Detection of DNA base variation and cytosine methylation at a single nucleotide site using a highly sensitive fluorescent probe

Article abstract of DOI:10.1039/c1cc11205h

A fluorescent DNA probe containing an anthracene group attached via an anucleosidic linker can identify all four DNA bases at a single site as well as the epigenetic modification C/5-MeC via a hybridisation sensing assay.

Full text of DOI:10.1039/c1cc11205h

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COMMUNICATION
Detection of DNA base variation and cytosine methylation at a single
nucleotide site using a highly sensitive fluorescent probewz
Jean-Louis H. A. Duprey,a Zheng-yun Zhao,a Dario M. Bassani,b Jack Manchester,a
Joseph S. Vylec and James H. R. Tucker*a
Received 28th February 2011, Accepted 19th April 2011
DOI: 10.1039/c1cc11205h
A fluorescent DNA probe containing an anthracene group
attached via an anucleosidic linker can identify all four DNA
bases at a single site as well as the epigenetic modification
C/5-MeC via a hybridisation sensing assay.
which results in conversion of cytosine sites to uracil, leaving
5-MeC untouched.6 However as with SNP genotyping, more
efficient and direct approaches to methylation profiling in
DNA are being actively pursued.7,8 Here we report a sensing
study using an anthracene-containing probe that is able to
distinguish between all four DNA bases and identify 5-MeC
through differences in the emissive responses generated by
duplex formation.
The establishment of convenient and rapid methods for
discriminating between various DNA bases is central to the
detection of single nucleotide polymorphisms (SNPs) in the
human genome, SNPs being chiefly responsible for genetic
diversity amongst the population.1 Among the various
approaches to DNA and SNP detection in the literature,1,2
there is active interest in techniques that do not rely on
differences in hybridisation efficiency.3,4 One of these is the
use of base-discriminating fluorescent probes in hybridisation
assays,3 where different duplexes generate different emissive
responses. Probes of this type can also find an application in
the detection of alterations to DNA bases, for example base
removal4a and base modification.4b A base modification in the
human genome apparently yet to be probed using this
approach is cytosine methylation at the 5-position (Fig. 1), which
in fact is present in 70–80% of 50-CpG-30 dinucleotides.5a
The oligonucleotide probes were designed containing an
alkoxy-anthracene group connected to the DNA backbone
via a D-threoninol unit. Threoninol has previously been shown
to be an effective anucleosidic group in various photo-active
oligonucleotides.9 It was conveniently coupled to known10
anthracene-containing carboxylic acids 1a and 1b (Scheme 1),z
with the products then converted through to phosphoramidites
4a and 4b to allow incorporation into DNA via standard
automated synthesis. The sequences of the DNA strands
synthesised are shown in Table 1. In this study it was decided
to explore the effect of varying the base directly opposite the
tether site, to include 5-MeC, the phosphoramidite of which is
commerically available. At the same time, the effect of varying
the length of the alkyl spacer connecting the DNA backbone
with the anthracene fluorophore could be examined by
comparing probes 5 (n = 1) and 6 (n = 4). All seven strands
were purified by reversed phase HPLC and characterised by
ESMS.z The latter technique allowed the target strands 8 and
The misregulation of this common epigenetic modification,
which can result in both hyper- and hypomethylated sites, is
associated with cancer development.5 Therefore there is
immense interest in methods that can establish the methylation
pattern of various regions of the genome. Of the assays that
are known, a common method is treatment with bisulfite,
a School of Chemistry, University of Birmingham, Edgbaston,
Birmingham, B15 2TT, UK. E-mail: j.tucker@bham.ac.uk;
Fax: +44 (0)121 414 4403; Tel: +44 (0)121 414 4422
b
´
Institut des Sciences Moleculaires, CNRS UMR 5255,
Universite Bordeaux 1, 351 Cours de la Liberation, F-33405 Talence,
´
´
France. E-mail: d.bassani@ism.u-bordeaux1.fr;
Fax: +33 (0)5 4000 6158; Tel: +33 (0)5 4000 2827
c School of Chemistry and Chemical Engineering, David Keir Building,
Queen’s University Belfast (QUB), Stranmillis Rd, Belfast BT9
5AG, UK. E-mail: j.vyle@qub.ac.uk; Fax: +44 (0)28 9097 6524;
Tel: +44 (0)28 9097 548
w This article is part of a ChemComm ‘Supramolecular Chemistry’
web-based themed issue marking the International Year of Chemistry
2011.
z Electronic supplementary information (ESI) available: Synthetic
procedures for phosphoramidite monomers, HPLC and MS character-
isation data for oligonucleotides, additional fluorescence data. See DOI:
10.1039/c1cc11205h
Fig. 1 Structure of C and 5-MeC within a 50-CpG-30 sequence of
DNA.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 6629–6631 6629

and 6 was replaced firstly with an abasic sugar unit and then
with a T nucleoside,z giving melting temperatures of 42 1C and
54.5 1C respectively for duplexes with 7. The increase in the
latter case is consistent with the introduction of an additional
H-bonding base pair.
Fluorescence measurements were then undertaken to assess
the effect of duplex formation on the emission spectrum of the
anthracene tether on each probe, with each giving a characteristic
signal centred at ca. 430 nm upon excitation at 360 nm.
Striking differences between the two systems were observed.
For probe 5 with the shorter linker, duplex formation at ca.
1 mM resulted in significant decreases in emission intensity for
all five targets 7–11, with the reduction at 426 nm very similar
in each case (ranging from ꢀ69 to ꢀ86%).z Only small
changes in intensity were observed beyond the addition
of one equivalent of target, consistent with the formation of
strongly bound 1 : 1 duplexes. In contrast, the response to
duplex formation with probe 6 was markedly different for
the four targets containing a different DNA base opposite the
tether site (Fig. 2).
Scheme 1 Synthesis of phosphoramidite monomers 4a and 4b with
linker lengths n = 1 and n = 4 respectively. Reagents (i) D-threoninol,
HOBt, DIPC, DIPEA (ii) dimethoxytrityl chloride, DMAP
(iii) DIPEA, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite.
The targets with purine bases A and G triggered significant
increases in emission intensity at 426 nm (6ꢁ7 +161%;
6ꢁ9 +98%), whereas for the pyrimidine bases C and T, relatively
smaller changes were observed, with an increase of +18% for 6ꢁ8
and a decrease of ꢀ43% for 6ꢁ10 observed. Such an identification
of each of the four bases in duplex DNA using a single probe is
relatively rare,3a,b with previous approaches largely relying on
base mismatch methodologies.3c–g
Table 1 Base sequences of the oligodeoxynucleotides synthesised
Sequence of oligodeoxynucleotidea
Probes
Targets
5
6
7
8
9
10
11
50–TGGACTC-a-CTCAATG–30
50–TGGACTC-b-CTCAATG–30
50–CATTGAG-A-GAGTCCA–30
50–CATTGAG-C-GAGTCCA–30
50–CATTGAG-G-GAGTCCA–30
50–CATTGAG-T-GAGTCCA–30
50–CATTGAG-MeC-GAGTCCA–30
The strands 8 and 11 containing C and 5-MeC respectively
were then compared by monitoring duplex formation via the
addition of aliquots of each target to probe 6. As shown in
Fig. 3, it is clear that the methylation status of this base can
also be distinguished, with methylation causing a decrease in
emission intensity (ꢀ15%) as opposed to an increase
(+18%).y In contrast, changes of ꢀ79% and ꢀ80% were
observed for probe 5 with strands 8 and 11 respectively.
Clearly the nature of the linker to the anthracene tag is an
important factor to consider in explaining these results. In fact
probe 5 in its single strand form is considerably more emissive
a
The letters a and b respectively represent the n =1 and n = 4
anthracene tethers (Scheme 1) incorporated into DNA via phosphor-
amidite synthesis; MeC in strand 11 denotes 5-MeC (Fig. 1).
Table 2 Tm data for duplexes (pH 7.0, 10mM phosphate buffer,
100 mM NaCl)
Tm/1C
a
Target
5
6
DTm
7
8
9
10
11
48.0
46.0
48.0
48.0
45.5
50
51
51.5
51.0
51.5
+2
+5
+3.5
+3
+6
a
DTm = Tm (probe 6 duplex) ꢀ Tm (probe 5 duplex).
11 to be conveniently distinguished from one another due to a
mass difference of 14 units.
The melting temperature data for the hybridised strands
(Table 2) indicate that each system forms a stable duplex at
room temperature. However it is also clear that probe 6, with
the longer alkyl linker, forms a slightly more stable duplex
with each target than probe 5, with DTm values ranging
between +2 and +6 degrees. This suggests that the longer
tether allows the anthracene to interact more effectively with
the duplex, possibly through a p-stacking interaction with
proximate base pairs.11 The presence of such an interaction
is supported by studies where the threoninol-based tag in 5
Fig. 2 Overlaid fluorescence spectra of 6 (dotted line) and (in
decreasing order of intensity) duplexes 6ꢁ7 (red), 6ꢁ9 (blue), 6ꢁ8
(purple), and 6ꢁ10 (green) (pH = 7, 10 mM phosphate buffer,
100 mM NaCl, ca. 293 K, lex = 360 nm).
c
6630 Chem. Commun., 2011, 47, 6629–6631
This journal is The Royal Society of Chemistry 2011

Support from the EPSRC (Leadership Fellowship to
JHRT), Advantage West Midlands, the University of
Birmingham, Peter Ashton (mass spectrometry) and Graham
Burns (HPLC) is gratefully acknowledged.
Notes and references
y Mixtures of targets (e.g. a 50 : 50 mixture of 8 and 11 with probe 6)
gave an additive fluorescent response which approximated to the
percentage composition of the mixture.
z An analogous probe to 5 with a L-threoninol unit has a much
smaller fluorescence quantum yield of 0.018, which indicates that the
stereochemistry of the linker has a strong bearing on the position of
the anthracene probe with respect to the DNA backbone.
8 Preliminary fluorescence lifetime studies indicate that several probes
and their duplexes give triexponential emission profiles, which suggests
that the emission behaviour of each system is governed by a rather
intricate interplay between various decay pathways. The results of a
detailed study will be published in due course.
Fig. 3 Titration showing the effect on the anthracene emission
intensity at 426 nm of forming duplexes 6ꢁ8 (purple, top) and 6ꢁ11
(orange, bottom), ca. 293 K, lex = 360 nm).
1 S. Kim and A. Misra, Annu. Rev. Biomed. Eng., 2007, 9, 289.
2 (a) D. J. Kleinbaum and E. T. Kool, Chem. Commun., 2010, 46,
8154 and references therein; (b) D. M. Kolpashchikov, Chem. Rev.,
2010, 110, 4709; (c) R. T. Ranasinghe and T. Brown, Chem.
Commun., 2005, 5487.
3 (a) R. W. Sinkeldam, N. J. Greco and Y. Tor, Chem. Rev., 2010,
110, 2579; (b) D. W. Dodd and R. H. E. Hudson, Mini-Rev. Org.
Chem., 2009, 6, 378; (c) Y. Xie, T. Maxson and Y. Tor, Org.
Biomol. Chem., 2010, 8, 5053 and references therein; (d) P. Cekan
and S. T. Sigurdsson, Chem. Commun., 2008, 3393 and references
therein; (e) N. Moran, D. M. Bassani, J.-P. Desvergne, S. Keiper,
P. A. S. Lowden, J. S. Vyle and J. H. R. Tucker, Chem. Commun.,
2006, 5003; (f) A. Okamoto, Y. Saito and I. Saito, J. Photochem.
Photobiol., C, 2005, 6, 108 and references therein; (g)
J.-L. H. A. Duprey, D. M. Bassani, E. I. Hyde, C. Ludwig,
A. Rodger, J. S. Vyle, J. Wilkie, Z. Zhao and J. H. R. Tucker,
Supramol. Chem., 2011, 23, 273.
than probe 6, as evidenced by fluorescence quantum yields
(FF = 0.053 and 0.011 respectively). This indicates that the
geometry of the short and relatively inflexible linker found in 5
precludes a significant quenching interaction with proximate
DNA bases,z which are known to quench the fluorescence of
anthracene derivatives.11 However duplex formation with 5
results in a general decrease in emission in all cases. Therefore
it appears that duplex formation per se, and the formation of
the B-DNA structure (shown by CD spectroscopy) dictates the
overall change in emission intensity for this probe, which
results in the probe being relatively insensitive to changes in
sequence in the target strand. In the case of probe 6 however,
clearly the identity of the base opposite the anthracene is the
dominant factor governing the emission profile of each duplex.
Overall, the results are consistent with the longer linker length
rendering the anthracene less emissive to begin with while
allowing it to interact more fully with the base pair stack. The
greater interaction raises the duplex melting temperature
(Table 2) and also makes the anthracene more sensitive to
local changes in the base environment. Previous work has
shown that various bases are able to quench the excited state
of organic fluorophores to a greater or lesser extent.12 Adenine
is generally the least effective as a quencher and pyrimidines
are the most effective, a trend that is consistent with these
results.8
4 (a) N. J. Greco and Y. Tor, J. Am. Chem. Soc., 2005, 127, 10784;
(b) N. J. Greco, R. W. Sinkeldam and Y. Tor, Org. Lett., 2009, 11,
1115.
5 (a) L. S. Kristensen, H. M. Nielsen and L. L. Hansen, Eur. J.
Pharmacol., 2009, 625, 131; (b) M. Ehrlich, Epigenomics, 2009, 1,
239; (c) E. A. Vucic, C. J. Brown and W. L. Lam, Pharmaco-
genomics, 2008, 9, 215; (d) For a recent special issue on epigenetics,
see A. Jeltsch and W. Fischle, ChemBioChem, 2011, 12, 183.
6 (a) L. S. Kristensen and L. L. Hansen, Clin. Chem., 2009, 55, 1471;
(b) F. Feng, H. Wang, L. Han and S. Wang, J. Am. Chem. Soc.,
2008, 130, 11338.
7 (a) Y. Yu, S. Blair, D. Gillespie, R. Jensen, D. Myszka,
A. H. Badran, I. Ghosh and A. Chagovetz, Anal. Chem., 2010,
82, 5012; (b) M. Ogino, Y. Taya and K. Fujimoto, Org. Biomol.
Chem., 2009, 7, 3163.
8 For two recent electrochemical approaches, see: (a) S. Liu, P. Wu,
W. Li, H. Zhang and C. Cai, Chem. Commun., 2011, 47, 2844;
(b) P. Wang, Z. Mai, Z. Dai and X. Zou, Chem. Commun., 2010,
46, 7781.
9 H. Kashida, X. Liang and H. Asanuma, Curr. Org. Chem., 2009,
13, 1065 and references therein.
In conclusion, these results demonstrate how a simple
fluorophore-tagged DNA strand can act as both a base-
discriminating probe and a signaller of an epigenetic change
(in this case signalling a site associated with hypomethylation),
illustrating the versatility of this direct approach to DNA
sensing. Further studies are now planned to rationalise these
results further and to assess the scope of these findings through
the targeting of particular base sequences in the human
genome.
10 Y. Molard, D. M. Bassani, J.-P. Desvergne, N. Moran and J. H.
R. Tucker, J. Org. Chem., 2006, 71, 8523.
11 C. V. Kumar, E. H. A. Punzalan and W. B. Tan, Tetrahedron,
2000, 56, 7027 and references therein.
12 M. Manoharan, K. L. Tivel, M. Zhao, K. Nafisi and T. L. Netzel,
J. Phys. Chem., 1995, 99, 17461.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 6629–6631 6631