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unsymmetrical squaraine dye in great purity (>90%, as esti- squaraine dye was detectable in the absence of a template; a
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mated by H NMR spectroscopy).
fluorescent signal characteristic of squaraine dyes (lem = 645 nm)
Having demonstrated that synthesis of squaraine dyes could was observed after 12 h. When raising the temperature up to
proceed in aqueous solutions (see Table 1, entries H and I), 40 1C, an 8-fold increase in the fluorescence intensity was
although in poor yields, and considering the fluorogenic properties observed after 3 h upon addition of the DNA template (when
of the reaction, we decided to investigate its suitability for optical compared to the non-templated reaction) (Fig. 3). The product of
sensing applications based on the concept of oligonucleotide- the OTR was finally characterised by MALDI, thus confirming
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templated reaction (OTR). Widespread in Nature, the concept of that the observed fluorescence at lem = 645 nm originated, as
OTRs has recently been applied to the highly sensitive and sequence- anticipated, from the formation of the PNA1–squaraine–PNA2
+
specific detection of nucleic acid biomarkers both in vitro and in vivo. conjugate ([M + H] m/z = 3990, yield o 5%, as estimated by
For such applications, oligonucleotide-templated fluorogenic reac- MALDI, see ESI†). Application and optimisation of this new
tions, for which the appearance of a characteristic fluorescent signal fluorogenic OTR in the sensing of nucleic acid secondary struc-
can be directly correlated to the sensing of the unique nucleic acid tures is currently underway in our laboratory.
target of interest, proved extremely popular.
We have demonstrated that symmetrical squaraine dyes can be
We recently reported the use of fluorogenic PNA probes for the synthesised under mild (and aqueous) conditions via carbodiimide-
detection of DNA secondary structures, based on an OTR of mediated activation of squaric acid (or of the semi-squaraine dye).
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2
cyanine dye (C3) formation. Herein, we investigated the applic- Although yields of reactions carried out in solution remain low
ability of our reaction of squaraine dye synthesis for DNA sensing. (up to 15%), near quantitative conversions are obtained when the
Two water-soluble PNAs functionalised at their N or C terminus reaction is carried out on a solid-support. We also show that the
with a semi-squaraine dye (PNA1) and with a 2-methylene- fluorogenic reaction of squaraine dye synthesis can be applied to
indoline moiety (PNA2), respectively, were synthesised on solid DNA sensing, using a strategy based on the concept of OTR.
phase (see ESI† for sequences and experimental details). Reac- Although more work is required to further improve the sensitivity
tions between PNA1 and PNA 2 (70 mM each) in potassium of the system, this first example of DNA-templated synthesis of a
phosphate buffer (15 mM, pH 7.4) containing an excess of EDCI squaraine dye emitting in the NIR from two non-fluorescent
(700 mM, 10 equiv.), in the absence and in the presence of a precursors is likely to have valuable applications for the develop-
complementary DNA strand (70 mM, 1 equiv.), were monitored by ment of new DNA (or RNA)-targeting probes.
fluorescence spectroscopy (lexc = 610 nm). Whilst at such low
This research was supported by the Leverhulme Trust (grant RPG-
concentration of PNA probes, no significant formation of the 2012-603) and by the EU FP7 (Career Integration Grant CIG 293981).
Notes and references
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Fig. 3 (bottom) DNA-templated synthesis of a squaraine dye from two
non-fluorescent precursors; and (top) fluorescence emission spectra of a
stoichiometric mixture of PNA1 and PNA2 (70 mM each) in potassium
phosphate buffer (15 mM, pH 7.4) in the absence (red circles) or in the
presence (black squares) of a DNA template. Spectra were recorded after
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2 K. Meguellati, G. Koripelly and S. Ladame, Angew. Chem., Int. Ed., 2010,
49, 2738; G. Koripelly, K. Meguellati and S. Ladame, Bioconjugate
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h at 40 1C and after a 6-fold dilution in water (lexc = 610 nm).
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290 | Chem. Commun., 2014, 50, 5288--5290
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