Articles
Nature Chemistry
1
5min at room temperature. The mixture was added to the cell culture medium
ꢄethods
in the absence of serum for 4h. Thereafter, cells were digested by trypsin-EDTA
(0.25%, Gibco), reseeded on a sterile 14-mm glass coverslip pre-treated with
matrigel matrix, and incubated in complete medium for 24h before PRIME
labelling.
Materials and reagents. ꢂe reagents used in this study are summarized in
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Supplementary Table 8.
LplA and
LplA were expressed in Escherichia
48
coli and puriꢁed according to a previously published method . For chemical
synthesis, 10-azidodecanoic acid (Az-9) and rel-(1R-4E-pR)-cyclooct-4-ene-
27
For primary rat hippocampal neuron culture, sterile 14-mm glass coverslips
1
-yl-N-pentanoic acid carbamate (4-TCO, previously described as TCO2 ) were
−1
26,27
were incubated with 20μgml poly-ꢄ-lysine (Sigma) solution at 37°C with 5%
synthesized as previously described
.
CO
incubated with 10μgml laminin mouse protein (Gibco) solution at 37°C with
% CO overnight. Thereafter, the coverslips were washed twice with ddH O and
2
for 24h in a 24-well plate. The coverslips were washed twice with ddH O and
2
−1
Chemical synthesis.
5
2
2
allowed to dry at room temperature. To isolate neurons, heads were separated from
neonatal Sprague–Dawley rat pups with scissors. The brain was dissected from the
skull and placed into a 35-mm dish with ice-chilled dissection solution (DMEM
with high glucose and penicillin-streptomycin antibiotics). The hippocampus was
isolated from the brains under a dissection scope, cut into small pieces (~0.5mm)
and incubated with 2ml of Trypsin-EDTA (0.25%, Gibco) at 37°C with 5% CO
2
Synthesis of 10-azidodecanoic acid (Az-9)
for 15min. The liquid was carefully aspirated and replaced with 1ml of DMEM
containing 10% FBS. The tissue fragments were dispersed by repeated pipetting
for 1min and incubated on ice for 5min. The sediment was discarded, and the
supernatant was collected and diluted by neuronal culture medium (Neurobasal
medium, B-27 supplement, GlutaMAX supplement and penicillin-streptomycin)
2
6
The synthesis of Az-9 has been described previously . Sodium azide (0.5g,
.7mmol) was added to 10ml N,N-dimethylformamide (DMF) containing
0-bromodecanoic acid (1.2g, 4.8mmol). The reaction was allowed to proceed at
7
1
room temperature overnight and then evaporated under reduced pressure. The
residue was dissolved in 15ml of 1M HCl and extracted with ethyl acetate (15ml)
three times. The organic layer, dried using magnesium sulfate, was evaporated
under vacuum. The crude product was purified by silica gel chromatography using
4
−1
to a final cell density of 6×10 cellsml . A 1-ml volume of cell suspension was
added to each well of a 24-well plate (with a pre-coated glass coverslip). Half of the
neuron culture medium was replaced with fresh medium once every four days.
Neurons were transfected on DIV7–9 (7–9 days in vitro). For each well of a
1
0–20% ethyl acetate in hexanes. Only 15ml of eluent was evaporated to afford
1
the product as a pale yellow oil (~0.15g). H NMR (400MHz, CDCl
3
, δ): 3.25 (t,
2
4-well plate, 250–500ng of plasmid DNA was mixed with 1μl of Lipofectamine
J=6.95Hz, 2H), 2.35 (t, J=7.49Hz, 2H), 1.61 (m, 5H), 1.32 (m, 9H). ESI-MS(−)
3
000 reagent in Neurobasal medium, before incubation with neurons for 45min.
−
−
calculated for C10
H
8
N
3
O
2
[M-H] : 212.14; found: 212.36.
The transfected neurons were labelled and imaged after 3–10 days. For wide-field
imaging assays, dissociated hippocampal neurons were nucleofected with
Asp81Cys
FCK-
Ace2 following the manufacturer’s protocol (Lonza V4XP-3024). For
synaptic transmission assays, dissociated hippocampal neurons were separately
Asp81Cys
nucleofected with FCK-
Ace2 and FCK-CheRiff-EGFP and co-plated in a 1:1
ratio into a well of a 24-well plate containing a pre-coated coverslip at a density of
5
1
4
.5×10 cells per well. The medium was replaced with 1ml of fresh medium after
Synthesis of rel-(1R-4E-pR)-cyclooct-4-ene-1-yl-N-pentanoic acid carbamate
h. Half of the medium was replaced by fresh medium every four days.
(
4-TCO)
2
7
The synthesis of 4-TCO has been described previously . The rel-(1R-
26
PRIME labelling. For PRIME-SPAAC labelling , hippocampal neurons were
4
E-pR)-cyclooct-4-enol (0.5g, 4.0mmol) and pyridine (0.47g, 6mmol) were
transfected with LAP-Ace(D81N) construct on DIV7–9 and labelled on DIV12–14.
dissolved in dichloromethane and cooled at 0°C. 4-Nitrophenyl chloroformate
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Transfected neurons were incubated with Tyrode’s buffer containing 5μM
LplA
(
0.96g, 4.8mmol) was added in multiple portions and the reaction was allowed to
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(
purified from bacterial culture as previously described ), 500μM Az-9, 2mM
reach room temperature. The mixture was stirred in the dark for 3h and poured
into water (15ml). The aqueous layer was extracted with diethyl ether (3×30ml).
The ether layer was combined, washed sequentially with aqueous acetic acid
ATP and 2mM magnesium acetate for 30min at 37°C. Cells were rinsed three
times with fresh Tyrode’s buffer. The neurons were then incubated with Tyrode’s
buffer containing 10μM DBCO-Cy3 for 10min at 37°C. Excess reagents were
removed by buffer replacement, three times, with fresh Tyrode’s buffer.
(
pH3, 3×30ml), saturated aqueous NaHCO (3×30ml) and saturated aqueous
3
2 4
NaCl (30ml). The organic layer was dried over Na SO , filtered, and the solvent
27
For PRIME-IEDDA labelling , transfected HEK293T cells or neurons were
was removed to give rel-(1R-4E-pR)-cyclooct-4-ene-1-yl (4-nitrophenyl)
rinsed with Tyrode’s salts solution (M&C Gene Technology) and then incubated
carbonate as a light-yellow solid.
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with Tyrode’s buffer containing 5μM
LplA, 100μM 4-TCO, 1mM ATP and
5
-Aminopentanoic acid (0.7g, 6mmol) and diisopropylethylamine (DIPEA,
1
mM magnesium acetate for 30min at 37°C. Cells were gently rinsed with Tyrode’s
1
4
.4ml, 8mmol) were dissolved in DMF (10ml) and cooled at 0°C. rel-(1R-
buffer three times, and subsequently labelled with 0.5μM tetrazine dyes in Tyrode’s
buffer for 10min. Excess reagents were removed from each well and cells were
rinsed three times before the voltage imaging experiment.
E-pR)-cyclooct-4-ene-1-yl (4-nitrophenyl) carbonate was added. The mixture
was stirred at room temperature in the dark, overnight. The DMF was vaporized
under reduced pressure and the resulting mixture was purified by column
chromatography using 5:1 petroleum ether and ethyl acetate followed by 20:1
Imaging apparatus and confocal microscopy. All of the fluorescence imaging
experiments were conducted on an inverted fluorescence microscope (Nikon-TiE)
equipped with a ×40, 1.3 NA oil immersion objective lens (except for wide-field
imaging in Extended Data Fig. 5, which used a ×20, 1.05 NA dry objective lens),
five laser lines (Coherent OBIS 488nm, 532nm, 561nm, 594nm and 637nm), a
spinning disk confocal unit (Yokogawa CSU-X1) and two scientific CMOS cameras
(Hamamatsu ORCA-Flash 4.0 v2). The microscope, lasers and cameras were
controlled with custom-built software written in LabVIEW (National Instruments,
dichloromethane and methanol to obtain the rel-(1R-4E-pR)-cyclooct-4-ene-
1
1
-yl-N-pentanoic acid carbamate (4-TCO) as a colourless oil (0.7g, 65%). H
NMR (400MHz, CD
3
OD, δ): 5.60–5.56 (m, 1H), 5.51–5.48 (m, 1H), 4.30–4.29
(
(
m, 1H), 3.09 (t, J=7.0Hz, 2H), 2.37–2.28 (m, 5H), 2.00–1.89 (m, 4H), 1.75–1.67
1
3
m, 2H), 1.64–1.57 (m, 3H), 1.53–1.47 (m, 2H). C NMR (100MHz, CD OD, δ):
3
1
3
5
77.35, 158.75, 136.10, 133.77, 81.57, 42.23, 41.23, 39.65, 35.18, 34.47, 33.49, 32.10,
+
+
0.42, 23.22. ESI-MS(+) calculated for C28
H
46
N
2
NaO
8
8
, [2M+Na] : 561.31; found:
−
−
61.41. ESI-MS(−) calculated for C H N NaO , [2M-H] : 537.32; found: 537.58.
2
8
45
2
15.0 version) and could switch between confocal and wide-field imaging modes. For
two-colour simultaneous imaging, a dual-view device (Photometrics DV2) was used
to split the emission into green/red and far-red fluorescence channels. The spectra
properties of the filters and dichroic mirrors for various fluorescent indicators
used in this study are summarized in Supplementary Table 10. The transfected
HEK293T cells or neurons on coverslips were transferred to Tyrode’s buffer before
imaging. Confocal images were acquired at 1×1 camera binning with an exposure
time of 100ms. Image analysis was performed in ImageJ/Fiji (version 1.52d).
Molecular cloning. Plasmids were constructed using the Gibson assembly
method. Briefly, the inserts and the vector were polymerase chain reaction (PCR)
amplified into linear double-stranded DNA with overlapping sequences at the
ends. These DNA fragments were mixed with Gibson assembly enzymes following
the manufacturer’s instructions (New England Biolabs). Successful clones were
verified by sequencing. Site-directed mutagenesis and linker insertion/deletion
were achieved by PCR-amplifying the vector with a pair of primers containing the
desired mutation/insertion/deletion. The CheRiff gene was a gift from A. Cohen at
Harvard University. The GCaMP6s, SF-iGluSnFR and ecliptic pHluorin genes were
provided by Y. Li at Peking University. The R-GECO1 gene was from H. Cheng at
Peking University. The Mac gene was a gift from L. Brown from the University of
Guelph. Primers used in this study are listed in Supplementary Table 9.
Electrophysiology. For single-cell electrophysiology recording, cultured neurons
were incubated in Tyrode’s buffer containing 20μM gabazine, 10μM NBQX and
25μM APV (Tyrode’s buffer containing 50nM 2-APB for HEK293T cells). The
electrophysiology experiments were performed at room temperature. Borosilicate
glass electrodes (Sutter) were pulled to a tip resistance of 2.5–5MΩ. The glass
electrode was filled with internal solution containing 125mM potassium
gluconate, 8mM NaCl, 0.6mM MgCl , 0.1mM CaCl , 1mM EGTA, 10mM
Cell culture and transfection. HEK293T cells were incubated in Dulbecco’s
modified Eagle medium (DMEM, Gibco) containing 10% vol/vol fetal bovine
2
2
−
1
serum (FBS, Gibco) at 37°C with 5% CO
2
. Cells were seeded in a 24-well plate and
HEPES, 4mM Mg-ATP, 0.4mM GTP·Na (pH7.3) and adjusted to 295mOsmkg
2
grown to 70–90% confluent for transfection. For each well, 500ng plasmid and 1μl
with 1M sucrose. The glass electrode’s position was adjusted by a Sutter MP285
micro-manipulator. The cells were clamped using an Axopatch 200B amplifier
Lipofectamine 2000 reagent were mixed in Opti-MEM medium and incubated for