In vitro selection of a photoresponsive RNA aptamer to hemin

Article abstract of DOI:10.1016/j.bmcl.2010.02.109

A photoresponsive RNA aptamer to hemin was selected in vitro from a random sequence library of RNAs with azobenzene residues. The aptamer bound to hemin under visible light irradiation and was released by ultraviolet light.

Full text of DOI:10.1016/j.bmcl.2010.02.109

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2964–2967
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro selection of a photoresponsive RNA aptamer to hemin
*
Mingzhe Liu, Hiroshi Jinmei, Hiroshi Abe, Yoshihiro Ito
Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-Shi, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A photoresponsive RNA aptamer to hemin was selected in vitro from a random sequence library of RNAs
with azobenzene residues. The aptamer bound to hemin under visible light irradiation and was released
by ultraviolet light.
Received 4 December 2009
Revised 24 February 2010
Accepted 27 February 2010
Available online 3 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Aptamer
Photo-responsive
In vitro selection
Azobenzene
Artificial control of dynamic molecular recognition is an impor-
tant and promising research area.1 Light has been used as a domi-
nant external stimulus to control various processes of dynamic
molecular recognition.2,3 For example, ‘photoresponsive crown
ethers’ and ‘light-powered molecular pedal’ were reported as
supramolecular systems.4 On the other hand, photoresponsive bio-
polymers (both DNA and peptide based) were also designed to
photocontrol the interaction among biologically active mole-
cules.5,6 In all of the cases described above, a rigorous molecular
design (commonly termed ‘rational design’) was performed, by
which a dynamic function induced by light was appended to the
molecules. However, when a structurally complicated or unknown
molecule (e.g., protein) was chosen as a guest, the design of the
photoresponsive host molecule was difficult. Moreover, an individ-
ual rational design method is usually required in each case.
Here, we propose another approach to achieve the photocontrol
of dynamic molecular recognition. Our strategy was to introduce
an ‘in vitro selection’ method into this research area. If a designed
component with a photoswitch (e.g., azobenzene or spiropyran)
can be applied to the in vitro selection process, a dynamic library
of molecules that respond to light can be constructed via enzy-
matic polymerization. From this library of constituents, photore-
sponsive host molecules will be selected against various guests
using the same approach. It is known that photoisomerization of
photoswitches can change the structure of nucleic acids.5,7
the last 15 years, the scope of the aptamers has been extended
from natural oligonucleotides to nonnatural oligonucleotides.10
So far, we have isolated several aptamers containing nonnatural
nucleotides using the in vitro selection method.11
Previously, we isolated hemin-binding RNA and DNA aptamers
by in vitro selection and demonstrated that these natural aptamers
also exhibited peroxidase activity by forming a complex with
hemin.12 Thus, as a mimic of peroxidase, the hemin-binding
aptamer can be used as a biocatalyst. On the other hand, the
hemin-binding aptamer is a promising tool for regulating and
understanding cell function, because heme is an essential molecule
that plays critical roles in numerous biological phenomena. There-
fore, in this study, we chose hemin as a target molecule for isolat-
ing a photoresponsive RNA aptamer by in vitro selection.
We synthesized a photoresponsive adenosine triphosphate
(azo-ATP, Fig. 1) and applied this azo-ATP to in vitro selection. A
double-stranded (ds) DNA library (ꢀ1011 molecules) was tran-
scribed into an azobenzene-modified RNA library using azo-ATP
instead of natural ATP. After visible light irradiation, the RNA
O
N
HN
N
N
N
O
P
O-
O
P
O-
O
P
O-
The in vitro selection (SELEX) method was developed in 1990.8
Numerous host molecules (aptamers) against various guest mole-
cules (target molecules), such as enzymes, proteins, and small mol-
ecules, have been developed using this method.9 Furthermore, in
N
N
-O
O
O
O
O
OH OH
azo-ATP
* Corresponding author.
E-mail address: y-ito@riken.jp (Y. Ito).
Figure 1. Chemical structure of the azo-ATP used in the transcription reaction.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.109

M. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2964–2967
2965
Scheme 1. In vitro selection of a photoresponsive RNA aptamer.
library was applied to a hemin-immobilized agarose column.
Bound RNA was eluted using a solution containing hemin, and
RT–PCR was then carried out to obtain a dsDNA library from the
recovered RNA for the next round of selection (Scheme 1).
Azobenzene-modified adenosine triphosphate (azo-ATP) was
synthesized as shown in Scheme S1. We confirmed that azo-ATP
was photoswitchable by analyzing the change of absorption
between 300 and 400 nm derived from azobenzene, either under
visible light irradiation or after UV irradiation (Fig. S1a).
of Fig. 2b). Finally, the maintenance of the sequence during
transcription and reverse-transcription reactions was confirmed
using azo-ATP. An oligoDNA was transcribed in the presence of
azo-ATP and the transcribed moiety (RNA) containing azobenzene
residues was reverse-transcribed to oligoDNA. The final sequence
of oligoDNA was the same as that of the original (Fig. S3).
After confirmation of these fundamental conditions, in vitro
selection was performed using azo-ATP according to Scheme 1.
An enhancement of the binding percentage was observed after five
rounds of selection, and the azobenzene content of the RNA library
became stable (Table S1). Therefore, the bound RNAs in the sixth
and eighth rounds were amplified by RT–PCR, and the resulting
dsDNA libraries were cloned and sequenced, respectively. In con-
trast to the sixth round, only two kinds of full-length sequence
(termed A8-6 and A8-10 RNA, see Table S2) were obtained after
eight rounds of selection.14
Therefore, we investigated the binding properties and photore-
sponsiveness of the A8-6 and A8-10 RNA molecules via the analysis
of spectral changes of the Soret band of hemin (Fig. 3).15 The black
lines show the Soret absorption spectra induced by hemin alone.
The green lines show the Soret absorption spectra when hemin
was added to the solution of RNA after visible light irradiation.
Subsequently, we irradiated the solutions containing RNA and
hemin with UV light. The Soret absorption spectra after UV irradi-
ation are shown as purple lines. In the presence of RNA, the absorp-
tion of the Soret bands increased after visible light irradiation
compared with the case of hemin alone. This hyperchromicity sug-
gests that both the A8-6 and A8-10 RNA molecules bound
hemin,12,16 as hyperchromicity of the Soret band is an indicator
of the hydrophobicity of the hemin environment.17 Interestingly,
UV irradiation of the A8-6 RNA led to a decrease in absorption
(purple line in Fig. 3a). This suggested that hemin was released
from the A8-6 RNA. However, in the case of A8-10 RNA, no signif-
icant decrease in absorption was observed after UV irradiation
Before the in vitro selection, we performed the following exper-
iments. First, isomerization from the cis-form to the trans-form was
investigated. Isomerization was so slow that there was almost no
spectral change over 10 h (Fig. S1b). As the cis-form of azobenzene
conjugated with ATP was thermally stable at 37 °C, it was consid-
ered possible to isolate stable isomers during each round of the
selection process. Next, we investigated whether azo-ATP can
serve as a substrate for T7 RNA polymerase instead of natural
ATP. As shown in Figure S2, azo-ATP can be efficiently incorporated
into RNA by T7 RNA polymerase, although transcriptional effi-
ciency was lower for azo-ATP than for natural ATP. It was also con-
firmed that in the absence of azo-ATP or ATP, transcription stopped
at position A (see Fig. S2), resulting in a truncated RNA. In addition,
it was verified that the azobenzene-tethered RNA can serve as a
template for reverse-transcription.13 We prepared an azoben-
zene-tethered RNA library by transcribing the dsDNA library using
azo-ATP instead of natural ATP, which was followed by gel purifi-
cation (Fig. 2a). The absorption band at 300–400 nm indicated that
azo-ATP was incorporated into the RNA (Fig. 2b). The apparent con-
tent of azobenzene incorporated into the RNA was defined as the
ratio of the absorbance at 335 nm (absorption of the trans-form
of azobenzene) over the absorbance at 260 nm (absorption of the
RNA). The content of azobenzene in the first-round RNA library
was about 18%. The RNA library also exhibited photoresponsive
properties, which depended on the wavelength of the light (inset

2966
M. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2964–2967
a
PCR primer / T7
PCR primer
ssDNAlibrary
5'-TAG-GGA-ATT-CGT-CGA-CGG-ATC-C -N59 -CTG-CAG-GTC-GAC-GCA-TGC-GCC-G-3'
PCR
dsDNA library
RNA library
-N59 -
Transcription withazo-ATPinstead of ATP
5'-G-GGA-AUU-CGT-CGA-CGG-AUC-C -N59 -CUG-CAG-GUC-GAC-GCA-UGC-GCC-G-3'
Random region
b
Figure 2. (a) Schematic illustration of the preparation of the azobenzene-tethered RNA library. (b) UV–vis spectrum of the first-round RNA library after visible light
irradiation. Inset, spectral changes of the RNA library after visible light or UV irradiation.
(purple line in Fig. 3b). Therefore, the A8-6 RNA aptamer was
photoresponsive against hemin. The A8-6 RNA aptamer binds
hemin after visible light irradiation and releases hemin after UV
irradiation.
We investigated the photoresponsiveness of the A8-6 RNA apt-
amer in another experiment. The peroxidase activity of the A8-6
RNA aptamer–hemin complex was investigated after visible and
UV light irradiation ( Fig. 4). Peroxidase activity was evaluated
according to the rate of ABTS oxidation as reported previously.12b
Figure 3. Evaluation of the photoresponsiveness of the A8-6 (a) and A8-10 (b) RNA
aptamers via the analysis of spectral changes at the Soret band of hemin. After
visible light irradiation, hemin was added to the solution of the RNA aptamer and
incubated for 30 min at room temperature (green lines). Subsequently, the solution
was UV irradiated for 3 min (purple lines). Black lines, hemin alone.15
Figure 4. Peroxidase activity of the A8-6 RNA aptamer–hemin complex under
visible light (green line) and UV light irradiation (purple line) conditions. H2O2 and
ABTS were added to the solution containing the A8-6 RNA aptamer and hemin. RNA,
0.25 lM; hemin, 0.25 lM; ABTS, 2.5 mM; H2O2, 0.75 mM.

M. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2964–2967
2967
As shown in Figure 4, the A8-6 RNA aptamer–hemin complex
References and notes
exhibited peroxidase activity after visible and UV light irradiation.
However, the rate of ABTS oxidation after visible light irradiation
(green line in Fig. 4) was faster than that after UV light irradiation
(purple line in Fig. 4). Because the aptamer–hemin complex is the
active catalyst in the peroxidase reaction, this difference in enzyme
activity indicates that the amount of aptamer–hemin complex
present was greater after visible light irradiation than after UV
light irradiation. The result demonstrated that the A8-6 RNA apt-
amer exhibited photoresponsiveness to hemin.
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Acknowledgments
We thank Dr. Yukishige Ito of RIKEN ASI for his support. This
study was financially supported in part by a Grant-in-Aid for Scien-
tific Research (A) (19200041) from the Ministry of Education, Cul-
ture, Sports, Science and Technology (MEXT) of Japan (Y.I.) and the
Special Postdoctoral Researcher Program of RIKEN (M.L.).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.109.