Detection of a single DNA base-pair mismatch using an anthracene-tagged fluorescent probe

Article abstract of DOI:10.1039/b611650g

A novel anthracene-tagged oligonucleotide can discriminate between a fully-matched DNA target sequence and one with a single mismatching base-pair through a remarkable difference in fluorescence emission intensity upon duplex formation. The Royal Society

Full text of DOI:10.1039/b611650g

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Detection of a single DNA base-pair mismatch using an anthracene-
tagged fluorescent probe{
Nina Moran,a Dario M. Bassani,*b Jean-Pierre Desvergne,b Sonja Keiper,c Philip A. S. Lowden,d
Joseph S. Vyle*c and James H. R. Tucker*a
Received (in Cambridge, UK) 14th August 2006, Accepted 23rd October 2006
First published as an Advance Article on the web 6th November 2006
DOI: 10.1039/b611650g
A novel anthracene-tagged oligonucleotide can discriminate
between a fully-matched DNA target sequence and one with a
single mismatching base-pair through a remarkable difference
in fluorescence emission intensity upon duplex formation.
The accurate detection of single-nucleotide polymorphisms (SNPs)
in DNA is the subject of intense research. SNPs are thought to be
the cause of genetic disorders (most human genetic variations
result from SNPs) and thus a fast, reliable and inexpensive sensor
is needed for use in DNA diagnostics.1 Of the many luminescence-
based methods used for the detection of mismatches in DNA,2 one
Scheme 1 Schematic drawing of the SNP sensing mechanism;
a
of the simplest is to tag a single fluorophore to an oligonucleotide
matching CG pair causes quenching whereas a CA mismatch enhances
emission.
probe and monitor changes in its emission intensity upon duplex
formation. By tagging pyrene to DNA in this manner, particulary
promising SNP sensors have been developed.3
Carbodiimide-mediated condensation of 2 with serinol afforded
the corresponding amide 3 bearing a 1,3-bis alcohol. Using
standard reaction protocols, these alcohol functions were sequen-
tially tritylated to the mono-protected racemate 4 and then
phosphitylated to afford the novel phosphoramidite 5 as a mixture
of diastereomers. This was then inserted at the central position of
15-mer oligonucleotides via automated solid-phase synthesis
applying extended DNA coupling conditions for the modification.
The two diastereomers (a and b) of Probe 1 (Table 1) could be
separated by RP-HPLC and were thus separately isolated and
characterised by MALDI-TOF mass spectrometry (see ESI). The
six oligonucleotides synthesised for this study, including a fully
In contrast to pyrene, only a few examples of anthracene-tagged
oligonucleotides have been reported.4 This is in spite of the well-
characterized properties of anthracene, these being its ability to
form excimers and undergo [4p + 4p] photocycloaddition, making
it a versatile and useful fluorophore.5 Here we report an
anthracene-tagged SNP sensor that can discriminate between fully
matched (cognate) and single base-pair mismatched target DNA
sequences through quenching and enhancement of fluorescence
emission respectively (Scheme 1).
Previous work on pyrene-based SNP sensors3 has involved
tagging the chromophore to a nucleobase. Here a non-nucleosidic
unit 5,6 containing a serinol linker previously identified as an
effective substitute for one nucleoside,7 was synthesized via the
route shown5c in Scheme 2.{
Thus ester 1 was prepared from anthrone and ethyl bromoa-
cetate. Subsequent saponification yielded the corresponding
anthracene carboxylic acid
2
following acidification.
aSchool of Chemistry, University of Birmingham, Edgbaston,
Birmingham, UK B15 2TT. E-mail: j.tucker@bham.ac.uk;
Fax: +44 (0)12 1414 4403; Tel: +44 (0)12 1414 4422
bLaboratoire de Chimie Organique et Organome´tallique, Universite´
Bordeaux 1, 351 Crs Libe´ration, F-33405, Talence, France.
E-mail: d.bassani@lcoo.u-bordeaux1.fr; Fax: 33(0) 540 00 6158;
Tel: 33(0) 540 00 2827
cSchool of Chemistry and Chemical Engineering, Queen’s University
Belfast (QUB), Stranmillis Road, Belfast, UK BT9 5AG.
E-mail: j.vyle@qub.ac.uk; Fax: +44 (0)28 9097 6524;
Tel: +44 (0)28 9097 5485
dSchool of Biological and Chemical Sciences and Institute of Structural
Molecular Biology, Birkbeck College, University of London, Malet
Street, Bloomsbury, London, UK WC1E 7HX
{ Electronic supplementary information (ESI) available: Experimental
procedures for the synthesis and characterisation of anthracene-tagged
oligonucleotides and thermal melting curves. See DOI: 10.1039/b611650g
Scheme 2 Reagents and conditions: (a) K2CO3, acetone, reflux, o/n,
35%; (b) i. 10% NaOH (aq) soln–EtOH (1 : 1), reflux, o/n; ii. HCl, 93%; (c)
HOBt, DIPC, DIEA, DMF, 40 uC, 40 h, 71%; (d) DMTrCl, DMAP,
pyridine, rt, o/n, 42%; (e) (i-Pr2N)PClO(CH2)2CN, DIEA, DCM, rt, 1.5 h,
76%.
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Table 1 Oligonucleotides synthesised in this study
Sequence (X = anthracene tagged unit)
Probe 1
FM1a
1MMa
2MMa
Za
59-TGGACTCXCTCAATG-39
59-CATTGAGAGAGTCCA-39
59-CATTGAGAAAGTCCA-39
59-CATTGAGAATGTCCA-39
59-TTAGGGAATTAGCG-39
59-TGGACTCACTCAATG-39
FM2a
a
Purchased from Operon Biotechnologies.
matching sequence, FM1 and sequences 1MM and 2MM with one
and two mismatches respectively, are presented in Table 1.
A series of titration experiments were performed on Probe 1
(aqueous phosphate buffer, pH 7) in order to the determine the
effect of DNA binding on the anthracene emission signal. As
depicted in Fig. 1, the addition of aliquots of the cognate target
FM1 to a 1 mM aqueous solution of Probe 1a{ resulted in a
significant decrease in anthracene emission intensity, with quench-
ing reaching approximately 85% once one equivalent of target was
added. No further decrease was observed in the presence of excess
amounts of target, which indicated the formation of a 1 : 1
complex, consistent with DNA duplex formation.
Fig. 2 Fluorescence titration plots at ca. 298 K for Probe 1a with 0.5,
1.0, 1.5 and 2.0 equivalents of targets FM1 (green), 1MM (light blue),
2MM (dark blue) and Z (pink) (0.01 M phosphate, 0.2 M NaCl, pH 7.0
aqueous buffer). lex = 350 nm.
Table 2 TM values for duplexes (pH 7.0 buffer, error margin ¡1 uC)
Duplex
TM/uC
Change/uC
As expected, a titration with a random mis-matching strand Z
resulted in no significant change in fluorescence emission.
However, in contrast to the fully matching system, studies with
targets 1MM and 2MM, containing one and two mis-matching
bases respectively, resulted in an increase in emission intensity
(Fig. 2). Once again, no additional changes were observed once
more than one equivalent of each target was added to the probe.
DNA melting studies{ confirmed that duplexes between Probe
1a and each of the three targets form at the temperature used for
the fluorescence studies. The Tm values of these three duplexes, as
well as that for FM1 and its complementary strand§ FM2, are
presented in Table 2.
FM1–FM2
52.0
50.0
39.5
36.0
—
FM1–Probe 1a
1MM–Probe 1a
2MM–Probe 1a
22
212.5
216
luminescence quenching, especially in GC rich regions.8a,8b
Anthracene intercalation within the FM1–Probe 1a duplex would
bring it into stacking contact with at least one GC unit. This would
explain the fluorescence changes and also account for there being
only a slight change in duplex stability when a stacking adenine
base in FM2 is replaced with a non-nucleosidic linker in Probe 1a.
On the other hand, it is less clear why duplex formation between
Probe 1a and either 1MM or 2MM leads to an increase in
emission intensity. Similar behavior has been observed when
anthracene intercalates in AT vs. GC-rich regions of DNA,8a but it
is unlikely that the tether in Probe 1a is sufficiently long to allow
the anthracene to bind distal to its point of attachment. Also, the
Tm values clearly indicate that a large decrease in duplex stability
results from the introduction of one and two base-pair mis-
matches, suggesting that for these duplexes, additional stabilisation
from anthracene intercalation is not present. Since anthracene is
only a weak intercalative DNA binder (K # 150 M21),8d it is
possible that the presence of a mismatch favours the anthracene
being placed in a hydrophobic pocket, which limits quenching by
stacking base residues and the aqueous environment.
In order to explain these results, it is probably significant to note
that the stability of the duplex FM1–Probe 1a, where anthracene
luminescence is quenched, is very similar to the stability of the
duplex FM1–FM2. It is well documented that anthracene can
intercalate into DNA8 and such an interaction not only increases
duplex stability, as evidenced by higher Tm values, but also causes
In conclusion, we have developed a novel anthracene-tagged
DNA probe that can detect a single base-pair mismatch in a target
sequence which is of relevance to the development of novel SNP
sensors. The unusual selectivity of the sensing signal (emission
either increasing or decreasing depending on the sequence) stems
from the local environment around the anthracene changing in
response to duplex formation, rather than it relying on the
thermodynamics of duplex formation per se.2a Studies with
different mismatching base sequences using a range of anthracene
tags are now planned in order to rationalise these findings further.
Fig. 1 Overlaid fluorescence spectra for the titration of up to 1 equiv.
FM1 with 1 mM Probe 1a (0.01 M phosphate, 0.2 M NaCl, pH 7.0
aqueous buffer). lex = 350 nm.
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We thank the BBSRC (BMS) for the award of a special
studentship (to NM), the EPSRC for the award of a research grant
(GR/S07438/02), and the French CNRS and Ministry of Research
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Notes and references
{ The other diasteroisomer of Probe 1 also appears to be capable of SNP
detection. Results will be reported elsewhere in due course.
§ To enable a comparison between the stability of different duplexes
formed with FM1, the non-nucleosidic base in Probe 1 was changed for a
mismatching adenosine in FM2 as an isosteric replacement.
6 For a very recent example of deletion nucleotide polymorphism sensing
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