Photoregulation of a peptide-RNA interaction on a gold surface

Article abstract of DOI:10.1021/ja071298x

The discovery of riboswitching has accelerated research on the interaction between RNA and small organic compounds. It will be important for biologists to artificially and reversibly control gene expression in vivo through the interaction of RNA and small

Full text of DOI:10.1021/ja071298x

Published on Web 06/20/2007
Photoregulation of a Peptide-RNA Interaction on a Gold Surface
Gosuke Hayashi, Masaki Hagihara, Chikara Dohno, and Kazuhiko Nakatani*
Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research (ISIR),
Osaka UniVersity, Ibaraki 567-0047, Japan
Received February 23, 2007; E-mail: nakatani@sanken.osaka-u.ac.jp
The discovery of riboswitching revealed novel paths for the
regulation of gene expression by the binding of a small molecular
ligand to RNA1 and encouraged the study of molecular switches
to modulate gene expression via the ligand-RNA interaction.2
Toward this end, we need pairwise combinations of a ligand
molecule and its target RNA in which interaction could be
controlled, preferably by external stimuli.3 While the molecular
design of ligands targeting a particular RNA is still a difficult task,
RNA aptamers that bind to the molecule of interest could be
developed by an in vitro selection method.4 We report here the
combination of a photoresponsive peptide and its RNA aptamer
pair as a molecular switching system, which could be turned on
and off by the light-induced structural change of azobenzene
incorporated into the peptide backbone.5-7 The peptide, covalently
immobilized on a gold surface, showed photoresponsive binding
to the target RNA. The method reported here has the potential to
provide a large number of ligand-RNA pairs that are useful for
molecular switching systems.
Figure 1. (a) Structures of the E- and Z-isomers of Lys-Arg-Az-Arg
(KRAzR). Available special orientations for the two guanidinium groups
(shown as the orange circles) were dependent on the configuration of
azobenzene (shown in the blue rectangles). (b) Illustrations of photoregu-
lation of KRAzR binding to its RNA aptamer (shown as a red line). The
dashed lines signify hydrogen bonds to the guanidinium groups and
hydrophobic interactions to the azobenzene chromophore.
The photoresponsive peptide KRAzR used in these studies
consisted of three basic amino acids and a photochromic amino
acid (Az)8 containing the azobenzene chromophore (Figure 1). The
RAzR motif was used with the expectations that (1) the guanidinium
groups in the two arginines may bind to RNA not only through
electrostatic interactions but also by hydrogen bonding to nucleotide
bases, and (2) the Az amino acid inserted between the two arginines
may produce a major effect from the structural change in azo-
benzene on the modulation of the spatial orientation of the two
guanidinium groups. The N-terminal lysine was used to immobilize
the peptide at the ꢀ-amino group to the agarose gel in aptamer
selection and the gold surface in the surface plasmon resonance
(SPR) assay. Photoisomerization of KRAzR effectively proceeded
at 360 nm from E to Z and at 430 nm from Z to E. The Z-isomer
was thermally stable at room temperature in the dark for 10 h but
gradually isomerized to the E-isomer under room light (Figure 2a).
The E/Z ratio of the peptide was determined by the peak area in
HPLC analysis with UV detection at the isosbestic point (282 nm)
in the E-Z isomerization. Under room light, the peptide was a
mixture of 75% E- and 25% Z-isomer (Figure 2b). Photoirradiation
at 360 nm for 5 min led to the photostationary state, where the E/Z
ratio was 5:95. Irradiation of the Z-predominant mixture at 430
nm for 5 min led to an E/Z ratio of 79:21.
Figure 2. Photochromic properties of KRAzR. (a) Photoisomerization of
E to Z by 360 nm irradiation followed by isomerization of Z to E under
room light monitored by UV spectroscopy. Key: (1) KRAzR left under
room light; (2-6) samples left under room light for 0, 15, 30, 45, and 60
min, respectively, after 360 nm irradiation for 5 min. (b) The E/Z ratio
determined by HPLC profiles. Key: (1) KRAzR left under room light; (2)
after 360 nm irradiation for 5 min; (3) subsequent irradiation at 430 nm for
5 min. HPLC detection was obtained at 282 nm.
The RNA aptamers binding to the peptide were obtained by in
vitro selection from the 70 random nucleotides (N70) sequence pool
of RNA (Figure 3a).4 The DNA pools consisting of a forward primer
sequence including T7 promoter and EcoR I restriction site, N70
random sequence, and a reverse primer sequence including the
BamH I site were transcribed to RNA pools, which were applied
to the agarose gel column of KRAzR. The RNA fractions retained
on the peptide column were recovered by competitive elution with
6 mM KRAzR. After eight cycles of the selection, the sequence
of aptamers binding to KRAzR was identified. No particular
Figure 3. (a) The structure of the DNA pool used for in vitro selection.
The gray areas are the sequence for hybridizing the forward (left) and reverse
PCR primers. (b) The N70 sequence of RNA aptamers.
consensus sequences were found for 33 aptamers obtained by the
selection (Table S1). The sequences of representative aptamers are
shown in Figure 3b. The dissociation constants of isolated aptamers
19 (Apt19), 23 (Apt23), and 40 (Apt40) determined by SPR were
0.71, 2.4, and 1.8 µM, respectively. The Kd values of these aptamers
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C O M M U N I C A T I O N S
of analysis (Figure 4d), whereas subsequent photoirradiation of the
same surface with 430 nm light restored the aptamer binding to
the surface. Photoregulation of the binding of KRAzR to Apt23
and Apt40 was also observed. Preliminary SPR imaging analyses
with KRAzR-immobilized gold surface clearly showed that the
association and dissociation of Apt19 to the surface is in fact fully
reversible with response to the photoirradiation (Figure S8). These
results indicate that the binding of KRAzR to its aptamers was
fully responsive to the photoisomerization of the azobenzene
chromophore and was controllable on the gold surface.
We here demonstrated that the in vitro selection toward a
photoresponsive peptide effectively provided the ligand-RNA pair,
in which the binding was controllable by photoirradiation both in
solution and on the gold surface. A more sophisticated approach
in terms of the design of the RNA pools and ligand molecules will
provide molecular systems that are useful for controlling RNA
functions by small molecular ligands with external stimuli.
Acknowledgment. This work was supported by a Grant-in-Aid
for Scientific Research (S) (18105006) for K.N. and by a Grant-
in-Aid for JSPS Fellows (18-3285) for G.H. from Japan Society
for the Promotion of Science (JSPS).
Figure 4. SPR assay for the binding of Apt19 to KRAzR immobilized on
the gold surface. (a) Competitive binding assay of Apt19 (1 µM) with
KRAzR (0, 1, 5, 10, 20, and 50 µM) in 50 mM Tris-HCl (pH 8.0), 250
mM NaCl, and 5 mM MgCl2. (b) As for (a) except KRGGR. (c)
Competitive binding assay of Apt19 (0.5 µM) with response to photo-
irradiation. Key: (1) Apt19 and KRAzR (75% E-isomer) (20 µM); (2)
Apt19 with photoirradiated KRAzR (95% Z-isomer) at 360 nm for 5 min.
(d) Effects of photoirradiation of KRAzR on the gold surface on the binding
of Apt19. Key: (1) gold surface left under room light; (2) the surface after
360 nm irradiation; (3) subsequent irradiation of the surface at 430 nm.
Supporting Information Available: Experimental procedures,
photochemical properties of KRAzR, and the sequence aptamers. This
material is available free of charge via the Internet at http://pubs.acs.org.
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are comparable to that of the reported aptamer for arginine, having
a Kd of 0.33 µM.4c
We then focused our attention on the aptamer binding in response
to the structural change upon photoisomerization. First, the
structure-activity relationship of the peptide on the binding to
Apt19 was investigated by competitive binding assay with the
KRAzR-immobilized SPR sensor. Increasing the concentration of
the peptide (0 to 50 µM) in the solution of Apt19 (1 µM) decreased
the intensity of the SPR signal, indicating that the peptides on the
gold surface and in the solution competitively bound to Apt19
(Figure 4a). Peptides having one to three glycines between two
arginines (KRGR, KRGGR, and KRGGGR) did not compete with
the immobilized KRAzR in the binding to Apt19 (Figure 4b). Both
the Az amino acid and arginine did not bind to Apt19 competitively
on the surface. In marked contrast to the E-isomer (75% E), the
photoirradiated KRAzR at 360 nm for 5 min consisting of 95%
Z-isomer could not competitively bind to Apt19 with the KRAzR-
immobilized surface (Figure 4c). These experiments showed that
the RAzR motif and the E-configuration of the azobenzene moiety
were necessary for the binding of KRAzR to Apt19. The binding
of KRAzR on the gold surface to Apt19 could be modulated by
photoirradiation of the surface.9Upon photoirradiation of the
KRAzR-immobilized surface at 360 nm for 5 min, the binding of
Apt19 to the surface was decreased by more than 90% after 110 s
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