z We cannot rule out the possibility that molecules in the
FRET melting experiments stabilise a non-quadruplex secondary
structure.
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Fig. 2 (Left) SPR binding curves for 1 binding to hTelo (%), c-kit
(K) and ds (m) DNA (running buffer: 50 mM Tris-HCl pH 7.4, 100
mM KCl). (Right) FRET assay for hTelo (%), c-kit (K) and ds (m)
DNA in the presence of 1 (buffer: 60 mM potassium cacodylate
pH 7.4).
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(X = N) always show higher stabilisation temperatures com-
pared to their benzene (X = CH) counterparts. For example,
16 shows moderate stabilisation of hTelo and c-kit (DTm
=
4 1C and 8 1C, respectively), whereas 17 shows significantly
higher stabilisation (DTm = 10 1C and 23 1C, respectively).
The simple replacement of an amine in 2 with oxygen (3) leads
to loss of detectable binding and stabilizing properties, sug-
gestive that protonation of the amine of 2 may be critical.
Replacement of the benzene ring in the 4-position of the
central pyridine of 2 with a thiazole (i.e. 4) results in 4100-
fold stronger binding to c-kit (Kd = 180 nM for 4) compared
to 2 (Kd 4 25 mM for c-kit). The origin of this may be steric
interactions between the hydrogens on the phenyl ring and the
central pyridine core of 2, which could cause the ring to reside
slightly out of plane, in contrast with 4 being able to adopt a
more planar structure owing to reduced steric interactions of
the heteroatoms (N and S) and smaller ring size. It was also
notable that some ligands were found to bind with 2 : 1
stoichiometry and others with 1 : 1 binding, suggestive of
more than one mode of binding. Overall it was found that the
ligand 4 is the tightest binder and has preference for c-kit
quadruplex, while ligands 11, 13 and 18 show preference for
hTelo quadruplex.
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15 G. W. V. Cave and C. L. Raston, J. Chem. Soc., Perkin Trans. 1,
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The TAPs are a new class of quadruplex binding ligands
that show versatility in their specificities, affinities and stabi-
lisation potential. These ligands do not bind to duplex DNA
and have provided proof of concept for the discrimination
between different genomic DNA quadruplexes by a small
molecule. Investigations into the biological activities of these
molecules are currently underway.
16 B. Tamami and H. Yeganeh, Polymer, 2000, 42, 415.
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We thank the BBSRC for project funding and CRUK for
program funding. We also thank the BBSRC for a studentship
(Z.A.E.W.) and the EPSRC Mass Spectrometry Service for
MS analysis. S.B. is a BBSRC Career Development Research
Fellow. We thank James Redman and Colin Raston for
discussions prior to this project.
Notes and references
z Above 25 mM, the TAPs were generally observed to adhere to the
SA Biacore sensor chip leading to data that could not be fitted;
thus 25 mM has been quoted as a lower limit for weak binding ligands.
y The Tm of c-kit by FRET melting (in 60 mM cacodylate buffer) was
found to be 71 ꢁ 1 1C and DTm 24 1C (i.e. Tm = 95 1C) is the
maximum DTm that can be confidently measured.
ꢀc
This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 1467–1469 | 1469