A wash-free SNAP-tag fluorogenic probe based on the additive effects of quencher release and environmental sensitivity

Article abstract of DOI:10.1039/c7cc01483j

A 1,8-naphthalimide-derived fluorogenic probe was reported to label SNAP-tag fusion proteins in living cells. The probe can rapidly label a SNAP-tag and exhibit a fluorescence increase of 36-fold due to the additive effects of environment sensitivity of fluorophores and inhibition of photo-induced electron transfer from O⁶-benzylguanine to the fluorophore. The labeling of intracellular proteins has been successfully achieved without a wash-out procedure.

Full text of DOI:10.1039/c7cc01483j

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DOI: 10.1039/C7CC01483J
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A Wash-Free SNAP-tag Fluorogenic Probe based on Additive
Effects of Quencher Release and Environmental Sensitivity
Shuang Leng,†ab Qinglong Qiao,†ac Lu Miao,*a Wuguo Deng,*bd Jingnan Cui,*c Zhaochao Xu*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A 1,8-naphthalimide-derived fluorogenic probe was reportd to Various fluorescent probes have been designed to rapidly and
label SNAP-tag fusion proteins in living cells. The probe can rapidly irreversibly react with SNAP-tag and widely applied in fields of
label SNAP-tag and exhibit fluorescence increase of 36-fold due to drug monitoring, 13 protein-protein interactions, 14 fluorescent
the additive effects of environment sensitivity of fluorophores and sensors15 and super-resolution fluorescence imaging.16
inhibition of photo-induced electron transfer from O6-
While these probes are very useful and well established, it
benzylguanine to the fluorophore. The labeling of intracellular is required to wash out the unreacted or nonspecifically bound
proteins has been successfully achieved without a wash-out probes before imaging. The washout process is tedious and
procedure.
time-consuming, and may potentially limit their applications in
real-time monitoring of molecular events such as receptor-
ligand binding, endocytosis, trafficking and so on. In addition,
the complete washout is difficult because of the
uncontrollable accumulation of synthetic probes in various
organelles. To overcome this limitation, fluorogenic probes for
SNAP tags based on different mechanisms have been
developed.17 The early reported probes DRBG-48818 and CBG-
549-QSY719 consist of a fluorescence quencher attached to the
C-8 position of the guanine ring. Upon reaction with the
tagged protein, the quencher group is dissociated to release
the fluorescence intensity of the fluorophore. However, these
fluorogenic probes need complex synthesis and the large
molecule size often displays low cell permeability. Notably, the
external quenchers reduce he reaction rate and cause a big
concern of toxicity. Tan et al. reported a fluorogenic probe
BGSBD based on another mechanism of fluorophore’s
environmental sensitivity.20 The attractive feature of this
mechanism is to retain the integrity of BG group and then
defend the reaction rate. Even this probe exhibits dramatic
fluorescence turn-on of 280-fold upon being labeled to SNAP-
tag, the brightness of this probe after binding to SNAP-tag was
Protein labeling plays an increasingly important role for
visualizing and manipulating proteins in living cells. Numerous
technologies to label proteins with chemical probes have been
validated, including genetically encoded fluorescent proteins
(FPs), site-specific incorporation of non-natural amino acids
and self-labeling protein tags. However, the application of FPs
in vivo is sometimes limited by their relatively large size and
the lack of near-infrared (NIR) fluorescence. Non-natural
amino acid technology suffers from both the complex
coexpression of tRNA synthetase and non-natural amino acid-
joined proteins and incorporation of excess non-natural amino
acids. The advantages of self-labeling protein tags have been
approved in practice, attributing to need of the only
expression of a fusion protein, a large variety of probes and
excellent rate of enzymatic reactions.1, To date, various
2
protein tags have been developed, such as HaloTag,3, 4 PYP-
tag,5-7 SNAP-tag,8, 9 TMP-tag10 and BL-tag11, 12. Among them,
SNAP-tag is one of the most prominent fusion tags, which
specially reacts with O6-benzylguanine (BG) derivatives.
weak (ε = 13000, Φf = 0.143), which was not ideal for living cell
a. Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. E-mail:
zcxu@dicp.ac.cn.
imaging. Therefore, fluorogenic probes based on
environmental sensitivity mechanism with bright fluorophores
are urgently required.
In this paper, we reported a new fluorogenic probe BGAN-
2C based on these two mechanisms of quencher release and
fluorophore’s environmental sensitivity. Among environment-
sensitive fluorophores, 1,8-naphthalimide is bright (ε ~15000-
b. Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, Dalian,
China.
c. State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian
116012, China. E-mail: jncui@dlut.edu.cn.
d. Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South
China; Collaborative Innovation Center of Cancer Medicine, Guangzhou, China. E-
mail: dengwg@sysucc.org.cn.
† These two authors contributed equally.
40000, Φf
~ 0.4-0.8 in nonpolar environment) and has been
Electronic Supplementary Information (ESI) available: [Synthesis of probes,
preparation of the SNAP-tag, experimental procedures, supplementary schemes
and Figures]. See DOI: 10.1039/x0xx00000x
proved to be a good candidate21,22 to sense protein dynamics
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and interactions with targets due to its small size to be The alky chains of different lengths oriented outside and may
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embedded into protein’s pocket and high polarity-sensitivity contribute differently to environmental sDeOnsI:it1i0v.1it0y3.9T/Co7CeCva0l1u4a83teJ
with significant fluorescence responses.23, 24 According to the the PET-induced fluorescence quenching from BG group to 1,8-
analysis of the crystal structure of SNAP protein, the BG- napthalimide, compounds AN-DM, AN-2C, AN-8C and AN-12C
binding activity center of the SNAP protein is a hydrophobic without BG ligand were synthesized as control compounds
narrow cavity.25 In addition, guanine itself is an electron-rich (Scheme S1).
group and well-known to quench the fluorescence of organic
fluorophores by photo-induced electron transfer (PET). Thus,
Table 1. Optical and Kinetic Properties of SNAP-tag Substrate.
a
b
e
we hypothesized that a short linker between BG group and
1,8-naphthalimide fluorophore would take 1,8-napthalimide
into the hydrophobic environment and simultaneously inhibit
BG-induced fluorescence quenching after 1,8-naphthalimide-
BG conjugates being labeled to SNAP-tag (Fig. 1). An obvious
fluorescence turn-on response can then be expected due to
the combinational effects of quencher (BG group) release and
environmental sensitivity of 1,8-naphthalimide. Notably, the
release of guanine can be considered to be biologically safe,
since guanine is a natural constituent of the diet.
λem
λem
k2
Φ푐
Φ푑
Compounds
[M−1 s−1
]
[nm]
[nm]
BGAN-DM
AN-DM
548
548
551
550
548
555
531
548
521
0.017±0.003 0.028±0.004
0.021±0.004
-
-
-
538
-
-
BGAN-2C
AN-2C
0.015±0.005 0.367±0.035 2031.7±63.7
0.286±0.015
0.018±0.005 0.275±0.022
0.087±0.012
0.012±0.007 0.101±0.010
0.025±0.007
-
-
BGAN-8C
AN-8C
531
-
105.2±6.0
-
-
-
-
BGAN-12C
AN-12C
514
-
-
aFluorescence emissions of compounds before and bafter binding to SNAP-
tag. cQuantum yields of compounds in PBS buffer before and dafter
incubating with SNAP-tag for 2h. ek2= second-order rate constant.
In order to evaluate environment-sensitive properties, the
fluorescence of these compounds in various solvents was
firstly studied. 4-Amino-1,8-naphthalimide is a typical push-
pull fluorophore. This type fluorophore ensures that after
absorbing light the charge is transferred from the electron
donor to the electron acceptor, which creates a highly dipolar
excited state. The latter relaxes through interaction with the
dipoles of solvent and thus its emission shifts to shorter
wavelengths in less polar solvents. As shown in Fig. S2, with
decreasing solvent polarity, significant blue-shifts in emissions
(from 550 nm to 505 nm, H2O/DCM) and fluorescence
increases (~ 100 fold, Table S1) were observed for all of these
four compounds. It demonstrated all of the probes displayed
typical solvatochromic properties.
Fig. 1 Structure of compounds BGAN-R and the reaction with SNAP-tag.
After covalently binding to the SNAP-tag protein, 1,8-naphthalimide
displayed increased fluorescence due to the localization into the
hydrophobic binding environment of the SNAP-tag and the release of
fluorescence quencher (guanine). Insert: computer simulations of the
hydrophobic SNAP-tag pocket binding with BGAP-R
.
To evaluate the fluorescence quenching effects of guanine
group, the fluorescence intensities of BGAN-2C, BGAN-8C,
Based on the above hypothesis, we designed and
synthesized several compounds (BGAN-R) with BG group
directly connected to 1,8-naphthalimide by an amide group
BGAN-12C and BGAN-DM in PBS buffer solutions were
compared with that of their control compounds, respectively.
As shown in Table 1, the fluorescence intensities of
compounds without BG group were 19, 5, 2 and 1.3 fold
stronger than those with BG group, respectively, which
(Fig. 1). 4-N,N-dimethylamino-1,8-naphthalimide (BGAN-DM
)
was expected to act as a molecule rotor and sense the
environmental changes by means of inhibiting twisted
intramolecular charge transfer (TICT) process. Secondary
amino group substituted 1,8-naphthalimide derivatives
demonstrated that guanine did act as
compounds BGAN-2C BGAN-8C BGAN-12C and BGAN-DM
Then, these compounds were examined to label SNAP-tag.
Compounds BGAN-2C BGAN-8C BGAN-12C and BGAN-DM all
a quencher in
,
,
.
(
BGAN-2C, BGAN-8C and BGAN-12C) were anticipated to
sense environmental changes only undergoing intramolecular
charge transfer (ICT). In order to optimize the environmental
sensitivity of 1,8-naphthalimide, alkyl chains of different
lengths were introduced to the electron-donor groups as
,
,
show weak fluorescence in PBS buffer solutions. After SNAP-
tag was labeled with these fluorophores, different degrees of
fluorescence enhancement were observed (Fig. 2a). The
shown in compounds BGAN-2C, BGAN-8C and BGAN-12C. The
fluorescence of BGAN-2C, BGAN-8C and BGAN-12C were
results of computer simulations showed that, after covalently
binding to SNAP-tag, 1,8-naphthalimide existed in the
hydrophobic pocket of the protein which positioned mainly by
Pro 138 and Pro 14025, and C-4 position of 1,8-naphthalimide
exposed at the outside of the cavity (inset of Fig. 1, Fig. S1).
increased after 2 h with a turn-on ratio of 36, 23 and 9 fold,
respectively. We noticed after much longer time the
fluorescence increases of BGAN-8C and BGAN-12C were all
over 30 fold while the fluorescence of BGAN-DM exhibited
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only 2 fold enhancements after covalently binding with SNAP- covalent binding between the SNAP-tag and theVBieGw AlritgicalenOdn.linIet
tag. These results demonstrated that the fluorescence suggests that fluorogenic probes for DpOrI:o1t0e.1in03s9/sCh7CoCu0ld148b3eJ
enchancement is a result of both the inhibition of guanine delicately designed. Above all, BGAN-2C was the much better
quenching and 1,8-naphthalimide’s environmental sensitivity. probe than other ones that we designed for no-wash SNAP-tag
For compound with a longer alkyl chain, the fluorescence labeling.
enhancement contributed from environmental sensitivity
becomes more significant. After being labeled to SNAP-tag, the
long alkyl chain of probe may increase the hydrophobicity and
decrease the polarity of the binding cavity. Unfortunately,
BGAN-8C and BGAN-12C displayed much lower reaction rate.
Fluorescence enhancement contributed from environmental
sensitivity can be further explained based on the polarity-
sensitivity of different compounds as shown in Fig. 2b. BGAN-
DM displays fluorescence increase only in low polarity
environments, but still weak intensity in medium polarity
environment where TICT dominates fluorescence. While other
three compounds BGAN-2C, BGAN-8C and BGAN-12C, only
governed by ICT, were much more sensitive to medium
polarity. It can be concluded that the interaction of BGAN-DM
with BG-binding cavity of SANP-tag was not so tight to inhibit
intramolecular twist, and polarity of this cavity is medium and
suitable for ICT fluorophores. Then molecular rotors based on
other fluorophores with larger size may have a chance to be
designed as fluorogenic probes for SNAP-tag.
The binding characteristics of the probes with SNAP-tag
were further investigated by SDS-PAGE (Fig. 2c). Purified SNAP-
tag was incubated with these four compounds and analysed.
Based on bands appeared in the gel and mass analysis (Fig. S3),
these compounds all reacted with SNAP-tag. But only two
strong fluorescence bands in accordance with BGAN-2C and
BGAN-8C were observed. These fluorescence responses were
consist with that in solutions. Then the labeling reaction of the
SNAP-tag-expression cell lysate with BGAN-2C was analyzed by
SDS-PAGE (Fig. 2d). The results showed that the reaction
between BGAN-2C and SNAP-tag is highly selective and
produced only one single product in the SNAP-tag-expression
cell lysate.
The detailed kinetic analysis of the protein labeling was
carried out by monitoring the fluorescence intensity of the
probes, in which the concentration of probes and the protein
were 5 μM (Fig. 2e). The time required for 50% labeling of
SNAP-tag was about 1.1 min for BGAN-2C, 46 min for BGAN-8C
and a much longer time for BGAN-12C (Fig. S4). None of the
Fig. 2 Fluorescence spectral, SDS-PAGE, and kinetic analyses of the labeling
reaction between SNAP-tag and probes. (a) Fluorescence spectra of BGAN-
2C (red lines), BGAN-8C (black lines), BGAN-12C (blue lines) and BGAN-DM
(pink lines) in the absence (solid lines) or presence (dotted lines) of SNAP-
tag. (b) The plots of quantum yield of the probes in various solvent (from
left to right: DCM, THF, EA, acetone, DMF, MeOH, and H2O, respectively). (c)
SDS-PAGE analysis of labeling reaction of purified SNAP-tag (lane 1) with
BGAN-2C (lane 3), BGAN-8C (lane 4), BGAN-12C (lane 5) and BGAN-DM
(lane 6), lane 2 was protein marker. (d) SNAP-tag expressed E. coli lysates
was labeled by BGAN-2C (lane 1), and the purified SNAP-tag covalently
bound with (lane 2) or without (lane 3) BGAN-2C. (e) Time course of
fluorescence intensity of probes in the presence of SNAP-tag. (f) Plots of
BGAN-2C concentration versus the pseudo-first-order rate constant.
Subsequently, no-wash live-cell fluorescent labeling of
intracellular proteins was carried out using BGAN-2C. HEK 293
cells which expressed SNAP-tag fusion proteins in cytosol,
mitochondria, and nucleus were investigated. First, 5 μM
BGAN-2C was added to the live-cells with non-specific SNAP-
tag expression and incubated for 20 min. The fluorescence
imaging was taken without washing the cells. An obvious
fluorescence was observed in cytosol (Fig. 3a-b), which
demonstrate the high cell-permeable of the probe that
benefiting for intracellular protein labeling. In contrast,
fluorescence was hardly detected in the non-transfected cells
(Fig. 3a-b). Toxicity test indicated that the probe is nontoxic to
the HEK 293 cells (Fig. S6). In the further investigation,
commercialized pSNAPf-Cox8A and SNAP-H2B (a gift from Prof.
Qingkai Yang) fusing SNAP-tag to cytochrome c oxidase
subunit 8 (Cox8A) and human histone H2B were transiently
probes displayed
a time-dependent alteration of the
fluorescence intensity in the absence of SNAP-tag. In further
kinetic analysis, the second-order rate constant (k2) for
reaction between BGAN-2C and SNAP-tag was determined to
be 2031.7±63.7 M−1 s−1 (Table 1), which was 20-fold higher
than BGAN-8C (105.2±6.0 M−1 s−1, Fig. S5). The labeling rate of
BGAN-2C is much faster than quencher-based SNAP-tag
fluorogenic probes, such as DRBG-488 (396±32 M−1 s−1)18 and
CBG-549-QSY7 (1027±457 M−1 s−1)19, and comparable with that
of BGSBD (7200 ± 1600 M−1 s−1). These results demonstrated
that the longer the hydrophobic carbon chains of the probes,
the slower the reaction rate with SNAP-tag. The reason may be
that most of hydrophobic cavity of proteins was easy to be
occupied by hydrophobic alkyl chains which restrained the
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transfected to cells. After 20 min incubation with 5 μM BGAN- promising to develop fluorogenic probes with dedicated
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DOI: 10.1039/C7CC01483J
2C, the cells were observed without washout of the probe. modifications to these fluorophores.
And clear green fluorescence was revealed without obviously
This work was supported by the National Natural Science
background fluorescence. This is in sharp contrast with Foundation of China (21422606, 21502189) and Dalian
conventional green SNAP-tag SNAP-Cell® 505-Star, which Cultivation Fund for Distinguished Young Scholars
required a lengthy post incubation washout period before (2014J11JH130 and 2015J12JH205).
effective visualization (Fig. S7). Co-staining of the probe with
red emission from Mitotracker Deep Red confirmed that the
probe localized in mitochondrial area. Meanwhile, specific
Notes and references
labeling of SNAP-H2B fusion proteins in the nucleus was also
observed. These results demonstrated that BGAN-2C can be
applied to visualize a target proteins at a specific location with
no-wash procedure required.
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Fig. 3 No-wash HEK 293 live-cell imaging of SNAP-tagged proteins labeled
with BGAN-2C. Cells were incubated with 5 µM BGAN-2C at 37 oC for 20
min. Row (a) shows HEK 293 cells without SNAP-tag expression. Row (b)
displays HEK 293 cells transiently expressing SNAP-tag for nonspecific
intracellular tagging, and row (c) and (d) present HEK 293 cells transiently
expressing SNAP-Cox 8A and SNAP-H2B, respectively. For each series: (i)
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In conclusions, a 1,8-naphthalimide-derived fluorogenic
probe BGAN-2C was designed on the additive effects of
quencher release and fluorophore’s environmental sensitivity.
BGAN-2C displayed 36 fold fluorescence increases after being
labeled to SNAP-tag. The high signal-to-noise ratio and
reaction rate ensure BGAN-2C a good probe capable of
labelling proteins in living cells without washout procedure.
Different substituents in C-4 position of 1,8-naphthalimide
were introduced to examine protein labelling properties. It’s
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concluded
that
ICT-governed
environmental-sensitive
fluorophores are more suitable to design fluorogenic probes
for SNAP-tag. For molecular rotor type fluorophores, it is
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