, and tobacco. Endogenous
4
6
sources of methylation include S-adenosylmethionine
and endogenous nitrosation products.
5,6
genomic loci of interest, such as oncogenes. Furthermore,
these methods provide little information regarding the
distribution of alkylation events in the genome, needed to
characterize causal relationships between adduct
occurrence and genetic mutations observed in cancer.
Therefore, a major standing goal for DNA adduct
detection is direct quantitative sequencing of genes
containing DNA adducts.
6
The formation of O -MeG is
a molecular
initiating event leading to carcinogenesis and cytotoxicity.
6
Upon encountering O -MeG, DNA polymerases often
7-9
preferentially incorporate thymidine.
The resulting
mismatch is a substrate for repair; however, the new
6
strand can persist and normal cells harboring O -MeG
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display a high rate of G→A mutations. Additionally,
when cancer cells are treated with methylating agents,
futile cycling of mismatch repair is exploited to induce
Several strategies are emerging for determining
the location of DNA adducts within the genome. First,
ligation-mediated polymerase chain reaction in
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cytotoxicity. G→A mutations have been causally related
with the development of tumors in experimental animals
treated with methylating carcinogens. The observation
combination with PAGE was used to map the distribution
of DNA lesions within gene sequences.
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21
This
of frequent G→A transition mutations in KRAS or p53
methodology can be used to examine the distribution of
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genes of human colon and lung tumors lacking O -MeG–
single strand breaks and alkali labile adducts (e.g. N -
6
23,24
MeG),
6
DNA methyltransferase, an enzyme that repairs O -MeG
but not to map the distribution of O -MeG.
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Figure 1. Schematic representation of the nanoprobe-based detection of the highly mutagenic O -MeG. The novel
synthetic nucleosides ExBenzi and ExBIM lead to the formation of more stable DNA duplexes when they are paired with
O -MeG than with G. Gold nanoparticles (AuNPs) functionalized with ordinary oligonucleotides serve as detection
nanoprobes while AuNPs functionalized with oligonucleotides containing a synthetic nucleoside (indicated by terminal
P) serve as discriminating nanoprobes. In the presence of O -MeG target DNA strands, the detection nanoprobes can
align in a tail-to-tail fashion with the discriminating nanoprobes. The hybridization of the target leads to aggregation of
AuNPs accompanied by a change in the color of the solution from red to purple.
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Sequencing of DNA with adducts was also achieved by
single-molecule real-time sequencing, which enables the
When
hydrophobic
perimidinone-
and
25
benzimidazole-derived nucleoside analogues (Benzi, BIM,
and Peri, Figure S1) were incorporated into
oligonucleotides, more stable DNA duplexes were formed
when the synthetic bases were placed opposite adducts
vs. unmodified bases. Ultimately, the larger nucleosides
Peri and Per were found to better stabilize O -BnG than
did smaller analogues. Nucleobases with large π surface
areas stack more strongly with neighboring bases than
their smaller counterparts since they have more area of
overlap with neighboring bases, as demonstrated for
benzene, naphthalene, and pyrene nucleosides
detection of an adduct on the basis of the slower rate of
polymerase-mediated nucleotide incorporation opposite
an adduct. More recently, a method based on the
enzymatic removal of oxidation adducts followed by the
introduction of marker nucleotides at the gapped sites
and subsequent PCR amplification and nanopore
30
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30
26
sequencing was reported. Another related approach was
based on the combination of DNA glycosylase-mediated
excision of specific DNA oxidation adducts with ligase-
mediated formation of deletion mutations at the sites of
the lesions, which could then be detected by DNA
37,38
and
39-42
size-expanded
natural
nucleobases.
Structural
27
sequencing. These strategies represent major conceptual
characterization studies with short duplexes indicated
that Per adopts a syn conformation and intercalates into
the duplex when placed opposite O -BnG, whereas
opposite G it adopts the anti conformation and forms a
and practical advances, but quantification of adduct
abundance remains to be addressed.
6
An approach that provides a plausible chemical
basis for quantitative in-gene detection of DNA alkylation
adducts is based on DNA adduct-directed nucleoside
probes containing artificial nucleobase surrogates that
complement DNA adducts. For example, a method for
43
less stable wobble pair. We proposed, therefore, to re-
orient the aromatic rings in Per and Peri in order to probe
the potential for their elongated surface to improve
hybridization performance.
6
site-specifically labeling O -MeG with fluorescent tags
New hydrophobic, elongated adduct-directed
nucleoside analogues 1’-β-[1-naphtho[2,3-d]imidazol-
2(3H)-one)]-2’-deoxy-D-ribofuranose (ExBenzi) and (1’-β-
[1-naphtho[2,3-d]imidazole]-2’-deoxy-D-ribofuranose
(ExBIM) (Figure 1) were synthesized and incorporated
into oligonucleotides. The modified oligonucleotides
formed more stable DNA duplexes when the artificial
nucleosides were placed opposite O -MeG vs. G.
Furthermore, oligonucleotides with ExBenzi and ExBIM
were conjugated to AuNPs to create nanoprobes effective
for colorimetric detection of O -MeG in cancer-related
hotspots of the KRAS oncogene. The colorimetric
detection of O -MeG is based on the distance-dependent
color change caused by AuNP aggregation upon
recognition of a target nucleic acid by the nanoprobes
(Figure 1).
transferred from complementary artificial nucleosides
28
was reported. It is not known whether this approach is
6
viable for sensing O -MeG in mixtures of targets. With
the goal of expanding alkylation adduct recognition, we
have devised a class of synthetic nucleosides with mixed
29-35
aromatic and H-bonding moieties.
The first example
6
was a perimidinone-derived nucleoside (Per, Figure S1)
that formed more stable DNA duplexes when paired with
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29
O -benzylguanine (O -BnG) than with G. In a recent
proof-of-principle study, we demonstrated that coupling
hybridization probes containing Per with gold
nanoparticles (AuNPs) allowed for the quantification of
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the model O -alkylG adduct O -BnG within a defined,
albeit non-biologically relevant, DNA sequence in the
36
presence of excess unmodified DNA strands.
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Figure 2. KRAS gene sequence studied herein. The target oligonucleotides were designed such that their middle base was
the middle base of either codon 12 or codon 13 (underlined bases). The length of the targets was varied for the evaluation
studies performed with targets centered on codon 13 (13-, 17-, 21-mer). The target was a 17-mer for the codon 12-based
studies.
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RESULTS AND DISCUSSION
containing G:C at the middle position had a T of 66.0 °C
m
6
(
Table 1). When O -MeG was paired with C, the Tm
Synthesis of elongated nucleoside analogues and
oligonucleotides
decreased to 55.2 °C. The diminished stability of this
duplex is consistent with previous studies suggesting that
ExBenzi and ExBIM were designed to explore the
influence of elongated nucleobase shape on adduct
recognition. ExBenzi is structurally similar to Benzi but is
extended by addition of a benzene ring (Figure 1).
Likewise, ExBIM is an extended version of BIM.
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O -MeG:C adopts a wobble configuration at physiological
pH and thus diminished stability could be attributed to
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the possibility for two instead of three hydrogen bonds.
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Additionally, the higher degree of lipophilicity of O -MeG
in comparison to
interactions within
complementary strands. In contrast, when the duplex
G
appears to disrupt stacking
both the modified and
Phosphoramidites of ExBenzi and ExBIM
nucleosides were newly synthesized as substrates for solid
phase DNA synthesis (Scheme S1). ExBenzi (1) was
prepared by heating neat 2,3-diaminonaphthalene and
5
2
6
contained ExBenzi opposite O -MeG, the T of the duplex
m
was slightly higher than when the duplex contained
4
4
urea, while ExBIM (2) was prepared by heating 2,3-
ExBenzi opposite G (55.0 °C vs 52.0 °C, ΔT =3.0 °C, Table
m
4
5,46
diaminonaphthalene in formic acid.
and 2 were glycosylated by nucleophilic displacement of
chloride from 1-(α)-chloro-3,5-di-O-p-toluoyl-2-
The nucleobases 1
1). Similarly, when the probe strand contained ExBIM, the
6
duplex containing O -MeG in the target strand had a
higher T compared to when there was a G in the target
m
6
deoxyribose to yield O-toluoylated nucleosides, which
were deprotected with NaOMe to yield nucleosides 3 and
strand (T =54.0 °C for ExBIM: O -MeG vs 51.7 °C for
m
ExBIM:G, Table 1). This preference may be rationalized on
the basis of more favorable hydrophobic interactions of
the nucleobase analogues with the alkylated base over G,
29,30,34
4.
Protection of the 5'-OH of 3 and 4 was initially
attempted using DMTCl, but very low yields of product
were obtained. Thus, DMT groups were installed with the
more reactive reagent DMT·BF to obtain 5 and 6.
5
3-58
consistent with previous observations.
Thus, the
4
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4
synthesized probes seem to discriminate between G and
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Finally, phosphoramidites 7 and 8 were formed by
alkylating the 3'-OH of 5 and 6 with chlorinated
O -MeG by destabilizing the duplexes containing G.
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Table 1. Melting temperatures of 13-mer DNA duplexes
phosphotidylating reagent.
These phosphoramidites
centered on KRAS codon 13 in which the target strand
were incorporated into oligonucleotides by solid-phase
DNA synthesis with an automated DNA synthesizer.
6
contains in the middle position either G or O -MeG and
the base in the middle position of the complementary
strand is C, ExBenzi, or ExBIM.
Thermal stability of DNA duplexes containing
ExBenzi or ExBIM opposite O -MeG
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a
Paired bases
C:G
T (°C)
ΔT (°C)
m
m
The capability of ExBenzi and ExBIM to stabilize
DNA duplexes containing
6
66.0±0.6
55.2±0.2
52.0±0.7
55.0±0.7
51.7±0.6
54.0±1.0
a
site-specific O -MeG
-
10.8±0.6
6
modification relative to those containing G was evaluated
in DNA duplexes having the sequence of the KRAS gene
surrounding codon 12 and codon 13 (Figure 2). The KRAS
oncogene is a gene frequently mutated in patients with
colorectal cancer and nearly 97% of all KRAS mutations
C:O -MeG
ExBenzi:G
+3.0±1.0
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ExBenzi: O -MeG
ExBIM:G
+
2.3±1.2
5
0
6
are localized to codons 12 and 13. The evaluation studies
were performed with 13-mer target oligonucleotides for
which the middle base of the target was the middle base
of KRAS codon 13. These targets contained at the middle
ExBIM: O -MeG
a
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ΔT = T (O -MeG duplex)- T (G duplex)
m
m
m
b
Data are mean ± SD from three independent
experiments
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base position either O -MeG or G (O -MeG_13mer target
and G_13mer target respectively, Table S1). Respective
complementary probe oligonucleotides contained
ExBenzi, ExBIM, or C at the middle base position
6
Colorimetric discrimination of O -MeG in KRAS
Encouraged by the oligonucleotide duplex
thermal stability data, we used oligonucleotides
containing ExBenzi and ExBIM to construct AuNP-based
(
ExBenzi_Probe_1, ExBIM_probe_1, C_Probe_1, Table S1).
The stability of DNA duplexes was investigated
6
by measuring their melting temperature (T ). The duplex
m
nanoprobes targeting O -MeG within the KRAS sequence
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context. The nanoprobes were constructed by
of the hybridized strands within the aggregates. The
aggregates exhibited exceptionally sharp melting
transitions characteristic of AuNP-DNA conjugates.
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functionalizing AuNPs (d=20 nm) with thiol-modified
oligonucleotides. Two types of nanoprobes were
prepared: a detection nanoprobe and discriminating
nanoprobes (Figure 1). The detection nanoprobe was
functionalized with a 5’-thiol-modified oligonucleotide
60
Aggregates of the ExBenzi nanoprobes and G target had a
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Tm of 29.0 °C (Table 2), while those from the O -MeG
target had a Tm of 32.0 °C (Table 2). In the case of the
ExBIM nanoprobes, aggregates formed in the presence of
(
5’-thiol_1, Table S1) with a 16-mer sequence comprised of
a (T)10 spacer and a 6-mer recognition sequence. The
discriminating nanoprobes were functionalized with 17-
mer oligonucleotides that were 3’-thiol-modified and
consisted of a (A)10 spacer and a 7-mer recognition
sequence ending with a 5’-ExBenzi or ExBIM (3’-
thiol_ExBenzi_1, 3’-thiol_ExBIM_1, respectively, Table S1).
The nanoprobes were designed such that the
discriminating nanoprobe (ExBenzi nanoprobe or ExBIM
nanoprobe) could hybridize to the target with its 5’-tail
and the detection nanoprobe could hybridize to the target
with its 3’-tail; therefore the nanoprobes were created
with the capability to align in a tail-to-tail fashion in
order to form a sequence complementary to the target
sequences (Figure 1).
modified target also had a higher T than the aggregates
m
formed in the presence of the natural target (32.7 °C vs
29.4 °C, Table 2).
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Table 2. Melting temperatures of AuNP aggregates
formed by either the ExBenzi or ExBIM nanoprobes and
6
the 13-mer KRAS codon 13 target (containing either O -
MeG or G in the middle position).
a
Paired bases
ExBenzi:G
T (°C)
ΔTm (°C)
m
29.0±0.0
32.0±0.0
29.4±0.6
32.7±0.6
+3.0±0.0
6
ExBenzi: O -MeG
ExBIM:G
+
3.3±0.8
6
ExBIM: O -MeG
The
characteristic surface plasmon resonance (SPR) band at
30 nm (Figure S16). In the absence of a matched target,
functionalized
AuNPs
exhibited
a
a
6
ΔT = T (O -MeG aggregates)- T (G aggregates)
m
m
m
b
Data are mean ± SD from three independent
experiments
5
the AuNPs remained dispersed, and the solution had a
red color. When a matched target was added, it
hybridized
to
the
covalently
attached
probe
The increased discrimination achieved with the
oligonucleotides, bringing detection and discriminating
nanoprobes in close proximity. Thus, a AuNP-DNA
network aggregate was formed causing a change in the
dielectric environment of the solution. The close
proximity of the AuNPs caused a coupling of their
individual localized plasmon fields and induced a red-to-
purple color change that could be quantified by
comparing the absorbance spectra of the solutions. Upon
AuNP aggregation, the 530-nm SPR peak was red-shifted
and the spectrum broadened, indicating a decrease in
inter-particle distance and an increase of aggregate size
nanoprobe system is noteworthy considering that the
concentration of target oligonucleotide was 110-fold lower
for the nanoprobe system in comparison to the free DNA
duplexes (20 nM vs 2.2 uM). The degree of discrimination
6
(
ΔTm = Tm probe:O -MeG - Tm probe:G) for ExBIM
nanoprobes was 3.3 °C vs. 2.3 °C measured for the
corresponding free duplexes. The sensitivity gained by
incorporating the AuNPs relates to their sharp melting
transitions. These are due to
a
combination of
60
cooperativity of nanoparticle dissociation (the network
of interconnected AuNPs gets progressively weaker as
multiple DNA linkers dissociate) and the extremely high
(
Figure S16). As a result, the absorbance at 530 nm (A530
)
8
-1
-1
decreased while absorbance in the 700-nm (A700) region
increased (Figure S16). Thus, the absorbance ratio at 700
molar extinction coefficient of AuNPs (9 × 10 L cm mol
61
for AuNPs with d= 20 nm). Moreover, in the nanoprobe
system, the detection and discriminating nanoprobes
bind to adjacent positions on the target forming a nicked
DNA duplex. This design allows better mismatch
discrimination from the standard DNA duplex format
through cooperative hybridization of the AuNP-
conjugated DNA strands to the target.
nm vs. 530 nm (A 00/A530) is indicative of the aggregation
7
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state.
Evaluation of thermal stability of AuNP probe:DNA
aggregates
To assess the capability of the nanoprobes to
distinguish between adducted and non-adducted target
strands, the detection nanoprobes (1 nM) were mixed
with discriminating nanoprobes containing either
ExBenzi or ExBIM (1 nM). (The mixture of the detection
nanoprobes with the discriminating nanoprobes
containing ExBenzi or ExBIM will hereafter be referred to
as ExBenzi nanoprobes and ExBIM nanoprobes,
respectively). ExBenzi and ExBIM nanoprobes were
6
O -MeG recognition in mixed targets
In the aforementioned studies either methylated
or undamaged DNA was targeted individually. To test
6
whether ExBenzi and ExBIM nanoprobes could sense O -
MeG in mixtures containing excess undamaged DNA
strands we investigated the magnitude of aggregation of
the nanoprobes when they were supplemented with a
mixture of different targets. Solutions containing either
ExBenzi (1 nM) or ExBIM nanoprobes (1 nM) were
supplemented with a mixture of G target and a non-
complementary 13-mer target (20 nM each, final
concentration). The non-complementary target (Table S1)
was included to mimic the presence of non-specific
targets in the mixture. Each mixture of nanoprobes and
6
supplemented with the same amount of either the O -
MeG or G target (20 nM final concentration). Upon
aggregation, the thermal stability of the aggregates was
evaluated; the Tm values of the AuNP probe:target
aggregates were determined by monitoring the
absorbance of solutions at 530 nm (A530) as a function of
temperature. Increased absorbance reflects denaturation
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Journal of the American Chemical Society
targets was further supplemented with either 2 pmol of
O -MeG target (20 nM final concentration) or 2 pmol of G
and ExBIM nanoprobes (Figure 4B) A700/A530 values
increased linearly as a function of the relative amount of
O -MeG target, indicating a corresponding increase in
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target (40 nM final concentration). Therefore the total
DNA concentration for the two supplemented solutions
was equal (60 nM) but the amounts of the two
aggregate size.
In order to compare the capability of the ExBenzi
6
6
competitive DNA strands were different (20 nM O -MeG
and ExBIM nanoprobes to detect O -MeG, we determined
6
and 20 nM G target vs 40 nM G target). Absorbance ratios
the limit of detection (LOD) for O -MeG target DNA
(A700/A530) were determined in order to assess the
strands (Figure 4). For the ExBenzi nanoprobes (Figure
6
6
magnitude of aggregation (Figure 3). For ExBenzi
nanoprobes, aggregates from solutions supplemented
4
A) the LOD was 2.3 % O -MeG target or 138 fmol O -
MeG target in the presence of 6 pmol DNA. For the
ExBIM nanoprobes (Figure 4B) the LOD was lower,
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with the O -MeG target exhibited higher A 00/A values
7
530
6
6
than the solutions supplemented with the G target
Δ(Α700/A530) = 0.31, Figure 3). Similarly, for the ExBIM
namely 1.6 % O -MeG target or 96 fmol O -MeG target in
the presence of pmol DNA. Absorbance ratios
A700/A530) were also plotted as a function of the relative
(
6
nanoprobes, aggregates from solutions supplemented
(
6
6
with the O -MeG target exhibited higher A 00/A values
7
530
concentration [O -MeG]/[G] and corresponding standard
curves appear in the Supporting Information (Figure S17).
These results demonstrate that both ExBenzi and ExBIM
nanoprobes, can be used for the competitive quantitative
than the solutions supplemented with the G target
(Δ(Α700/A530)= 0.36, Figure 3). These results indicated that
6
the selective recognition of O -MeG by ExBenzi or ExBIM
6
lead to larger aggregates of the corresponding ExBenzi
detection of O -MeG, with ExBIM being approximately
6
and ExBIM nanoprobes when mixed with the O -MeG
1
.4-fold more sensitive in the tested sequence context,
target even in the presence of the competing G target.
and that they are effective in the presence of G target
6
DNA strands even when O -MeG target is a minor
component of the mixture.
Figure 3. Absorbance ratios of aggregates formed from
ExBenzi or ExBIM nanoprobes and target mixtures (20
nM of each G and non-complementary target, control)
6
6
supplemented with either the O -MeG (control + O -
MeG, 20 nM final concentration) or G target (control +
G, 40 nM final concentration). Aggregates formed from
the initial mixture (control) served as the control. Data
are mean±SD from three independent experiments.
Significant differences indicated as ** for P<0.01 and ***
for P<0.001.
6
Sensitivity of nanoprobes for detecting O -MeG in
KRAS sequence: ExBenzi vs ExBIM nanoprobes
The sensitivity of the nanoprobes for detecting
O -MeG in the KRAS sequence was evaluated by
6
Figure 4. Absorbance ratios (Α700/A530) as a function of
measuring aggregation in samples containing decreasing
6
6
6
relative O -MeG target concentrations ([O -MeG] / [total
target DNA] for aggregates formed from (A) ExBenzi or
(B) ExBIM nanoprobes. Data points indicate mean ± SD
from three independent experiments.
concentrations of the O -MeG target in the presence of
competitive strands. Aggregates containing either the
ExBenzi or ExBIM nanoprobes (1 nM each) were mixed
with the G and non-complementary targets (20 nM each)
6
and supplemented with O -MeG and G targets at different
It is notable that the nanoprobes distinguish the
presence of a single methyl group in one of the 13 bases of
a target strand, and that the selection is achieved without
heating and under non-stringent salt conditions, which
are often needed for mismatch discrimination for single
6
ratios (6 pmol in total) so that the final relative O -MeG
6
concentration ([O -MeG]/[total DNA]) ranged from 0-13.3
%
(
(Table S2). Upon aggregation, absorbance ratios
A700/A530) were measured. For both ExBenzi (Figure 4A)
ACS Paragon Plus Environment

Journal of the American Chemical Society
nucleotide polymorphism (SNP) detection. A feature of
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these nanoprobes that suggests their further potential in
bioanalytical applications is their compatibility with
AuNP-based techniques currently used for SNP detection
6
2-66
in unamplified genomic samples.
In this study, the
increase in AuNP aggregation associated with the
6
presence of the O -MeG target was monitored by UV-Vis
spectroscopy. It can be anticipated that with further
engineering, the adduct-sensitive AuNP aggregation
could be monitored with analytical readouts that are
significantly more sensitive than absorbance, such as
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5,66
62,64
light-scattering,
detection
scanometric
currently applied for SNP detection in
unamplified genomic samples.
Data from melting studies of the DNA duplexes,
or
electrical
63
Figure 5. Absorbance ratios (Α700/A530) as a function of
6
6
relative O -MeG target concentrations ([O -MeG] / [total
target DNA] for aggregates formed from ExBIM
nanoprobes in the presence of fragmented human
genomic DNA. Data points indicate mean±SD from three
independent experiments.
as well as AuNP-DNA aggregates, together with data from
the colorimetric ratiometric detection studies suggest
that both ExBenzi and ExBIM have a higher affinity for
6
O -MeG than for G. The relative performance of the two
6
novel nucleoside analogues is compared in Table S3. In
duplex DNA, there appears to be a slight increase in
discrimination in the case of ExBenzi, whereas the
detection capacity of the nanoprobe system is slightly
improved in the case of ExBIM. The similarity between
the two analogues suggest a lack of relevant hydrogen
bonding interactions between the adduct and probes
since the analogues differ only in H-bonding capacity.
From a synthetic perspective, however, ExBIM can be
produced with consistently higher yields primarily due to
better solubility characteristics during work-up and
purification.
O -position of guanine was also examined. ExBIM
nanoprobes (1 nM) were mixed with the same amount (20
nM, final concentration) of a 13-mer KRAS sequence
6
6
target that contained either O -MeG or O -BnG at the
6
middle position (O -BnG_13mer target, Table S1). The
6
color of the O -MeG-supplemented suspension turned
6
purple, whereas the color of the O -BnG-supplemented
suspension remained red (Figure S19). This observation
suggested that the O -BnG target did not induce any
aggregation. To further confirm the accuracy of this
simple visual observation, the solutions were gradually
heated and A530 was monitored as a function of
6
6
temperature. Characteristic sharp melting transitions
Detection of O -MeG oligonucleotide targets in the
6
were observed for O -MeG whereas the A
did not
30
5
presence of human genomic DNA
6
change for O -BnG, further indicating the absence of
We investigated the performance of the ExBIM
nanoprobes for detecting O -MeG oligonucleotide targets
in the presence of human genomic DNA. Genomic DNA
extracted from SW480 colon carcinoma cells was
fragmented by ultrasonication (Figure S18). ExBIM
nanoprobes (1 nM) were combined with fragmented
genomic DNA (100 ng) and O -MeG oligonucleotides.
The same target sequence with G at the modification
position was also added as a direct competitor. Ratios of
aggregates and confirming the naked-eye inspection
6
(
Figure S20). These results indicate a lack of affinity of
6
ExBIM for O -BnG. Furthermore, the convenience of
visually judging aggregation due to color changes
associated with the SPR phenomenon suggests a future
utility of the assay for screening diverse analogues with
the goal of optimizing base surrogates for selection
toward different DNA adducts.
6
6
Effect of target length
added O -MeG to G targets where the same as when the
sensitivity of the nanoprobes was assessed with non-
complementary oligonucleotides as background. The final
relative concentration of O -MeG in the sample ([O -
Hypothesizing that longer target sequences
would result in lower discrimination, we investigated
thermal stability differences for longer DNA duplexes
6
6
6
MeG]/[total DNA]) ranged from 0-2.8 % (Table S4). Upon
containing ExBIM placed opposite either O -MeG or G.
target-induced aggregation, absorbance ratios (A700/A530
)
Therefore, we synthesized longer target oligonucleotides
(17 and 21-mer) for which the middle base of the target
was the middle base of KRAS codon 13 (Figure 2) and
were measured. Ratiometric absorbance values A700/A530
6
increased linearly as a function of O -MeG target
6
concentration (Figure 5), indicating a corresponding
increase in aggregation. The LOD for O -MeG DNA
strands with genomic DNA as background (Figure 5) was
contained in the middle position either O -MeG or G
(Table S1). The corresponding complementary probe
oligonucleotides containing ExBIM in the middle position
6
0
.24 %, indicating that the presence of a large background
were also synthesized (Table S1) and the T
duplexes formed from the probe and target strands was
determined. For the 17-mer DNA duplex the ΔT was 1.6
°C (Table S5). For the 21-mer duplex the ΔT was 1.3 °C
Table S5). It should be noted that the melting analysis
m
of the DNA
of fragmented genomic DNA did not interfere with the
sensing response of the nanoprobes and quantification of
O -MeG.
m
6
m
(
Specificity of ExBIM nanoprobes
experiments for the longer targets (17 and 21-mer) were
performed with a lower DNA concentration (1 uM) than
for the 13-mer targets (2.2 uM) to ensure that the A260
6
The specificity of ExBIM towards O -MeG as
opposed to adducts containing other alkyl groups on the
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Journal of the American Chemical Society
6
measurements were in the linear response range of the
UV/Vis spectrophotometer.
containing O -MeG in the presence of different
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competitor strands and human genomic DNA. To the best
of our knowledge, this is the first sensing strategy for in-
gene quantification of the highly mutagenic DNA adduct
O -MeG in a complex mixture. Studies to elucidate the
molecular origin of discrimination of O -MeG from G by
To test the capability of ExBIM to distinguish
6
between O -MeG and G in longer DNA strands using
6
nanoprobes, we prepared detection and discriminating
ExBIM nanoprobes designed to hybridize with either the
6
ExBIM and ExBenzi on the basis of their orientation in
DNA and stacking capacity are anticipated. Finally,
integration of the nanoprobes developed in this study
with hybrid capture techniques regularly used for
targeted enrichment of nucleic acids prior to next
generation sequencing and with well-established
ultrasensitive AuNP-based DNA detection technologies
used for SNP detection in unamplified genomic samples
1
7-mer or 21-mer KRAS codon 13 targets. Thus, the same
type of AuNPs (d=20 nm) were functionalized with longer
’-thiolated oligonucleotides (5’-thiol_2 and 5’-thiol_3,
5
Table S1) to yield detection nanoprobes targeting the 17-
and 21-mer target, respectively. Similarly, discriminating
ExBIM nanoprobes were produced by functionalizing
AuNPs with longer 3’-thiolated oligonucleotides (3’-
thiol_ExBIM_2 and 3’-thiol_ExBIM_3, Table S1). The
detection and discriminating ExBIM nanoprobes (1 nM
each) were mixed with the same amount (20 nM final
concentration) of either O -MeG or G target, and the Tm
values for the resulting aggregates were determined. For
the 17-mer target, aggregates formed in the presence of
O -MeG target exhibited a higher Tm than the aggregates
formed in the presence of G target (47.7 °C for O -MeG vs
4
mer target, aggregates formed in the presence of O -MeG
exhibited a higher Tm than aggregates formed from the
ExBIM nanoprobes and G target (62.0 °C for O -MeG vs
0
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may enable monitoring the in-gene abundance of O -MeG
in biological samples.
6
ASSOCIATED CONTENT
Supporting
Information.
Detailed
experimental
6
procedures, synthesis, chemical characterization, additional
figures and tables. This material is available free of charge via
the Internet at http://pubs.acs.org.
6
4.1 °C for G, ΔT = 3.6 °C, Table S6). Similarly, for the 21-
m
6
AUTHOR INFORMATION
Corresponding Authors
6
6
0.2 °C for G, ΔT = 1.8 °C, Table S6). The thermal stability
m
*
*
ioannis.trantakis@hest.ethz.ch
sturlas@ethz.ch
of AuNP-DNA aggregates is influenced by the
interparticle distance, which is modulated by
a
Funding Sources
combination of the length of the interconnecting
oligonucleotides and efficiency of hybridization.
Hybridization efficiency, in turn, is influenced by the
ratio of mismatched to matched bases. Thus, as
hypothesized, discrimination diminished for the longest
target. However, balancing the combination of
interparticle distance and hybridization efficiency
influences, discrimination for the 17-mer target was no
worse than for the 13-mer. The 17-mer target was
therefore preferred for further studies because there was
no loss in discrimination.
This work was supported by the European Research Council
(260341) and the Swiss National Science Foundation
(
31003A_156280).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Larissa Roth for synthesizing oligonucleotides and
for performing DNA duplex melting analyses. We thank Dr.
Laura Wyss for helping with the synthesis of
phosphoramidites and writing.
6
Detection of O -MeG in KRAS cod12
To address the potential influence of sequence
context, ExBIM nanoprobes targeting KRAS codon 12
were also developed. Thus, 17-mer target oligonucleotides
in which the middle base (either O -MeG or G) was the
middle base of KRAS codon 12 were synthesized (Figure 2,
Table S7). Aggregates formed between KRAS codon 12-
targeting nanoprobes in the presence of the O -MeG
target (20 nM final concentration) exhibited a higher Tm
than those formed in the presence of the G target (ΔT =
2
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