New quinoline-based caging groups synthesized for photo-regulation of aptamer activity

Article abstract of DOI:10.1016/j.jphotochem.2010.02.009

A series of quinoline-based photo-removable protecting groups for photo-regulation of thrombin aptamer (HD1) activity were synthesized with improved caging and uncaging efficiency. Among them, 8-bromo-2-diazomethyl-7-hydroxyquinolinyl (BHQ-diazo, 1) chromophore was found to cage the HD1 with highest caging and restoration efficiency. Moreover, on the basis of the RP-HPLC and SPR analysis, BHQ was demonstrated to regulate HD1s specific affinity to target molecule with 3-fold photolysis sensitivity and about 40% percent higher uncaging efficiency than Bhc (6-bromo-7-hydroxycoumarin-4-ylmethyl) group. It was proposed that the development and use of quinoline derivative may provide a general strategy to photo-regulate oligonucleotide's activity with improved caging and uncaging efficiencies by the convenient non-site-specific caging method.

Full text of DOI:10.1016/j.jphotochem.2010.02.009

Journal of Photochemistry and Photobiology A: Chemistry 211 (2010) 129–134
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
New quinoline-based caging groups synthesized for photo-regulation of
aptamer activity
Yi Ming Lia, Jing Shia,∗, Rong Caia, XiaoYun Chena, Zhao Feng Luob, Qing Xiang Guoa
a Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei 230026, China
b Hefei National Laboratory for Physical Sciences at Microscale and School of Life Science, University of Science and Technology of China, Hefei 230026, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of quinoline-based photo-removable protecting groups for photo-regulation of thrombin
aptamer (HD1) activity were synthesized with improved caging and uncaging efficiency. Among them,
8-bromo-2-diazomethyl-7-hydroxyquinolinyl (BHQ-diazo, 1) chromophore was found to cage the HD1
with highest caging and restoration efficiency. Moreover, on the basis of the RP-HPLC and SPR analy-
sis, BHQ was demonstrated to regulate HD1s specific affinity to target molecule with 3-fold photolysis
sensitivity and about 40% percent higher uncaging efficiency than Bhc (6-bromo-7-hydroxycoumarin-
4-ylmethyl) group. It was proposed that the development and use of quinoline derivative may provide
a general strategy to photo-regulate oligonucleotide’s activity with improved caging and uncaging effi-
ciencies by the convenient non-site-specific caging method.
Received 7 November 2009
Received in revised form 6 January 2010
Accepted 12 February 2010
Available online 21 February 2010
Keywords:
Quinoline-based caging group
Aptamer
Photo-regulation
© 2010 Elsevier B.V. All rights reserved.
Photolysis sensitivity
Caging (uncaging) efficiency
Site-specific caging
1. Introduction
ciencies could be realized. Unfortunately, most single photoactive
groups proved to be too stable, requiring longer irradiation time
Photochemical approaches for controlling bio-molecule func-
tions have become increasingly important in elucidating complex
biological processes [1–5]. Generally, molecules were encapsulated
in an inactive form by being caged and subsequently, restored to
display biological activity with irradiation. Recently, many stud-
ies on “up- and down-photoregulating” DNA and RNA functions,
including DNA hybridization [6,7], polymerase [8–10], RNase H
activity [11,12], RNA interference [13–17], siRNA [16,18] with
exact spatiotemporal resolution, have already been carried out.
In 1999, Monroe et al. [19] provided the first example of target-
ing gene expression with light in a biological system by none
site-specific (multiple) caging strategy using DMNPE. Ando et al.
[20,21] applied the similar strategy to control gene expression
in zebrafish embryos by Bhc protecting group and 23% activity
was restored after 20 s irradiation with high intensity UV light.
Recently, Monroe [6,15] further accomplished photoregulation of
DNA hybridization and RNA interference in vitro and in vivo with
a better uncaging efficiency. Simultaneously, site-specific caging
strategy was developed and performed by Heckel [2,7,16,22,23],
Dmochowski [1,8,11,24–28], Deiters [9,29–33], Chen [34] groups
by using a single photoactive group. Through complicated and
time-consuming caging processes, higher caging and uncaging effi-
(at least 5 min) for releasing the compounds, which should be the
primary obstacle to this method [35].
Photo-regulation of aptamer (HD1) activity was firstly pro-
cessed by Heckel and Mayer using site-specific method [23]. HD1,
derived from an evolution process called SELEX (Systematic Evolu-
tion of Ligands by Exponential enrichment), shows a high affinity
and specific binding to target molecules and inhibits their biological
functions. These features make HD1 a promising probe for molec-
ular recognition as well as diagnostic and therapeutic applications
[36,37]. In the previous work, thrombin HD1, a 15mer ssDNA
molecule (5ꢀ-GGTTGGTGTGGTTGG-3ꢀ), was caged by one-photon
compound o-nitrophenylpropyl (NPP) or o-nitrophenylethyl (NPE)
by solid-phase approach [23]. This site-specific caging method
gains control over the spatially and temporally high resolution
availability of the HD1s function. However, due to the drawback
described above, the excessively longer irradiation time (25 min)
was required for activity restoration. Besides, this strategy is
limited by complicated processes and unavailable for potential
two-photon uncaging.
Recently, our group developed a method to photo-regulate
thrombin HD1 activity by Bhc caging strategy. Through co-
incubation of Bhc-diazo with HD1 within 2 h, the specific activity of
HD1 was successfully blocked and the effect was partially restored
with subsequent 1 min illumination [38]. However, insufficient
uncaging efficiency (less than 60%) was observed presumably due
to various negative properties of Bhc group and therefore, became
∗
Corresponding author. Tel.: +86 551 3607476; fax: +86 551 3606689.
E-mail address: shijing@ustc.edu.cn (J. Shi).
1010-6030/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotochem.2010.02.009

130
Y.M. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 211 (2010) 129–134
Fig. 1. The structures of compounds 1–5 (a) and the synthesis procedures of compound 1 (b).
the primary obstacle to biological studies. As Dmochowski group
reported, this incomplete uncaging was caused by the difficulty of
removing most of the caging group with non-toxic exposures to
UV light [35]. To solve these problems, we prefer to develop some
modified compounds with less reactive equivalency and better
photolysis sensitivity for improvements of blockade and restora-
tion efficiency [15,38]. Among previously reported groups for
none site-specific caging strategy, 8-bromo-7-hydroxyquinolinyl
chromophore (BHQ), which was firstly synthesized by Dore and
co-workers for photo-releasing acetate, phosphates, and diols-
functional groups [39–41], was found to have a larger two-photon
across section, a higher photolysis sensitivity, and an improved
water solubility.
In the present study, we synthesize the diazomethane form of
BHQ (BHQ-diazo) to cage with thrombin HD1 and further photo-
regulate its activity. Moreover, through changing the position of
bromine and hydroxyl groups, a series of quinoline-based deriva-
tives were developed for the purpose of selecting the higher pho-
tolysis sensitivity compounds for multiple caging with nucleic acid.
2.2. Synthesis
2.2.1. The synthesis of BHQ-diazo (1)
On the basis of above proposal, we synthesized compound 1–5.
To provide an example of synthetic procedures, BHQ-diazo (1)
was prepared from 7-Hydroxyquinaldine and described as follows
(Fig. 1). Compounds 2–5 were synthesized by using similar syn-
thetic procedures.
8-Bromo-2-methylquinolin-7-ol (6): The solution of bromine
(6 mmol) in the glacial acetic acid (20%) was added to the solu-
tion of the 7-hydroxyquinaldine (5.5 mmol) in the glacial acetic acid
(15 mL) dropwise with vigorous stirring. The precipitate appeared
during reaction. The mixture was stirred for 8 h, then mixture was
diluted with CHCl3 and washed with saturated NaHCO3, water, and
brine. The organic layer was dried with Na2SO4 and evaporated
which was purified by silica gel with EtOAc/petroleum ether (1:4)
to give product 6 (78% yield).
8-Bromo-2-methylquinolin-7-yl acetate (7): The compound
6 (4 mmol) and DMAP (0.4 mmol) were dissolved in pyridine
(20 mL) under argon atmosphere. Followed by the acetic anhy-
dride (6.0 mmol) slowly added to the reaction mixture, the reaction
was stirred for 4 h at room temperature. The mixture was diluted
with CHCl3, washed by 15% citric acid, water, and brine, and the
organic layer was dried over Na2SO4. The solvent was removed by
rotary evaporation, and the crude product was purified over silica
gel using EtOAc/petroleum ether (1:2) to give product 7 (83% yield).
8-Bromo-2-formylquinolin-7-yl acetate (8): The selenium diox-
ide (2.04 mmol) was dissolved in 1,4-dioxane (4 mL), and a solution
of compound 7 (2.0 mmol) in 1,4-dioxane (2.5 mL) was added to the
mixture at 60 ◦C under argon. The temperature was allowed to rise
to 80 ◦C, and the mixture was stirred for 8 h. The precipitate was
removed by filtration, and the filtrate was evaporated to give the
crude product which was purified by flash column chromatography
(EtOAc/petroleum ether; 1:4) to gave 8 (78% yield).
2. Experiments
2.1. Chemistry and instrumentation
All solvents used for reactions were purchased as anhydrous
grade from Fluka and were used without further processing.
Solvents for extractions, column chromatography, and TLC were
commercial grade and distilled before use. TLC was performed on
Merck TLC aluminum sheets (silica gel 60 F254). Spots were visu-
alized with UV light or through staining with phosphomolybdic
acid or KMnO4. Flash column chromatography (FC) was performed
using Fluka silica gel 60 for preparative column chromatography
(40–63 mm), unless specifically noted otherwise. Proton NMR
spectra were obtained on a Varian Unity 400 (400 MHz) spec-
trometer with use of CDCl3 as solvent and tetramethylsilane as
an internal standard. Carbon-13NMR spectra were performed on
a Varian Unity 400 (100 MHz) spectrometer with use of CDCl3 as
solvent. Carbon-13 chemical shifts are referenced to the centre of
the CDCl3 triplet (d 77.0 ppm).
8-Bromo-2-((2-tosylhydrazono)methyl)quinolin-7-yl acetate
(9): The compound 8 (1 mmol) was dissolved in EtOH (6 mL). p-
Toluenesulfonyl hydrazine (1.06 mmol) was added to the mixture.
The reaction was stirred for 19 h at 40 ◦C. The precipitate was

Y.M. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 211 (2010) 129–134
131
collected by filtration, washed with EtOH, and dried under vacuum
to yield 9 (76% yield).
mine the spectral changes following photoactivation, some of the
light-activated HD1 products were further purified with the Micro-
con YM-3 filters following light exposure to remove the released
compound, and then scanned as described. These samples are men-
tioned as purified light-activated HD1.
8-Bromo-2-(diazomethyl)quinolin-7-ol (1): The compound 9
(3.0 mmol) was dissolved in MeOH (7 mL) at room temperature.
Et3N (6.0 mmol) was then added to the suspension. The reaction
was stirred for 2 days. Then the precipitate was collected by filtra-
tion, washed with CH3OH, and dried under vacuum to yield 1 (66%
yield).
2.2.5. Gel electrophoresis of caged HD1
Three species of HD1 (250 ng) were run in a 12% polyacrylamide-
urea denaturing gel in Tris-borate (TBE) buffer (100 mM tris-borate,
2 mM EDTA pH 8.5) at 80 V for 100 min. Gels were stained after
electrophoresis with 1× Gel-red nucleic acid gel stain (Molecular
Probes) in NaCl buffer (100 mM) for 8–10 min.
2.2.2. The analytical data of compounds 1–5
The 1H NMR (400 MHz, DMSO, ı ppm) of 1: 11.33 (s, 1H), 8.27
(s, 1H), 7.93 (d, J = 8.7, 1H), 7.68 (q, J = 9.2, 2H), 7.35 (d, J = 8.7, 1H);
The 13C NMR (100 MHz, DMSO, ␦ppm) of 1: 157.2, 133.6, 131.8,
129.8, 127.7, 126.3, 119.3, 115.9, 112.45, 96.8. HRMS calcd for
2.2.6. Determination of photolysis efficiency
10H6N3O79Br: 262.9694; found: 262.9702.
RP-HPLC was used to evaluate the control, caged, and light-
activated HD1. Each sample type was injected (1.25 mg, 25 mL) in
HPLC-grade water into a C18 analytical column, ODS2 spherisorb
5 ␮m (Waters Corporation, Milford, MA). Separation was obtained
using a gradient of 1% TEAA in HPLC water to 1% TEAA in 40% ace-
tonitrile over 15 min.
C
The 1H NMR (400 MHz, DMSO, ␦ppm) of 2: 10.74 (s, 1H), 8.24
(s, 1H), 8.01 (d, J = 2.0, 1H), 7.94 (d, J = 8.7, 1H), 7.70 (d, J = 9.2, 1H),
7.60 (d, J = 9.2, 1H), 7.19 (dd, J = 8.7, 2.0, 1H); The 13C NMR (100 MHz,
DMSO, ␦ppm) of 2: 159.6, 132.4, 132.0, 130.9, 127.2, 126.9, 117.3,
116.5, 111.4, 100.2. HRMS calcd for C10H7N3O: 185.0589; found:
185.0586.
The 1H NMR (400 MHz, DMSO, ␦ppm) of 3: 10.16 (s, 1H), 8.52
(d, J = 8.8, 1H), 8.23 (s, 1H), 7.75 (d, J = 9.3, 1H), 7.66 (d, J = 9.3, 1H),
7.33 (dt, J = 8.8, 2.5, 2H); The 13C NMR (100 MHz, DMSO, ␦ppm) of 3:
156.5, 130.8, 127.6, 126.5, 125.5, 124.7, 119.7, 117.0, 115.4, 112.4.
HRMS calcd for C10H7N3O: 185.0589; found: 185.0596.
The 1H NMR (400 MHz, DMSO, ␦ppm) of 4: 11.02 (s, 1H), 8.59 (d,
J = 9.1, 1H), 8.31 (s, 1H), 7.90 (s, 2H), 7.51 (d, J = 9.1, 1H); The 13C NMR
(100 MHz, DMSO, ␦ppm) of 4: 153.8, 130.6, 127.9, 125.4, 125.0,
124.3, 119.4, 117.1, 116.3, 107.5. HRMS calcd for C10H6N3O79Br:
262.9694; found: 262.9701.
2.2.7. Study the specific affinity of HD1 by SPR assay
SPR sensorgrams measurements were carried out at 25 ◦C using
BIAcore 3000 (GE Healthcare). Before immobilization, PBS was used
as the running buffer. The carboxyl groups on the chip surface were
activated by injecting a freshly prepared solution of 50 mM NHS
and 200 mM EDC in distilled water for 20 min. Then 0.2 M ethy-
lene diamine in 50 mM sodium borate buffer (pH 8.5) was injected
for 20 min to react with the activated chip surface. The remaining
active groups on the chip surface were then deactivated by injecting
1 M ethanolamine hydrochloride solution in distilled water (pH 8.5)
for 10 min. Subsequently, thrombin was diluted with PBS (PH 7.4)
to 25 ng/␮l and immobilized onto the CM5 sensor chip (GE Health-
care). In each measurement, control, caged or light-activated HD1
was passed through sensor chip for 2 min at 10 ␮l/min to test the
response units (RU). All experiments were performed at least in
triplicate.
The 1H NMR (400 MHz, DMSO, ␦ ppm) of 5: 11.56 (s, 1H), 8.52
(s, 1H), 8.23 (s, 1H), 8.03 (d, J = 9.6, 1H), 7.90 (d, J = 9.6, 1H); The
13C NMR (100 MHz, DMSO, ı ppm) of 5: 168.8, 134.4, 133.2, 128.0,
127.2, 124.8, 120.3, 119.7, 118.0. HRMS calcd for C10H6N3O79Br2:
340.8799; found: 340.8797.
The NMR spectrums of compounds 1–5 were shown in e-
component.
3. Results and discussion
2.2.3. HD1 caging with compound
Thrombin HD1 (5ꢀ-GGTTGGTGTGGTTGG-3ꢀ) was synthesized as
previously described [23]. A 5 ␮l volume of the 1–5 (25 ␮g/␮l
in DMSO) solution was mixed with 5 ␮l HD1 (1 ␮g/␮l in 4:1
DMSO–aqueous) and the solution was incubated for 6–8 h at room
temperature respectively. To remove excess caging compound,
buffer and organic solvent from the reaction, the mixture was
purified using Microcon YM-3 (3000 MW cutoff) centrifugal fil-
ters (Millipore, Bilerica, MA). The solution (10 ␮l) was diluted with
DMSO (240 ␮l) and water to achieve a 1:1 DMSO–aqueous solu-
tion and spun at 12,000 × g for 100 min. Then the caged HD1 was
re-suspended with aqueous solutions (4× 500 ␮l) and spun at
12,000 × g for another 200 min. All products were stored at 4 ◦C
and protected from light.
3.1. Spectrophotometer and HPLC analysis of compounds (1–5)
caging with HD1
We execute multiple-caging strategy to cage with thrombin
HD1 by using compounds (1–5) as previously described [20]. Each
caging group was co-incubated with HD1 within 6–8 h respec-
tively and then purified using Microcon YM-3 (3000 MW cutoff)
centrifugal filters [6,15,38]. As shown in Fig. 2a, the spectropho-
tometric analysis exhibited two characteristic absorbance peaks
of BHQ-HD1 and 7-HQ-HD1 in addition to main peak at 260 nm,
whereas others showed none of the additional peaks. In general,
the excess unattached compounds have been filtered and the char-
acteristic absorbance peaks should attribute to the attachment of
compounds. To further determine whether HD1 could be actu-
ally caged by BHQ-diazo and other compounds, reversed phase
high performance liquid chromatography (RP-HPLC) method was
performed. One part of BHQ-HD1 retained longer than their con-
trol counterparts, suggesting the binding of BHQ group with HD1.
We consider that another elution profile similar as control HD1
was possibly caused by a heterogeneous product mixture of var-
ious degrees of caging. However, the elution peak of 6-HQ-HD1,
7-HQ-HD1 and 5-BHQ-HD1 only have a shoulder which probably
means less conjugates between HD1 and caging group. 6-DBHQ-
HD1 showed only single elution profile similar with control HD1
and therefore indicated the less reactive efficiency of these BHQ-
diazo derivatives which alter the position of substituent to cage
2.2.4. Light-activation and spectrophotometric analysis of caged
HD1
Absorption spectrophotometric analysis of caged species was
used to estimate the degree of caging. Native (noncaged) and caged
HD1 were dissolved in aqueous in separate cuvettes and scanned
for absorbance from 240 to 500 nm using a spectrophotometer (U-
2810; Hitachi). Light-activated HD1 was exposure to ultraviolet
light by illumination with the 100-W mercury lamp of a micro-
scope (Carl Zeiss). The intensity of the light is nearly 10% of the
maximum intensity of mercury lamp and the does is approximately
100 mJ/cm2. The selected spectrum of the light is 365 5 nm and
scans with similar methods as described above [20,42]. To deter-

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Y.M. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 211 (2010) 129–134
Fig. 2. Spectrophotometric (a) and RP-HPLC (b) analysis of compounds 1–5 caging with HD1.
with HD1 (Fig. 2b). Thus we considered that the position of bromine
and hydroxyl on quinoline-based may play a key role in caging reac-
tion. Whatever swapping the bromine substituent for other groups
or exchanging the position of bromine or hydroxyl, the caging group
was hardly to form a covalent bond with the phosphate moiety of
the sugar-phosphate backbone of nucleic acid. Moreover, it should
presume that the additional absorbance peak of 7-HQ-HD1 was
caused by the higher self-absorbance of remainder unattached 7-
HQ. After further purified by centrifugal filters, the additional peak
subsequently disappeared (data not shown).
HD1. Likewise, RP-HPLC results indicate that upon exposure to
light, the elution profile of the light-activated species resembled
the elution profile of control HD1, suggesting the removal of the
caging groups (Fig. 3b). The result of denaturing gel electrophore-
sis further confirms characteristic changes in mobility and intensity
of band corresponding to the addition and removal of the BHQ
caging groups (Fig. 3c). All the above results clearly demonstrated
that BHQ-diazo could cage with HD1 through forming a covalent
bond with the phosphate moiety of nucleic acid during simply
co-incubation and compounds were subsequently released upon
light-activation (Fig. 4).
3.2. Spectrophotometric, gel electrophoresis and HPLC
confirmation of photo-regulation of BHQ-HD1
3.3. RP-HPLC evaluation of photolysis sensitivity of BHQ-HD1
After confirming HD1 was caged by BHQ-diazo using none
site-specific method, we subsequently examine the photolysis sen-
sitivity of BHQ-HD1. Upon irradiation with 365 nm light, caged HD1
was released more than 90% within 30 s. This result indicates that
the photolysis sensitivity of BHQ-HD1 improved 3-fold than Bhc at
least which should contribute to lower cellular damage (Fig. 5).
Then, we further explore whether irradiation could release
the HD1 from caging group as soon as possible. As shown in
Fig. 3a, following illumination with 365 5 nm ultraviolet light
and purification, the additional peak of BHQ-HD1 disappeared
and the photocleaved product resembled the control (non-caged)
Fig. 3. Spectrophotometric (a) RP-HPLC (b) and gel electrophoresis (c) analysis of caged and light-activated HD1. All samples were processed in parallel. Inset of (a) shows
the attachment of the BHQ to the phosphate backbone of HD1, considered to be the predominant site of caging.

Y.M. Li et al. / Journal of Photochemistry and Photobiology A: Chemistry 211 (2010) 129–134
133
Fig. 4. Reaction of HD1 caged by BHQ-diazo.
related with the specific affinity between HD1 and thrombin pro-
tein. This result clearly demonstrated that illumination ensured
the caged HD1, which was encapsulated into an inactive form and
lost affinity to target molecule, mostly recover to natural state
and might act well to complete its natural function. Compared
with Bhc caging (support information), BHQ exhibited bout 40%
percent higher restoration (uncaging) efficiency. Likewise, the dis-
sociation constants (KD) of natural, caged and light-activated HD1
were performed under different concentrations with a steady state
affinity method, and the result is 3.1 × 10−7 M, 6.3 × 10−6 M and
3.5 × 10−7 M, respectively. We have further optimized the caging
and uncaging efficiency by systematically increasing the reactive
equivalents of BHQ. For this experiment, 1- (25 ␮g/␮l, 5 ␮l), 2-
and 3-folds of BHQ-diazo relative to duplex were performed to
cage target HD1 and result in the best effect on 2 equivalents of
caging compound (support information). Besides, the caging effi-
ciency of 6-HQ, 7-HQ and 5-BHQ analyzed by SPR was less than 35%
which might partly caused by lower reactive efficiency (support
information).
Fig. 5. Time-course of photolysis of BHQ-HD1, Bhc-HD1.
As mentioned in pioneering work, the lower uncaging efficiency
of this none site-specific caging method was due presumably to
the difficulty of removing most of compounds with short irra-
diative period and thereby, acted as the primary obstacle to its
further application [35]. Thus, we consider that the less amount of
BHQ group for caging reaction and its higher photolysis sensitivity
should primarily contribute to the improved uncaging efficiency.
3.4. SPR measurements of caged HD1
Furthermore, the effect of the BHQ caging HD1 on the specific
binding to target molecule was investigated by surface plasmon
resonance (SPR) strategy [43]. We prefer to measure the fluctua-
tion of affinity between HD1 and thrombin protein as our group
previously reported [38]. As shown in Fig. 6, the response unit
(RU) of caged HD1 decreased to 12% of control sample (named
caging efficiency equal to 88%), and restored to more than 85%
with subsequent photoirradiation within 30 s. The nonspecific
binding between other oligonucleotides with thrombin can be
eliminated. It was well known that the response unit is closely
4. Conclusion
In summary, BHQ-diazo (1) could photo-regulate HD1s activ-
ity during concise caging process and transitory photolysis period.
It substantially improves the restoration efficiency and shows 3-
fold photolysis sensitivity than previously reported Bhc group. We
anticipate that the further development of higher photolysis sensi-
tivity compounds for multiple caging with nucleic acid may provide
a general strategy to improve uncaging efficiency and enlarge
the application of this concise caging method to oligonucleotides.
Moreover, better two-photon property of BHQ should contribute
to potential photo-regulation of oligonucleotides’s function under
2PE-mediated (two-photon excitation) with exact spatiotemporal
resolution.
Acknowledgements
This study was supported by the National Natural Science Foun-
dation of China (Grant Nos. 90713009 and 20932006).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jphotochem.2010.02.009.
Fig. 6. SPR analyses of the affinity fluctuating of nature, caged and light-activated
HD1.

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