A new dark quencher for use in genetic analysis

Article abstract of DOI:10.1039/b300934c

A novel, long-wavelength, non-fluorescent quencher (LQ), based on 1,4-diaminoanthraquinone, has been incorporated at the 3′ and 5′-termini of oligonucleotides. The quencher has been used in Molecular beacons, efficiently quenching the long wavelength fluo

Full text of DOI:10.1039/b300934c

A new dark quencher for use in genetic analysis
Jonathan P. May,a Lynda J. Brown,b Ivo Rudloffb and Tom Brown*a
a Department of Chemistry, University of Southampton, Highfield, Southampton, UK. SO17 1BJ.
E-mail: tb2@soton.ac.uk; Fax: +44 (0)23 80592991; Tel: +44 (0)23 80592974
b Oswel Research Products Ltd, Biological Sciences Building, University of Southampton, Bassett Cresent
East, Southampton, UK SO16 7PX; Tel: +44 (0)23 80592984
Received (in Cambridge, UK) 23rd January 2003, Accepted 6th March 2003
First published as an Advance Article on the web 18th March 2003
A novel, long-wavelength, non-fluorescent quencher (LQ),
based on 1,4-diaminoanthraquinone, has been incorporated
at the 3A and 5A-termini of oligonucleotides. The quencher
has been used in Molecular beacons, efficiently quenching
the long wavelength fluorophore, Cy5.
moiety at the 1-position of deoxyribose whilst the 3A position is
attached to controlled pore glass resin (CPG). This resin-bound
molecule allows 3A labelling of oligonucleotides with the
quencher.
The quencher molecules (LQ1 and LQ2) were used to
prepare two labelled poly-T oligonucleotides, ODN1 labelled at
the 5A and ODN2 at the 3A (Table 1).
The oligonucleotides were cleaved from the resin with conc.
NH3 (aq) (30 min, room temp.) then immediately evaporated to
dryness, and purified by RP-HPLC. In each case the major peak
which was blue in colour was collected. Following desalting,
the oligonucleotides were used to determine optimum condi-
tions for removal of the heterocyclic base protecting groups of
the DNA. An aliquot of each oligonucleotide was heated in
conc. NH3 (aq) at 55 °C for 4 h and then analysed by RP-HPLC.
ODN1 gave one major peak and mass spectrometry (MALDI-
TOF) confirmed the expected mass of the desired product
(expected: 3337; found: 3338). This indicates that conventional
deprotection conditions are suitable for oligonucleotides la-
belled at the 5A with LQ1. However, the chromatogram for
ODN2 showed three significant peaks and closer examination
of the products by mass spectrometry revealed degradation
products. The amide bonds linking the sugar to the dye were
partially cleaved (Fig. 1 red) and masses corresponding to loss
Current detection methods in genetic analysis require high
performance fluorescent oligonucleotide probes. In particular,
real-time detection of PCR products is commonly achieved with
fluorescent probes such as Molecular beacons,1,2Scorpion
primers™3–5 and TaqMan®.6,7 These probes are single-stranded
oligonucleotides possessing a fluorescent dye and a quencher
molecule. In their “dark state” they adopt a conformation where
the quencher is close enough to the fluorescent dye to absorb its
fluorescence. However, upon hybridisation to a target sequence
these probes either undergo a conformational change (Molec-
ular beacons and Scorpion primers™) or enzymatic cleavage
(TaqMan® probes) separating the quencher and fluorophore
allowing a fluorescent signal to be detected. Advances in
genetic analysis have increased the demand for a wider range of
novel efficient fluorophore–quencher pairs.8 Ideally, new
quenchers possess no native fluorescence but have large
extinction coefficients for maximal spectral overlap with large
range of fluorophores.
We have developed a novel long-wavelength fluorescence
quencher based on the 1,4-diaminoanthraquinone chromophore.
This quencher (LQ) is designed to be complementary to the first
dark quencher to be reported by our laboratories, Methyl Red
(MR).3–5 Methyl Red (an isomer of dabcyl) has a large spectral
absorption range of 350–550 nm and LQ has an absorption
range of 500–700 nm. Both of these quenchers possess no native
fluorescence (i.e. are dark quenchers) and between them they
are able to quench the majority of commonly utilised fluores-
cent dyes. Experiments to determine the optimal synthesis,
deprotection and purification methods for oligonucleotides
labelled with the novel LQ quencher are described. We have
synthesised Molecular beacons using the LQ and MR quench-
ers, and their relative quenching characteristics have been
compared.
Two quencher molecules (LQ1 and LQ2, Fig. 1), based on
1,4-diaminoanthraquinone, were synthesised. LQ1 is a phos-
phoramidite monomer that can be incorporated into an oligonu-
cleotide at the 5A teminus during standard solid-phase oligonu-
cleotide synthesis.
It was made by phosphitylation of hydroxyethyl diaminoan-
thraquinone. LQ2 (Scheme 1) is used to position the quencher
Scheme 1 Synthesis of LQ2–CPG. Reagents and conditions: i, DEAD
(1.1eq.), PPh3 (1.1eq.), phthalimide (1.1eq.), THF, rt, 1.5 h, then hydrazine
monohydrate (5 eq.), CH2Cl2: MeOH (1+1), rt, 15 h, 90% over two steps; ii,
succinic anhydride (1.1eq.), DMAP (0.1eq.), pyridine, rt, 1.5 h, 90%; iii,
DIPEA (6 eq.), HOBT (1.1 eq.), EDC (1.1 eq.), 3 (1.1 eq), DMF, rt, 15 h,
60%; iv, succinic anhydride (1.2 eq.), DMAP (0.1 eq.), pyridine, rt, 18 h
then 60 °C, 5 h, 94%; v, LCAA–CPG, EDC (5 eq.), DIPEA (5 eq.), CH2Cl2:
DIPEA (1%), rt, 4 h, loading = 25 µmol g21
Table 1
ODN1
ODN2
5A-LQ-TTT TTT TTT T-3A
5A-TTT TTT TTT TTT TTT-LQ-3A
Fig. 1 Structures of LQ monomer and cpg
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CHEM. COMMUN., 2003, 970–971
This journal is © The Royal Society of Chemistry 2003

of the chromophore from the sugar were observed (results not
shown).
To determine suitable deprotection conditions for oligonu-
cleotides labelled with the 3A quencher, samples of ODN2 were
exposed to the conditions described in Table 2, and examined by
RP-HPLC (Fig. 2). Results showed that heating the oligonu-
cleotide in conc. aq. ammonia caused significant degradation
(expt 1) even with shorter exposure times. However, using
t
water+methanol+ butylamine (2+1+1) or ammonium hydrox-
Fig. 3 Fluorescence melting spectra for the Molecular beacons containing
MR and LQ. MR (red); LQ (blue). Dotted line = no target; solid line = with
target. Fluorescence traces have been normalised i.e. F/Fo, where F =
fluorescence intensity, Fo = maximum fluorescence intensity for each
Molecular beacon.
ide+conc. aq. methylamine (1+1) mixtures. a much cleaner
product was obtained (expts. 2–4).
Table 2
Expt.
Temp. (°C)
Time (h)
Reagents
maximum fluorescence with target/minimum fluorescence
without target). Fig. 4 illustrates that with the shorter wave-
length fluorophore FAM, there is comparable quenching by LQ
and MR. However, when the longer wavelength fluorophore,
CY5, is used there is a significant difference in signal-to-noise
ratio, indicating that LQ is a much more effective quencher for
this fluorophore.
1
2
3
4
70
70
25
25
2
2
24
1
conc. aq. NH3
H2O+MeOH+BuNH2 (2+1+1)
t
H2O+MeOH+ BuNH2 (2+1+1)
NH4OH+MeNH2 (aq) (1+1)
Fig. 2 RP-HPLC chromatograms for experiments 1, 2, 3 and 4 (Table 2).
It should be noted that deprotection using water+metha-
Fig. 4 Signal-to-noise ratios for the Molecular beacons at 30 °C.
t
nol+ butylamine (2+1+1) requires dmfdG amidite to be used
during oligonucleotide synthesis and ammonium hydrox-
ide:conc. aq. methylamine, (1+1) requires acetyldC.
The synthesis and purification of both 5A and 3A LQ labelled
oligonucleotides has been achieved. Molecular beacons con-
taining the new quencher have been shown to perform
efficiently with two fluorophores. LQ performed significantly
better than MR for the long wavelength dye Cy5, whilst both
quenchers show very similar results with the shorter wavelength
dye, FAM.
To assess the performance of the new quencher, Molecular
beacons were synthesised using the LQ1 phosphoramidite and
either fluorescein (FAM, lem = 520 nm) or Cy5 (Amersham
Pharmacia, lem = 670 nm). Beacons of identical sequence but
terminating in methyl red (MR) were also prepared in order to
compare the signal-to-noise ratios of different fluorophore–
quencher pairs. The sequences used are shown in Table 3.
The fluorescence properties of the Molecular beacons
(ODN3-6) were examined by fluorescence melting, with and
without their target strand present in the hybridisation buffer
(ODN7). In the presence of a target strand a large increase in
fluorescence was observed for all Molecular beacons (Fig. 3).
This change in fluorescence was used to calculate the signal-to-
The authors would like to thank the EPSRC and Oswel
Research Products Ltd. for funding this work.
Notes and references
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2 S. Tyagi, D. P. Bratu and F. R. Kramer, Nature Biotech., 1998, 16, 49.
3 D. Whitcombe, J. Theaker, S. P. Guy, T. Brown and S. Little, Nature
Biotech., 1999, 17, 804.
noise ratio for each fluorophore–quencher pair (Fmax/Fmin
=
4 N. Thelwell, T. Millington, A. Solinas, J. Booth and T. Brown, Nucleic
Acids Res., 2000, 28, 3752.
Table 3
5 A. Solinas, L. J. Brown, C. McKeen, J. M. Mellor, J. T. G. Nicol, N.
Thelwell and T. Brown, Nucleic Acids Res., 2001, 29, e96.
6 P. M. Holland, R. D. Abramson, R. Watson and D. H. Gelfand, Proc.
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7 K. J. Livak, S. A. J. Flood, J. Marmaro, W. Giusti and K. Deetz, PCR
Methods and Applications, 1995, 357.
ODN3
ODN4
ODN5
ODN6
ODN7
LQ-GCAGCCGTTTCCTCCACTGTTGCGCTGC-FAM
MR-GCAGCCGTTTCCTCCACTGTTGCGCTGC-FAM
LQ-GCAGCCGTTTCCTCCACTGTTGCGCTGC-Cy5a
MR-GCAGCCGTTTCCTCCACTGTTGCGCTGC-Cy5a
GCAACAGTGGAGGAAACG
8 S. A. E. Marras, F. R. Kramer and S. Tyagi, Nucleic Acids Res., 2002, 30,
e122.
a Cy5 at 3A-end was a functionalised dT resin.
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