Conformational switching of G-quadruplex DNA by photoregulation

Article abstract of DOI:10.1002/anie.201002290

Bend and stretch. . . bend and stretch. . . An azobenzene derivative was used to induce reversible stretching and folding of G-quadruplex DNA upon photoirradiation (see picture). The G-quadruplex formed in the presence of the trans isomer was dissociated

Full text of DOI:10.1002/anie.201002290

Angewandte
Chemie
DOI: 10.1002/anie.201002290
DNA Structures
Conformational Switching of G-Quadruplex DNA by
Photoregulation**
Xiaolin Wang, Jing Huang, Yangyang Zhou, Shengyong Yan, Xiaocheng Weng, Xiaojun Wu,
Minggang Deng, and Xiang Zhou*
Native, self-assembling nucleic acid nanomachines that can
walk, move, or rotate have been developed.[1] Owing to their
ability to form diverse secondary structures, for example, by
the highly sequence-specific hybridization of complementary
sequences, the hybridization of DNA and RNA through
Watson–Crick H bonds, and the assembly of triplexes through
Hoogsteen bonds, nucleic acids are ideal building blocks for
the construction of nanodevices. Quadruplex architecture is a
nucleic acid secondary structure that plays an important role
in nanomachine research, particularly in the control of
reversible folding and extension of the G quadruplex of
DNA in the presence of external stimuli.[2] Mergny and co-
workers reported that a copper(II)-mediated structural switch
with a flexible ligand could regulate the conformation of the
G quadruplex.[3] Nanodevices based on a quadruplex-to-
duplex-transition that rely on the use of single-stranded
DNA as fuels have been shown to perform rotary move-
ments.[4] Among external stimuli, such as temperature,[5]
pH value,[6] electrical-field strength,[7] and molecular recog-
nition,[8] photoregulation is particularly advantageous for
controlling movement and conformation. For example, pho-
toregulation does not require any additional components and
does not cause undesirable side reactions. Irradiation is an
accurate and simple method, and the timing, location, and
strength of light can be controlled readily. Moreover, photo-
regulation provides a clean source of energy and can be
repeated many times without loss of efficiency.[9–12]
The introduction of a photochromic group into biomol-
ecules, such as peptides, oligonucleotides, sugar scaffolds, and
phospholipids, can cause conformational changes that alter
the photochemical properties of the biomolecule. Accord-
ingly, various biological processes involving modified biomol-
ecules can be regulated in a straightforward manner by
irradiation.[13] Recently, Ogasawara and Maeda demonstrated
the successful photoregulation of G-quadruplex formation
through isomerization of a photochromic nucleobase, 8FVG,
incorporated in aptamers.[14] Spada and co-workers intro-
duced a photoactive moiety at the C8 position of a lipophilic
guanosine derivative to regulate the existence of G quar-
tets.[15] However, all these photocontrollers are photochromic
modified nucleobases. Specific molecules have not been
shown to function as G-quadruplex photocontrollers; thus,
we became interested in designing a photoswitch to regulate
the formation of G-quadruplex DNA.
The azobenzene moiety is widely used as a photorespon-
sive molecular tool[16] because it possesses excellent photo-
chemical characteristics. Specifically, azobenzene isomerizes
to predominantly trans and cis forms under visible (Vis) and
ultraviolet (UV) light, respectively. In this study, we synthe-
sized the azobenzene derivative 1 (Scheme 1) to control the
movement and conformation of a G quadruplex by irradi-
ation. Our results suggest that the formation and dissociation
of G-quadruplex DNA was induced by interconversion of the
trans and cis forms of compound 1.
Compound 1 was synthesized by treating 4,4’-dihydrox-
yazobenzene with 1-(2-chloroethyl)piperidine hydrochloride
[*] X.-L. Wang,[+] J. Huang,[+] Y.-Y. Zhou, S.-Y. Yan, X.-C. Weng, X.-J. Wu,
M.-G. Deng, Prof. X. Zhou
College of Chemistry and Molecular Sciences
Key Laboratory of Biomedical Polymers, Ministry of Education
Wuhan University, Hubei, Wuhan 430072 (P.R. China)
Fax: (+86)27-8733-6380
E-mail: xzhou@whu.edu.cn
Prof. X. Zhou
State Key Laboratory of Natural and Biomimetic Drugs
Peking University and
State Key Laboratory of Applied Organic Chemistry
Lanzhou University (P.R. China)
[+] These authors contributed equally.
[**] We thank Prof. Guangfu Yang (College of Chemistry, Central China
Normal University, Hubei, Wuhan) for providing Accelrys Discovery
Studio 2.1 software. X.Z. thanks the NSFC (National Science Fund
of China, No. 90813031, 30973605) and the National Key Founda-
tion for Infectious Diseases (protection against and treatment of
AIDS and hepatitis, 2008ZX10003-005).
Scheme 1. Synthesis of compound 1: a) 1-(2-chloroethyl)piperidine
hydrochloride, dry acetone, K2CO3, argon, reflux, 59%; b) CH3I, CHCl3,
458C, 49%.
Supporting information for this article is available on the WWW
under http://dx.di.org/10.1002/anie.201002290.
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to give 2, which was subsequently transformed into the final
tra clearly indicated that the chemical shifts of the aromatic
hydrogen atoms in trans and cis azobenzenes are distinct.
Before and after illumination at 350 nm, we recorded the
1H NMR spectra of compound 1 in H2O/D2O (1:9; see the
Supporting Information). Irradiation of 1 (trans isomeric
form) for 30 minutes completely shifted the signals for 3-H, 3’-
H, 5-H, 5’-H (azobenzene meta hydrogen atoms) and 2-H, 2’-
H, 6-H, 6’-H (azobenzene ortho hydrogen atoms) from d = 7.0
and 7.7 ppm to d = 6.8 and 6.9 ppm, respectively. The result-
ing chemical shifts correspond to the cis isomeric state of
compound 1. However, when compound 1 was irradiated with
visible light following UV irradiation, the 1H NMR spectra of
1 were not restored to their original state (as observed prior to
irradiation), in agreement with the results of UV/Vis spec-
product by methylation (Scheme 1). Compounds 1 and 2 were
fully characterized by NMR spectroscopy and HRMS meth-
ods (see the Supporting Information).
Initially, the isomerization of compound 1 was investi-
gated by photoillumination. The azobenzene moiety is known
to undergo trans-to-cis isomerization under UV/Vis light.
UV/Vis and 1H NMR spectroscopy were used to characterize
the isomerization of compound 1.
The absorption spectra of the trans and cis azobenzene
were confirmed to be distinct.[17,18] As shown in Figure 1,
photoisomerization depends on the duration of UV/Vis
1
troscopy. According to the H NMR spectra, a mixture of
trans and cis forms was present following irradiation with
visible light, and the efficiency of photoconversion was
approximately 84.6%.
Next, the folding and stretching motions of the G qua-
druplex of the proposed photocontroller were evaluated.
Circular dichroism (CD) is a reliable method for determining
the conversion of the quadruplex into the unfolded confor-
mation. Unfolded telomere DNA (d(TTAGGG)4) appears as
a positive CD peak at 257 nm. In the presence of the trans
form of compound 1, the CD spectrum of d(TTAGGG)4
indicated a parallel G-quadruplex structure, which is charac-
terized by a positive peak at 265 nm and a negative peak at
240 nm (Figure 2a). Upon irradiation with UV light at 350 nm
for 1 minute, formation of the cis form led to the disappear-
ance of the signal at 265 nm. The open and closed forms of the
d(TTAGGG)4 DNA were interconverted by UV/Vis photo-
regulation (Figure 2b). Figure 2c shows the spectra of d-
(TTAGGG)4 throughout 10 continuous cycles of CD during
UV/Vis photoillumination. No changes in absorptivity were
observed for the signal at 265 nm, which suggested that the
exchange process was totally reversible, even after 30 cycles.
Thus, the conformation of the G quadruplex alternated
between the folded and unfolded forms. These results
revealed that the proposed nanodevice converted light
directly into mechanical work.
The results indicated that the folding and stretching
motion of G-quadruplex devices could be induced by UV/Vis
photoillumination (Figure 3). The folding–stretching motion
of telomere DNA was initiated by formation of the G qua-
druplex in the presence of compound 1. Next, the folded
conformation of the G quadruplex was dissociated by irradi-
ation of the solution with UV light at 350 nm for 1 minute.
The stretched oligomer was folded into the G quadruplex by
irradiation with visible light for 1 minute. By irradiating the
solution alternately with UV and visible light, each time for
1 min, the folding and stretching of the DNA could be
repeated (Figure 2d).
Figure 1. UV/Vis spectra of compound 1 (25 mm) in 10 mm Tris/HCl
buffer with EDTA (1 mm) at pH 7.4. a) UV/Vis absorbance of com-
pound 1 under UV irradiation at 350 nm at various reaction times (0–
90 s). b) UV/Vis absorbance of compound 1 prior to irradiation (top
line) and under visible light at various reaction times (0–90 s)
following UV irradiation at 350 nm. EDTA=ethylenediaminetetraacetic
acid, Tris=2-amino-2-hydroxymethylpropane-1,3-diol.
irradiation. Upon the irradiation of a dilute solution of 1
(25 mm in 10 mm Tris/HCl buffer with EDTA (1 mm), pH 7.4)
at 350 nm for approximately 60 s, compound 1 underwent
isomerization to the cis form. However, irradiation with
visible light, did not completely reisomerize the cis to the
trans form, even when irradiation was continued for more
than 30 minutes. The reaction system attained a photosta-
tionary state after irradiation with visible light for about
60 seconds. These results are similar to those of previous
studies[9,17,19] and suggested that repeated photoconversion
between the cis form and the photostationary state could be
induced by irradiating 1 alternately with visible and UV light
(350 nm), each time for 1 minute.
The thermal stability of the telomere DNA (d-
(TTAGGG)4) in the presence of the trans and cis forms of 1
was determined by monitoring the melting point of the
oligomer. The results suggested that the thermal stability of
DNA was dependent on the configuration of compound 1:
further evidence for conformational changes in the G qua-
druplex. In these experiments, the CD signal at 265 nm was
Direct evidence for the cis and trans ligand in the
photostationary state of compound 1 was provided by
1H NMR spectroscopy. In previous studies,[20] 1H NMR spec-
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also monitored. In a solution of trans-1, d(TTAGGG)4
showed a large variation in absorbance over a wide range of
temperatures (Tm = 55.88C). This behavior indicated the
formation of a G-quadruplex secondary structure. In a
solution of the cis form of 1, the melting profile of d-
(TTAGGG)4 resembled that of control DNA and thus
indicated that the G-quadruplex structure had completely
dissociated (data shown in the Supporting Information).
We hypothesized that UV/Vis light could act as a switch
for exonuclease I, which catalyzes hydrolysis reactions, on the
basis of the conversion of a folded G quadruplex into an
unfolded conformation by photoillumination. The exonuclea-
se I hydrolysis assay described by Tan and co-workers was
used to evaluate the stability of the quadruplex in the
presence of compounds known to interact with DNA.[21a]
When the 3’ end of T24G4 was folded into a G quadruplex
in the presence of K+ ions, exonuclease I could not degrade
DNA (Figure 4). Moreover, in the presence of compound 1
Figure 4. Quadruplex formation by T24G4 and resistance to hydrolysis
by exonuclease I. Lane 1: T24G4; lane 2: negative control (without
T24G4); lanes 3–5: T24G4 treated with K+ (100 mm), trans-1
(1.25 mm), and cis-1 (1.25 mm), respectively; lane 6: T24RG4; lane 7:
negative control (without T24RG4); lanes 8–10: T24RG4 treated with
K+ (100 mm), trans-1 (1.25 mm), and cis-1 (1.25 mm), respectively.
and visible light, the reaction was inhibited as a result of G-
quadruplex formation. However, upon irradiation with UV
light at 350 nm, the exonuclease was switched on, and
hydrolysis occurred. The assay was also conducted with
mutant T24RG4, which is not able to form a secondary
structure. In this case, the digestion of DNA was not affected
by irradiation. Overall, our results suggested that the folding
and unfolding of the G quadruplex are regulated by photo-
illumination.
Figure 2. CD spectroscopic studies of telomere DNA in 10 mm Tris/
HCl buffer with EDTA (1 mm) at pH 7.4. a) Titration of trans-1
(r=[compound 1]/[DNA strand]). b) Spectra of telomere DNA in the
absence or presence of compound 1 (r=5) without or with photo-
irradiation. The irradiation time with UV or visible light was 1 min.
c) Reversible stretching and folding motion of the G quadruplex upon
photoirradiation (10 cycles). d) Cycling of the photomediated structural
conversion of telomere DNA by irradiation alternately with UV and
visible light, each time for 1 min, as monitored by the CD intensity at
265 nm.
The binding affinity of the isomers for the quadruplex was
determined through a UV/Vis-absorption titration experi-
ment. The binding data determined by UV titration were
Figure 3. Reversible folding and stretching motion of the G quadruplex was induced by the photoresponsive compound 1: a) the telomere DNA
was induced to form a G quadruplex; b) the folded G quadruplex was disassociated by irradiation with UV light at 350 nm for 1 min (stretching);
c) the stretched oligomer folded into the G quadruplex again upon illumination with visible light for 1 min (folding).
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Communications
1H NMR spectroscopic experiments with the ligands for config-
uration analysis were performed on Varian Mercury 300 spectrom-
eters and carried out at 208C in an H2O/D2O (1:9) mixture containing
Tris/HCl (10 mm) and EDTA (1 mm; pH 7.4). A standard jump–
return pulse sequence was used for water suppression with a
relaxation delay of 2 s. The corresponding changes in the molecular
analyzed quantitatively by using the Scatchard equation[22]
(see the Supporting Information). The results showed that
trans-1 induced significant hypochromicity in the telomere-
DNA titration system, and that the binding constant of trans-1
and the telomere DNA was about (3.9 Æ 0.5) ꢀ 106 mÀ1
.
1
Finally, the trans and cis forms of compound 1 were
optimized on the basis of the PM3 method of Gaussian03. The
primitive molecular-modeling result was used for calculations
with Accelrys Discovery Studio 2.1, which provided more
information on the binding interactions between the isomers
of 1 and telomere DNA (see Figure S4 in the Supporting
Information). The trans isomer displayed a planar azoben-
zene core, which participated in p–p stacking with the
guanine quartets (see Figure S4C in the Supporting Informa-
tion). The positively charged side chains linked to the
azobenzene core displayed a high degree of relaxation and
approached the phosphate backbone. In the cis isomer, the
benzene rings of the azobenzene core formed a v-shaped
groove. This configuration reduced p–p stacking and the
rotary flexibility of the side chain. Thus, the cis isomer and
telomere DNA could not interact sufficiently to effect a
conformational change.
configuration upon photoillumination were determined by H NMR
spectroscopy.
The samples for CD spectroscopy were recorded at 208C with a
quartz cell with an optical path length of 1 mm. All CD spectra were
baseline-corrected for signal contributions of the buffer. Neither the
trans nor the cis form of compound 1 contributed to the CD signal
between 220 and 320 nm under our experimental conditions. For the
melting-temperature experiment, the signal at 265 nm was monitored
between 20 and 908C.
Previously reported conditions were used for the exonuclease I
hydrolysis experiments.[21] A mixture of T24G4 (5’-TAMRA-T24-
(G3T2A)3G3-3’;
0.25 mm)
or
T24RG4
(5’-TAMRA-
T24GTGTGAGTGGAGGTGTGAGGT-3’; 0.25 mm) and compound
1 was heated to 948C and then cooled down. The mixture of DNA and
compound 1 was irradiated with UV light at 350 nm to form the cis
configuration. After irradiation, all procedures were performed in the
dark.
Received: April 18, 2010
Published online: June 22, 2010
In summary, the azobenzene derivative 1 was synthesized,
and its photoisomerization properties were evaluated by UV/
Vis and 1H NMR spectroscopy. CD spectroscopy, thermal
denaturation studies, a UV/Vis-absorption titration experi-
ment, and an exonuclease I hydrolysis assay were performed
to demonstrate that the formation and dissociation of G-
quadruplex DNA could be photoregulated successfully by
compound 1. In agreement with our hypothesis, a trans
azobenzene moiety induced the formation of G quadruplexes,
whereas a cis azobenzene moiety caused dissociation of this
conformation. Compound 1 is the first small molecule that has
been used to induce reversible stretching and folding in a
G quadruplex on the basis of photoirradiation. This nano-
device directly converts light into mechanical work and could
be applied as a nanounit that moves or works by photo-
regulation. Thus, compound 1 may enable the development of
new DNA-nanodevice applications.
Keywords: azobenzene · circular dichroism · DNA ·
G-quadruplexes · photoregulation
.
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