Photoswitchable molecular glue for DNA

Article abstract of DOI:10.1021/ja074325s

DNA molecular glue is a small synthetic ligand that can adhere two single-stranded DNAs that do not spontaneously hybridize with each other. For reversible control of DNA hybridization by an external light stimulus, we have developed a photoswitchable molecular glue for DNA. The photoswitchable molecular glue, NCDA, consists of two guanine-recognizing naphthyridine moieties connected with a photochromic azobenzene unit. Azobenzene undergoes a reversible cis/trans isomerization by photoirradiation, which changes the relative orientations and positions of the naphthyridine moieties, resulting into photoswitching of NCDA binding to the DNA containing GG-mismatch. NCDA in the cis configuration binds to a GG-mismatch sequence and induces the formation of the DNA duplex. Using the photoswitchable binding property of NCDA, the hybridization event of two natural unmodified DNAs can be reversibly controlled by an external light stimulus. Copyright

Full text of DOI:10.1021/ja074325s

Published on Web 09/12/2007
Photoswitchable Molecular Glue for DNA
Chikara Dohno, Shin-nosuke Uno, and Kazuhiko Nakatani*
Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research (ISIR),
Osaka UniVersity, Ibaraki 567-0047, Japan
Received June 14, 2007; E-mail: nakatani@sanken.osaka-u.ac.jp
Spontaneous hybridization between two complementary DNAs
or RNAs is the unique and indispensable property of nucleic acids
and triggers many biological functions. In addition, the sequence-
specific hybridization of DNA is effectively used in constructing
DNA-based materials and nanoarchitectures.1 To regulate the
diverse functions triggered by DNA hybridization, studies toward
controlling or modulating the DNA hybridization in response to
external stimuli have drawn considerable interest.2-5 Here, we
describe a photoswitchable molecular glue for DNA which ac-
complished the reversible control of hybridization of two natural
unmodified DNAs by external light stimuli.
We have developed a series of synthetic small molecules which
can specifically bind to mismatch-containing DNAs.5-8 Since the
mismatch binding ligands (MBL) increased the apparent thermo-
dynamic stability of the mismatched DNA duplex, MBL can
function as a molecular glue to adhere two single-stranded DNAs
(ssDNA) that do not spontaneously hybridize with each other.5
Figure 1. Illustration of the concept for photoswitching of DNA hybridiza-
tion by NCDA. NCDA in the cis configuration binds selectively to a GG-
mismatch site and induces formation of the DNA duplex. Formation and
dissociation of the cis-NCDA-stabilized dsDNA from two unmodified DNAs
can be reversibly controlled by an external light stimulus.
While the function of MBL in the first generation was the
unidirectional control of DNA hybridization, pseudo-bidirectional
control was achieved by thermally degradable molecular glue that
can drive the DNA hybridization/dehybridization cycle by irrevers-
ible decomposition on heating followed by refilling the molecular
glue.5b
To achieve completely reversible and bidirectional control of
DNA hybridization, we have investigated the incorporation of a
photoresponsive unit into MBL to drive hybridization/dehybrid-
ization cycle by external light stimuli. Naphthyridine carbamate
dimer, which binds to DNA duplex containing a GG-mismatch,5-6
was integrated into photoswitchable molecular glue (NCDA) by
incorporating a photochromic azobenzene between two base-
recognizing naphthyridine heterocycles (Figure 1). We anticipated
that a reversible cis/trans isomerization of azobenzene by photo-
illumination changes the relative orientations and positions of the
naphthyridine moieties, and therefore, results into the switching of
the NCDA ability to adhere two ssDNAs containing the GG-
mismatch (Figure 1).
NCDA was synthesized by reductive amination of 4,4′-diformy-
lazobenzene with naphthyridine carbamate derivative. The azoben-
zene moiety of NCDA exists exclusively in the trans form under
ambient light conditions. Photoirradiation of NCDA in sodium
cacodylate buffer (pH 7.0) at a wavelength of 360 nm for 5 min
produced the photostationary state consisting of a nearly equimolar
mixture of cis- and trans-NCDA. The mixtures were reverted
exclusively to the trans form upon irradiation of visible light of
430 nm (Supporting Information, Figure S1).
Figure 2. (a) Thermal melting profiles of DNA duplex 1‚2 containing GG-
mismatch (4.5 µM) in the presence of NCDA (18.2 µM). The absorbance
at 260 nm was measured in 10 mM Na‚cacodylate buffer (pH 7.0) containing
100 mM NaCl. Key: The plots before irradiation, open circles; plots after
photoirradiation at 360 nm for 5 min, filled circle; subsequent photoirra-
diation at 430 nm for 5 min, open triangle; subsequent photoirradiation at
360 nm for 5 min, filled triangle. (b) CSI-TOF MS of duplex DNA1‚2
(20 µM) in the presence of NCDA (40 µM) before (upper panel) and after
photoirradiation at 360 nm for 5 min (lower panel).
a 2:1 stoichiometry (MBL/DNA).5-6 The Tm of DNA1‚2 was
25.6 °C, and 32.7 °C in the presence of trans-NCDA (Figure 2a,
circle). After irradiation of the solution at 360 nm for 5 min, the
Tm value increased by 15.2 °C (Tm ) 48.0 °C, Figure 2a, filled
circle). The fraction of the cis-isomer in the photoirradiated solution
was 75% (Figure S2). That was higher than the 50% fraction
obtained in the absence of the mismatch DNA. Tm measurements
of DNA having all possible combinations of matched and mis-
matched base pairs showed that the GG mismatch duplex was
specifically stabilized after the photoirradiation of 360 nm light
(Table S1). The Tm of the GG mismatch duplex was found to reduce
to the original level by the second photoirradiation of 430 nm light
(Figure 2a, triangle). All the data suggested that (1) cis-NCDA
stabilized the GG mismatch DNA more strongly than trans-NCDA
The stabilization of mismatch DNA duplexes by NCDA-binding
was evaluated by the melting temperature (Tm) of an 11-mer duplex
DNA 5′-(CTAA CGG AATG)-3′/5′-(CATT CGG TTAG)-3′ con-
taining a GG mismatch in a CGG/CGG sequence (Figure 2a,
DNA1‚2). The CGG/CGG sequence is the most preferential binding
site for the naphthyridine carbamate dimer to form a complex with
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C O M M U N I C A T I O N S
difference image before and after photoirradiation (Figure 3a). The
DNA4 and trans-NCDA were added in the bulk solution and
circulated between a photoreaction cell and a SPR cell equipped
with the DNA3X-immobilized gold surface. Strong SPR signals
due to the hybridization of DNA4 were observed selectively at the
spots of DNA3C having the fully complementary sequence to the
overhang sequence (Figure 3b, state 1). The circulating solution
was irradiated in the photoreaction cell with 360 nm light to
isomerize NCDA from its trans to cis form. Immediately after
photoirradiation, SPR signals started to emerge at the spots of
DNA3G with the intensity of SPR signal being increased as the
irradiation of 360 nm light continued (Figure 3b,c, state 2, X )
G). SPR signals were not detected at the spots of DNA3A and
DNA3T, indicating that hybridization with DNA4 selectively
occurred on DNA3G with the assistance of cis-NCDA. Subsequent
irradiation of the circulating solution with 430 nm light resulted in
the disappearance of SPR signals at the DNA3G spots (state 3).
The decrease of the concentration of cis-NCDA in the bulk solution
by photoisomerization caused the dissociation of cis-NCDA bound
to the GG mismatch, eventually leading to the dehybridization of
DNA4 from DNA3G. These results clearly showed that the
photoisomerization of NCDA in fact controlled the hybridiation
and dehybridization of the DNA duplex containing GG-mismatch
in response to external light stimuli.
Figure 3. SPR difference imaging experiments showing photoswitching
of DNA hybridization by NCDA: (a) Sequences of DNA oligonucleotides
and schematic illustration of the experiments. The probe DNA3X possessing
a terminal sulfhydryl group were immobilized on a gold chip via A15 spacer
sequence. (b) SPR difference images of a DNA3X array. The DNA3X array
(X ) A, C, G, and T) was prepared on a gold chip in the pattern depicted.
Key: state 1, image collected after addition of 52-mer DNA4 (1 µM) and
NCDA (5 µM); state 2, after 360 nm photoirradiation of state 1 for 40
min; state 3, after 430 nm photoirradiation of state 2 for 20 min. (c) Time
course of intensities of the spots shown in panel b. Horizontal arrows above
the plots represent the irradiation periods. DNA4 and NCDA were injected
at 0 and 6 min, respectively.
In conclusion, incorporation of the photochromic azobenzene
linkage into MBL permits reversible control of DNA hybridization
by external light stimulus both in homogeneous solution and on
the gold surface. NCDA is a new class of molecules that functions
as a photoswitchable molecular glue for DNA and will be useful
for controlling the biological functions triggered by DNA hybrid-
ization and reversible construction of DNA-based nanoarchitectures.
and (2) the cis-NCDA-DNA complex was disassembled upon
isomerization of cis-NCDA by 430 nm photoillumination.
The formation of the cis-NCDA-DNA complex was confirmed
by the cold-spray ionization time-of-flight mass spectrometry (CSI-
TOF MS). With trans-NCDA, ions corresponding to the DNA were
the only detectable peaks (Figure 2b, upper panel). Upon photoir-
radiation at 360 nm, the ion peak at m/z 1633.28 corresponding to
[2NCDA+DNA1‚2]5- (calcd: 1632.0) was clearly detected (Figure
2b, lower panel). The 2:1 stoichiometry between cis-NCDA and
the duplex is in good agreement with the previously characterized
complexes of the naphthyridine carbamate dimer binding to the
CGG/CGG sequence.5-7 On the other hand, complexes of trans-
NCDA and DNA were not detected under the conditions. This is
most likely because the folded cis-azobenzene linkage in NCDA
allows two naphthyridine moieties to be placed in the appropriate
positions for the binding, whereas the extended trans-azobenzene
does not.
The reversible control from ssDNA to dsDNA, and vice versa,
by NCDA was further investigated by the surface plasmon
resonance (SPR) imaging with the DNA immobilized on the gold
surface.9 A large Tm difference before and after photoirradiation
that is necessary for spontaneous DNA hybridization/dehybridiza-
tion was attained by incorporating two CGG/CGG sites in 15 mer
DNA (Figure S6). Probe DNA3X 5′-(TAA CXG AAA CXG AAT)-
3′, where X is A, C, G, and T, were immobilized through A15 spacer
on a gold surface for the SPR imaging measurement. The
hybridization of the hairpin DNA4 having an overhang sequence
of 5′-(ATT CGG TAT CGG TTA)-3′ was monitored by the SPR
Acknowledgment. This work was supported by Grant in Aid
for Scientific Research (S) (18105006) for K.N. and Grant in Aid
for Young Scientists (B) (18710187) for C.D. from the Japan
Society for the Promotion of Science. K.N. gratefully acknowledges
the support from the Toray Science Foundation.
Supporting Information Available: Synthetic details of NCDA.
HPLC profiles for isomerization, UV, and CSI-TOF MS analysis. This
material is available free of charge via the Internet at http://pubs.acs.org.
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