Photoregulation of thrombin aptamer activity using Bhc caging strategy

Article abstract of DOI:10.1016/j.bmcl.2009.07.128

Thrombin aptamer was attempted to cage with Bhc (6-bromo-7-hydroxycoumarin-4-ylmethyl) group for controlling its specific affinity to target molecular through photolysis. By multiple-caging strategy, aptamer could be rendered biologically inert and partia

Full text of DOI:10.1016/j.bmcl.2009.07.128

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5368–5371
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Photoregulation of thrombin aptamer activity using Bhc caging strategy
b
b
a
a
b
YiMing Li a, Jing Shi a, , ZhaoFeng Luo , Hao Jiang , XiaoYun Chen , FengLiang Wang , Xu Wu ,
*
QingXiang Guo a,
*
a Department of Chemistry, University of Science and Technology of China, 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:
Thrombin aptamer was attempted to cage with Bhc (6-bromo-7-hydroxycoumarin-4-ylmethyl) group for
controlling its specific affinity to target molecular through photolysis. By multiple-caging strategy, apt-
amer could be rendered biologically inert and partially restored with subsequently illumination. This pro-
vides a convenient method for photoregulating aptamer activity with exact spatiotemporal resolution.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 7 May 2009
Revised 26 July 2009
Accepted 28 July 2009
Available online 6 August 2009
Keywords:
Aptamer
Caged compound
Light-activated
Bhc
Activity regulation
Chemical approaches for regulating nucleic acid activity within
living cells offer exciting possibilities for elucidating complex bio-
logical processes.1,2 The term ‘caging’ refers to installation of a
photoremovable group on a biologically active molecule and encap-
sulates molecule in an inactive form. Irradiation with light removes
the caging group and restores biological activity.3 Monroe and co-
workers firstly demonstrated the photoregulation of GFP expression
in HeLa cells by labeling DNA plasmid with multiple 1-(4,5-dime-
thoxy-2-nitrophenyl)ethyl (DMNPE) groups.4 In other pioneering
works, light-activated (caged) oligonucleotides including DNA
hybridization,5–7 polymerase,8–10 RNase H activity,6,11 RNA interfer-
ence,12,13 as well as gene expression in cells and embryos,3,4,12,14–16
provide unique possibilities for controlling oligonucleotides func-
tion with high spatial and temporal resolution.15,17
Aptamers are small single-stranded nucleic acids that fold into a
well defined three-dimensional structure.18,19 It shows a high
affinity and specificity to target molecules and inhibits their bio-
logical functions. These properties make aptamer very interesting
tool for molecular biology as well as diagnostic and therapeutic
applications.20,21 Thus, the regulation of their specific function
using light is a challenging and significant issue.18 Previous effort
was carried out by post-selective and site-specific caging method.
One or two of the six thymidine residues of thrombin aptamer was
modified with photolabile protecting groups, such as o-nitrophe-
nylpropyl (NPP) or o-nitrophe-nylethyl (NPE), and further synthe-
sized through solid-phase approach.7,18 This strategy gains control
over the spatially and temporally high resolution availability of the
aptamer’s function. However, due to complicated and time-con-
suming processes of site-specific caging nucleotide and solid-phase
synthesis, the use of this technology was limited. Likewise, since
different caging position results in varying de-caging efficiency,
the optimal position might be determined primarily by comparing
the efficiency of each caged nucleotide. Based on this principle, the
more nucleotides that an aptamer includes, the less suitable for the
method applied. Moreover, we assumed that NPP and NPE group
own weak two photon properties and unsuitable for further used
in living cell or tissue. Considering the aforementioned drawbacks,
it is necessary to trace some simple methods to fulfill the task.
It has been demonstrated that the Bhc (6-bromo-7-hydroxy-
coumarin-4-ylmethyl) is an outstanding two photon caged com-
pound.16,24,25 It owns several advantages such as high photolysis
efficiency and biologically useful cross-sections upon two-photon
excitation.22 Comparing with nitrobenzyl- and DMNPE-caged mol-
ecules, Bhc-caged molecules are several-fold more sensitive to illu-
mination with ultraviolet light and ꢀ30-fold more sensitive to
photolysis by pairs of coincident infrared photons. These positive
properties not only contribute to minimize cellular-induced re-
sponses and reduce possibility of photodamage to neighboring
cells, but also permit better three-dimensional spatial localization
and greater penetration into scattering or absorbing tissues.23 In
general, Bhc-diazo (6-bromo-4-diazomethyl-7-hydroxycoumarin)
was used as the caging reagent, which forms a covalent bond with
the phosphate moiety of the sugar-phosphate backbone of nucleic
acid. The whole caging process was completed within 1–2 h at
room temperature just through co-incubation of the caging reagent
* Corresponding authors.
E-mail address: shijing@ustc.edu.cn (J. Shi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.128

YiMing Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5368–5371
5369
80
Aptamer
Caged
Purified caged-light
60
40
20
0
7
8
9
Retention time (minutes)
Figure 3. RP-HPLC elution profiles for caged and light-activated aptamer (deter-
mined by 260 nm UV–vis). Two peaks of the caged sample indicate the aptamer in
various caging degrees.
Figure 1. Bhc bind aptamer and resist affinity to target molecule until released with
light.
with biologically active molecule.16 This approach has been used to
cage mRNA by statistically (multiple) labeling method, thereby
perturbing mRNA structure and controlling induction of gene
expression in zebrafish embryos spatially and temporally.24,25
Thrombin aptamer is a 15mer ssDNA molecule (50-GGTTGGTGTG
GTTGG-30) that shows a high affinity and specificity to R-thrombin,
which has already been well studied.18 Herein, we would like to
try this multiple-caging strategy by using Bhc-diazo for controlling
its activity in exact time and space with illumination (Fig. 1).
Bhc-diazo was synthesized in five steps as previously described
and stored at ꢁ20 °C under dark condition.22 The method of caging
reaction was carried out by co-incubating aptamer and Bhc-diazo
within 2 h and then purified using Microcon YM-3 (3000 MW cutoff)
centrifugal filters.5,16 Firstly, we estimated the caging efficiency of
Bhc-diazo binding with aptamer. The spectrophotometric analysis
exhibited two characteristic absorbance peaks at 333 nm and
383 nm in addition to main peak at 260 nm. The appearance of the
additional peaks was attributed to the attachment of Bhc, as the
Bhcgroupshowsabsorbancepeaksat333 nmand383 nminproton-
ated and ionized states, respectively.16 Following illumination with
365 5 nm ultraviolet lightand purification, the photocleaved prod-
uct resembles the control (non-caged) aptamer (Fig. 2). Measure-
ment of the intensity of these additional peaks and the main peak
for aptamer (260 nm) gives an estimated binding of 6.8 Bhc cage
groups per 15mer aptamer strand on average.16
We subsequently characterized the caged and light-activated
aptamer using reversed phase high performance liquid chromatog-
raphy (RP-HPLC).26 The elution profile of aptamer shown in the
representative chromatogram was determined by standard UV–
vis at 260 nm (the characteristic absorbance of aptamer) (Fig. 3).
Aptamer control samples eluted quickly with a sharp peak elution
profile. One part of caged apatmer gave the peak at 7.5 min, which
retained longer than their control counterparts. Upon exposure to
light, the elution profile of the light-activated species resembled
the elution profile of control aptamer, suggesting the removal of
the caging groups. Because of incomplete caging efficiency of this
method, caged-aptamer showed another elution profile similar
with control sample which indicated some remained unreactive
aptamer. Other two chromatograms determined by 333 nm and
383 nm UV–vis represent the characteristic elution profile of cag-
ing compound (Fig. S1). Likewise, the result of denaturing gel elec-
trophoresis further confirmed characteristic changes in mobility
and intensity of band corresponding to the addition and removal
of the Bhc caging groups (Fig. S2).
Next, we prefer to investigate the effect of the caged and light-
activated aptamers on the specific binding to target molecule.
Therefore, the fluctuation of affinity between aptamer and throm-
bin was measured using surface plasmon resonance (SPR) strat-
egy.27,28 As shown in Figure 4A, the responds unit (RU) of caged
aptamer decreased to 14% of control sample and restored to more
than 60% with subsequent photoirradiation within 30 s. Likewise,
the dissociation constants (KD) of aptamer, caged and light-acti-
vated aptamer were performed under the different concentrations
with a steady state affinity method, and the result is 3.2 ꢂ 10ꢁ7 M,
6.1 ꢂ 10ꢁ6 M, 3.9 ꢂ 10ꢁ7 M, respectively. Furthermore, the nonspe-
cific binding between other oligonucleotides with thrombin can be
eliminated. Above results clearly demonstrated that, comparing
with nonspecific oligonucleotides, illumination ensured the caged
aptamer which was encapsulated into an inactive form and lost
affinity to target molecule most recovery to natural state and
might act well to complete an aptamer’s function. Actually, caged
aptamer still had about 14% affinity toward thrombin, while re-
'
5
O
BASE
OH
O
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
O
P
O
O
O
BASE
Br
O
HO
O
O
O
OH
'
3
Aptamer
Caged aptamer
Caged - light
Purified caged - light
250
300
350
400
450
Wavelengh (nm)
Figure 2. Spectrophotometric analysis of caged and light-activated aptamer. All
samples were processed in parallel. Inset shows the attachment of the Bhc to the
phosphate backbone of aptamer, considered to be the predominant site of caging.

5370
YiMing Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5368–5371
24
Aptamer
Caged
Purified caged-light
Nonspecific ODN
22
20
18
16
14
12
10
8
A
50
40
30
20
10
0
70
60
50
40
30
20
10
0
Aptamer
Caged
light for15s
30s
1min
5min
Purified caged-light
6
0
50 100 150 200 250 300
Photoirradiation time
(
s
)
-10
60
80
100
120
140
Time (s)
-10
80
100
120
140
B
Aptamer
Caged
Purified caged-light
45
40
35
30
25
20
15
10
5
Time (s)
Figure 5. Effect of cumulative photoactive time on affinity restoration of caged
aptamer.
activity recovery. Comparing with 30 min irradiation for DMNPE-
caged oligonucleotides, Bhc is more light-sensitivity and poten-
tially contributes to lower cellular-induced responses. The results
of gel electrophoresis and spectrophotometric analysis indicate
the similar trends (Fig. S3).
In summary, our results demonstrated that the spatial and tem-
poral regulation of bioactive aptamer by multiple attachments of
the photolabile compound Bhc is possible. This non-site specific cag-
ing species resisted the specific affinity of aptamer to target mole-
cule and the effect was partially reversible with the application of
illumination. We have further indicated that this effect can be mod-
ulated by varying the time of photodeprotection. Indeed, comparing
with site-specific caging, this style of inactive reaction leads to
incomplete blockade and restoration of aptamer’s activity.26 How-
ever, the concise caging process and moderate reaction condition
would contribute to expand the application of photoregulating apt-
amer activity. Furthermore, transitory light-active period may lead
to less phototoxicity of biomolecules. Additional potential advan-
tage is more available of Bhc for two-photon uncaging than other
site-specific caging group. This approach could minimizes cellular
damage and increases targeting precision for liberating caged mole-
cule.22,29 In order to maximize caging efficacy, further modifications
might be obtainable with alternative caged compounds such as BHQ,
NHQ, CHQ, and CyHQ that own higher quantum yields and biologi-
cally useful two-photon cross sections.30,31 It should anticipate that
better caging group would result in improvements of blockade and
subsequent restoration efficiency.
0.2
0.4
0.6
0.8
1.0
Conc. of aptamer (uM)
Figure 4. SPR analyses of the affinity fluctuating in the presence and absence of
caged and light-activated aptamers.
stored sample showed just 60% RU compared with control aptamer
after the irradiation. We consider that the remaining affinity was
due to incomplete caging, which was matched with the remained
unreactive aptamer shown in Figure 3. It is supposed that, if the
caging compound doesn’t fully bind with the specific position of
aptamer, which plays key roles for specific affinity to target mole-
cules, the partly remaining affinity is possible. Likewise, we ana-
lyze that nearly 40% loss of restored efficiency might result from
the partial removal of Bhc groups.
It was necessary to further examined whether caging and resto-
ration efficiency should be potential influenced by the change of
concentration. For this experiment, we used each sample from
0.2 to 1
l
M for SPR assay. The results indicated the similar caging
All the results presented here provide a possible and convenient
strategy for regulating aptamer’s function under illumination with
own advantages. This light-controllable method has potential
applications in precisely probing biological systems and eventually
enabling targeted therapeutics in the cellular and organic
environments.32
and restoration efficiency of 0.8 and 1
the affinity decreased to 14.5–14%, and restored to 63–64.7% of
control aptamer. (Fig. 4B). Simultaneously, higher concentration
samples (more than 5 lM) were used but led to invariable results
l
M sample. In maximum,
(data not show). These findings confirmed that the range of con-
centration we chose was suit for distinguishing the fluctuation of
affinity between caged and light-activated samples. To further
determined the proper time of irradiation required for rescuing
the activity of caged aptamer, time course experiments from 15 s
to 5 min were carried out. As shown in Figure 5, the affinity of
caged aptamer restored from 14% to 63% under first 1 min illumi-
nation. In theory, increasing the time of irradiation should lead to a
complete restoration of active aptamer.5 We have found, however,
that prolonged irradiation beyond 1 min barely contributes to far-
ther affinity recovery but a little decrease.26 Therefore, we con-
clude that less than 1 min photoreleased period is available for
Acknowledgements
This study was supported by the National Natural Science Foun-
dation of China (Grant Nos. 90713009, 30727001).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.128.

YiMing Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5368–5371
5371
17. Richards, J. L.; Tang, X. J.; Turetsky, A.; Dmochowski, I. J. Bioorg. Med. Chem. Lett.
2008, 18, 6255.
References and notes
18. Heckel, A.; Mayer, G. J. Am. Chem. Soc. 2005, 127, 822.
19. Mayer, G. Angew. Chem., Int. Ed. 2009, 48, 2672.
20. Tuerk, C.; Gold, L. Science 1990, 249, 505.
21. Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818.
22. Furuta, T.; Takeuchi, H.; Isozaki, M.; Takahashi, Y.; Kanehara, M.; Sugimoto, M.;
Watanabe, T.; Noguchi, K.; Dore, T. M.; Kurahashi, T.; Iwamura, M.; Tsien, R. Y.
ChemBioChem. 2004, 5, 1119.
23. Furuta, T.; Wang, S. S. H.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E.
M.; Denk, W.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1193.
24. Ando, H.; Okamoto, H. Methods Cell Sci. 2003, 25, 25.
25. Ando, H.; Kobayashi, M.; Tsubokawa, T.; Uyemura, K.; Furuta, T.; Okamoto, H.
Dev. Biol. 2005, 287, 456.
26. Blidner, R. A.; Svoboda, K. R.; Hammer, R. P.; Monroe, W. T. Mol. Biosyst. 2008, 4,
431.
27. Homola, J. Anal. Bioanal. Chem. 2003, 377, 528.
28. Boozer, C.; Kim, G.; Cong, S. X.; Guan, H. W.; Londergan, T. Curr. Opin. Biotech.
2006, 17, 400.
1. Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900.
2. Tang, X. J.; Dmochowski, I. J. Mol. Biosyst. 2007, 3, 100.
3. Tang, X. J.; Maegawa, S.; Weinberg, E. S.; Dmochowski, I. J. J. Am. Chem. Soc.
2007, 129, 11000.
4. Monroe, W. T.; McQuain, M. M.; Chang, M. S.; Alexander, J. S.; Haselton, F. R. J.
Biol. Chem. 1999, 274, 20895.
5. Ghosn, B.; Haselton, F. R.; Gee, K. R.; Monroe, W. T. Photochem. Photobiol. 2005,
81, 953.
6. Tang, X. J.; Dmochowski, I. J. Angew. Chem., Int. Ed. 2006, 45, 3523.
7. Krock, L.; Heckel, A. Angew. Chem., Int. Ed. 2005, 44, 471.
8. Young, D. D.; Lusic, H.; Lively, M. O.; Yoder, J. A.; Deiters, A. Chembiochem. 2008,
9, 2937.
9. Asanuma, H.; Tamaru, D.; Yamazawa, A.; Liu, M. Z.; Komiyama, M.
Chembiochem. 2002, 3, 786.
10. Tang, X.; Richards, J. L.; Peritz, A. E.; Dmochowski, I. J. Bioorg. Med. Chem. Lett.
2005, 15, 5303.
11. Matsunaga, D.; Asanuma, H.; Komiyama, M. J. Am. Chem. Soc. 2004, 126, 11452.
12. Shah, S.; Rangarajan, S.; Friedman, S. H. Angew. Chem., Int. Ed. 2005, 44, 1328.
13. Mikat, V.; Heckel, A. RNA 2007, 13, 2341.
14. Shestopalov, I. A.; Sinha, S.; Chen, J. K. Nat. Chem. Biol. 2007, 3, 650.
15. Tang, X.; Swaminathan, J.; Gewirtz, A. M.; Dmochowski, I. J. Nucleic Acids Res.
2008, 36, 559.
29. Piston, D. W. Trends Cell Biol. 1999, 9, 66.
30. Pelliccioli, A. P.; Wirz, J. Photochem. Photobiol. Sci. 2002, 1, 441.
31. Davis, M. J.; Kragor, C. H.; Reddie, K. G.; Wilson, H. C.; Zhu, Y.; Dore, T. M. J. Org.
Chem. 2009, 74, 1721.
32. Casey, J. P.; Blidner, R. A.; Monroe, W. T. Mol. Pharm. 2009, 6, 669.
16. Ando, H.; Furuta, T.; Tsien, R. Y.; Okamoto, H. Nat. Genet. 2001, 28, 317.