A cell-penetrating artificial metalloenzyme regulates a gene switch in a designer mammalian cell

Article abstract of DOI:10.1038/s41467-018-04440-0

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin-streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells.

Full text of DOI:10.1038/s41467-018-04440-0

A R T I C L E
DOI: 10.1038/s41467-018-04440-0
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1 Department of Chemistry, University of Basel, Mattenstrasse 24a, BPR 1096, CH-4058 Basel, Switzerland. 2 Department of Biosystems Science and
Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland. 3 Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan. 4 Department of Organic Chemistry, University of Geneva, CH-1211 Geneva, Switzerland. These authors contributed equally: Yasunori
Okamoto, Ryosuke Kojima, Fabian Schwizer. Correspondence and requests for materials should be addressed to S.M. (email: Stefan.Matile@unige.ch)
or to M.F. (email: martin.fussenegger@bsse.ethz.ch) or to T.R.W. (email: thomas.ward@unibas.ch)
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n recent years, there has been an increasing effort to exploit extracellular environment and producing output molecules on
the cell as a test-tube to complement the biochemical reaction demand (Fig. 1b)29–31. Thus far, only genetically encodable
networks with abiotic reactions1, 2 (Fig. 1a). With this goal in modules have been integrated into designer mammalian cells.
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mind, both organometallic complexes and nanoparticles have This limits the available intracellular reaction repertoire to those
been shown to catalyze abiotic reactions in Escherichia coli3–7
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labeling of a protein) or a loss-of-function (i.e. drug-release that enzymatic catalysts (Fig. 1c and Fig. 2)32, 33. Most recently, the
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function) by an intracellular abiotic reaction has been next step, we aim to integrate an ArM in a designed mammalian
reported26, 27
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cell to upregulate the expression of a reporter protein via a
In addition to these efforts, metabolic engineering in E. coli, complex reaction cascade (Fig. 1c). To achieve this goal, the
yeast, and higher organisms has received increasing attention following challenges need to be addressed: (i) the efficient uptake
thanks to its enormous potential to produce either high-added of an ArM into mammalian cells; (ii) the assembly of a gene
value products and biofuels or to cure diseases28. In this latter switch that senses and responds to the product of the ArM
context, the assembly of gene switches allows for the construction resulting in (iii) the intracellular upregulation of the gene switch
of engineered mammalian cells that are capable of sensing the by the ArM.
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Herein we demonstrate the proof-of-principle of an intracel-
OH
O
OH
NH2
lular abiotic reaction enabled by an ArM modified with a cell-
penetrating module. This cell-penetrating ArM is taken up into
mammalian cells and catalyzes an abiotic reaction, leading to the
upregulation of a designed gene circuit.
1, 2 or 22⊂Sav
HN
l
l
I
I
O
PBS buffer (pH 7.5)
37 °C, 16 h
HO
O
l
HO
O
I
6
7
TONRu
15 20
Results
0
•
5
10
25
•
30
35
Design of a cell-penetrating artificial metalloenzyme. Thus far,
most of the reported intracellular abiotic catalysis has relied on
the catalysts’ inherent cell permeability: only few reports have
used cell-permeable carrier modules13, 16, 19, 20. In the absence of
a cell-permeable moiety, the efficiency of the catalyst’s cellular
uptake has been a matter of controversy14, 35. To achieve efficient
delivery of an organometallic catalyst into cells, we capitalize on
the homotetrameric nature of a streptavidin scaffold to combine
an abiotic biotinylated catalyst with a biotinylated cell-penetrating
moiety. Additional attractive features of ArMs based on the
biotin–streptavidin technology include: (i) the possibility to
optimize the catalytic performance using genetic means6, 36 and
(ii) protection of the precious metal cofactor against detrimental
cellular components6, 37. We hypothesized that this Sav-based
approach to assemble a cell-permeable ArMs may provide a
versatile tool for the introduction of synthetic catalysts into cells.
With the aim of combining a gene switch with an abiotic
reaction catalyzed by an ArM in a designer mammalian cell, we
No catalyst •
1
•
•
2
•
•
WT
A
22⊂Sav
•
•
•
•
C
E
•
•
•
•
•
H
K
•
•
•
•
M
R
T
•
•
•
•
•
Y
•
•
A
•
•
•
C
E
•
combined a ruthenium catalyst for the intracellular O-allyl
•
carbamate cleavage12,
with a gene switch that is
14, 19, 35
•
F
•
upregulated in the presence of the thyroid hormone, triiodothyr-
onine (T3)38. The T3 hormone is known to affect thermogenesis,
carbohydrate metabolism, and lipid homeostasis in all tissues.
Our T3-responsive gene switch has been shown to work in
various cell lines, including HEK-293T, Hela, immortalized
human mesenchymal stem cells (hMSC-TERT), HT-1080, and
CHO-K1 cells38. Among these cell lines, we selected HEK-293T
cells, which is the most responsive. Building on the ruthenium
complex 1 introduced by Meggers and co-workers14, the
biotinylated ruthenium complex 2 was prepared in situ by
mixing [CpRu(NCCH3)3](PF6) 3 and the biotinylated ligand 4 in
a 1:1 ratio. (Fig. 2). We selected the cell-penetrating poly
(disulfide) (CPD) 5, which was previously developed by us, as a
cell-permeable module (Fig. 2)39, 40. CPD is taken up via dynamic
covalent disulfide exchange with thiols on mammalian cell
surfaces. The presence of glutathione in the cytosol leads to
depolymerization of the CPD, thus alleviating continual
membrane-perturbing activities and reducing cytotoxicity com-
•
H
M
P
•
•
•
•
•
•
R
S
•
•
•
•
T
•
•
W
Y
•
•
•
Fig. 3
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7
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.
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pared to traditional arginine-rich cell-penetrating peptides40, 41
.
None of the screened double mutants outperformed 22 ⊂ Sav
S112A, which was selected for subsequent studies.
Relying on the versatility of the CPD as demonstrated for Hela
cells39 and Drosophila S2 cells40, we hypothesized that these
would be applicable for HEK-293T cells as well.
Designer cells with a T3-gene switch. In order to monitor the
In vitro optimization of the ArM. To ensure efficient coupling output of the reaction cascade, which is reflected by the transgene
between the reaction catalyzed by the ArM and the gene switch, expression level resulting from the T3-gene switch, we slightly
the performance of the ArM 22 ⊂ Sav (the subscript indicates the adapted a previously reported thyroid hormone-responsive gene
number of biotinylated catalyst moieties 2 added to the homo- switch in HEK-293T cells (Fig. 4a–c)38. This modified thyroid
tetrameric Sav) was optimized by single point mutations of the hormone-responsive gene switch consists of a set of two plasmids,
Sav scaffold for the O-allyl carbamate cleavage of the caged pSP27 and pYO1. The plasmid pSP27 codes for a synthetic
hormone (AT3) 6, to yield T3 7 (Fig. 3, Supplementary Figure 1). thyroid hormone receptor (TSR), which is a fusion protein
The ruthenium complex 1 (ref.14) displayed higher activity than comprised of the DNA-binding domain of yeast Gal4 and the T3-
both the biotinylated ruthenium complex 2 and the corre- binding domain of human thyroid receptor α (PhCMV-TSR-pA)
sponding ArM 22 ⊂ Sav wild type in vitro. Genetic optimization (PhCMV: human cytomegalovirus promoter). The plasmid pYO1
of the catalytic performance of 22 ⊂ Sav was achieved by site- comprises a Gal4-specific operator sequence (PUAS5) connected to
directed mutagenesis at position Sav S112X or K121X, which are a minimal hCMV promoter (PhCMVmin) that drives expression of
the putative closest lying amino acid residues36. The mutant 22 ⊂ a secreted nanoluc (sec-nluc), a potent bioluminescence reporter
Sav S112A had a 2.5-fold higher TON (turnover number) than (PUAS5-sec-nluc-pA). In the absence of T3 7, TSR binds to PUAS5
the wild-type ArM and a comparable activity to the catalyst 1. and recruits endogenous corepressors, silencing mediators for
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
(
2
0
1
8
)
9
:
1
9
4
3
D
O
I
:
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
1
8
-
0
4
4
4
0
-
0
w
w
w
.
n
a
t
u
r
e
.
c
o
m
/
n
a
t
u
r
e
c
o
m
m
u
n
i
c
a
t
i
o
n
s
3
|
|
|

A
R
T
I
C
L
E
N A T U R E C O M MU N ICA T IO N S | D O I: 1 0 . 1 0 3 8 / s 41 46 7 - 0 1 8 -0 4 44 0 -0
retinoid or thyroid hormone receptors (SMRT)/nuclear receptor promoter). Hydrolysis of p-nitrophenylphosphate 11 affords p-
corepressor 2 (NcoR2), leading to histone deacetylation. As a nitrophenolate 12, which can be monitored spectro-
result, the expression of sec-nluc is repressed (Fig. 4b). In the photometrically at 405 nm (Fig. 4a–c).
presence of T3 7, the TSR interaction with T3 recruits coactiva-
Transport of native T3 7 into cells usually relies on a
tors: steroid receptor coactivator 1 (SRC-1) and a 220-kDa transporter-mediated process42. As it was not clear whether the
thyroid hormone receptor-associated protein complex compo- AT3 6 is transported into cell via this pathway, we modified AT3 6
nent (TRAP 220). These coactivators trigger histone acetylation to increase its cell permeability. For this purpose, we esterified the
to initiate sec-nluc expression, which is monitored by the lumi- carboxylic group of AT3 6 with an acetoxymethyl (AM) group to
nescent conversion of furimazine 9 into product 10 (Fig. 4a). As yield AM-AT3 8 (refs.43, 44). Upon passive diffusion of AM-AT3 8
an indicator of the cell viability, a human placental secreted into the cytosol, endogenous esterases rapidly hydrolyze it to
alkaline phosphatase (SEAP), was co-expressed constitutively by produce the allyl-carbamate protected AT3 6 (Fig. 4b, c).
pSEAP2-control (PSV40-SEAP-pA) (PSV40: similian virus 40
O
O
a
O
O
N
O
O
NH
N
N
O
O
pSP27
+ O2
N
H
N
+
CO2
pYO1
O
O
Light
HN
I
I
9
10
HO
O
I
pSEAP2-control
8
O–
2x 5y⊂Sav S112A
O–
P
O
Incubation (1 h) Cell wash
Incubation (24 h)
O–
O
+ HPO4–
O N11
12
2
Assay
Transfection
Catalyst loading
Substrate loading
b
c
– 2x 5y⊂Sav
sec-nluc expression OFF
SEAP expression ON
– 2x 5y⊂Sav
sec-nluc expression ON
SEAP expression ON
8
8
6
6
Esterase
PhCMV
Esterase
PhCMV
7
pA
pA
TSR
(pSP27)
TSR
7
TRAP220
SRC-1
NCoR2/
SMRT
7
7 7
RNA Pol II
pA
pA
PUAS5 PhCMVmin
PUAS5 PhCMVmin
(pYO1)
sec-nluc
sec-nluc
PSV40
PSV40
pA
pA (pSEAP2-control)
SEAP
SEAP
d
e
2000
1500
1000
500
0
(3,1)
4
3
2
1
0
•
•
•
(2,1)
(2,2)
•
•
•
•
•
•
•
(1,1)
•
•
•
•
•
•
•
(1,2)
• •
•
(1,3)
•
•
•
•
•
•
•
• •
•
•
•
•
•
•
• •
•
•
•
•
•
•
•
••
•
•
f
1.5
1.0
0.5
•
•
•
•
•
•
•
•
•
• •
•
•
•
•
•
•
•
•
•
•
•
•
•
•
••
•• •
•• •
••
(1,1)
•
•• •
(x,y) (–,–)
(1,1)
(1,2)
(1,3)
0
Ru / μM
5 / μM
2 / μM
5 / μM
Sav / μM
0
0
0
0.50
0.50
0.50
0.25
0.50
0.25
0.17
0.50
0.17
0.50
0.50
0.50
0
0
0
0.17
0.50
0.17
0.25
0.50
0.25
0.50
0.50 0.50 0
0.50 0.25 0
1.00
0.50
0.50
1.50
0.50
0.50
0
0
0
0
0
0
0
0
Sav / μM
No catalyst
2x 5y⊂Sav S112A, (x,y)
1
4
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
( 2 0 1 8 ) 9 : 1 9 4 3
D O I : 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 1 8 - 0 4 4 4 0 - 0 w ww .n a t u r e . c o m / n a t u re c o m m u n i c a t i o n s
| |
|

N
A
T
UR
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
D
O
I
:
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
1
8
-
0
4
4
4
0
-
0
A R T I C L E
First, we investigated the dose-dependence of T3 7 and AM- catalyst 1 (Fig. 4e). Although its activity in vitro is comparable to
AT3 8 against the T3-responsive gene switch (pSP27 and pYO1) that of the ArM 22 ⊂ Sav S112A (Fig. 3), the use of complex 1 did
and the pSEAP-2 control (Supplementary Figure 2). When using not lead to higher luminescence intensities than when the
T3 7, the luminescence intensity derived from the expressed sec- designer cells were treated with the substrate AM-AT3 8 but in
nluc activity reached a plateau at very high gene expression level the absence of any Ru-catalyst (Fig. 4e). The cofactor 2 alone and
(i.e. >6 nM T3 7). At concentrations below 500 nM, AM-AT3 8 the ArM 21 ⊂ Sav S112A were also examined and found not to
did not induce sec-nluc expression. This highlights the efficient turn on the gene switch (Supplementary Figure
7 and
caging effect of the allyl carbamate moiety of AM-AT3 8. Supplementary Discussion). This corroborates the flow cytometry
Gratifyingly, the phosphatase activity of SEAP did not decrease in observations, highlighting the limited uptake in HEK-293T cells
the presence of AM-AT3 8: we thus conclude that it does not when low Ru-concentrations are used (e.g. <1.5 μM)35. These
exhibit cytotoxicity at these concentrations.
results confirm that only cell-penetrating ArMs turn on the T3-
responsive gene switch by producing T3 7 via the intracellular
uncaging of AT3 6. Increasing the Ru-catalyst 2 to CPD 5 ratio
led to higher luminescence intensities: the highest activity was
observed with the ArM 2351 ⊂ Sav S112A. This highlights the
importance of the “membrane permeability” and the “inter-
nalization” features of the cell-penetrating ArM. The cell viability,
reflected by the SEAP activity, is essentially constant for all
conditions tested. This confirms that there is no critical
cytotoxicity for the intracellular reaction cascade (Fig. 4f).
Intracellular abiotic reaction. After the in vitro identification of
the most active ArM for the allyl-carbamate cleavage of AT3 6
(see Fig. 3), we next assembled a cell-penetrating ArM 2x5y ⊂ Sav
S112A by sequential incorporation of the ruthenium complex 2
and biotinylated CPD 5 into Sav S112A (Fig. 2, Supplementary
Figure 3 and Supplementary Discussion). Capitalizing on the four
biotin-binding sites present in Sav, the ratio of the ruthenium
catalyst 2 and CPD 5 was optimized. Based on a previous report40
and a preliminary screening experiment (Supplementary Fig-
ure 4), the concentrations of CPD 5 and AM-AT3 8 were set to Discussion
0.5 and 4 μM, respectively. These concentrations were selected to Herein, we have demonstrated the versatility of a complex
maximize the cellular uptake of the ArM with the minimum cell intracellular reaction cascade that results from combining a cell-
toxicity and minimize the background luminescence caused by penetrating ArM with a synthetic gene circuit. The uncaging
AM-AT3 8.
reaction catalyzed by the ArM leads to the exquisite control of a
The designer HEK-293T cells transfected with the T3- designer cell, ultimately upregulating the activity of a genetically
responsive gene switch were treated with ArM 2x5y ⊂ Sav encoded sec-nluc. The observed output of this system is unam-
S112A. Following incubation and careful washing of the cells to biguous. However, further improvement would be desirable for
remove surface-adsorbed ArM, the substrate AM-AT3 7 (4 μM) practical use. For this purpose, in vivo directed evolution of ArMs
was added (Fig. 4a). We monitored the ArM’s uptake in the have proven versatile6. Also, it may be possible to stably integrate
designer cell by the confocal microscopy (Supplementary the gene switch into designer cells to allow for the selection of the
Figure 5) and flow cytometry (Fig. 4d) before and after the best performing single cell clone45.
reaction respectively. For comparison, the CPD 5 was replaced by
Although cell-targeting modules have previously been com-
Atto565-Biotin, which exhibits similar spectroscopic features to bined with organometallic moieties to catalyze intracellular
CPD 5 but is not internalized by covalent-mediated translocation transformations19, the use of a Sav-based ArM combines several
through the cell-membrane (Supplementary Figure 6 and attractive features including: (i) a straightforward means to
Supplementary Discussion), to afford 21(Atto565-Biotin)1 ⊂ Sav. assemble and optimize the ArMs by chemical and genetic stra-
In the case of 21(Atto565-Biotin)1 ⊂ Sav, the fluorescence derived tegies, (ii) easy modulation of the catalyst:CPD ratio to fine-tune
from Atto565-Biotin was not detectable either by confocal the uptake and the activity of the ArM, (iii) a means to visualize
microscopy or flow cytometry. This indicates that the ArM by fluorescence the internalization of the ArM, and (iv) the
devoid of CPD 5 is not cell-permeable. In contrast, the cellular shielding of the precious metal moiety within a protein scaffold37.
uptake of ArMs 2x5y ⊂ Sav was confirmed by the pronounced
increase in fluorescence within the cells caused by the presence of that critically relies on a reaction requiring components that are
TAMRA moieties on the ArMs. not genetically encoded. This strategy may be extended to new-
This proof-of-concept highlights the gain of cellular function
As for the intracellular reactions, the luminescence intensities to-nature reactions based on related ArMs: Suzuki-coupling,
resulting from the induced sec-nluc activity were markedly higher metathesis and C–H activation33. This may allow to endow
in the presence of the ArM 2151 ⊂ Sav S112A than with model mammalian cells with highly versatile catalytic function. We thus
6
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believe that this study may offer a novel means to engineer cel- incubated in the medium C containing AM-AT3 8 (4 μM) with 0.5% DMSO for 24
h. Expression of sec-nluc and SEAP was quantified in the supernatant as described
in the following section. Then, cells were detached using 0.05% trypsin-EDTA. The
cell populations were analyzed using a Becton Dickinson LSRII Fortessa Flow
lular function via highly modular cell-penetrating ArMs.
Methods
Cytometer (Becton Dickinson) (conditions: filter set for monitoring red fluores-
cence; 561-nm laser, mirror: 570 LP, Filter: 586/15 BP). Data were analyzed by
FlowJo. Populations of living cells (23963 to 25336 events) were extracted from
30,000 events based on SSC and FSC.
Synthesis of chemical compounds. Details are collected in the Supplementary
Information (Supplementary Figures 8–17 and Supplementary Methods)
Stock solutions of Sav, 1, 2, ArM 2x ⊂ Sav, and ArM 2x5y ⊂ Sav. Sav-stock
solutions (stock A) were prepared in phosphate-buffered saline (PBS) (pH 7.5) or
water to yield 800 μM free biotin-binding site (referred to as FBBS). A stock
solution (stock B) of the ruthenium complex 1 (2 mM) was prepared in dimethyl
sulfoxide (DMSO). A stock solution (stock C) of the ruthenium complex 2 (5 mM)
was prepared by mixing [CpRu(NCCH3)3](PF6) 3 (10 mM in DMF) and the bio-
tinylated ligand 4 (Supplementary Figure 9) (10 mM in DMF) in a 1:1 ratio. These
stock solutions were freshly prepared for each experiment. The ArM 2x ⊂ Sav was
prepared using the conditions listed in Supplementary Tables 1–3, respectively.
Stock C was incubated with water for 10 min prior to the addition of stock A. The
ArM 2x5y ⊂ Sav was prepared using the conditions collected in Supplementary
Tables 4–9. After mixing the ArM 2x ⊂ Sav and water, CPD 5 was added and
incubated for 2 h.
Activity assays of SEAP and sec-nluc. SEAP: The supernatant was transferred
into a 96-well plate (100 μL/well) and incubated at 65 °C for 30 min to heat-
inactivate endogenous alkaline phosphatases. After cooling, 80 μL of the treated
supernatant was mixed with 100 μL of 2× SEAP buffer (20 mM homoarginine, 1
mM MgCl2, 21% (v/v) diethanolamine, pH 9.8) and 20 μL of 720 μM of p-nitro-
phenylphosphate. Immediately thereafter, the absorbance was measured at 405 nm
at 37 °C using the EnVision 2104 Multilabel Reader. The measurement was per-
formed over 61 repeats of 30 s/repeat and the plate was shaken for 5 s at 900 rpm at
the beginning. From the time-course increase of the absorbance at 405 nm, derived
from the enzymatic activity of SEAP, the activity of SEAP (in U/L) was determined.
To avoid saturation effects, the linear part of the time-course plots was used for the
determination of the slope. The correlation between the slope and the corre-
sponding activity was determined using a standard.
Sec-nluc: The supernatant was transferred into a black 384-well plate (7.5 µL/
well), and the same amount of nanoglo assay solution (Promega). After 5 min
incubation, the luminescence was measured with Tecan Infinite M1000 Pro.
Screening Sav for the uncaging of AT3 6. Prior to Sav mutant screening,
potassium isocyanoacetate was tested as an irreversible inhibitor for the catalytic
activity of the ruthenium complexes 1, 2 and ArM 22 ⊂ Sav (see Fig. 3 and Sup-
plementary Figure 1). Allyl carbamate caged amino-coumarin 13 (see Supple-
mentary Figure 18a) was used as a substrate. The reaction conditions are collected
in Supplementary Table 10. Experiments were performed at 37 °C in a 96-well
plate. The fluorescence intensity at 450 nm derived from the produced 7-amino-4-
methylcoumarin 14 (excitation at 375 nm) was monitored with a Tecan Infinite
M1000 Pro (Supplementary Figure 18b). Potassium isocyanoacetate was found to
efficiently inhibit the catalytic activity of ruthenium complexes 1, 2 and ArM 22 ⊂
Sav. Based on this, potassium isocyanoacetate was added to the reaction mixture to
quench the reaction during the genetic optimization of the ArM’s activity. For the
screening of Sav variants, AT3 6 was used as a substrate. Reaction conditions are
listed in Supplementary Table 11 and were performed at 37 °C in an HPLC vial.
Potassium isocyanoacetate (500mM in water, 4 μL), tryptophanamide hydro-
chloride (10mM in water, 20μL) as an internal standard, acetonitrile (800μL), and
PBS (400 μL) were added to the reaction mixture. The resulting solution was
centrifuged (10,000g) and analyzed by UPLC-MS (Supplementary Figure 19).
TONs were determined based on the calibration curve of T3 7 (Supplementary
Figure 20).
Data availability. The authors declare that all data supporting the findings of this
study are available either in the paper and in the Supplementary Information or
from the authors upon reasonable request.
Received: 28 February 2018 Accepted: 24 April 2018
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Acknowledgements
The authors thank P. Saxena for providing the plasmids. The authors thank the Swiss
National Science Foundation for generous funding via the NCCR Molecular Systems
Engineering. Additional funding was provided by an ERC (the DrEAM) to T.R.W., the
NCCR Chemical Biology to S.M., a JSPS Overseas research fellowship to Y.O., Human
Frontier Science Program (HFSP) long-term postdoctoral fellowship to R.K. and the
Universities of Basel and Geneva, and ETH Zurich.
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Additional information
Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-
018-04440-0.
Competing interests: The authors declare no competing interests.
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