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S. A. Teichmann, Science 350, aaa2245 (2015).
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We thank C. Robinson, J. Schnell, P. Kukura, D. Staunton (all at
University of Oxford), and B. Metzger (University of Chicago) for
helpful discussions. We acknowledge access to B21 and help from
M. Tully and J. Doutch at the Diamond Synchrotron (J.L.P.B. for
SM9384-2); and the ARCUS cluster at Advanced Research
Computing, Oxford. We thank the following funding sources:
Engineering and Physical Sciences Research Council (G.K.A.H. for a
studentship, J.L.P.B. for EP/J01835X/1); Carl Trygger’s Foundation
(E.G.M.); Swiss National Science Foundation (M. T.D. for P2ELP3_155339)
and Biotechnology and Biological Sciences Research Council (A.J.B.
for BB/J014346/1, J.L.P.B. for BB/K004247/1 and BB/J018082/1);
National Institutes of Health (E. V. for RO1 GM42761); Massachusetts
Life Sciences Center (E.V. for a New Faculty Research Award);
and the Royal Society (J. L.P. B. for a University Research Fellowship).
All data necessary to support the conclusions are available in the
manuscript or supplementary materials and are deposited with
Materials and Methods
Figs. S1 to S15
Tables S1 and S2
Data Files S1 and S2
8 January 2017; resubmitted 25 September 2017
Accepted 8 January 2018
imaging of deep tissue in freely
Satoshi Iwano,1 Mayu Sugiyama,1 Hiroshi Hama,1 Akiya Watakabe,2 Naomi Hasegawa,2
Takahiro Kuchimaru,3 Kazumasa Z. Tanaka,4 Megumu Takahashi,5 Yoko Ishida,5
Junichi Hata,6 Satoshi Shimozono,1 Kana Namiki,1 Takashi Fukano,1 Masahiro Kiyama,7
Hideyuki Okano,6 Shinae Kizaka-Kondoh,3 Thomas J. McHugh,4 Tetsuo Yamamori,2
Hiroyuki Hioki,5 Shojiro Maki,7 Atsushi Miyawaki1,8*
Bioluminescence is a natural light source based on luciferase catalysis of its substrate
luciferin. We performed directed evolution on firefly luciferase using a red-shifted
and highly deliverable luciferin analog to establish AkaBLI, an all-engineered
bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that
were brighter by a factor of 100 to 1000 than conventional systems, allowing
noninvasive visualization of single cells deep inside freely moving animals. Single
tumorigenic cells trapped in the mouse lung vasculature could be visualized. In
the mouse brain, genetic labeling with neural activity sensors allowed tracking of
small clusters of hippocampal neurons activated by novel environments. In a
marmoset, we recorded video-rate bioluminescence from neurons in the striatum,
a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light
source to spur unprecedented scientific, medical, and industrial applications.
Bioluminescence imaging (BLI) is based on the detection of light produced by the en- zyme (luciferase)–catalyzed oxidation re- action of a substrate (luciferin) (1, 2). In vivo BLI is a noninvasive method for mea-
suring light output from luciferase-expressing
cells after luciferin administration in living ani-
mals (3), and this method typically employs
firefly luciferase (Fluc) and the natural substrate
D-luciferin (Fig. 1A, left) that produces longer-
wavelength (green-yellow) light and is more
stable for enzymatic reaction after administra-
tion than the other commonly used luciferase
substrate, coelenterazine (4–7). However, due to
its relatively low tissue permeability, D-luciferin
has a heterogeneous biodistribution in the body
(8). The low affinity (high Michaelis constant,
KM) of D-luciferin for Fluc also suggests un-
even saturation of the Fluc reporter enzyme with
substrate in vivo. In particular, in vivo BLI in
the brain has been hampered due to low pas-
sage of D-luciferin through the blood-brain bar-
rier (BBB) (8). In recent years, synthetic analogs
of D-luciferin were reported (9–11), including
AkaLumine (Fig. 1A, right), that when catalyzed
by Fluc produces near-infrared emission peak-
ing at 677 nm, which can penetrate most animal
tissues and bodies. We previously demonstrated
that AkaLumine hydrochloride (AkaLumine-
HCl) has favorable biodistribution to access
Fluc-expressing cells in deep organs such as the
lung and can saturate Fluc more effectively than
We hypothesized that Fluc is not enzymatically optimal for AkaLumine-HCl; therefore, we
performed directed evolution on the luciferase
gene through successive rounds of mutagenesis,
screening, and validation to develop an enzyme
that could strongly pair with AkaLumine-HCl.
We constructed gene libraries encoding variants of three luciferases (13)—Fluc, emerald luciferase (Eluc), and crick beetle red luciferase
(CBRluc)—and screened them by selecting for
bacterial colonies with brighter emission in the
presence of AkaLumine (fig. S1A). Candidates in
the Fluc-based library were iteratively screened
with multiple cycles of random mutagenesis (fig.
S1B) to produce Akaluc, which has 28 amino acid
substitutions relative to Fluc (fig. S1C). Application of D-luciferin and AkaLumine on bacterial colonies expressing Akaluc or Fluc enabled
bright near-infrared emission with AkaLumine/
Akaluc and very weak emission with the other
combinations (fig. S2). In vitro experiments with
purified luciferases (Fig. 1B) showed that Akaluc
catalyzed AkaLumine ~7 times more efficiently
than Fluc to produce emissions with a maximum
at 650 nm (fig. S3). Their catalytic activity was
equally pH-sensitive (fig. S4) and, in addition,
Akaluc exhibited higher thermostability than
Fluc (fig. S5).
Akaluc-expressing HeLa cells (HeLa/Akaluc
cells) were constructed after transfection with
a cDNA encoding Venus-Akaluc and purification by flow cytometry for Venus fluorescence.
Likewise, HeLa/Fluc cells were also prepared.
We examined the comparative BLI performance of the four luciferin/luciferase combinations (Fig. 1C). To compare the performance
of Akaluc and Fluc for AkaLumine in cultured
cells, we additionally performed a comparative
experiment using HeLa/Akaluc cells and HeLa/
Fluc cells exposed to 500 mM AkaLumine (fig.
S6A). The cellular BLI signal ratio of Venus-Akaluc to Venus-Fluc was ~52. In addition,
we analyzed Venus-luciferase–expressing HeLa
cells by flow cytometry and quantified the expression level ratio of Venus-Akaluc to Venus-Fluc to be ~4. Accordingly, we assigned the
~13-fold improvement of Akaluc over Fluc to
an improvement intrinsic to luciferase’s catalytic activity. Akaluc showed a 3.79 ± 0.20 (n =
58) times higher expression level compared
1Laboratory for Cell Function and Dynamics, Brain Science
Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198,
Japan. 2Laboratory for Molecular Analysis of Higher Brain
Function, Brain Science Institute, RIKEN, 2-1 Hirosawa,
Wako-city, Saitama 351-0198, Japan. 3School of Life
Science and Technology, Tokyo Institute of Technology,
4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
4Laboratory for Circuit and Behavioral Physiology, Brain
Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama
351-0198, Japan. 5Department of Morphological Brain
Science, Graduate School of Medicine, Kyoto University,
Yoshida-Konoe-Cho, Sakyo-ku, Kyoto 606-8501, Japan.
6Laboratory for Marmoset Neural Architecture, Brain Science
Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198,
Japan. 7Graduate School of Informatics and Engineering,
The University of Electro-Communications, 1-5-1 Chofugaoka,
Chofu-city, Tokyo 182-8585, Japan. 8Biotechnological
Optics Research Team, Center for Advanced Photonics,
RIKEN, 2-1 Hirosawa, Wako-city, Satitama 351-0198, Japan.
*Corresponding author. Email: firstname.lastname@example.org