Photoresponsive Formation of an Intermolecular Minimal G-Quadruplex Motif

Article abstract of DOI:10.1002/anie.201510269

The ability of three different bifunctional azobenzene linkers to enable the photoreversible formation of a defined intermolecular two-tetrad G-quadruplex upon UV/Vis irradiation was investigated. Circular dichroism and NMR spectroscopic data showed the formation of G-quadruplexes with K⁺ ions at room temperature in all three cases with the corresponding azobenzene linker in an E conformation. However, only the para-para-substituted azobenzene derivative enables photoswitching between a nonpolymorphic, stacked, tetramolecular G-quadruplex and an unstructured state after E-Z isomerization.

Full text of DOI:10.1002/anie.201510269

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International Edition: DOI: 10.1002/anie.201510269
German Edition: DOI: 10.1002/ange.201510269
G-Quadruplexes
Photoresponsive Formation of an Intermolecular Minimal
G-Quadruplex Motif
Julie Thevarpadam+, Irene Bessi+, Oliver Binas+, Diana P. N. GonÅalves, Chavdar Slavov,
Hendrik R. A. Jonker, Christian Richter, Josef Wachtveitl, Harald Schwalbe,* and
Alexander Heckel*
In memory of Gerhard Quinkert
Abstract: The ability of three different bifunctional azoben-
zene linkers to enable the photoreversible formation of
a defined intermolecular two-tetrad G-quadruplex upon UV/
Vis irradiation was investigated. Circular dichroism and NMR
spectroscopic data showed the formation of G-quadruplexes
with K+ ions at room temperature in all three cases with the
corresponding azobenzene linker in an E conformation.
However, only the para–para-substituted azobenzene deriva-
microscopes, allows for very precise spatiotemporal and dose
control,[15–18] far beyond the precision of the injection of
trigger compounds, as recently shown for blood clotting and
miRNA.[19,20]
The ability to control a process by light can be introduced
either by using photolabile groups or photoswitches such as
azobenzene.[24,25] The use of photolabile groups has been
applied to G-quadruplexes which were either formed or
destroyed irreversibly upon light irradiation.[21–23]
tive enables photoswitching between
a nonpolymorphic,
stacked, tetramolecular G-quadruplex and an unstructured
state after E–Z isomerization.
In the G-quadruplex field, Ogasawara and Maeda have
used stilbene-type substituents to control the formation of G-
quadruplexes by light-induced E–Z isomerization.[26] Spada
et al. controlled the self-assembly of guanosine monomers in
a similar fashion.[27] The formation of G-quadruplexes can
also be induced by small molecules that act as molecular
chaperons in the absence of cations.[28–30] Zhou et al. devel-
oped azobenzene-containing small-molecule chaperones to
regulate G-quadruplex formation.[31] Tan et al. synthesized an
azobenzene-modified antisense DNA linked to a thrombin-
binding aptamer to regulate G-quadruplex formation.[32]
Herein, we present a minimal light-switchable DNA
module enabling the formation of an intermolecular and
conformationally well-defined G-quadruplex structure with
a photoswitchable azobenzene residue as part of the back-
bone structure (Figure 1).
G
-Quadruplexes are important DNA secondary structures.
The structures form in an intramolecular manner or by
association of multiple strands and can exist in many
polymorphic forms.[1–4] G-Quadruplexes are important regu-
latory elements in the genome[5,6] but they are also selected in
SELEX procedures as versatile aptamers (SELEX = system-
atic enrichment of ligands by exponential amplification).[7]
For example a simple G-rich DNA 15mer can inhibit blood
clotting.[8] In DNA nanoarchitectures, G-quadruplexes can
act as interesting structural scaffolds.[9–14]
Light is an ideal external trigger signal that can be highly
selective and superior to changes in temperature and
pH value. Localized irradiation, for example in laser scanning
Azobenzene derivatives Az1, Az2, and Az3 (Figure 2a)
were employed as photoswitchable linkers between two sets
of two consecutive guanosine moieties and were introduced
using DNA solid-phase synthesis. The size and substitution
patterns on the azo linkers were chosen to offer a suitable
balance between the rigidity and flexibility of the overall
structure, such that the photoswitch in the E conformation
should permit the formation of a G-quadruplex, whereas in
the Z conformation no such G-quadruplex formation should
be possible. Simple predictions suggested that Az1 can bridge
a distance of 13.2–13.6 Š in the E conformation and 7.7–
[*] J. Thevarpadam,[+] Dr. D. P. N. GonÅalves, Prof. Dr. A. Heckel
Goethe University Frankfurt
Institute for Organic Chemistry and Chemical Biology
Buchmann Institute for Molecular Life Sciences
Max-von-Laue-Strasse 9, 60438 Frankfurt (Germany)
E-mail: heckel@uni-frankfurt.de
Homepage: http://photochem.uni-frankfurt.de
I. Bessi,[+] O. Binas,[+] Dr. H. R. A. Jonker, Dr. C. Richter,
Prof. Dr. H. Schwalbe
Institute for Organic Chemistry and Chemical Biology
Center for Biomolecular Magnetic Resonance (BMRZ)
Max-von-Laue-Strasse 9, 60438 Frankfurt (Germany)
E-mail: schwalbe@nmr.uni-frankfurt.de
Homepage: http://schwalbe.org.chemie.uni-frankfurt.de
Dr. C. Slavov, Prof. Dr. J. Wachtveitl
Institute for Physical and Theoretical Chemistry
Max-von-Laue-Strasse 7, 60438 Frankfurt (Germany)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201510269.
Figure 1. Sequence and numbering of the azobenzene derivative used
and characterized in this study, with the azo unit (Az1) shown in gray.
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65% of its maximum value, whereas at a 5 mm K+ ion
concentration, the signal intensity was at 10% of its maximum
(Figure 3b). In absence of K+ ions there is no detectable
interaction between the nucleobases, indicating that neither
G-quadruplexes nor any other aggregates of the oligonucleo-
tide strands are formed. Additionally, at Na+ concentrations
of up to 500 mm, we did not detect the formation of any
secondary structure (Figures 3c and 3d). In this case, G-
quadruplex formation was induced only after adding an
additional 100 mm of K+ ions (Figure 3d; Figure S39).
For another recent example of ion selectivity in G-
quadruplexes and possible application in nanotechnology, see
also Ref.[36]. Cation selectivity has also been reported for the
thrombin-binding aptamer G-quadruplex,[37] whose G-tetrad
core adopts the same folding topology as we have determined
for GG-Az1-GG (see Figure 4 and the corresponding dis-
cussion). We propose that the smaller size of the Na+ ion is
not optimal to coordinate all eight O6 atoms in the two-tetrad
cavity, which is necessary to keep the GG-Az1-GG in a stable
quadruplex structure. Furthermore, the azobenzene linkers
may introduce additional strain to the ionic channel, leading
to the observed ion selectivity.
Figure 2. a) The structures of the azo units employed in this study.
b) Imino regions of the 1D 1H NMR spectra of GG-Az1-GG, GG-Az2-
GG, and GG-Az3-GG in the presence of KCl (100 mm). Experimental
conditions: DNA (50 mm), Tris-HCl buffer (50 mm; pH 7.4), 298 K,
600 MHz.
11.5 Š in the Z conformation between the two oxygen atoms
adjacent to the azobenzene core. Going from a para–para to
a para–meta substitution pattern in Az2, these values change
À
to 11.1–13.6 Š and 9.0–11.9 Š, respectively. The O O dis-
tance at equivalent positions in the narrow grooves in G-
quadruplex structures can reach 11–12 Š (see for example
PDB 2GKU). We also included the double homologue Az3
(8.8–14 Š in the E conformation and 7.3–12.4 Š in the
Z conformation). Clearly, minor changes in the structure of
the azo linker result in significant changes of the “hinge
qualities” of the photoswitch linkers. All of these consider-
ations should be considered with reservation given the highly
polymorphic nature of G-quadruplex structures and their
respective structural flexibility. We specifically refrained from
using longer homologues so as not to dissipate the perturba-
tion induced by the E–Z transition into too many internal
degrees of freedom.
We investigated structural changes upon photoswitching
of the azobenzene units. Two distinguishable sets of CD
spectra could be obtained upon irradiation with either UV or
visible light (Figure 3e). After an initial irradiation with
UV light for 5 min, the CD signal at l = 295 nm disappeared
completely. Over the course of 30 min, the system reached
thermal equilibrium (red data points in Figures 3e–g).
Whereas the thermal Z–E isomerization occurs over the
course of several days (Figure S42), irradiation with visible
light for a short period of time (2 min) led to the almost
complete recovery of the initial CD signal after 15 min of
equilibration (Figure 3 f) which could also be paralleled with
corresponding NMR experiments (Figure 3h; Figure S60a).
1H NMR spectroscopy showed the complete disappearance of
signals for the imino protons upon irradiation with UV light
and their almost complete recovery after irradiation with
visible light (Figure 3i, 100 mm of K+). We found that the
degree of recovery after irradiation with visible light is
a function of the DNA concentration: although at 50 mm the
recovery of signals attributable to a G-quadruplex structure
after irradiation with visible light is basically complete,
a sample containing 125 mm DNA shows 75% recovery of
resonance signals for the G-quadruplex 15 min after irradi-
ation. However, the recovery of the G-quadruplex structure is
complete after thermal equilibration (Figure S60a).
We speculate that at DNA concentrations concentrations
greater than 50 mm, after UV illumination unspecific aggre-
gates are formed (indicated by a broad signal in the aromatic
region of the 1H NMR spectrum) that slowly convert into the
quadruplex folded state and/or to the completely unfolded
state.
No effects of UV degradation (such as the photo-
oxidation of guanine) were detected in the aromatic region
of the 1D 1H NMR spectra after repetitive UV/visible-light
irradiation cycles (Figure S60b). Additionally, UV/Vis differ-
ence spectra (Figure S6) showed the differential absorption
signature typical for G-quadruplexes.[38]
Initial 1H NMR (100 mm K+) characterization of the short
azobenzene-linked sequences chosen for this investigation
showed signals in the imino region of the spectrum, typical for
Hoogsteen-type hydrogen bonds (Figure 2b), suggesting the
formation of G-quadruplex structures. Circular dichroism
(CD) studies were performed to assess the conformational
properties of the modified G-rich sequences. After the
addition of 25 mm of K+ ions, a positive signal around l =
295 nm and a negative signal near l = 260 nm were detected
for all three systems, indicating formation of antiparallel G-
quadruplex structures (see Figure 3a for GG-Az1-GG and
the Supporting Information for GG-Az2-GG and GG-Az3-
GG).[33,34] Interestingly, 1H NMR analysis of the different
derivatives revealed structural polymorphism for GG-Az2-
GG and GG-Az3-GG which was not detectable by CD. In all
three cases, no formation of higher order aggregates was
detected (see Figure S58 in the Supporting Information). For
details on the thermal stability of the structures formed by the
three sequences, please see Figure S5.
1
Only four H NMR signals for imino groups, indicating
a highly symmetric G-quadruplex structure, were detected
after addition of K+ to GG-Az1-GG. The 1H NMR spectra of
GG-Az1-GG also showed that at a K+ ion concentration of
25 mm, the intensity of the set of imino signals was already
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Figure 3. Spectroscopic characterization of the GG-Az1-GG sequence. a) CD spectra recorded with increasing K+ concentrations (0, 5, 25, 50,
1
100 mm). b) 1D H NMR spectra showing the imino region recorded in the presence of different KCl concentrations. c) CD spectra with
1
increasing concentrations (0, 50, 100, 200, 300, 400, 500 mm) of Na+. d) Imino region of the 1D H NMR spectra in the presence of increasing
amounts of NaCl and KCl. e) CD spectra of GG-Az1-GG with photoirradiation (5 min UV, 2 min visible light) and f) corresponding time course of
the signal at l=295 nm under the indicated treatment. Cycling of the photoresponsive structural conversion of GG-Az1-GG by alternate
irradiation with UV (15 min) and visible (4 min) light as monitored g) by CD at l=295 nm and h) by 1H NMR ([DNA]=125 mm, [KCl]=100 mm)
at d=11.55 ppm (green line; monitoring the intensity of the signal for the imino proton of residue G5 in the E conformation) and at 6.64 ppm
(red line; monitoring the intensity of the signal for the aromatic proton from the azobenzene moiety in the Z conformation). Absolute NMR peak
intensities are referenced to an internal standard. i) Imino region of the 1D 1H NMR spectra ([DNA]=50 mm, [KCl]=100 mm) showing spectral
changes with photoirradiation (30 min UV, 2 min visible light). j) IR difference spectra (bottom); IR transient absorption data recorded from GG-
Az1-GG after excitation of the azobenzene moiety at l=335 nm (top left); decay-associated spectra from the global lifetime analysis[35] of the
transient absorption data (top right).
Photoswitching of GG-Az1-GG was also evident in the
FTIR difference spectra (Figure 3j, bottom; Figure S53). The
azobenzene isomerization and the subsequent disruption of
the G-quadruplex structure led to a bathochromic shift
ruplex, residual conformational dynamics occur on longer
time scales.
For studies of GG-Az1-GG derivatives elongated or
shortened at the 3’- and 5’-end and for the results obtained
with the sequence GGG-Az1-GGG, please see Figures S40/
S41 and S57, respectively.
À1
[39–41]
(1675 cmÀ1!1667 cm ) of the C O stretching band
=
and to batho- and hypsochromic shifts of the bands associated
=
=
with the C C and C N purine ring vibrations (1550–
1590 cmÀ1 range),[39,41] features reported in the literature to
be characteristic of G-quadruplex melting.[39,41] Ultrafast UV-
pump/mid-IR-probe experiments[42] in the carbonyl stretching
vibration range (Figure 3j, top left; Figure S54–55) were
performed to investigate the dynamics of the G-quadruplex
after E azobenzene excitation (l = 335 nm). In the first
picoseconds after laser excitation, the transient absorption
To elucidate the molecular structure of the homogene-
ously folded GG-Az1-GG, 2D NMR experiments (2D
1H–1H NOESY, 1H–13C HSQC, and 1H–13C HMBC) were
conducted to obtain the complete proton chemical shift
assignment. Distance restraints were derived from NOE data
and additional angular restraints were obtained from high-
resolution 1H–13C HSQC, 2D 1H–1H P.E.COSY, and 1H–
31P TOCSY experiments.[43] Assignment and J-coupling anal-
ysis are reported in the Supporting Information.
=
data are dominated by the cooling dynamics of the C O
stretching band (1645–1680 cmÀ1) with a lifetime of circa
11 ps. The absorption and bleach bands for the product state
become visible after circa 10 ps (see the infinity spectrum in
Figure 3j, top right). The last spectrum in the transient
absorption data is essentially a Z–E IR spectrum of GG-Az1-
GG at about 1800 ns. Evidently, this spectrum does not fully
match the corresponding FTIR difference spectrum (Fig-
ure 3j, bottom), which indicates that despite the nearly
instantaneous disruption of the FTIR features of a G-quad-
The folding topology of GG-Az1-GG was determined on
the basis of NOESY data. As indicated by intra-tetrad H1–H8
connectivities (Figures S44), GG-Az1-GG adopts a symmet-
ric, antiparallel G-quadruplex structure with tetrads com-
posed as shown in Figure 4a,b. The edgewise loops containing
the azobenzene moieties are located above the G2–G4 tetrad.
The anomeric-aromatic region of the NOESY spectrum
(Figure S45) indicated a syn conformation for the glycosidic
bond of residues G1 and G4 and an anti conformation for
residues G2 and G5.
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Table 1: Statistics of the structure calculation.[a]
Intriguingly, the structure of GG-Az1-GG resembles very
much the ones of the G-quadruplex formed by the thrombin-
binding aptamer (PDB: 148D, NMR; and PDB: 4DII, X-
ray)[37,44] and in the promoter region of the B-raf gene
(PDB: 4H29; see Figure S59 for an overlay).[45]
NOE distance restraints
Unambiguous NOEs:
intra-residue
193
146
87
42
1
sequential
long-range
Hydrogen–deuterium exchange experiments (Figure S46)
revealed that imino protons belonging to residues G1 and G5
are protected from solvent exchange. This result suggests
a tetrameric G-quadruplex consisting of two dimeric units
with the previously defined topology, in which the G1–G5
tetrads of both dimers face each other (Figure 4c). The
stacking of two dimeric G-quadruplex units is supported by
DOSY data (Figure S47). The structure of the tetramer
(Figure 4d) was calculated using ARIA (details in the
Supporting Information). The tetrameric arrangement was
further supported by the fact that structure calculations as
either monomer or dimer led to NOE violations. A significant
number of NOEs are unambiguously assigned for the dimer
and tetramer (Table 1). The prevalent conformation of the
sugar moieties of G1, G2, and G4 is C2’-endo (confirmed by
dimer
tetramer
Ambiguous NOEs:
intra-residue
4
12
47
5
22
7
3
8
2
14
4
intra-monomer
monomer or dimer
monomer or tetramer
dimer or tetramer
mono-, di-, or tetramer
Distance restraints
intra-monomer hydrogen bonds
inter-monomer hydrogen bonds
potassium site coordination
Base planarity
4
6
4
2
intra-monomer
inter-monomer
Torsion angles
2
35
13
3
4
15
0
3
typical strong NOE cross-peaks and J coupling constants).
backbone
b (from 3J(H5’1,2,P))
glycosidic (c)
Only the 3’-terminal G5 sugar moiety is less well-defined, in
part caused by resonance overlap, but manual inspection of
the NOE cross-peaks (which are weaker than for the others)
also suggest that this sugar possibly interconverts between the
C3’-endo and C2’-endo conformations. Therefore no addi-
tional torsion angle restraints were included for the G5
nucleoside. The final bundle of structures was refined in
explicit water, including three coordinated potassium ions
within the tetrads. The structure is well-defined with an
average root-mean-square deviation (RMSD) to mean for all
atoms of 0.60 Š.
sugar pucker
Violations
distances (>0.3 Š)
dihedral angles (>58)
RMSD (average to mean)
monomer (all atom)
tetramer (all atom)
0
0
0.55 Š
0.60 Š
[a] Statistics per monomer.
In summary, we have developed a photoswitchable G-
quadruplex module and have characterized its photochemical
behavior and its 3D conformation. Out of three investigated
photoswitchable linkers in a number of sequence contexts,
only GG-Az1-GG showed a defined and robust structural
behavior. The system shows excellent photocontrol by UV/
Figure 4. a) The G2–G4 tetrad and b) the G1–G5 tetrad with color coding corresponding both to the structure shown in (c) and to residue
numbering given in Figure 1. c) Representation of the structure of the tetramer, with each monomer displayed in a different color. Conformations
of the bases (syn or anti) are indicated. d) NMR solution structure of the G-quadruplex, showing the best representative of the bundle (indicated
faintly in the background). K+ ions are shown as gray spheres. PDB-code: 2N9Q; BMRB-code: 25915.
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Keywords: azobenzene · DNA structures · G-quadruplexes ·
IR spectroscopy · NMR spectroscopy
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