In vitro selection of a photo-responsive peptide aptamer using ribosome display

Article abstract of DOI:10.1039/c2cc36618e

A photo-responsive peptide aptamer against microbeads immobilized streptavidin was isolated using in vitro selection combined with photo-manipulation. This is the first example of the introduction of a peptide aptamer in the photo-control of dynamic molecular recognition.

Full text of DOI:10.1039/c2cc36618e

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COMMUNICATION
In vitro selection of a photo-responsive peptide aptamer using ribosome
displaywz
Mingzhe Liu,yz Seiichi Tada,z Mika Ito, Hiroshi Abe and Yoshihiro Ito*
Received 12th September 2012, Accepted 16th October 2012
DOI: 10.1039/c2cc36618e
A photo-responsive peptide aptamer against microbeads immo-
bilized streptavidin was isolated using in vitro selection combined
with photo-manipulation. This is the first example of the intro-
duction of a peptide aptamer in the photo-control of dynamic
molecular recognition.
photo-responsive host molecules are selected against a target.
We previously isolated photo-responsive hemin-binding RNA
aptamers by in vitro selection and demonstrated that the
aptamers exhibited photo-responsive peroxidase activity by
forming a complex with hemin.9 However, a peptide-based
photoreactive aptamer has not been developed; although the
greater diversity of polypeptide functional groups when compared
with oligonucleotides may increase the possibility of higher
affinity and specificity. In this communication, we isolated a
photo-responsive peptide aptamer which recognizes a target by
using ribosome display.
Artificial control of dynamic molecular recognition is an
important and promising research area.1 Light has been used
as a dominant external stimulus to control various processes of
dynamic molecular recognition.2 For example, ‘‘photo-
responsive crown ethers’’,3 ‘‘a photo-chemically driven molecular
machine’’4 and ‘‘a light-powered molecular pedal’’5 have been
reported as supramolecular systems. A light-triggered switch has
also been developed in dendritic polymers,6a material surfaces,6b
catalyst systems,6c and bulk material structures.6d In addition,
photo-responsive biopolymers (both DNA and peptide based)
were also designed to photocontrol the interaction between
biologically active molecules.7 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. Recently several researchers
reported peptides or oligonucleotide recognizing photo-responsive
targets.8 However, the design of the photo-responsive
host molecule for arbitrary targets, which is structurally
complicated or unknown, is difficult and was not achieved.
We have proposed an approach to achieve the photocontrol
of dynamic molecular recognition with an arbitrary target by
using an in vitro selection method. In this method, a random
sequence oligomer library containing a photo-isomerizable
molecule (e.g. azobenzene or spiropyran) is prepared and
Phage display has been employed by many researchers in the
selection of a peptide aptamer.10 However, it is impossible to
incorporate non-canonical amino acids into the display system.
In order to incorporate non-canonical amino acids into the selection
system, other selection systems using in vitro translation such as
ribosome-display11 or mRNA-display12 have been developed.
Using the molecular level peptide display, Roberts and
coworkers13 selected biocytin containing peptides against
streptavidin and a hybrid drug-peptide aptamer against S. aureus
penicillin-binding protein PBP2a from a random sequence
polypeptide library that was chemically modified after translation.
Subsequently, it was recently reported that artificial amino acids
carried by tRNA were incorporated into a peptide library using a
cell-free protein synthesis system and peptide aptamers were
selected from the library.14 These methods enable the selection
of peptide aptamers against arbitrary targets from a peptide
library which contains non-canonical amino acids.
Here we used the incorporation of non-canonical amino
acids in ribosome display15 for the preparation of a photo-
responsive peptide aptamer. tRNA carrying an amino acid
coupled with an azobenzene residue was prepared in order to
provide photo-responsiveness to the peptide aptamer. The
selection protocol is schematically shown in Fig. 1 and
explained in detail in the ESI.z First, tBoc-e-(azobenzoyl)-
lysine was synthesized and coupled with 50-O-phosphoryl-20-
deoxycytidylyl-(30-50)adenosine (pdApC). Subsequently, the
e-(azobenzoyl)-lysine–pdApC was ligated with truncated tRNA
carrying the amber anticodon. Similar to the random sequence
library, an amber codon-containing random sequence library of
DNA, (NNN)3TAG(NNN)7 and (NNN)7TAG(NNN)3 where
N = G, C, T, or A, was prepared.
Nano Medical Engineering Laboratory, RIKEN Advanced Science
Institute, 2-1, Hirosawa, Wako-Shi, Saitama 351-0198, Japan.
E-mail: y-ito@riken.jp; Fax: +81 (48)467-9300;
Tel: +81 (48)467-5809
w This article is part of the ‘Nucleic acids: new life, new materials’ web
themed issue.
z Electronic supplementary information (ESI) available: Synthesis of
azobenzene-lysine; preparation of azobenzoyl-lysine-tRNA; in vitro
selection; subcloning and sequencing; primer sequences; chemical
synthesis of the peptide; binding assay. See DOI: 10.1039/c2cc36618e
y Present address: Key Laboratory of Structure-Based Drugs Design &
Discovery of Ministry of Education, School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, Shenyang 110016,
China.
After in vitro transcription of the random sequence DNA
library, a cell-free translation using the PURE SYSTEM in the
z M. L. and S. T. contributed equally.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 11871–11873 11871

recovered mRNA. The DNA was employed for the next step
of the selection process.
After five rounds of these selection processes, the selected
sequence was cloned and the sequences were analyzed. The
sequences are listed in Table S1, ESI.z Seventy-seven kinds of
sequences were found from 81 clones. Two sequences VLIM-
VAVXASS (LA37, 48 and 91) and HSCXVTIDVFF (LA40,
43 and 93) were derived from three clones. In addition, there
are five sequences containing two azobenzene-containing
lysine residues. Here, two peptides derived from plural clones
and one peptide containing two azobenzene groups were
chosen, and these peptides were chemically synthesized by a
solid phase method (see ESIz). The former was considered to
be major population of the selected peptides. The latter was
considered to be more photo-responsive, because two photo-
isomerizable moieties were present. Taking into consideration
the low incorporation efficiency of non-canonical amino acids,
the two incorporation sites-containing sequence was considered
to be a high-binding candidate. In addition, the sequence was
considered to be water-soluble because it had plural arginine
residues which contribute to water-solubility.
Fig. 1 The principle of in vitro selection using ribosome display and
misacylated tRNA. A DNA library was transcribed into an mRNA
library. The mRNA library and azo-Lys-tRNA (tRNA carrying
azobenzene-coupled lysine) were then introduced into a cell-free
translation system, resulting in the production of an azobenzene-
modified peptide displayed on the surface of the ribosome. After
affinity selection, the bound peptides were eluted by UV irradiation.
The eluted complexes were collected and subsequently dissociated. The
mRNA that encodes the peptide sequence was recovered and used for
the next round of selection after RT-PCR.
Peptides containing the sequence of LA37, LA40 and LA81
with DYKDDDDKA (FLAG sequence) were synthesized
(Table 1) and the binding behavior onto the microbeads was
assayed by staining with a fluorescein isothiocyanate (FITC)-
labeled anti-FLAG-antibody (Fig. 2). Higher binding affinity
was observed for LA81 than found for LA37 or LA40, as
shown in Fig. 2a. The dissociation constant (Kd) of the LA81
peptide bound to the microbead was calculated to be 6.31 mM
by curve-fitting to a Langmuir isotherm. In addition, LA81
adsorbed onto the microbead under visible light irradiation
but this adsorption was significantly reduced by UV irradiation
(Fig. 2b). In contrast, a scrambled sequence peptide (control)
did not bind and showed no photo-responsiveness.
presence of the tRNA carrying azobenzene-coupled lysine was
performed to prepare the azobenzene-containing peptide library.
The translation yield was about 10-fold lower due to the
addition of the tRNA. The library forms a complex with each
coding mRNA and ribosome by losing the stop codon and
processing at low temperature, which is called the ribosome
display system.10 The library was incubated with magnetic
microbeads immobilized with streptavidin for 30 min under
dark conditions, in which almost azobenzene took trans-form,
and the microbeads were washed with the same buffer. Sub-
sequently, the beads were irradiated with ultraviolet light for
10 min. The irradiation of ultraviolet light was carried out by
using a UV spot light instrument (from Hamamatsu Photonics)
with a UV-D36B filter (from Asahi Technoglass). The intensity
of the UV light was 6 mW cmꢀ2. In this step, peptides with cis-
form azobenzenes can be released from beads, since conformational
changes of the peptides by the trans–cis isomerization
of azobenzenes are accompanied by the changes in affinity
to streptavidin. Thus, the irradiated solution was recovered
and the complex was dissociated by the addition of ethylene-
diaminetetraacetic acid. The recovered mRNA was collected
and a reverse transcription (RT)-polymerase chain reaction
(PCR) was performed for the amplification of DNA from the
The conformational change of LA81 in response to photo-
irradiation was observed by ultraviolet (UV) and circular
dichroism (CD) measurements (Fig. 3). The spectral change
of LA81 (Fig. 3a) was similar to that of the monomer that
is tBoc-e-(azobenzoyl)-lysine (Fig. S1, ESIz), whereas the
efficacy of trans–cis isomerization is significantly different from
each other. About 70% of trans-azobenzenes were isomerized
to cis-form in the case of tBoc-e-(azobenzoyl)-lysine, but only
Table 1 Peptides synthesized by the solid phase method
Abbreviation
Sequencea
Fig. 2 Binding experiments on selected peptides. (a) Binding of
peptides containing LA37, LA40 and LA81 sequences. The incubation
was carried out at 20 1C for 1 h in TBS-T buffer (20 mM Tris, 137 mM
NaCl, 0.1% Tween 20, pH 7.6). The bound microbead was stained
with an FITC-labeled anti-FLAG-antibody and then quantified by a
microplate reader. (b) Photo-responsive binding of peptides containing
LA81 or a scrambled sequence. White bar: under the visible light
irradiation conditions; grey bar: under the UV irradiation conditions.
LA37
LA40
LA81
QAVLIMVAVXASSGQLGQFEGSDYKDDDDK
QAHSCXVTIDVFFGQLGQFEGSDYKDDDDK
QAGVTXRRFIXYVGQLGQFEGSDYKDDDDK
Scramble peptide CYSCGSQVAFRLSLFGCSFGGDYKDDDDK
a
Bold letters indicate peptide sequences selected from the library.
Underlined letters indicate FLAG sequences. X indicates Lys residues
coupled with azobenzene.
The intensity of the UV light was 6 mW cmꢀ2
.
c
11872 Chem. Commun., 2012, 48, 11871–11873
This journal is The Royal Society of Chemistry 2012

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This is the first example of the introduction of a peptide
aptamer in the photo-control of dynamic molecular recogni-
tion. The application of the strategy proposed in this study
renders in vitro selection of photo-responsive host molecules
against various guests as promising. The photo-responsive
peptide sequence will be available as a protein-tag for the
purification of biologically significant protein. Moreover, the
photo-responsive peptide that binds nanomaterials such as carbon
nanotubes could also be applicable in particular photo-switching
nanodevices.
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Chem. Commun., 2012, 48, 11871–11873 11873