stability of the xanthine/8-oxoguanine-tetrad-containing
quadruplexes was determined, both the parallel folds
(observed at 265 nm) and the anti-parallel folds (observed at
290 nm) showed significantly lowered melting temperatures of
40 1C or below, see Fig. 2B. Although the incorporation of an
8-oxoguanine/xanthine-tetrad makes it possible to favor one
quadruplex topology over the other, incorporation of the
unnatural tetrad slightly destabilizes the quadruplex structure
compared to a conventional tetrad made up from guanines.
In conclusion, the use of 8-oxoguanosine in combination
with xanthosine makes it possible to program the overall
topology of quadruplexes by redesigning constructive hydro-
gen bonding patterns. Notably, the strategy should even make
it possible to program topologies very specifically. For exam-
ple, the basket, chair, and dogear conformations of intra-
molecular quadruplexes differ in the guanosines participating
in the external tetrads and hence should be individually
programmable using this approach. The present study proves
the viability of the concept whereas more elaborate structural
studies will be necessary in order to differentiate between the
mentioned conformations.
sequences,22 it is remarkable that the difference of four out
of in total 24 possible hydrogen bonds (eight per tetrad) is able
to tip the folding equilibrium between anti-parallel and paral-
lel quadruplexes to one side or the other (folding of the non-
programmed conformation results in the loss of two hydrogen
bonds in two tetrads together with potential steric hindrance in
one tetrad, see ESIw). Besides stabilizing factors such as
nucleobase stacking and hydrogen bonding, electrostatic in-
teractions play an important role, particularly in quadruplex
structures.23 Hence, it will be interesting to characterize the
behavior of the modified sequences with respect to different
concentrations and types of cations. Anyway, only small
differences in energetic stability of anti-parallel and parallel
telomeric sequences have been found which could explain the
viability of the presented approach.5,11
We thank Elmar Weinhold for sparking discussions, Astrid
Joachimi and Vicki Lee for excellent technical assistance. JSH
gratefully acknowledges the VolkswagenStiftung for funding a
Lichtenberg-Professorship.
Notes and references
A variety of nucleobase analogs participating in quadruplex
structures have been studied before. For example, Mergny and
co-workers have characterized several guanosine derivatives in
the context of tetrameric quadruplexes, finding mostly negative
effects on stability and assembly kinetics of the four-stranded
structures.16 In addition, 8-bromoguanosine-modified G-rich
sequences showed altered quadruplex structures.17,18 Incor-
poration of adenosine analogs has been reported to result in
changed conformations in tetrameric quadruplexes.19 Incor-
poration of ribonucleosides into DNA quadruplexes has been
shown to restrict the possible conformations to parallel strand
orientations. The likely reason for this observation is the
preference of the anti-conformation of the ribonucleoside
glycosidic bond.3 In contrast, substitutions in the 8-position
favor the syn-conformation.20 Nevertheless, the anti-conforma-
tion of the glycosidic bond is still possible with guanosine
analogs modified at C8.17,21 The parallel conformation of the
natural telomeric sequence in its K+ form contains only anti-
conformations of the glycosidic bond.8
The basket-type Na+ form of the natural telomeric se-
quence displays alternating anti–syn–anti–syn conformations
of guanines 1, 6, 7, and 12 of the first tetrad referring to the
numbering in Fig. 1.9,10 Since 8-oxo-G favors the syn-con-
formation, one out of the two 8-oxo-Gs is in the disfavored
anti-conformation in both HT-ap as well as HT-ap (inv). In
the parallel form, both 8-oxo-Gs would adopt the disfavored
glycosidic conformation. This would represent an explanation
for the decreased stabilities of all modified quadruplexes
compared to the natural sequence as shown in Fig. 2B. Never-
theless, the question of whether the structures of the modified,
programmed sequences adopt the exact conformations of the
natural sequences can only be addressed with more sophisti-
cated structural studies. For a detailed review of the glycosidic
conformations in vertebrate telomeric quadruplexes see ref. 2.
In addition, the presented findings are interesting with
respect to the contribution of hydrogen bonds to the overall
stability of quadruplex sequences. Since stacking interactions
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a
major role in stabilizing four-stranded
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4012 | Chem. Commun., 2008, 4010–4012