Nanosculpting reversed wavelength sensitivity
into a photoswitchable iGluR
Rika Numanoa,b,1,2, Stephanie Szobotaa,c,1, Albert Y. Laud, Pau Gorostizaa,3, Matthew Volgrafe, Benoit Rouxd,
Dirk Traunere,4,5, and Ehud Y. Isacoffa,f,5
Departments ofaMolecular and Cell Biology andeChemistry andcBiophysics Graduate Program, University of California, Berkeley, CA 94720;bLaboratory
Animal Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan;dDepartment of
Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637; andfDivisions of Material and Physical Bioscience, Lawrence Berkeley National
Laboratory, Berkeley, CA 94720
Edited by Lily Y. Jan, University of California School of Medicine, San Francisco, CA, and approved February 24, 2009 (received for review
November 25, 2008)
Photoswitched tethered ligands (PTLs) can be used to remotely
control protein function with light. We have studied the geometric
and conformational factors that determine the efficacy of PTL
gating in the ionotropic glutamate receptor iGluR6 using a family
of photoiosomerizable MAG (maleimide-azobenzene-glutamate)
Experiments and molecular dynamics simulations of the modified
proteins show that optical switching depends on 2 factors: (i) the
relative occupancy of the binding pocket in the 2 photoisomers of
MAG and (ii) the degree of clamshell closure that is possible given
the disposition of the MAG linker. A synthesized short version of
on the point of attachment. This yin/yang optical control makes it
possible for 1 wavelength of light to elicit action potentials in one
set of neurons, while deexciting a second set of neurons in the
same preparation, whereas a second wavelength has the opposite
effect. The ability to generate opposite responses with a single PTL
and 2 versions of a target channel, which can be expressed in
different cell types, paves the way for engineering opponency in
neurons that mediate opposing functions.
glutamate receptor ? ion channel ? optics ? photoswitch
attractive approach is to use light as both an input and output for
interrogation and manipulation of the functional state of pro-
teins. Although there has been significant progress in optical
detection of protein function over the last 20 years, the devel-
opment of optical remote control has accelerated recently. Much
of the effort has been focused on cell signaling, with a particular
emphasis on controlling the activity of ion channels. Several
naturally photosensitive channels and pumps have been cloned
and used in a variety of biological preparations (1). In parallel,
3 classes of chemical–biological approaches have been used to
obtain optical control over biological signaling: (i) free photo-
labile ‘‘caged’’ ligands, (ii) photoisomerizable (photochromic)
free ligands (2), and (iii) photoswitched tethered ligands (PTLs)
(3–6). Of these, the most molecularly focused are the PTLs,
endow them with sensitivity to light.
PTLs have been used in ion channels to conditionally present
an agonist to an allosteric regulatory site, originally in the
nicotinic acetylcholine receptor (7–10), and recently in a kainate
receptor, iGluR6 (LiGluR) (11–13) or to conditionally present
a blocker to the Shaker K?channel (SPARK) (14, 15). PTLs
contain the ligand (agonist/antagonist or blocker) at one end, a
reactive group that attaches covalently to the protein at the other
end, and a linker in the middle that contains a photoisomerizable
moieity, such as azobenzene. The PTL is anchored in a site-
directed manner to the protein of interest, usually at a cysteine
that is introduced by mutagenesis, near the ligand-binding site.
major challenge in biology is to develop new ways of
determining how proteins function in cells and how their
Two wavelengths of irradiation are used to isomerize the azo-
benzene back and forth between an extended trans state and a
bent cis state, projecting the ligand at an angle and shortening
the end-to-end distance by 0.35 nm or more, depending on the
length of the PTL (26). Optical control is obtained when the
ligand preferentially binds in one of the states.
The PTL blocker for the Shaker K?channel, MAQ (male-
imide-azobenzene-quaternary ammonium), was designed based
on the structure of the homologous KcsA K?channel (16) and
on previous knowledge that a tethered quaternary ammonium
compound dangling from a passive linker could permanently
block the outer end of the channel when conjugated to a cysteine
located close enough for the extended linker to allow the ligand
to reach the pore (17). Insertion of an azobenzene into the linker
enabled the PTL to be long enough for the QA to reach and
block the pore in the trans state but not in the cis state, when the
linker is shorter (14). This design is expected to apply generally
for proteins in which there is a ‘‘line of sight’’ from the PTL
anchoring position to the ligand-binding site.
to an allosteric regulatory domain. In a clamshell domain, in
which ligand binding stabilizes a closed conformation to activate
the protein, the cysteine attachment site is better placed outside
of the conserved clamshell ‘‘mouth’’ to avoid altering residues
mean a loss of line of sight from the anchoring site to the binding
site as suggested for the first version of LiGluR and its PTL,
maleimide azobenzene glutamate (MAG) (11, 12).
To elucidate the structural basis of optical switching of
iGluR6, we tested 3 MAGs, including a newly synthesized short
MAG0 [see supporting information (SI) Appendix], at a series of
attachment sites surrounding the glutamate-binding pocket. We
find that the cis state is the activating state for 1 or more of the
Author contributions: R.N., S.S., A.Y.L., P.G., M.V., B.R., D.T., and E.Y.I. designed research;
R.N., S.S., and A.Y.L. performed research; M.V. and D.T. contributed new reagents/analytic
tools; R.N., S.S., A.Y.L., P.G., B.R., and E.Y.I. analyzed data; and R.N., S.S., A.Y.L., P.G., M.V.,
B.R., D.T., and E.Y.I. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1R.N. and S.S. contributed equally to this work.
Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1
Hirosawa, Wako, Saitama 351-0198, Japan.
3Present address: Institucio ´ Catalana de Recerca i Estudis Avanc ¸ats and Institut de Bioeng-
inyeria de Catalunya, Parc Científic de Barcelona, C/Josep Samitier 1-5, 08028 Barcelona,
4Present address: Department of Chemistry and Biochemistry, University of Munich,
D-81377 Munich, Germany.
5To whom correspondence may be addressed. E-mail: email@example.com
This article contains supporting information online at www.pnas.org/cgi/content/full/
April 21, 2009 ?
vol. 106 ?
MAGs at most of the sites. However, some combinations
activated in trans. All-atom molecular dynamics (MD) simula-
tions of iGluR6 with tethered MAG0 in explicit solvent, includ-
ing an analysis of the occupancy of the glutamate-binding site
and the degree of clamshell closure that is possible given the
location of the linker, provided insight into the mechanism of
photoswitched activation and the basis of its state dependence.
Strikingly, 1 of the PTLs, MAG0, was found to activate in cis at
some attachment sites but in trans at others. This could be used
to drive 2 populations of neurons in the same preparation to fire
in a complementary manner, paving the way toward engineering
opponency into distinct subgroups of neurons.
Strategies for Optical Control. To endow a protein of interest with
sensitivity to light, the simplest design is for a PTL whose
ligand-binding pocket is within an unhindered line of sight to a
nearby cysteine anchoring site, so that photoisomerization
changes linker length, and only 1 of the lengths is compatible
with ligand binding (Fig. 1 A and B). The design is more
complicated for controlling agonist binding deep in a regulatory
domain, especially in clamshell domains where it may not be
possible to install the anchoring cysteine within a line of sight of
the ligand-binding pocket. In the first light-gated version of
iGluR6 (11), the PTL, maleimide azobenzene glutamate 1
(MAG1), was attached to the ‘‘lip’’ of the clamshell, facing away
from the glutamate-binding site, and the cis state was the
activating state, consistent with the notion that at this site the
linker needs to turn a corner to point the ligand into the binding
site (Fig. 1C). We explored the mechanism of optical switching
of iGluR6 conjugated site-specifically with 3 variants of MAG,
including a newly synthesized short MAG0 (Fig. 1F).
Functional Assays with a Family of MAGs at a Series of Attachment
Sites. In an initial screen to measure the activity of iGluR6 in
response to free glutamate and optical switching of MAG, we
took advantage of the channel’s calcium permeability by per-
forming calcium imaging in HEK293 cells. Sixteen cysteine
mutants were examined around the glutamate binding site (Fig.
2A and Table S1). The sites were chosen to ring the binding
pocket and lie on the surface of the protein where they would be
expected to be accessible to MAG and where mutation to
cysteine and conjugation of MAG would be expected to produce
a minimal disruption to structure and function. Of the 16 sites
tested, 11 yielded functional channels that showed glutamate-
activated rises in internal calcium. Three MAG variants, MAG0,
MAG1, and MAG2 (Fig. 1E), were tested at these sites. Six of
the cysteine mutants showed optical responses to 1 or more of
the MAGs. (Table S1). Interestingly, site 486 and 2 nearby sites
in the upper lobe, 482 and 484, were found to be more strongly
activated by trans-MAG0 than cis-Mag0.
(A–D) Models of photoswitching of iGluR6 by MAG. (A–B) Line-of-sight and
matched length. Two conceptual versions of how isomerization from trans
In B, cis is too short, but trans is long enough to reach. (C) Turning the corner.
Another difference between cis and trans, in that cis is bent and can turn a
For an attachment site that is outside the binding pocket, as illustrated, the
bent cis state is favored to orient the glutamate into the binding pocket. (D)
may interfere with clamshell closure. (E) Structures of photoisomerizable
azobenzene in cis state under illumination at 380 nm and in trans state in the
dark and at 500-nm illumination. (F) Three MAG variants contain a maleimide
(G) agonist at the other end and differ only in linker length by number of
Models of state-dependent liganding by MAG and MAG structures.
of light responses in HEK293 cells expressing iGluR6 with cysteine substitution sites conjugated to MAG. Currents are elicited by near-saturating (300 ?M)
glutamate (blue bars) and by changes in wavelength from 380 to 500 nm during constant illumination (black traces). Time bars represent 25 s. (B) Two cysteine
sites activated by the cis state of the longest MAG. (C) Three cysteine sites activated by the trans state of the shortest MAG, including the sites in B.
Locations of MAG attachment and patch clamp current recordings of light responses. (A) Location of 16 residues around the glutamate-binding pocket
Numano et al.PNAS ?
April 21, 2009 ?
vol. 106 ?
no. 16 ?
Five of the cysteine mutants were selected for further analysis
by whole-cell patch clamping. Inward currents were measured in
voltage clamp in response to exposures to glutamate and to
illumination at 380 and 500 nm (Fig. 2 B and C). The patch clamp
analysis confirmed the calcium imaging for all of the sites
examined, with cis being more active than trans in all cases except
for MAG0 at 482, 484, and 486, where the greater activation was
in trans (Fig. 2 B and C).
Although comparing optical and glutamate responses reports
which isomer activates more strongly, it does not reveal the
degree of channel opening induced by either isomer. To deter-
mine this, we measured dose–response curves for inhibition of
MAG activation by the competitive antagonist DNQX (Fig. 3).
Responses to glutamate, 380-nm light, and 500-nm light were
measured in the absence of DNQX, and then various concen-
trations of DNQX (ranging over 3 orders of magnitude, up to the
millimolar solubility limit) were washed on, and photocurrents
were measured under illumination at 380 and 500 nm (Fig. 3 A
and B). The fractional currents, normalized to the glutamate
response, were plotted against DNQX concentration (Fig. 3 C
and D). As can be seen from this analysis (Fig. 4), some sites,
such as 439, activated almost exclusively in 1 state for all 3 of the
MAGs, whereas other sites, such as 482, were strongly and
almost equally activated by both isomers for all 3 of the MAGs.
Site 439 was the best of the cis activators and site 486 was the best
preference (Figs. 2–4). iGluR6(439C)-MAG0 showed the big-
gest change in effective concentration that we observed of all of
the sites, with an ?120-fold-higher DNQX IC50 in cis than in
trans (Fig. 3C).
Whereas at 2 sites (482 and 486) the short MAG0 activated in
trans and the longer MAGs activated in cis, as one might expect
for a ‘‘reach’’ mechanism (Fig. 1B), at other sites (e.g., 439) the
cis isomer activated well for all 3 of the MAGs (Fig. 5 and Table
S1). This illustrated that a more sophisticated structural analysis
is required to understand the mechanistic basis for photoacti-
vation. Because the opposite behavior (cis activating vs. trans
activating) observed for Mag0 at sites 439 and 486 is interesting
from the point of view of engineering opponency into neuronal
populations (see below), we decided to investigate further the
structural basis for photoswitching by MAG0 using molecular
MD Simulations of Ligand Docking and Clamshell Closure. All-atom
MD umbrella sampling simulations with explicit solvent were
used to compute the ‘‘free energy landscape,’’ or ‘‘potential of
mean force’’ (PMF), for docking a tethered MAG0 molecule
into the iGluR6-binding site. The relative free energy between
conformational states of MAG0, in this case docked vs. un-
docked, is a measure of the probability of finding MAG0 in those
states (Eq. 1). The iGluR6 LBD was constrained to an open
conformation, and the MAG0 azobenzene was constrained to
either a cis or trans configuration. PMFs were computed for the
2 isomers at attachment sites 439 and 486. For each of the sites
and isomers both the R/S stereoisomers of the bond between the
maleimide of MAG and the cysteine were modeled. Consistent
with the experimental observations, PMFs for 439C-MAG0
showed that cis MAG0 is more likely to bind than trans MAG0
(Fig. 5 A, B, and E, Fig. S1, and Table 1). However, PMFs for
486C-MAG0 also indicated that MAG0 is more likely to bind in
cis (Fig. S1 and Table 1), demonstrating that additional factors
must contribute to the state dependence of activation.
An additional factor that needs to be considered to account
closure also increases the apparent agonist binding affinity.
Comparing the extents of LBD closure for different conforma-
tions of 439C-MAG0 and 486C-MAG0 (Fig. S2) with (i) various
GluR2 LBD ligand-complex crystal structures (Table 1 and Fig.
S3), and (ii) free energies associated with GluR2 clamshell
closure (18) (Table 1), suggested the relative activation between
cis and trans forms (Eq. 3 and Table 2). In agreement with the
experimental results, the cis form was estimated to activate more
traces of iGluR6(439C)-MAG0 (A) and iGluR6(486C)-MAG0 (B) in response to
switching from illumination at 380 and 500 nm in the presence of different
concentrations of DNQX (?M). DNQX can be seen to reduce inward current at
both wavelengths. p2/g2, cis photocurrent normalized to glutamate-evoked
current; p3/g2, trans photocurrent normalized to glutamate-evoked current.
Red bars indicate 300 ?M glutamate. (C and D) DNQX titration curves of
fits) and trans (green symbols and fits). Currents are calculated as defined in
Error bars are SEM.
DNQX titrations for cis-on and trans-on channels. (A and B) Current
5 attachment sites. Bar graph showing amplitudes of current responses
500 nm (trans). Photocurrents are calculated and normalized to current
evoked by 300 ?M glutamate, as in Fig. 3. Bars are means of 3–4 experiments.
Error bars are SEM. Note that MAG0 activates more in trans at sites 482, 484,
and 486, with relative potency of trans/cis ? 486 ? 484 ? 482. Note also that
all of the MAGs activate more in cis at 439.
Summary of patch clamp recordings of photoswitching by 3 MAGs at
www.pnas.org?cgi?doi?10.1073?pnas.0811899106Numano et al.
strongly for 439C-MAG0 and the trans form to activate more
strongly for 486C-MAG0. Thus, one can account for the polarity
of photoswitching of LiGluR by MAG by taking into account 2
factors: (i) the isomer dependence of docking of the glutamate
end into the open LBD, as illustrated in Fig. 1 A–C and (ii) the
ability of the LBD clamshell to close on the docked ligand, given
the disposition of the linker (Fig. 1D), which has the effect that
greater closure increases both channel activation and ligand-
binding free energy.
Yin/Yang Remote Control of Neuronal Activity with a Single Photo-
switch. Having found that a single MAG, MAG0, can activate
from some sites in cis and other sites in trans, we explored the
possibility of driving 2 subsets of neurons to fire in opposing
manners within the same preparation with this 1 PTL. We chose
the variants with the most dramatic preference for 1 state of
MAG0, i.e., 439C for cis and 486C for trans (Figs. 3–5). Hip-
pocampal neurons were transiently transfected with
iGluR6(439C) and then later with iGluR6(486C). Because of the
low (?1–2%) efficiency of transfection, neurons that were
successfully transfected expressed one or the other but not both
of these cysteine versions of iGluR6 (Fig. 6A). We labeled the
neurons with MAG0 and then carried out both optical and
elelctrophysiological recordings of the transfected cells, which
were identified by a coexpressed fluorescent protein. Calcium
imaging showed that, as expected, illumination at 380 nm
increased calcium in neurons expressing iGluR6(439C) and
decreased it in neurons expressing iGluR6(486C), whereas illu-
mination at 500 nm had the opposite effect (Fig. 6B). Untrans-
fected neurons did not respond to the illumination. Whole-cell
patch recordings under current clamp mode showed that short
(B) MAG0 docked in the trans configuration. (C and D) MAGs may covalently
attach to a cysteine residue to yield 2 different R/S stereoisomers. MAG0
ment site. The asterisks indicate the asymmetric carbon in the succinimide
(E) Docking PMF for MAG0 with (R)-configuration at the attachment site
restrained to either the cis or trans configuration at site 439. The order
parameter for the PMF is the distance z between the guanidinium group of
iGluR6 R523 and the ?-carboxyl group of the MAG0 glutamate moiety. In our
docking free energy calculations, the MAG0 glutamate is considered to be
docked when z ? 6 Å. All other docking PMFs are shown in SI Appendix.
Table 1. MAG0 docking, LBD closure, and channel activation
estimated from MD (Materials and Methods and Fig. S3). Gclosureis the free
Gtb? Gdock? Gclosureis the free energy of total binding.
Table 2. Relative activation between cis and trans forms
6.5 ? 105
cases shown in Table 1, the unfavorable docking free energy is mitigated by
the favorable free energy of domain closure, which becomes more favorable
with increasing domain closure. For attachment site 439, cis activates more
than trans for (R), but trans activates more than cis for (S). Given equal
proportions of (R) and (S) at site 486, trans would activate more than cis
MAG at 2 different attachment sites. (A) Neurons in the same culture express-
ing either iGluR6(486C) or iGluR6(439C) are loaded with Fluo4-AM for calci-
um-imaging and labeled with MAG0. (B) Illumination with 380-nm light
(purple bars) causes an increase in calcium (and consequently Fluo4-AM
emission intensity) in cells expressing iGluR6(439C)-MAG0 and a decrease in
cells expressing iGluR6(486C)-MAG0. When illuminated with 500-nm light
(green bars), the opposite occurs. The scale bar for Fluo4-AM emission inten-
sity is ?F/F. (C) Action potentials can be precisely stimulated with brief pulses
(2–7 ms) of moderately intense light (?6 mW/mm2). Neurons expressing
iGluR6(439C) and labeled with MAG0 are activated by 375-nm light (purple
arrows), whereas neurons expressing iGluR6(486C) and labeled with MAG0
are activated by 488-nm light (green arrows).
Opposite polarity photoswitching of neuronal activity with same
Numano et al.PNAS ?
April 21, 2009 ?
vol. 106 ?
no. 16 ?
pulses of light at 375 nm elicited action potentials precisely and
reproducibly in neurons expressing iGluR6(439C), whereas
pulses of light at 488 nm elicited action potentials in neurons
expressing iGluR6(486C) (Fig. 6C). Thus, our photoswitch
MAG0 could provide complementary, opposing control of
neural activity in subsets of neurons expressing either
iGluR6(439C) or iGluR6(486C).
We experimentally tested 3 MAGs of different lengths at a
series of attachment sites on the iGluR6 LBD. These sites were
situated on the ‘‘lips’’ of the clamshell in a ring around the
glutamate-binding pocket. From this analysis, we identified
several sites with large gating responses to light. The efficacy
and cis/trans dependence of the optical gating differed between
attachment sites and the MAG variants. MD simulations
revealed the mechanism of photoswitching by MAG, showing
that channel activation depends on 2 factors: (ii) the fraction
of rotamers of a cysteine-anchored MAG that enable the
glutamate to orient in the binding site of the open LBD and
(ii) the ability of the clamshell to close, given the location of
the linker. Greater clamshell closure was shown to contribute
in 2 ways: increasing the opening of the gate (19) and
increasing the affinity for the glutamate (18). It should
be noted that despite the fact that the maleimide linkage can
result in unequal probabilities of R/S stereoisomers and that
labeling may not reach saturation for all of the subunits,
clear on/off effects are found in living cells. Thus, it seems
that a kind of ‘‘binary filtering’’ may be going on by the
A new short version of MAG0 emerged as the best photoswitch
in 2 regards. First, MAG0 has the biggest change in effective
concentration seen to date: ?120-fold higher in cis than in trans,
based on competition with the antagonist DNQX. Second, MAG0
sites in cis but at other sites in trans. When the best cis activator
[iGluR6(439C)] and best trans activator [iGluR6(486C)] are ex-
labeling with MAG0 endows the 2 sets of cells with opposite
sensitivity to light. Neurons expressing iGluR6(439C) fire in re-
sponse to pulses of 380-nm light and those expressing
to use 1 PTL simultaneously to excite 1 subpopulation of neurons
into distinct subgroups of neurons, such as, for example, ON and
OFF retinal ganglion cells, which have overlapping receptive fields
but opposite responses to light.
Materials and Methods
Synthesis of MAG0. MAG synthesis and chemical analysis is described in SI
Site-Directed Mutagenesis. Cysteine point mutations were substituted outside
of the glutamate-binding site of the iGluR6 LBD. The iGluR6 cDNA containing
(Colorado State University, Fort Collins, CO). Cysteine point mutations were
kit (Stratagene). The forward and reverse oligonucleotide primer sequences
of the cysteine point mutations are shown in Table S2. The following PCR
profilewasused:1cycle(95 °Cfor30s);20cycles(95 °Cfor30s,55 °Cfor1min,
68 °C for 12 min); 1 cycle (68 °C for 12 min).
FBS on polyl-lysine-coated glass coverslips at ?3 ? 106cells per milliliter and
transiently cotransfected with various iGluR6 plasmids and EYFP at a ratio of
9:1 by using Lipofectamine 2000 (Invitrogen). Calcium imaging or patch
clamping was performed 12–48 h after transfection.
Dissociated postnatal rat hippocampal neurons (P0-P5) were prepared and
transfected as described previously (13). Serial transfections of iGluR6(486C)
and iGluR6(439C) were performed identically but separated by 24 h.
Ca2?Imaging and Patch Clamp Recordings. Ca2?imaging and patch clamp
recordings were as described in SI Appendix.
MD Simulations and Analysis. The atomic model for the iGluR6 LBD was
constructed from the X-ray crystal structure PDB ID 1S50 (20). All simulations
were performed by using the program CHARMM (21) with the all-atom
potential energy function PARAM27 for proteins (22) and the TIP3P potential
energy function for water (23). The software package Antechamber (24) was
used to obtain GAFF (generalized amber force field) parameters (25) for
MAG0 adapted for use in CHARMM. Additional details are described in SI
The total effective receptor activation (ERA) is predicted from the proba-
bility of finding the tethered ligand in the binding site when the clamshell is
open (‘‘ligand docking’’), times the probability of the LBD to adopt a closed
effectiveness of receptor activation once the LBD has reached its maximum
closure. The free energy of ligand docking onto an open clamshell, Gdock, is
obtained as follows:
to occur at z ? 6 Å, where z is the distance between the guanidinium group
of iGluR6 R523 and the ?-carboxyl group of the MAG0 glutamate moiety. The
open. The free energy associated with the closure of the clamshell onto a
computed with all-atom MD simulations (18). The total binding free energy is
the extent of LBD closure. This is predicted by using an empirical relationship
derived from the change in the clamshell distance ?d taken from available
crystal structures relative to the glutamate-bound crystal structure and the
observed peak current for GluR2:
Act(?d) ? exp(?3.492 ? ?d),
where ?d must be ?0 (Fig. S3). Domain closure is measured in terms of the
2-dimensional order parameter (?1, ?2), where ?1is the distance between the
distance between residues 439–441 (Lobe 1) and 721–722 (Lobe 2). ??12is the
GluR6 bound to glutamate, where ?12? (?1??2)/2. ???12? was calculated from
It follows that, for a given construct, the total effective receptor activation
the tethered ligand in the binding site times the probability of finding the
?ERA?(Actcis?Acttrans) ? exp? ? ?Gtb-cis? Gtb-trans??kBT?,
where Gtb? Gdock? Gclosure.
for guidance on calcium imaging, and Harald Janovjak for helpful discussion.
This work was supported by Human Frontiers Science Program Grant RGP23-
2005 and National Institutes of Health (NIH) Nanomedicine Development
Center for the Optical Control of Biological Function Grant PN2 EY018241 as
well as postdoctoral fellowships from the Japan Society for the Promotion of
Science and the Institute of Tokyo Vascular Disease (to R.N.) and the Gener-
alitat de Catalunya (Nanotechnology Program), Ministerio de Educacio ´n y
Novartis and Roche Biosciences for support. A.Y.L and B.R were supported by
NIH Grant GM-62342.
www.pnas.org?cgi?doi?10.1073?pnas.0811899106Numano et al.
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