Ligand-driven G-quadruplex conformational switching by using an unusual mode of interaction

Article abstract of DOI:10.1002/anie.200605075

Under control: An anthracene polyammonium derivative induces folding of the highly polymorphic human telomeric DNA into a single parallel G-quadruplex conformer through an unusual mode of interaction. The sequential use of this ligand and a porphyrazine allows controlled conformational switching of the quadruplex between parallel and antiparallel conformations. (Figure Presented).

Full text of DOI:10.1002/anie.200605075

Angewandte
Chemie
DOI: 10.1002/anie.200605075
DNA Folding
Ligand-Driven G-Quadruplex Conformational Switching By Using an
Unusual Mode of Interaction**
Raphal Rodriguez, G. Dan Pantos¸, Diana P. N. Gonçalves, Jeremy K. M. Sanders, and
Shankar Balasubramanian*
We report herein the successful design of the first molecule
that induces the folding of the parallel human telomeric G-
quadruplex from single-stranded DNA in the absence of
added cation.[1] Ammonium ions stabilize quadruplex struc-
tures,[2] and K+ ions have been shown to be separated by
approximately 3.3 Š in the crystal structure of the human
single,parallel,quadruplex conformation. After addition of
10 equivalents of 1,an induced CD signal at the absorbance of
achiral 1 (253 nm) was observed as a result of 1 sensing the
DNA chirality in the complex. In control experiments,we
observed that the addition of 20 equivalents (200 mm) of
diethylene triamine (DETA) to a solution free of added
cation,but containing telo24,did not induce quadruplex
structures as seen by CD spectroscopy. Furthermore,ligand 2,
in which an amide linkage reduces the protonation state and
geometrical freedom of the molecule,also did
telomeric quadruplex.[3] Therefore,we synthesized
1
(Scheme 1) based on the hypothesis that the anthracene
moiety would stack onto guanines,forming one of the
not induce quadruplex formation under sim-
ilar conditions (see the Supporting Informa-
tion). These results suggest that the poly-
amine component of 1 is involved in a rather
specific interaction with the DNA quadru-
plex.
Quadruplex-specific stabilization by cat-
ions has an order of preference of K+ >
NH4+ > Na+ > Li+ as a result of the relative
free energies,hydration state,and ionic radius
Scheme 1. Ligands studied for G-quadruplex induction.
for optimal sphere coordination within the
external quartets and allowing the ammonium centers of 1,
which are separated by approximately 3.4 Š,to induce the
DNA folding through hydrogen bonding and cation–dipole
interactions,mimicking K + ions within the central quadruplex
channel.[4]
Upon the addition of 1 to a solution containing the human
telomeric DNA d[TTAGGG]4 (telo24) at room temperature
and free of added cation,we observed positive and negative
CD signals at 263 and 240 nm,respectively,within 30 s
(Figure 1). This resultant CD spectrum is characteristic of a
parallel quadruplex.[5] The human telomeric quadruplex is
highly polymorphic and can exist as parallel,[3] antiparallel,[6a]
and mixed-type parallel/antiparallel[6b] structures depending
on strand orientation. However,ligand 1 appears to induce a
Figure 1. CD spectra of telo24 (10 mm) with 0 (c), 2 (a), 3 (+), 5
( ), 10 ( ), 12.5 ( ), and 15 equivalents ( ) of 1 in Tris-HCl (10 mm,
pH 7.4; Tris=tris(hydroxymethyl)aminomethane).
~
~
*
*
[*] Dr. R. Rodriguez, Dr. G. D. Panto¸s, Dr. D. P. N. Gonçalves,
Prof. J. K. M. Sanders, Dr. S. Balasubramanian
Department of Chemistry
University of Cambridge
Lensfield Road, Cambridge CB21EW (UK)
Fax: (+44)1223-336-913
E-mail: sb10031@cam.ac.uk
quadruplex cavity.[2] The more-general cation-induced stabi-
lization owing to shielding of charge repulsion of the
phosphate backbone is less sensitive to the nature of the
monovalent cation. To explore the proposal that the poly-
amine of 1 is involved in a threading mode of interaction with
the quadruplex,we studied the competition of 1 with specific
monovalent cations. The presence of a large excess of K+ ions
[**] This study was supported by Cancer Research UK, the EPSRC and
The Portuguese Science Foundation. S.B. is a BBSRC Career
Development Research Fellow.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Communications
should prevent the interaction of 1 with the DNA by
saturating cation binding sites within the quadruplex core.
The addition of 1 to a solution of telo24 preannealed with Li+
induced changes in the DNA conformation,which are
characteristic of a parallel conformer,within 2 min at room
temperature with the emergence of signals at 263 and 240 nm
(Figure 2). The addition of 1 to a solution of telo24
Figure 3. 1H NMR spectra at 500 MHz of 1 (bottom), telo24 (top), and
of telo24 with various mole equivalents of 1 (spectra in between).
Conditions: 0.5 mm telo24 in D2O/H2O (1:9), pH 6.5–7.0, recorded at
298 K.
Figure 2. CD spectra of telo24 (10 mm) annealed with 100 mm Li+ ( )
averaged set of the CH2 signals between bound and free
ligands progressively shifted upfield,indicating rapid
exchange on the chemical-shift time scale. These observations
are supportive of our threading model and highlight the
dynamic character of the telomeric DNA.[8]
*
+
~
*
followed by the addition of 20 equivalents of 1 ( ); 100 mm Na ( )
+
~
followed by the addition of 20 equivalents of 1 ( ); 100 mmK (c)
in Tris-HCl (10 mm, pH 7.4).
We recently reported that porphyrazine 3[9] can induce
and stabilizes the antiparallel quadruplex conformation in
both the absence and presence of added cations. Given the
ability of 1 and 3 to induce folding of the human telomeric
DNA quadruplex preferentially into parallel or antiparallel
structures,respectively,we reasoned that it may be possible to
reversibly switch quadruplex conformations by sequential
addition of both ligands. Figure 4a depicts a possible dynamic
process of ligand-driven conformational switching as a result
of two competing modes of interaction. The changes in the
CD spectrum after the addition of 15 equivalents of 3 to a
mixture of telo24 and 1 are illustrated in Figure 4b. We
observed the disappearance of signals at 263 and 240 nm and
the appearance of signals at 298 and 247 nm within 9 min of
the addition of 3,confirming a conformational switch from
parallel to antiparallel structures. The opposite phenomenon
was observed by the addition of 3 to telo24,followed by the
addition of 1 (Figure 4c).[10] This demonstrates that the
conformation of folded quadruplex DNA can be directed by
one ligand,then subsequently switched by the addition of a
second ligand.
A number of flat aromatic ligands have been reported that
can stack onto the terminal G-tetrad of a prefolded quad-
ruplex and thereby stabilize the structure.[11] Herein,we have
described the first example of a molecule (1) capable of
selectively inducing parallel quadruplex formation from
unfolded DNA in the absence of added cations. Furthermore,
this probably involves a combination of threading and
stacking modes of interaction. This molecule was used along
with a complementary ligand to controllably switch quad-
ruplex conformations between the parallel and antiparallel
preannealed with Na+ led to a slow conformational switch
of the DNA from the antiparallel to the parallel conformer.
After 2 h incubation at room temperature,we observed that
positive signals at 298 and 247 nm and a negative signal at
[6]
265 nm,characteristic of the antiparallel conformer,
decreased slowly while signals characteristic of the parallel
conformer increased. The addition of 1 to a solution of telo24
preannealed with K+ did not increase the signals character-
istic of the parallel conformer even after two days at room
temperature. Thus the apparent ability of 1 to bind and induce
conformational changes in monovalent cation-complexed
quadruplex DNA is in the order Li+ (easier) > Na+ > K+
(difficult),reflecting the ease with which cations coordinated
within the quadruplex core,rather than the phosphate
backbone,can be displaced. These results are consistent
with a mode of binding whereby the polyamine of 1 is
threading through the quadruplex core,directly competing
with and mimicking the central cations that stabilize the
folded quadruplex.
To gain further insights into the interaction between 1 and
the DNA,we carried out 1H NMR spectroscopic studies in
the absence of added cations (Figure 3). Upon the addition of
1 equivalent of 1 to telo24,we observed that all CH 2 signals of
the ligand shifted downfield ( ꢀ 0.3 ppm) compared with the
free ligand. The similarities in the Dd observed are consistent
with the presence of essentially identical hydrogen-bonding
interactions occurring between the 12 guanine O6 lone pairs
available from the DNA and the 8 methylene hydrogen atoms
from the protonated ligand.[7] Upon increasing the concen-
tration of 1 beyond 1 equivalent,we observed that a time-
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Angewandte
Chemie
Keywords: conformation analysis · DNA · G-quadruplex ·
.
self-assembly · supramolecular chemistry
[1] For a general review on quadruplexes,see: J. T. Davis, Angew.
Chem. 2004, 116,684; Angew. Chem. Int. Ed. 2004, 43,668.
[2] For a report on cation-templating quadruplexes,see: J. L.
Mergny,J. Gros,A. De Cian,A. Bourdoncle,F. Rosu,B.
Saccà,L. Guittat,S. Amrane,M. Mills,P. Alberti,M. Takasugi,
L. Lacroix in Quadruplex Nucleic Acids (Eds.: S. Neidle,S.
Balasubramanian),RSC Biomolecular Sciences,Cambridge,
2006,pp. 31 – 80 and N. V. Hud,J. Plavec in Quadruplex Nucleic
Acids (Eds.: S. Neidle,S. Balasubramanian),RSC Biomolecular
Sciences,Cambridge, 2006,pp. 100 – 130.
[3] G. N. Parkinson,M. P. H. Lee,S. Neidle, Nature 2002, 417,876.
[4] Liu and co-workers identified triethylene tetraamine as G-
quadruplex ligand (see: F. Yin,J. Liu,X. Peng, Bioorg. Med.
Chem. Lett. 2003, 13,3923) and suggested that the polyamine
“inserts into the cavity of G-quadruplex” and stabilizes the
antiparallel conformation of telo24. In our hands a triethylene
tetraamine induced UV melting transition characteristic of
quadruplex structure was not observed in the absence of K+.
[5] R. Jin,B. L. Gaffney,C. Wang,R. A. Jones,K. J. Breslauer, Proc.
Natl. Acad. Sci. USA 1992, 89,8832.
[6] a) Y. Wang,D. J. Patel, Structure 1993, 1,263; b) A. Ambrus,D.
Chen,J. Dai,T. Bialis,R. A. Jones,D. Yang, Nucleic Acids Res.
2006, 34,2723.
[7] In pioneering work on synthetic pseudorotaxanes involving alkyl
ammonium complexed with crown ethers,Stoddart and co-
1
workers described similar H NMR Dd owing to weak C-H···O
interactions between the CH2 located next to the ammonium and
oxygen lone pairs of the crown ether (see: P. R. Ashton,P. J.
Campbell,E. J. T. Chrystal,P. T. Glink,S. M. D. Philip,N.
Spencer,J. F. Stoddart,P. A. Tasker,D. J. Williams,
Angew.
Chem. 1995, 107,1997; Angew. Chem. Int. Ed. Engl. 1995, 34,
1865).
Figure 4. a) Schematic illustration of DNA conformational switching.
b) CD spectra of telo24 (10 mm) with 5 equivalents of 1 (c),
followed by the addition of 5 equivalents of 3 after 3 min (+), then
[8] The changes in the imino proton resonances of the quadruplex
preannealed with 100 mm NaCl upon addition of 1 are given in
Figure S4 in the Supporting Information. Owing to the highly
dynamic character of the complex formed between 1 and the
DNA,we did not observe NOESY correlations,which is
supportive of our model between exchangeable guanine imino
protons and the CH2 of 1.
*
after a further 3 min the addition of 5 equivalents of 3 ( ), then the
~
addition of 5 equivalents of 3 after a further 3 min ( ) at RT. c) The
above experiment repeated with an inverse ligand addition (3 then 1).
[9] D. P. N. Gonꢀalves,R. Rodriguez,S. Balasubramanian,J. K. M.
Sanders, Chem. Commun. 2006,4685.
[10] Owing to system poisoning,we could not monitor more than one
conformational switch for each experiment.
[11] For a recent report on G-quadruplex ligands,see: M. S. Searle,
G. D. Balkwill in Quadruplex Nucleic Acids (Eds.: S. Neidle,S.
Balasubramanian),RSC Biomolecular Sciences,Cambridge,
2006,pp. 131 – 153.
forms in either direction. Given the apparent prevalence of
DNA quadruplexes in biology[12] and their application in
nanoscience,[13] ligands such as 1 and 3 present the possibility
of controlling biological or nanomolecular mechanisms by
ligand-induced conformational switching.
[12] J. L. Huppert,S. Balasubramanian, Nucleic Acids Res. 2005, 33,
2908.
[13] a) J. J. Li,W. Tan, Nano Lett. 2002, 2,315; b) P. Alberti,J. L.
Mergny, Proc. Natl. Acad. Sci. USA 2003, 100,1569.
Received: December 15,2006
Revised: May 9,2007
Published online: June 11,2007
Angew. Chem. Int. Ed. 2007, 46, 5405 –5407
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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