Supplemental Information
Molecular engineering of single opsins and White-opsin
The vector maps of ChR2, ReaChR, and White-opsin are shown in Fig. S1a-c. The gel
electrophoresis images of ChR2, ReaChR and White-opsin are shown in Fig. S1d. In Fig. S1eg, we show the absorption spectra of the cloned ChR2, ReaChR and White-opsin plasmids.
AmpR(bla) Pbla
RSV
LTR
5’-R/U5
PBS
Psi+
(a)
pUC Ori
SV40 Early PolyA
Blasticidin Resistance
(b)
hSyn1
RRE
EM-7
pLenti-EF1ahChR2-EYFP-WPRE
EF1a
F1-Ori
cPPT
hChR2
SV40 PolyA
WPRE
WPRE
(d)
Ladder
attB3
ChR1
ReaChR
attB4
EYFP
hChR2
White
Opsin
E122T E162T VChR1 TS EYFP
ReaChR
LTR-DeltaU3
Citrine
3’LTR
EYFP
TETO
ReaChR
F1-Ori
BGH-PolyA
LTR-R/U5
pAAV-hSynReaChR-Citrine
Amp
ChR2
SV40-Ori
(c)
5’LTR
Citrine
CMV
pENTR-LaclWhite -Opsin
attB1
attR3
Kanamycin
T2
T1
pUC Ori
(e)
(f)
(g)
10
15
10
5
0
ReaChR
A b s o r p t io n ( a .u .)
ChR2
A b s o r p t io n ( a .u .)
A b s o r p t io n ( a .u .)
15
10
5
250
275
300
W a v e le n g t h ( n m )
325
350
6
4
2
0
0
225
White-opsin
8
225
250
275
300
W a v e le n g t h ( n m )
325
350
225
250
275
300
325
350
W a v e le n g t h ( n m )
Supplementary Figure S1. Molecular engineering of single opsins and White-opsin. (a-c)
Vector maps of (a) ChR2, (b) ReaChR, and (c) White-opsin. (d) Gel electrophoresis images of
ChR2 (digested by restriction enzymes NotI and BamH1 to fragment sizes of 1.5, 3.3 and 5.6
kb), ReaChR (digested by NotI and BglII to fragment sizes of 3 and 3.3 kb) and White-opsin
(digested by EcoRI to fragment sizes of 6.4 and 4.2 kb). (e-g) Absorption spectra of cloned
ChR2, ReaChR and White-opsin plasmids.
Visualizations of the insertion of White-opsin plasmids into HEK293 cells via optoporation or
lipofection are shown in Fig. S2. An increase in intracellular fluorescence (and a few distinct
specks attributed to the propidium iodide staining of White-opsin plasmids) after optoporation
was observed after optoporation (Fig. S2c). Similarly, propidium-iodide-stained White-opsin
plasmids were also observed in the cytoplasm of the lipofected cells (Fig. S2e and S2f).
(d)
(a)
(b)
0 min
(e)
25 min
(c)
1 min
(f)
35 min
Supplementary Figure S2. Visualizations of the insertion of White-opsin plasmids into
HEK293 cells. (a-c) Optoporation: (a) Bright field and (b) fluorescence images of propidiumiodide-stained White-opsin plasmids in the extracellular medium and (c) the increase in
intracellular fluorescence (and a few distinct specks, attributed to the propidium iodide staining
of the plasmids) after optoporation (at the site marked by the arrow in a).(d-f) Lipofection: (d)
Bright-field image of multiple HEK293 cells and fluorescence images of propidium-iodidestained White-opsin plasmids after (e) 25 min and (f) 35 min of incubation of the cells with the
Lipofectamine-plasmid mixture. Scale bar: 5 µm.
Fig. S3 shows a representative photocurrent profile induced by white light at an intensity of 0.12
Photocurrent (pA)
mW mm-2 (pulse width: 100 ms) in a ChR2-transfected HEK293 cell.
100 ms
0
ChR2
-50
-100
0.0
0.2
0.4
0.6
0.8
1.0
Time (s)
Supplementary Figure S3. White-light-induced inward photocurrent in a ChR2transfected HEK293 cell. Representative inward photocurrent profile generated in response to
white-light optogenetic stimulation (0.12 mW mm-2) at pulse width of 100 ms.
In Fig. S4, we show confocal imaging results for White-opsin-transfected HEK293 cells treated
via lipofection. The XY, XZ and YZ cross sections of the White-opsin-expressing cells are
shown in Fig. S4a. The cell morphology is observed to be compromised by the transfection
process. Fig. S4b-d show the distributions of White-opsin and the nuclear staining dye Hoechst
33242 in live White-opsin-transfected HEK293 cells.
XZ
(a)
XY
(b)
(c)
YZ
(d)
Supplementary Figure S4. Confocal imaging of HEK293 cells transfected with Whiteopsin via lipofection. (a) The XY, XZ and YZ cross sections of the White-opsin-expressing
cells. (b-d) Distributions of (b) White-opsin, (c) the nuclear staining dye Hoechst 33242 in live
White-opsin-transfected HEK293 cells and (d) composite image. Scale bar: 5 µm.