A light-driven supramolecular optical switch

Full text of DOI:10.1002/anie.200903251

Communications
DOI: 10.1002/anie.200903251
DNA Hybridization
A Light-Driven Supramolecular Optical Switch**
Shin-nosuke Uno, Chikara Dohno, Holger Bittermann, Vladimir L. Malinovskii, Robert Hꢀner,*
and Kazuhiko Nakatani*
The potential of DNA for use as a functional material[1–3] in
applications beyond its natural role as carrier and guardian of
genetic information is well known. By forming extended
stacks of aromatic base pairs, nucleic acids adopt well-defined
multidimensional structures.[4–7] Thus, nucleic acids represent
an exceptionally useful and versatile template for the precise
placement of functional auxiliaries.[8–12] The potential of
nucleic acids, combined with well-established DNA chemistry
that permits the facile synthesis of a large range of modified
oligonucleotides, prompted a rapid growth of research in the
area of functionalized DNA. Auxiliary functions, such as
chromophores,[13,14] metal binding ligands,[15–17] and other
groups, have been introduced into DNA either directly by
chemical modification of the nucleotides or by complete
replacement of the natural base pairs by other functional
elements.[8,18] The characteristic chemical and physical prop-
erties of these auxiliaries often exhibit sensitive responses to
hybridization states.
mismatches that are recognized by a photoresponsive ligand
led us to propose a light-driven, DNA-based switching device
(Figure 1a), which reversibly changes the fluorescence of a
pyrene derivative from blue (monomer) to green (excimer)
emission or vice versa by forward and reverse DNA hybrid-
ization controlled by a light-induced structural change of the
mismatch-recognizing azobenzene ligand ndca.
One of our research groups has investigated the structural
and spectrophysical effects of polyaromatic molecules on
DNA by using non-nucleosidic aromatic building blocks as
surrogates for base pairs.[19,20] The chromophoric molecules
assembled within DNA are strongly affected by the helical
structure and the hydrophobic environment of the inner DNA
space, thus resulting in significant changes of the spectro-
scopic properties of the chromophores.[21,22] The other
research group has focused on the reversible control of
hybridization states by external light stimuli.[23] The sponta-
neous and thermodynamically favorable duplex formation
can be reversed by the introduction of mismatches and
addition of a photoresponsive ligand that selectively binds to
the mismatched sites after activation by light. The combina-
tion of pyrene-based, fluorescent base-pair surrogates with
[*] Dr. H. Bittermann, Dr. V. L. Malinovskii, Prof. Dr. R. Hꢀner
Departement fꢁr Chemie und Biochemie, Universitꢀt Bern
Freiestrasse 3, 3012 Bern (Switzerland)
E-mail: robert.haener@ioc.unibe.ch
Homepage: http://www.dcb-server.unibe.ch/groups/haener/
S.-n. Uno, Dr. C. Dohno, Prof. Dr. K. Nakatani
Regulatory Bioorganic Chemistry
The Institute of Scientific and Industrial Research
Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047 (Japan)
E-mail: nakatani@sanken.osaka-u.ac.jp
Figure 1. a) Representation of a light-driven supramolecular optical
switch. The optical input (excitation at 400 nm) is converted to an
optical output (pyrene fluorescence), which can be switched back and
forth between blue (monomer) and green (excimer) fluorescence by
modulating light, thus inducing cis–trans isomerization of the photo-
switchable, mismatch-recognizing ligand ncda. cis-Ncda enables pair-
ing of two mismatched, pyrene-modified single strands. Formation of
a cis-ncda-stabilized duplex leads to excimer fluorescence. b) Pyrene-
modified DNA sequences (py-mis and py-full; W denotes the dipropy-
nylpyrene unit. c) GG-mismatch binding ligands ncda and ncd.
Homepage: http://www.sanken.osaka-u.ac.jp/labs/rbc/
[**] Financial support by the Swiss National Foundation (to R.H., grant
200020-117617) and by the Japan Society for the Promotion of
Science (to K.N., Grant in Aid for Scientific Research (S) 18105006)
is gratefully acknowledged.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200903251.
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Replacement of base pairs by non-nucleosidic dipropynyl
pyrene building blocks (W, Figure 1b) has little influence on
duplex stability (see the Supporting Information). Interstrand
stacking of the pyrene building blocks results in excimer
formation[24,25] with a fluorescence maximum at 520 nm,
whereas the monomer emission maximum is at 430 nm.
Thus, the hybridization of two DNA strands modified with
dipropynyl pyrene building blocks changes the emission from
blue (monomer) to green (excimer). As the hybridization
occurs spontaneously at ambient temperature, it is not
possible to change the emission from green to blue at the
same temperature.
Naphthyridine-derived ligands bind strongly and selec-
tively to distinct mismatches and increase the thermodynamic
stability of mismatch-containing duplexes.[26–29] The ligand ncd
(Figure 1c, right) was previously demonstrated to bind to G–
G mismatches flanked by G–C and C–G base pairs (CGG/
CGG; C = cytosine, G = guanine) with a stoichiometry of
2:1.[30,31] Analysis by NMR spectroscopy showed that a total
of four naphthyridine heterocycles that originate from two
ncd molecules form hydrogen bonds to the four guanine bases
in the CGG/CGG mismatch, and that the widowed cytosine
units are flipped out from the p stack.[32] If two CGG/CGG
sequences exist in proximity in the sequence, the resulting
duplex should be substantially destabilized in the absence of
ncd, thus favoring the single-stranded state if the oligonu-
cleotides are relatively short. In that case, ncd should function
as ꢀmolecular glueꢁ that promotes duplex formation from the
two single strands. Incorporation of a photochromic azoben-
zene moiety allowed the further elaboration of ncd.[23] The
resulting photoswitchable mismatch binding ligand (ncda,
Figure 1c, left) can be reversibly isomerized from trans to cis
or vice versa by photoirradiation. Only cis-ncda can bring two
single-stranded DNA sequences that contain a CGG/CGG
mismatch together.
We designed two oligomer sequences that contain one
pyrene building block in the centre of 14 natural bases. Each
sequence contains two CGG units, one on each side of the
pyrene unit. The duplex that results from the two modified
oligonucleotides (py-mis; see Figure 1b for oligonucleotide
sequences) is expected to have a very low melting temper-
ature (Tm) because of the presence of two GG mismatches.
We anticipated that by changing the cis/trans ratio of ncda by
external light stimuli, the hybridization of py-mis could be
controlled, and, as a result, the emission of py-mis between
monomer and excimer fluorescence could be reversibly
modulated.
Figure 2. Evaluation of ncd binding to py-mis. a) Thermal melting
profiles of py-mis (1.54 mm) in the absence (blue) and presence (red)
of ncd (12.3 mm). The absorbance at 260 nm was measured in 10 mm
sodium cacodylate buffer (pH 7.0) containing 100 mm NaCl. b) CD
spectra of py-mis (1.54 mm) with increasing ncd concentrations (0–8
molar equivalents) in 10 mm sodium cacodylate buffer (pH 7.0) and
100 mm NaCl. c) Fluorescence spectra of py-mis in the temperature
range 13–508C in the absence of ncd (inset: fluorescence spectrum of
py-full at 238C); d) Fluorescence spectra of py-mis with increasing ncd
concentrations (0–8 molar equivalents; 238C).
300 nm grow with increasing ncd from zero to eight molar
equivalents. The signals at 315 and 349 nm are assigned to
DNA-bound ncd, whereas those at 391 and 436 nm originate
from interstrand stacked pyrene molecules. The CD spectrum
of py-full showed no dependence on ncd (see the Supporting
Information), thus confirming that ncd selectively binds to py-
mis and that the pyrene units are optimally arranged in the
formed duplex.
Temperature-dependent fluorescence spectra of py-mis
are shown in Figure 2c. At 138C, and in the absence of ncd,
py-mis exhibits predominantly monomer fluorescence at
430 nm, and weak excimer fluorescence at 520 nm. As the
temperature increases, the excimer fluorescence gradually
decreases further as the fluorescence intensity at 430 nm
increases. At 508C, the excimer fluorescence has completely
disappeared. In contrast, py-full exhibits mainly excimer
fluorescence at 238C. These results show that py-mis pre-
dominantly exists in single-stranded form at ambient temper-
ature.
To elucidate whether the binding of ncd to py-mis drives
excimer formation, the fluorescence spectra of py-mis were
measured in the presence of different concentrations (0–8
equivalents) of the mismatch-binding ligand at 238C (Fig-
ure 2d). Evidently, monomer fluorescence largely decreases
and excimer fluorescence increases on raising the molar ratio
of ncd. In the presence of 8 equivalents of ncd, the excimer
fluorescence is more intense than the monomer fluorescence.
Ncd binding to py-mis results in the emission of green
fluorescence, which is comparable to the fluorescence of py-
We first examined the hybridization modulation of py-mis
by the non-photoresponsive ligand ncd. Thermal denatura-
tion profiles were measured in the absence and presence of
ncd (Figure 2a). The Tm was below 258C in the absence of ncd
and increased by more than 308C in the presence of ncd. The
observed Tm of 548C was comparable to the Tm (54.38C) of
the fully matched DNA duplex (py-full). The presence of ncd
had no influence on the Tm of py-full (see the Supporting
Information). Thus, ncd effectively stabilizes py-mis by
forming an ncd-bound complex at room temperature.
Changes in the CD spectra of py-mis were measured upon
titration with ncd at 238C (Figure 2b). The CD bands above
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full (see the Supporting Information). By taking these data
show pH dependence, trans-ncda is favored under acidic and
cis-ncda under basic conditions. The proportion of cis-ncda
reached a maximum value of 80% at pH 8.0 or higher. After
further optimization, the proportion of cis-ncda was improved
to 63% in the presence of py-mis at the photostationary state
at pH 8.5 by irradiation at 360 nm (Figure 3a bottom and the
Supporting Information).[33]
We subsequently examined the reversible light-driven
switching of the pyrene fluorescence. Fluorescence spectra of
a mixture of the two pyrene-modified oligonucleotides (py-
mis) and ncda were measured before and after irradiation at
360 nm for 10 minutes (Figure 4a). While the monomer
into account, one can conclude that 1) pyrene-modified 15-
mer oligonucleotides with two mismatched CGG sequences
exist in the single-stranded form at ambient temperature;
2) ncd effectively supports the hybridization of two partially
mismatched pyrene-modified single strands; 3) the pyrene
building blocks in ncd-stabilized py-mis form well-ordered
aggregates; and 4) the pyrene fluorescence changes from blue
(monomer) to green (excimer) with increasing ncd concen-
trations.
Encouraged by these results, we examined the light-driven
modulation of the py-mis hybridization state with the photo-
responsive ligand ncda. Preliminary experiments showed that
the photoinduced isomerization from trans- to cis-ncda is
influenced by the presence of DNA as well as the pyrene
chromophore. Initial attempts to isomerize ncda in the
presence of py-mis were unsatisfactory under standard
conditions (100 mm NaCl, pH 7.0; see the Supporting Infor-
mation) and the fraction of cis-ncda did not exceed 26% in
the presence of py-mis, as determined by HPLC analysis
(Figure 3a top and the Supporting Information). Further
attempts revealed that cis-ncda was highly pH-dependent
(Figure 3b). While the control compounds az-OH and az-
cation, which do not have the naphthyridine units, did not
Figure 4. Reversible photoswitching by light-driven photo-isomeriza-
tion of ncda. a) Fluorescence spectra of py-mis in the presence of ncda
after iterative irradiation at 360 nm (10 min) and 430 nm (3 min).
Conditions: 0.4 mm py-mis, 24 mm ncda, 500 mm NaCl, 100 mm Tris-
HCl buffer (pH 8.5; excitation at 400 nm). b) Repetitive fluorescence
changes of py-mis in response to photoirradiation; blue: intensity of
the monomer emission (430 nm); green: intensity of the (green)
emission (520 nm).
emission (430 nm) was predominant before irradiation, the
excimer fluorescence at 520 nm was significantly higher and
the 430 nm emission was lower after irradiation. Subsequent
irradiation with modulating light at 430 nm for 3 minutes to
induce isomerization from cis- to trans-ncda completely
restored the monomer fluorescence. This light-controlled
switching of fluorescence emission, during which the fluores-
cence spectra remained essentially unchanged, was repeat-
able at least five times (Figure 4a,b). Thus, the system
presented here can effectively and reversibly be switched
between two states, each characterized by light emission at a
different wavelength that corresponds to an information
content of 1 bit.
Light-controlled DNA hybridization was previously
addressed by engineering photoresponsive units into
DNA.[34,35] The light-driven optical switch described here
advances these first-generation systems, since the function
that is installed on the DNA and the switch that changes the
DNA hybridization state are distinctly separated from each
other. The switch is based on the on–off hybridization of two
DNA strands in the presence of an effector ligand. This type
of switching module should also be applicable for the
regulation of other auxiliary functions installed on DNA.
Molecular machines based on the hybridization–dehybridiza-
tion event of DNA strands reported to date[36–38] are generally
Figure 3. a) HPLC analyses of the photoisomerization of ncda in the
presence of py-mis. The solution was irradiated at 360 nm for 10 min.
Conditions: (top) 12.3 mm ncda, 0.77 mm py-mis, 100 mm NaCl, 10 mm
sodium cacodylate buffer (pH 7.0); (bottom) 12.3 mm ncda, 0.40 mm
py-mis, 500 mm NaCl, 10 mm tris(hydroxymethyl)aminomethane hy-
drochloride buffer (Tris HCl; pH 8.5). Full HPLC profiles are provided
in the Supporting Information. b) pH dependence of photoisomeriza-
tion of ncda and azobenzene reference compounds (az-cation and az-
OH). Ligands were photoirradiated at 360 nm for 10 min in 20 mm
sodium cacodylate (pH 5.0–7.0) or Tris-HCl buffer (pH 7.5–9.0) prior
to HPLC analysis. Fractions of cis-ncda were determined by integration
of HPLC signals at 260 nm.
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Received: June 16, 2009
Revised: July 21, 2009
Published online: September 2, 2009
Keywords: DNA · fluorescence · nucleobases · photochromism ·
.
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