A General Strategy to Control Viscosity Sensitivity of Molecular Rotor-Based Fluorophores

Article abstract of DOI:10.1002/anie.202011108

Molecular rotor-based fluorophores (RBFs) have been widely used in many fields. However, the lack of control of their viscosity sensitivity limits their application. Herein, this problem is resolved by chemically installing extended π-rich alternating carbon-carbon linkages between the rotational electron donors and acceptors of RBFs. The data reveal that the length of the linkage strongly influences the viscosity sensitivity, likely resulting from varying height of the energy barriers between the fluorescent planar and the dark twisted configurations. Three RBF derivatives that span a wide range of viscosity sensitivities were designed. These RBFs demonstrated, through a dual-color imaging strategy, that they can differentiate misfolded protein oligomers and insoluble aggregates, both in test tubes and live cells. Beyond RBFs, it is envisioned that this chemical mechanism might be generally applicable to a wide range of photoisomerizable and aggregation-induced emission fluorophores.

Full text of DOI:10.1002/anie.202011108

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: A General Strategy to Control Viscosity Sensitivity of Molecular
Rotor-Based Fluorophores
Authors: Songtao Ye, Han Zhang, Jinyu Fei, Charles H. Wolstenholme,
and Xin Zhang
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10.1002/anie.202011108
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RESEARCH ARTICLE
A General Strategy to Control Viscosity Sensitivity of Molecular
Rotor-Based Fluorophores
Songtao Ye [a], Han Zhang [a], Jinyu Fei [a], Charles H. Wolstenholme [a] and Xin Zhang*[a,b]
[a]
[b]
S. Ye, H. Zhang, J. Fei, C.H. Wolstenholme, Prof. X. Zhang
Department of Chemistry, Pennsylvania State University
University Park, PA 16802, USA
E-mail: xuz31@psu.edu
Prof. X. Zhang
Department of Biochemistry and Molecular Biology
Pennsylvania State University
University Park, PA 16802, USA
Supporting information for this article is given via a link at the end of the document.
Abstract: Molecular rotor-based fluorophores (RBFs) have been
widely used in many fields. However, it remained enigmatic how to
rationally control their viscosity sensitivity, thus limiting their
application. Herein, we resolve this problem by chemically installing
extended p-rich alternating carbon-carbon linkage between the
rotational electron donor and acceptor of RBFs. Our data reveal that
the length of p-rich linkage strongly influences the viscosity sensitivity,
likely resulted from varying height of the energy barriers between the
fluorescent planar and the dark twisted configurations. This
mechanism allows for the design of three scaffolds of RBF derivatives
that span a wide range of viscosity sensitivities. Application of these
RBFs is demonstrated by a dual-color imaging strategy that can
differentiate misfolded protein oligomers and insoluble aggregates
both in test tubes and live cells. Beyond RBFs, we envision that this
chemical mechanism might be generally applicable to a wide range of
photoisomerizable and aggregation-induced emission fluorophores.
conformational change of RBF. Despite extensive efforts to
chemically regulate spectra and fluorescence quantum yields of
these fluorophores[5], it remains a challenge how the viscosity
sensitivity can be rationally controlled via chemical modifications
on the wide range of fluorophores.
Introduction
Photoisomerizable fluorophores, molecular rotor-based
fluorophores and aggregation-induced emission fluorophores are
emerging classes of molecules that rotate along specific bonds at
excited state[1]. This feature has enabled their broad applications
in biological systems, membrane chemistry and material
science[2]. In solvents wherein rotation between electron donor
and acceptor is allowed, fluorophores undergo twisted
conformations wherein intramolecular charge-transfer rapidly
occurs at excited state to effectively quench fluorescent emission.
When rotation is restricted, non-radiative decay is voided,
resulting in significant fluorescence emission. As one major family
of these fluorophores, molecular rotor-based fluorophores (RBFs)
have been appreciably used as fluorogenic molecules in different
fields, particularly as biosensors because of their ability to exhibit
enhanced fluorescence signal in viscous environments[3].
Structurally, most RBFs possess an electron donor, electron
acceptor and linkages to allow charge transfer. When RBFs are
buried in viscous environments, inhibition of the low energy
twisted intramolecular charge transfer (TICT) state leads to
enhanced fluorescence[4]. One of the most featuring properties of
RBF is viscosity sensitivity, which defines RBF's transition from
bright to dark state that is linked to the viscosity-dependent
Figure 1. Installation of p-rich alternating linkages enhances the rotational
energy barrier and changes the viscosity sensitivity of RBFs. (a) Comparison
between commonly used simple RBFs with single bond rotational axis and
extended RBFs possess p-rich alternating linkages. The p-rich linkages include
both polyene and polymethine type. (b) The proposed Jablonski diagram
showing the role of p-rich linkages to enhance the inherent rotational energy
barrier (Ea) of RBFs. (c) Fluorescence of extended RBFs can be activated in
lower a viscosity environment compared to simple RBF analogs, likely because
the increased Ea restricts the rotation-induced non-radiative decay.
In this work, we demonstrate a new mechanism to control the
excited state rotational barrier leading to the twisted conformation,
thus controlling RBF’s emission in response to environmental
viscosity as a rotational restriction. As an attempt to control the
fluorogenic behavior of RBFs, we chose to modify the simplest
fragment—linkage, by installing p-rich alternating bridges
between the two rotational moieties (Figure 1a). As described by
the Jablonski diagram[6] (Figure 1b), excited RBFs could either
return to the ground state through fluorescence or through internal
rotation which leads to a twisted, non-radiative configuration[7].
We hypothesize that the chemical modification of linkages could
1
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Figure 2. Viscosity sensitivity of RBFs is significantly and systematically affected by introduction of extended linkages. (a-c) Structure of a library of 7 RBFs from 3
scaffolds. (d-f) Fluorescence intensity of RBFs in a series of ethylene glycol/glycerol mixture with defined viscosities as shown in Table S1. (g-i) Viscosity sensitivity
calculated based on the fluorescence intensity in ethylene glycol and glycerol mixture. (See Experimental Procedures in SI regarding the measures and calculations)
change the rotational energy barrier leading to the twisted dark
state, therefore control the population of each energy dissipation
pathway, hence tuning the fluorogenic behavior of RBFs.
Traditionally, many RBFs often incorporate a single bond
Enabled by this method, RBFs with finely tuned fluorogenic
properties were further used to detect protein aggregations with
different compactness both in vitro and in live cells.
between electron donor and acceptor. In
a non-viscous
environment, rotations around this single bond will experience
minimal resistance because of the low inherent rotational barrier.
As a result, they are poorly fluorescent as the excited state energy
is constantly dissipated through non-radiative TICT process. Only
in a rigid environment (polymer films[8], protein aggregates[9], etc.),
the internal rotation of simple RBFs would be restricted, leading
to an enhanced fluorescence. Installing p-rich alternating bridge,
on the other hand, forces excited RBFs to undergo trans-cis (E-
Results and Discussion
p
Introduction of extended -rich alternating linkages
reduces the viscosity sensitivity of RBFs
We started by incorporating varying linkers in three
representative RBFs: benzothiazolium, 4-hydroxybenzylidene-
imidazolinone (HBI) and 2-dicyanomethylene-3-cyano-2,5-
dihydrofuran (DCDHF) (Figures 2a-2c; Figure S1-S4; Table 1).
These linkers contain polymethine structures, thus enabling
multiple modes of rotation and isomerization. Thioflavin-T (ThT),
a benzothiazole-based fluorophore, has been extensively used as
a fluorogenic probe to detect amyloid fibers[12]. Modification on the
ThT was achieved by introducing a methine group between the
electron-donating aminobenzene group and electron-withdrawing
benzothiazolium ring, affording 1a, a stilbene type analog of ThT.
Further extension of the methine bridge led to 1b. For HBI-based
fluorophores, 2a and 2b were synthesized mimicking the
chromophore core structure of the green fluorescent protein
Z) isomerization in order to adopt a twisted conformation[10]
.
Unlike single bond rotation, such E-Z isomerization was
previously suggested to have a high energy barrier because the
p-electrons along the alternating carbon-carbon bonds are largely
equalized, making rotations along these bonds to be difficult[11]
.
As a result, compared to simple RBF analogs, extended RBFs
could fluoresce in less viscous environments, in which the
inherent rotational energy barrier plays the dominant role to
restrict internal rotation. Through photophysical characterization,
we demonstrated that the installation of short alternating p-rich
bridges may act as a general modification to control the
fluorogenic behavior of RBFs via enhanced energy barrier.
2
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Table 1. Photophysical properties of fluorophores measured in 100% glycerol.
Stokes shift
ε
Brightness
(B = ε x ϕ)
Viscosity
sensitivity (x)
RBFs
λAbs (nm)
λEm (nm)
QY (ϕ)
(nm)
(M-1cm-1)
ThT
1a
423
531
595
450
485
500
600
492
600
714
518
630
540
650
69
30,000
49,000
17,000
24,000
30,000
87,000
86,000
0.16
0.75
0.51
0.03
0.08
0.19
0.65
4,800
36,750
8,670
720
0.79
0.51
0.32
0.68
0.26
0.72
0.52
69
1b
2a
119
68
2b
3a
145
40
2,400
16,530
55,900
3b
50
(GFP)[9a, 13], wherein the aminobenzene electron donor and
imidazolinone electron acceptor were connected by one methine
group for 2a and two methine groups for 2b. For the DCDHF-
based fluorophores 3a, a single methine-based linker was used
to connect the aminobenzene electron donor and DCDHF
electron acceptor, resulting 3b. As shown by fluorescence
spectroscopic measurements, all of these fluorophores emit
stronger fluorescence in the viscous glycerol compared to non-
viscous water and non-polar 1,4-dioxane (Figures S3-S5, Table
S2), suggesting that the restriction of internal rotation
accompanied by enhanced viscosity is the major cause for the
fluorogenic response of these newly resolved fluorophores.
Furthermore, we carried out the fluorescence lifetime
measurement was carried out using a time-correlated single
photon counting (TCSPC) experiment (Figure S6 and Table S3).
Except for ThT and 2a, other fluorophores exhibited modestly
elongated lifetime when viscosity of solvent increased from 30 to
1078 mPa•s, suggesting the inhibition of TICT in high viscosity.
Next, we sought to evaluate the viscosity sensitivity of the
newly synthesized RBFs. Viscosity sensitivity (x) is a quantitative
measure for RBFs to describe the relationship between
fluorescence intensity and viscosity[14]. Based on the Föster-
Hoffmann equation (Eq 1),
mPa·s, followed by a 2.8-fold change for 1a and a 1.8-fold change
for 1b. In agreement with the fluorescence intensity measures,
ThT showed the highest x value, reaching 0.79. For 1a and 1b
that incorporate extended linkages, their x values decreased with
increasing number of double bonds in the linkages: 0.50 for 1a
and 0.32 for 1b. The x values were also measured for the HBI and
DCDHF scaffolds. In both cases, RBFs that bear extended
linkages exhibited smaller x values (x = 0.68 for 2a, x = 0.26 for
2b; x = 0.72 for 3a, x = 0.52 for 3b). Additionally, we measured
fluorescence quantum yields (Φ) in a series of methanol and
glycerol mixture (Figure S7). As the viscosity decreased, the Φ
values of RBFs with extended linkages declined less significantly
than RBFs with a single bond linkage, further confirming the
smaller x values for RBFs bearing extended linkages. Taken
together, these results suggest the installation of π-rich
alternating linkages could reduce the viscosity sensitivity of RBFs,
therefore change their fluorogenic behaviors.
Extended linkages increase the excited state energy
barrier for RBFs
Here, we attempted to understand the underlying
mechanism that governs the viscosity sensitivity of RBFs with
varying p-rich linkages. Most RBFs quench fluorescent emission
through a rapid non-radiative TICT process[4b]. Consequently,
restriction of internal rotation between a high-energy planar (or
near planar) configuration and a low-energy twisted configuration
in the excited state could determine the viscosity sensitivity for
RBFs. Herein, we hypothesized that the introduction of p-rich
linkage altered the rate of internal rotation (krot) between the
planar and the twisted configuration of RBFs. According to the
Arrhenius equation (Eq 2), krot can be quantified as a function of
the rotational energy barrier Ea in comparison with thermal
fluctuation kBT,
ꢀꢁꢂ∅ = ꢃꢀꢁꢂꢄ + ꢅ
(1)
the fluorescence intensity maxima (proportional to quantum yield
Φ) were measured and plotted against the solvent viscosities in a
double logarithmic scale to give a linear correlation, whose slope
defines the value of x. The larger the x value is, the steeper
fluorescence intensity changes as a function of viscosity. In this
regard, we measured the fluorescence intensity of these RBFs in
a series of binary mixtures of ethylene glycol and glycerol (EG/G)
with known viscosities (Figures 2d-2e)[5b], and derived x values for
each RBFs (Figures 2h-2i). Given that ethylene glycol and
glycerol have similar polarity, the fluorescence intensity measured
in EG/G mixtures should depend solely on solvent viscosity. For
the benzothiazole scaffold, ThT exhibited a 4.7-fold increase of
fluorescence intensity when viscosity changes from 81 to 621
!"
#
&
ꢆ!"# = ꢇ(ꢄ)ꢈ$%
(2)
3
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where A is a pre-exponential factor that is solely viscosity
dependent. Given that all factors in the Arrhenius equation are
identical except Ea when comparing simple and extended RBFs
in the same solvent, the difference of their viscosity sensitivity is
ultimately coming from Ea.
We measured fluorescence emission intensity of RBFs at
varying temperatures and viscosities. ThT and 1a were chosen
as an initial set of RBFs, with the expectation that their linkages
could generate either low or high Ea values. For ThT with a single
bond as linkage, we observed that the fluorescence measure at
different temperatures overlaps well (Figure 3b, left panel),
despite minor deviations. This result showed that the internal
rotation of ThT at excited state operates in a temperature-
independent manner, indicating a minimal Ea as the model I
suggested. A previous study calculated Ea of ThT to be close to
thermal fluctuation (kBT)[12a], which further corroborate our finding.
When the same experiments were carried out using 1a with an
extended linkage (Figure 3b, right panel), results were more
consistent with the model II. The fluorescence intensity difference
of 1a at various temperatures became greater as we increased
solvent viscosity, suggesting
a high Ea for 1a. Besides
benzothiazole scaffold, similar trend was observed in both HBI
scaffold and DCDHF scaffold (Figure S8). This Ea change,
accompanied by incorporating p-rich alternating linkages, would
significantly alter the viscosity sensitivity of RBFs, through
rerouting the energy dissipation pathway at excited state. When
buried in high viscosity environment, RBFs dissipate most
absorbed photon energy through fluorescence since internal
rotations are inhibited by local compactness. Even when the
environmental viscosity is reduced, RBFs with high Ea would
maintain a higher fraction of fluorescence compared to their low
Ea analogs, because high Ea would hinder the rotation process
leading to non-radiative internal conversion. Consequently, RBFs
with high Ea exhibit a low viscosity sensitivity x. Collectively, these
results suggested that the introduction of p-rich linkage will
increase the Ea for RBFs, therefore reduce their viscosity
sensitivity.
Previously, the incorporations of extended p conjugation were
reported on other fluorophores with different turn-on mechanism.
Ashoka et al. reported an enhanced polarity sensitivity of
dioxaborine probe with extended p conjugation, indicating the
extended p conjugation might increase the charge transfer
character for solvatochromic dye[16]. Vu et al. revealed the
extended conjugation in phenyl-BODIPY fluorophore—a class of
RBF operates with photoinduced electron transfer (PET)
mechanism—exhibited reduced viscosity sensitivity and
enhanced temperature sensitivity[17]. These works, along with our
investigation for RBFs with extended p linkages, should provide a
comprehensive understanding as how extended p conjugation
modification would alter the photophysical properties of different
fluorophores, and facilitate future design of fluorophores with
controlled fluorogenicity.
Figure 3. Temperature dependence reflects the magnitude of the rotational
energy barrier (Ea) of RBFs. (a) Proposed models suggest that heights of
rotational energy barriers determine the fluorescence intensity pattern in the
solvents with different viscosity at different temperatures. While two ideal cases
are listed as a low energy barrier and a high energy barrier, the real RBFs
usually are in between these two ideal cases. (b) Fluorescence pattern of ThT
and 1a.
The Arrhenius equation allows us to predict how fluorescence
intensity of RBFs is dependent on temperature and viscosity. In
this regard, two models are envisioned for RBFs with low and high
Ea values. In the first model, where Ea is minimal (<kBT) (Figure
3a, top panel), the above Arrhenius equation could be simplified
to ꢆ!"# = ꢇ(ꢄ), suggesting that the kinetics of internal rotation is a
temperature-independent process. Therefore, when we measure
the fluorescence intensity in methanol and glycerol mixtures at
different temperatures, we expect to observe that the
fluorescence intensity is dependent solely on viscosity but not
temperature. In other words, an overlap of fluorescence intensity
on different temperatures is expected[15]. In the second model,
where a high Ea (>>kBT) is expected (Figure 3a, bottom panel),
both solvent viscosity and temperature could affect krot, and
therefore expected results are dependent on viscosity of the
experiments. When solvent viscosity is low, the pre-exponential
factor ꢇ(ꢄ) was small that only marginal krot differences are
expected at different temperatures. As a result, we predicted a
good overlapping for fluorescence intensity in the low viscosity
region at different temperatures. As the solvent viscosity
increases, the pre-exponential factor ꢇ(ꢄ) becomes more
RBFs with extended linkers enable an early detection of
low-viscosity misfolded protein oligomers in-vitro
RBFs have been widely applied as powerful fluorogenic
probes that can report on biological events that involve viscosity
changes. For instance, RBFs have been extensively used to study
protein aggregation—a process that starts from the formation of
soluble protein oligomers with low-viscosity and gradually evolves
into insoluble protein aggregates with high-viscosity[9]. Given that
the viscosity sensitivity of RBFs was tunable by varying linkages,
we asked whether this structural change could affect the detection
of protein aggregation. Because RBFs with extended linkers
dominant. In this scenario,
a
temperature dependence
phenomenon is projected for RBFs with high Ea at high viscosity.
4
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exhibit lower x values, therefore we expect them to maintain a
strong fluorescence in misfolded protein oligomers that have low
viscosity. By contrast, RBFs with higher x values are envisioned
to only emit strong fluorescence in a high viscous environment
such as insoluble aggregates, as their high x values — a steeper
fluorescence intensity change as a function of viscosity — would
result in low fluorescence quantum yield in the less viscous
environment, losing their resolution to resolve protein oligomers.
(See Table S4, Figure S9 and Note S1 regarding the resolution of
fluorophore in different viscosity regions).
During protein aggregation, a reduced local polarity is
accompanied with enhanced viscosity because of the exposed
hydrophobic protein cores. Without careful characterization, this
would complicate the analysis since most fluorophores with
electron “push-pull” nature often exhibit solvatochromic
behavior[18]. Here, benzothiazolium class fluorophores are chosen
as model compounds to detect protein aggregations as their
fluorogenic response are predominantly coming from enhanced
folded monomers into amyloid fibers via the formation of
misfolded oligomers, termed protofibrils[22]. Despite that ThT was
able to monitor the fibrilization of a-syn[23], it showed minimal
fluorescent signal from 0-18 h (Figure S10a), largely because ThT
cannot detect protofibrils that have been shown to be the primary
species accumulated between 4-18
h
under identical
experimental conditions[9a]. Although 1a failed to detect a-syn
protofibrils (Figure S10b), its fluorescent intensity increased faster
than that of ThT, suggesting a better resolution to detect early
stages of fibrilization compared to ThT. By contrast, 1b exhibited
enhanced fluorescence as early as 5 h when protofibrils start to
appear. A 29-fold fluorescence intensity increase was further
observed at 13 h (Figure S10c). This observation indicates that
the fluorescence signal for 1b is predominantly coming from low
viscosity oligomeric protofibrils. Given that the x values decrease
in the sequence of ThT, 1a and 1b, these results collectively
support the notion that RBFs with higher Ea values (corresponding
to lower x values) are more suited to detect low-viscosity protein
viscosity, not from reduced polarity (Figures S3 and S5, Table S2). oligomers and early-stage fibrilization species. Whereas, RBFs
with lower Ea values (corresponding to higher x values) only
detect high-viscosity insoluble aggregates and late-stage fibrils.
Beyond proteins that form amyloid fibrils, we also examined
whether a similar trend would be observed for proteins that form
amorphous aggregates. Following an established AggTag
method that can report POI aggregation through enhanced
fluorescence[9], we genetically fused HaloTag onto protein-of-
interest (POI) whose aggregation is known to form amorphous
aggregates (Figure 4b). Subsequently, we installed HaloTag
reactive warhead to 1a and 1b (Figure 4c), resulting in P1a and
P1b as HaloTag substrates that can covalently react with the POI-
HaloTag fusion protein (Figure 4b). Both P1a and P1b incorporate
a rigid cyclohexane linker to avoid undesired fluorescence
response as a result of interacting with HaloTag surfaces (Figures
S11 and S12). When P1a or P1b is covalently conjugated to the
POI-Halo protein, fluorescence of probes is quenched. Formation
of misfolded oligomers and/or insoluble aggregates of the POI,
however, inhibits quenching and yields turn-on fluorescence via
intra-molecular interactions with probes (Figure 4c).
We first chose superoxide dismutase 1 (SOD1), mutations of
which lead to protein aggregation that have been reported to
damage motor neurons and associate with familial amyotrophic
lateral sclerosis (fALS)[24]. In particular, SOD1 (A4V) mutation is
commonly found in North America and cause rapid disease
progression[25]. Upon incubating P1b conjugated SOD1(A4V)-
Halo at 59˚C, the fluorescence of P1b reached a maximum at 5
min, surpass the kinetics of P1a, whose fluorescence plateaued
at 12.5 min. Notably, both probes showed faster kinetics than
turbidity, a signal that is predominantly coming from insoluble
Figure 4. Extended RBFs enable the detection of misfolded protein oligomers
protein aggregates. In addition to SOD1(A4V), a destabilized
in vitro. (a) Detection of a-synuclein aggregation by ThT, 1a and 1b. (b) Scheme
mutant of firefly luciferase Fluc(R188Q) was tested with the
identical method[26]. Similarly, fluorescence of P1b was activated
of the AggTag method to monitor protein aggregation. (c) Structure of P1a and
P1b. (d-e) Aggregation of (d) SOD1(A4V) at 59 ºC and (e) Fluc(R188Q) at 57
ºC are monitored by the fluorescence intensity of P1a and P1b, as well as the
earlier than P1a, indicating that RBFs with lower x values are
increased turbidity. In both experiments, 42 µM proteins were incubated with 5
suitable to detect early events, in particular the intermediate
µM probes prior to heat-induced aggregation.
misfolded oligomers during protein aggregation. Collectively,
these data suggest that tuning down x values through attaching
extended linkers may serve as a novel approach to modify RBFs
Previously, benzothiazolium class fluorophore has been
to enable enhanced sensitivity towards biological events with low
reported to detect amyloid fibers which are associate with
viscosity.
neurodegenerative diseases, including a-synuclein[19]
,
Tau
protein[20], amyloid ß[21], etc. To this end, we monitored the
aggregation of a-synuclein (α-syn) with ThT, 1a and 1b under
prolonged agitation (Figure 4a). Aggregation of a-syn turned
5
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RBFs with extended linkers enable an enhanced
sensitivity towards low-viscosity misfolded protein
oligomers during live-cell imaging
To demonstrate this two-color imaging strategy, we chose Htt-
polyQ, which code for the Huntingtin exon 1 protein with
expansion of a polyglutamine tract within its N-terminal domain[28]
.
The aggregation of Htt-polyQ that contains long-repeats of polyQ
(> 36Q) is well known for its association with Huntington's disease.
To test how P2a and P2b activate their fluorescence emission in
misfolded oligomers and/or insoluble aggregates, we expressed
HaloTag-fused Htt-46Q and Htt-110Q in HEK293T cells and
treated cells with equal concentration (0.5 µM) of P2a and P2b
during protein translation. Overexpression of Htt-46Q has been
found to form soluble oligomers in the cytosol in mammalian cells,
Finally, we applied RBFs with distinct x values in live-cell
imaging to differentiate biological events that bear varying
viscosities. As a demonstration, we elected to develop an imaging
strategy to differentiate misfolded oligomers (low viscosity) from
insoluble aggregates (high viscosity) in live cells[27]. To this end,
we synthesized HBI-based HaloTag substrates P2a (x = 0.68)
and P2b (x = 0.26) (Figure 5a). The zero net charge has enabled
a good cell membrane permeability for HBI derivatives (P2a and
P2b) compared to the positively charged benzothiazolium
derivatives (P1a and P1b), whose fluorescent signal was mostly
found when binding with extracellular cell debris in live cell
imaging applications (Figure S13). Similar to P1a and P1b, the x
values of P2a and P2b determine how they can differently activate
fluorescence in response to local viscosity. Because P2a has a
higher x value than P2b, we expect that the viscosity to activate
fluorescence of P2a would be much higher than that of P2b. More
importantly, the fluorescence responses for HBI class
fluorophores are predominantly resulted from enhanced viscosity
during protein aggregation, as their fluorescence showed minimal
response in solvents with different polarities (Figures S4 and S5,
Table S2). Given that P2a and P2b exhibit non-overlapping
fluorescence spectra (Figure S2), we envision a dual-probe
imaging strategy wherein P2b emits fluorescence as soon as
misfolded oligomers form and P2a only is activated by the much
more tightly packed insoluble aggregates.
while Htt-110Q coalesce into dense insoluble aggresomes[29]
.
While P2a showed notable fluorescence signal in Htt-110Q-Halo
(middle panel in Figure 5c), no significant fluorescence was
detected in cells expressed Htt-46Q (top panel in Figure 5c). This
quenched P2a fluorescence was not caused by the low
expression level of Htt-46Q-Halo, whose expression level was
confirmed by the fluorescence signal from P2b (top panel in
Figure 5c). Thus, these data suggest that fluorescence of P2a is
unable to be activated by misfolded oligomers as these oligomers
tend to be loosely packed. P2b, on the other hand, not only
showed punctuated fluorescence in cells expressed Htt-110Q-
Halo (middle panel in Figure 5c) but also exhibited a strong
diffusive fluorescent structure for Htt-46Q-Halo (top panel in
Figure 5c). Such turn-on fluorescence of P2b was not observed
in cells expressing HaloTag (known not to misfold or aggregate,
bottom panel in Figure 5c). Compared to the HaloTag, Htt-46Q-
Halo induced a 4-fold fluorescence increase (Figure S14),
suggesting that the diffusive P2b fluorescence of Htt-46Q-Halo
arises from misfolded oligomers. In summary, these results
indicated that controlling viscosity sensitivity of RBFs could
enable a two-color imaging strategy to distinguish misfolded
oligomers from insoluble aggregates in live cells, thus potentiate
an unprecedented imaging capacity to examine how small
molecule proteostasis regulators can intervene the process of
protein aggregation. Going beyond the AggTag method, a similar
two-color imaging strategy with non-covalent recognition of
aggregates of different proteins can be achieved by combining
RBFs with varying p-rich linkages with established recognition
motif towards aggregates of specific proteins.
Conclusion
In this work, we demonstrated that the installation of extended
p-rich linkage between electron donor and acceptor is a general
method to control the viscosity sensitivity of RBFs. Compared to
RBFs with a simple single bond linkage, RBFs with extended p-
rich linkages harbor higher rotational energy barriers (Ea) between
the planar fluorescent configuration and the dark twisted
configuration. As a result, RBFs with extended linkers exhibit
lower x values as a measure of their viscosity sensitivity and could
activate their fluorescence emission at much lower viscosities.
This mechanism allows for the design of three scaffolds of RBF
derivatives that span a wide range of viscosity sensitivities (x =
0.26 to 0.79). Using RBFs with desired viscosity sensitivities and
distinct emission spectra, we were able to present a dual-color
imaging strategy that can differentiate misfolded protein
oligomers and insoluble aggregates both in test tubes and live
cells. Because this control is exerted via the linkage between
electron donor and acceptor of fluorophores, we envision that this
Figure 5. Live cell imaging to demonstrate that extended (P2b) and simple
(P2a) RBFs detect the misfolded protein oligomers and insoluble aggregates,
respectively. (a) Structure of P2a and P2b. (b) Scheme of how P2b and P2a
detect the less viscous misfolded protein oligomers (as the diffusive signal) and
highly compact insoluble protein aggregates (as the punctate signal),
respectively. (c) Images of Htt-46Q (top panel), Htt-110Q (middle panel) and
WT Halo (bottom panel) using P2a and P2b. HEK293T cells were treated with
0.5 µM of P2a and P2b during transfection of desired proteins. Proteins were
expressed for 48 h and excess probes were washed away prior to confocal
fluorescence imaging. Scale bar: 10 µm.
6
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10.1002/anie.202011108
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RESEARCH ARTICLE
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We thank support from the Burroughs Welcome Fund Career
Award at the Scientific Interface 1013904 (X.Z.), Paul Berg Early
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10.1002/anie.202011108
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RESEARCH ARTICLE
Entry for the Table of Contents
In this work, we reported a novel method to rationally control the viscosity sensitivity of molecular rotor-based fluorophores (RBFs)
by installing π-rich linkage between the electron donor and acceptor of RBFs. The outcome of this work generates RBFs that span a
wide range of viscosity sensitivity and allow detection of protein aggregation with different compactness both in vitro and in live cells.
Institute and/or researcher Twitter usernames: kfzhangxin
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