Fluorophore-Promoted RNA Folding and Photostability Enables Imaging of Single Broccoli-Tagged mRNAs in Live Mammalian Cells

Article abstract of DOI:10.1002/anie.201914576

Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence. Spinach/Broccoli-DFHBI complexes exhibit high fluorescence in vitro, but they exhibit lower fluorescence in mammalian cells. Here, computational screening was used to identify BI, a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands. BI prevents thermal unfolding of Broccoli at 37 °C, leading to more folded Broccoli and thus more fluorescent Broccoli-BI complexes in cells. Broccoli-BI complexes are more photostable owing to impaired photoisomerization and rapid unbinding of photoisomerized cis-BI. These properties enable single mRNA containing 24 Broccoli aptamers to be imaged in live mammalian cells treated with BI. Small molecule ligands can thus promote RNA folding in cells, and thus allow single mRNA imaging with fluorogenic aptamers.

Full text of DOI:10.1002/anie.201914576

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Fluorophore-promoted RNA folding and photostability enable
imaging of single Broccoli-tagged mRNAs in live mammalian cells
Authors: Xing Li, Hyaeyeong Kim, Jacob Litke, Jiahui Wu, and Samie
Jaffrey
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201914576
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10.1002/anie.201914576
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RESEARCH ARTICLE
Fluorophore-promoted RNA folding and photostability enable
imaging of single Broccoli-tagged mRNAs in live mammalian
cells
Xing Li,[a] Hyaeyeong Kim,[a] Jacob L. Litke,[a] Jiahui Wu,[a] and Samie R. Jaffrey*[a]
Abstract: Spinach and Broccoli are fluorogenic RNA aptamers that
bind and activate the fluorescence of DFHBI, a mimic of the green
fluorescence protein chromophore. Although Spinach/Broccoli-
DFHBI complexes exhibit high fluorescence in vitro, their
fluorescence is considerably lower in cells. Here we used an in silico
screen to identify BI, a DFHBI derivative that binds Broccoli with
higher affinity and leads to markedly higher Broccoli fluorescence in
cells compared to other DFHBI-related ligands. We show that BI
prevents thermal unfolding of Broccoli at 37°C in mammalian cells,
leading to substantially more folded Broccoli and subsequently more
fluorescent Broccoli-BI complexes. Additionally, Broccoli-BI
complexes are significantly more photostable due to impaired light-
induced photoisomerization, and rapid unbinding of photoisomerized
cis-BI. These optimized fluorescence properties enable single mRNA
transcripts containing 24 Broccoli aptamers in the 3’UTR to be
imaged in cells treated with BI. These studies reveal that small
molecule ligands can promote RNA folding and function in cells, and
show the feasibility of single mRNA imaging with fluorogenic
aptamers.
Spinach and/or Broccoli to be used as a tag for imaging single
mRNA molecules in living cells.
Spinach and Broccoli bind DFHBI, a small molecule
mimic of the fluorophore found in GFP.[1b, 6] Upon irradiation,
DFHBI can undergo a light-induced cis-trans isomerization to a
low fluorescence trans- form.[5] The trans-fluorophore
subsequently unbinds from the aptamer. Fluorescence is only
restored when a cis-fluorophore from solution binds the aptamer.
The fluorescence output could be increased if (1) the ability of
the fluorophore to isomerize to the trans isoform could be
suppressed, (2) if the low-fluorescence trans-fluorophore would
unbind from the aptamer more quickly, and/or (3) if the rate of
rebinding of cis-fluorophore could increase.
Here we describe the structure-guided design of BI, a
DFHBI derivative that binds Broccoli and produces markedly
increased brightness and photostability of Broccoli in cells. BI
functions by promoting Broccoli folding in cells, resulting in a
substantial increase in the fraction of Broccoli that is folded in
cells. In addition to this effect, BI overcomes the key problems
that have resulted in photobleaching of Broccoli in previous
experiments. As a result of these improved properties, single
mRNAs containing an optimized tandem Broccoli tag can be
imaged in live mammalian cells.
Introduction
Fluorogenic aptamers are RNA sequences that bind otherwise
nonfluorescent small molecules and induce their fluorescence
while bound to RNA.[1] By tagging an RNA with a fluorogenic Results and Discussion
aptamer, fluorescence can be genetically encoded into the RNA,
and the endogenously expressed tagged RNA can be detected
by fluorescence microscopy. Since only the RNA-fluorophore
complexes are fluorescent, fluorescence signals derive from
tagged RNAs, while the unbound fluorophores are
nonfluorescent.
Fluorogenic aptamers have been used for diverse
applications, including monitoring transcription rates[1a] or for
imaging RNA aggregates such as aggregates of the 5S RNA[1b]
or trinucleotide repeat RNA,[3] or mRNA aggregates in stress
granules.[4] RNA aggregates are relatively easy to image since
they contain a large number of tagged RNAs, resulting in a
concentrated signal that can be readily detected by fluorescence
microscopy.
Structure-guided identification of DFHBI derivatives with
higher affinity for Spinach
In order to optimize the fluorescence of Spinach and
Broccoli for imaging individual mRNAs, we wanted to reduce
photobleaching and develop an approach to accelerate the
recovery
of
fluorescence
after
photobleaching.[5]
Photobleaching of Spinach-DFHBI begins with light-induced
isomerization of DFHBI from the cis to the trans form.[5] This
results in termination of Spinach fluorescence. The trans-DFHBI
remains bound as a low-fluorescence Spinach-DFHBI complex.
This causes extends the duration of the low-fluorescence state
of Spinach. Next, the trans-DFHBI unbinds. Lastly, fluorescence
is restored when a cis-DFHBI fluorophore binds Spinach (Figure
1a).[5]
We reasoned that photoisomerization and the
persistence of a bound low-fluorescence trans isomer could be
resolved with an improved fluorophore that would either (1) be
less susceptible to light-induced cis-trans isomerization; or (2)
would exhibit rapid unbinding when it isomerizes to the trans-
fluorophore.
In contrast, imaging single mRNA molecules using
fluorogenic aptamers is challenging. In the case of the Spinach
and Broccoli fluorogenic aptamers, photobleaching occurs
rapidly, which limits the total fluorescence that can be generated
from
an
aptamer-tagged
mRNA.
Thus
overcoming
photobleaching would substantially enhance the ability of
To suppress cis-trans isomerization, we sought to
generate a derivative of DFHBI that would bind with higher
affinity. Higher affinity may reflect more contacts with the RNA,
which may suppress isomerization (Figure 1b).
To increase the unbinding rate of the trans-fluorophore,
we reasoned that a bulky substituent on the imidazolinone ring
[a]
Dr. X. Li, Mrs. H. Kim, Mr. J. Litke, Dr. J. Wu, Prof. Dr. S. R. Jaffrey
Department of Pharmacology, Weill Cornell Medicine, Cornell
University, New York, NY 10065 (USA)
E-mail: srj2003@med.cornell.edu
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201914576
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Figure 1 Structure-guided identification of BI, a fluorophore that leads to higher brightness in Broccoli-expressing mammalian cells. (a) Schematic of
the molecular steps that account for photobleaching of Spinach/Broccoli and the resoration of fluorescence. (b) A bulky substituent on the N1 (blue atom) may
enable rapid dissociation of trans-fluorophore. Upon isomerization the bulky substituent would cause the fluorophore to no longer fit in Broccoli, causing rapid
ejection. (c) Orientation of DFHBI in the Spinach cystal structure.[2] The N1 position faces the solvent-accessible opening. The Spinach surface representation
is colored according to electrostatic potential, from red (negative) to blue (positive). (d) Chemical structure and docking score of DFHBI-1T and DFHBI
derivatives identified by structure-guided screening. Shown are the top-binding hits. Each of these molecules have higher docking scores that than DFHBI-1T.
(e) HEK293T cells expressing circular Broccoli RNA or control mCherry mRNA were imaged in the presence of 10 μM DFHBI-1T or BI. Exposure times: 100
ms for FITC filter, 50 ms for DAPI filter. Scale bar, 100 μm. (f) Quantification of average brightness of Broccoli-expressing cells (left) incubated with 10 μM
DFHBI-1T or BI. Background fluorescence (right) measured in cells lacking Broccoli was similar in 10 μM DFHBI-1T or BI-treated cells. Error bars indicate
standard error of the mean (s.e.m.) for n=150 cells per condition. ****P < 0.0001. NS, not significant. (g) Excitation and emission spectra of Broccoli bound to
either DFHBI-1T (grey) or BI (green). Excitation (Ex, dotted line) and emission (Em, solid line) spectra were measured using in vitro transcribed and purified
Broccoli RNA (10 μM) and the indicated fluorophore (0.5 μM) in buffer containing 1 mM MgCl2.
(Figure 1b) would cause the fluorophore to be rapidly ejected
upon isomerization since the trans- fluorophore would not fit in
the DFHBI-binding pocket of Spinach. This contrasts with DFHBI,
which can remain bound upon isomerization since both the cis-
or trans- form can fit in the DFHBI-binding pocket.
would be unable to fit in the DFHBI-binding pocket of Spinach.[2,
7]
Each substituent contained a minimum of one methylene
carbon between the N1 nitrogen and the chemical moieties in
the library. We used Glide (grid-based ligand docking with
energetics) from Schrödinger.[8] In Glide, a docking score is
generated for each molecule based on an energy minimization
calculation in which each library member is docked in diverse
conformations. The docking score is related to the expected
binding affinity.[8a, 8b] We selected three molecules for synthesis
and for in vitro binding assays based on their strong docking
scores and synthetic accessibility (Figure 1d).
To identify DFHBI derivatives that meet these criteria,
we virtually screened libraries of DFHBI derivatives for their
docking scores in the Spinach fluorophore-binding pocket. This
library contained 817 substituents appended to the N1 position
of the imidazolinone portion of the DFHBI (Figure 1b). The N1
position faces a solvent-accessible opening in the Spinach
aptamer (Figure 1c), so these DFHBI derivatives would be
expected to fit into Spinach. In contrast, substituents elsewhere
We first asked if the compounds are cell permeable and
have low background fluorescence in mammalian cells. We
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either used HEK293T cells expressing mCherry as a control, or
HEK293T cells expressing Broccoli[6] using the Tornado
expression system, which results in high level expression of
Broccoli as a circular RNA.[9] We used Broccoli since its
fluorophore-binding pocket is essentially identical to that of
Spinach[10], but it produces improved fluorescence in cells due to
Since this increase in quantum yield is less than the
~10.5-fold increase in brightness of BI compared to DFHBI-1T in
cells, the increased quantum yield cannot explain the markedly
improved performance of BI in cells.
Additionally, circular Broccoli expression was essentially
identical in vehicle-, DFHBI-1T-, and BI-treated cells (Figure S3).
Thus, the enhanced brightness in BI-treated cells is not due to
increased expression of circular Broccoli.
a
lower dependence on magnesium for folding.[6] Any
fluorescence seen in cells lacking Broccoli is by definition
background fluorescence, including nonspecific fluorescence
activation by cellular constituents.
BI suppresses thermal denaturation of Broccoli
Fluorescence brightness is usually defined as
proportional to the product of the extinction coefficient (ɛ) and
the quantum yield (). However, the brightness of an RNA-
fluorophore complex involves an additional factor, i.e., the
fraction of the RNA that is folded. Thus, for a fluorogenic
aptamer, brightness = ɛ ×  × % folded. We therefore considered
the possibility that BI could increase the fraction of Broccoli that
is folded in the cell.
Cells were pretreated for 2 h with each DFHBI derivative
(10 µM) or DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-
2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one, 10 µM),
which exhibits low fluorescence in cells.[11]
The different
compounds exhibited low background fluorescence in cells
lacking Broccoli, similar to DFHBI-1T (Figure 1e, f). When each
derivative was applied to circular Broccoli-expressing HEK293T
cells, increased fluorescence was seen (Figure 1e).
Although Broccoli folding has been measured in vitro[12]
,
Surprisingly, one derivative, BI (referring to the
benzimidazole substituent), caused a ~10.5-fold increase in
cellular fluorescence compared to DFHBI-1T (Figure 1f).
The marked increase in fluorescence was lost when the
linker to benzimidazole was lengthened, or if benzimidazole was
exchanged with pyridine (Figure S1). Furthermore, derivatives
with different linker lengths or attachment points on the
benzimidazole (Figure S2a) did not further improve the
fluorescence in Broccoli-expressing cells (Figure S2b).
The increased brightness cannot be explained by
enhanced photostability since the increased brightness of BI-
treated cells can be detected immediately upon imaging.
Therefore, we first wanted to understand the mechanism of the
unusual degree of fluorescence enhancement seen with BI.
measurements of the percent of a folded RNA in cells is not
straightforward. Nevertheless, our in vitro studies previously
showed that Broccoli and Spinach are not completely folded
when synthesized in vitro.[12]
Furthermore, like all RNA
aptamers, both Broccoli and Spinach are thermally unstable at
6]
37°C, the imaging temperature used in mammalian cells.[3,
Thus, folding is a major problem that limits brightness for
fluorogenic aptamers in cells.
We therefore performed several tests to determine if BI
influences the folding of Broccoli. First, we noted that BI binds
with a KD of 51 nM (Table 1 and Figure 2b, c), compared to 305
nM for DFHBI-1T. The increased affinity of BI for Broccoli
compared to DFHBI suggests that the benzimidazole moiety in
BI makes contacts with the Broccoli RNA, as predicted in the
docking study. These additional contacts may create a Broccoli-
BI complex that is more thermally stable.
The enhanced cellular brightness of Broccoli-BI cannot be
fully explained by enhanced photophysical properties
We measured the extinction coefficient and quantum
yield of this complex in vitro in a 200 nM solution of Broccoli-
fluorophore complex prepared by incubating 200 nM BI or
DFHBI-1T with 10 µM in vitro transcribed Broccoli. The
Although Broccoli folding has been measured in vitro[12]
,
measurements of the percent of a folded RNA in cells is not
straightforward. Nevertheless, our in vitro studies previously
showed that Broccoli and Spinach are not completely folded
when synthesized in vitro.[12] Furthermore, like most RNA
aptamers, both Broccoli and Spinach are thermally unstable at
37°C, the imaging temperature for mammalian cells.[3, 6] Thus,
fluorescence calculation (i.e., quantum yield
× extinction
coefficient) show that Broccoli-BI complexes are 1.88-fold
brighter than Broccoli-DFHBI-1T complexes at 25°C (Table 1).
Table 1. Photophysical and Binding Properties of Fluorophore-Broccoli Complexes
Extinction
Coefficient
(M-1, cm-1)[a]
Excitation
(nm)
Emission
(nm)
Quantum
Yield
KD
(nM)
Structure
RNA-Fluorophore
DFHBI-1T
Brightness[b]
428
470
425
470
500
505
500
505
35,400
28,900
42,500
33,600
0.00041
0.414
0.121
100
N/A
305±39
N/A
Broccoli-
DFHBI-1T
BI
0.00048
0.669
0.171
188
Broccoli-
BI
51±5
[a]. Extinction coefficients were all measured in buffer containing 40 mM HEPES pH 7.4, 100 mM KCl, 1 mM MgCl2. Experiments were
performed at 25°C in order to compare with previous studies.[1] [b]. Brightness (extinction coefficient × quantum yield) is relative to Broccoli-
DFHBI-1T.
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10.1002/anie.201914576
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misfolding limits brightness for fluorogenic aptamers in cells.
We therefore performed several tests to determine if BI
fluorescence after injection of 0.5 µM DFHBI-1T or BI into a
cuvette containing a solution of 25 µM Broccoli at 37°C (Figure
2e). Since cells contain ~0.2 mM free Mg2+,[13] these
experiments were performed at 37°C in buffer with 0.2 mM
MgCl2, in contrast to the other in vitro experiments which used 1
influences the folding of Broccoli. First, we performed a thermal
denaturation analysis of Broccoli (25 µM) in the presence of 0.5
µM of DFHBI-1T or BI (Figure 2d). Broccoli fluorescence was
measured as the temperature was increased from 20°C and
37°C. While ~77% of Broccoli-DFHBI-1T fluorescence was lost
at 37°C, only ~32% Broccoli-BI fluorescence was lost at 37°C.
Overall, Broccoli-BI exhibited a ~12°C shift in the Tm relative to
Broccoli-DFHBI-1T (Figure 2d, left). Importantly, BI exhibits
mM MgCl2. As expected, there was
a rapid phase of
fluorescence increase, reflecting the on-rate of DFHBI-1T or BI.
DFHBI-1T showed a plateau in fluorescence by ~25 sec.
However, showed
a second slow phase of fluorescence
increase. The overall rate of fluorescence increase could be
~6.4-fold higher fluorescence than DFHBI-1T at 37 °C (Figure 2d, modeled with two rate constants, k1 and k2, with k1 representing
right).
the initial rate of BI binding, and k2 representing the rate of a
further increase in fluorescence (Figure 2e). One possible
explanation for this second phase of fluorescence increase is
that BI binds a partially folded Broccoli RNA that is normally not
able to activate BI fluorescence. However, BI may convert this
into a properly folded form. Although a speculative model, this
effect may contribute to the overall brightness of BI in Broccoli-
expressing cells.
Overall, these data suggest that BI can promote and
stabilize the folded form of Broccoli, which may account for the
substantial fluorescence enhancement in cells.
Broccoli-BI complexes exhibit increased photostability
To determine if Broccoli-BI complexes also exhibit
increased photostability, we measured total fluorescence in
HEK293T cells during continuous irradiation in an
epifluorescence microscope. As expected, DFHBI-1T (10 µM)-
treated cells showed a 50% loss of fluorescence in ~0.6 sec
(Figure 3a-b). In contrast, BI-treated cells exhibited increased
photostability, with a 50% loss in fluorescence at ~2.9 sec
(Figure 3a and 3b).
In both cases, a fluorescence plateau was reached
(Figure S4), since fluorescence is restored when cis-fluorophore
rebinds the aptamer after ejection of the trans- fluorophore.[5a]
We next asked if the increased photostability reflects
reduced photoisomerization of BI. The photoisomerization rate is
determined by measuring the initial rate of fluorescence loss
upon irradiation[5b] (see Supplementary Note 1) in cells and in
vitro. In cells irradiated using an epifluorescence microscope,
the photoisomerization rate of Broccoli-BI complexes was 0.283
sec-1, which was ~3.3-fold slower than Broccoli-DFHBI-1T
complexes (0.947 sec-1) (Figure 3c, left). In vitro, using a
fluorimeter, the photoisomerization rate of Broccoli-BI complexes
was 0.008 sec sec-1, which was ~5-fold slower than Broccoli-
DFHBI-1T complexes (0.041 sec-1) (Figure 3c, right). Therefore,
based on both in-cell and in vitro photostability experiments, BI
is less susceptible to photoisomerization than DFHBI-1T when
bound to Broccoli.
Figure 2 BI stabilizes Broccoli. (a) Model for how BI could enhance overall
cellular fluorescence by stabilizing Broccoli in folded form. (b) Broccoli (50 nM)
was titrated with either DFHBI-1T or BI at 25°C and then fitted using the Hill
equation as described previously.[1a] BI (KD= 51 nM), DFHBI-1T (KD= 305 nM).
(c) kon and koff were calculated from the linear fit (solid line) of kobs vs.
concentration at 25°C. The slope provides kon and the intercept gives koff
.
kon=14,900 M-1 s-1, koff=0.0036 s-1 for DFHBI-1T and kon=13,600 M-1 s-1,
koff=0.0006 s-1. (d) The fluorescence of a solution containing 25 μM RNA and
500 nM BI or DFHBI-1T was measured in 1 mM MgCl2 buffer while gradually
increasing the temperature. In the left image, the fluorescence is normalized
so that both Broccoli-BI and Broccoli-DFHBI-1T start at 1.0. Broccoli-BI
exhibited a ~12°C shift in the Tm relative to Broccoli-DFHBI-1T (left). On the
right, the fluorescence is normalized by the fluorescence level of Broccoli-
DFHBI-1T at 37°C. (e) Fluorescence intensity upon mixing Broccoli and BI or
DFHBI-1T (final concentration: 25 μM Broccoli, 0.5 μM fluorophore) at 37°C
(0.2 mM MgCl2 buffer). Upon adding BI to Broccoli, an initial (0-25 sec) rapid
increase (k1=0.06771 s-1) in fluorescence was observed (expected binding
based on a pseudo-first order binding reaction indicated as a dotted line, the
actual observed fluorescence increase is shown in solid color). BI exhibited a
continued increase in fluorescence consistent with a second mode of binding
(k2=0.02756 s-1) with a plateau at ~200 sec. The rate constants (k1 and k2)
were modeled by a single exponential fit in Origin software.
The other factor that contributes to Broccoli
photobleaching is the unbinding rate of trans-DFHBI, the low-
fluorescence form of the fluorophore.[5] If trans-fluorophore
unbinding rate is slow, then fluorescence cannot be quickly
restored by binding cis-fluorophore. In this case, the rate-limiting
step for restoring fluorescence is the unbinding of trans-
fluorophore. However, if trans-fluorophore unbinding is fast, then
the rate-limiting step is the rate of binding of cis-fluorophore.
Overall, these data suggest that BI stabilizes Broccoli at
37°C , which therefore results in more Broccoli-BI complexes
than Broccoli-DFHBI-1T complexes (Figure 2a). This effect,
coupled with the moderate increase in the extinction coefficient
and quantum yield relative to DFHBI-1T, may explain the ~10.5-
fold increase in brightness of BI in Broccoli-expressing cells.
To further explore if additional mechanisms may mediate
the effects of BI, we monitored the time-dependent acquisition of
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RESEARCH ARTICLE
concentrations of DFHBI-1T (Figure 3d, right). This suggests
that BI and DFHBI-1T are fundamentally different: when BI is
used, rebinding of cis-BI is rate limiting since trans-BI unbinds
rapidly. In contrast, when DFHBI-1T is used, unbinding of the
trans-DFHBI-1T is rate limiting, and therefore increasing the
rebinding rate of cis-DFHBI-1T by increasing DFHBI-1T
concentration has no effect.
Taken together, the overall increase in photostability of
Broccoli-BI complexes reflects (1) its reduced susceptibility to
photoisomerization and (2) its rapid unbinding upon
photoisomerization, allowing Broccoli fluorescence to be
restored quickly by cis-BI in solution. These findings explain the
different plateaus seen when using BI and DFHBI-1T: since both
DFHBI-1T and BI show similar rebinding rates (Figure 2c), the
difference in the plateaus is due to their different
photoisomerization rates and different rates of unbinding of the
trans-fluorophore.
Notably, the plateau seen in vitro (Figure 3c, right) and in
cells (Figure 3d) is different due to the different ways in which
the experiments were performed. The in vitro experiments were
performed in a cuvette in which only a small part of the sample
in the cuvette is exposed the excitation light. In cells,
fluorescence was tested in a microscope using a light source
that irradiates the entire cell. Since this leads to
photoisomerization throughout the entire cell, the loss of
fluorescence is more substantial, leading to a lower plateau
(Figure 3d).
Figure 3 Broccoli-BI complexes exhibit increased photostability in vivo
and in vitro. (a) In vivo photostability of Broccoli bound to flurophores (10 μM)
was assessed by continuous imaging of Broccoli-expressing cells (100 ms per
frame). Scale bar, 50 μm. (b) Quantification of in vivo photostability. Broccoli-
DFHBI-1T loses 50% of signal within ~0.6 sec; Broccoli-BI lost 50% of signal
within ~2.9 sec. Error bars indicate s.e.m. for n=10 cells per condition. (c)
Broccoli-BI exhibits reduced photoisomerization compared to Broccoli–DFHBI-
1T. Shown is the initial light-induced rate of fluorescence loss in cells (left) and
in vitro (right). In the cellular experiment, flurophore concentration was 10 μM.
In the in vitro experiments, we used solutions containing 0.1 μM BI or DFHBI-
1T and 1 μM Broccoli. (d) To determine if trans-fluorophore unbinding is rate
limiting, we measured photobleaching during continuous illumination of
Broccoli-expressing HEK293 cells in a microscope. Fluorescence diminishes
over time and reaches a plateau. If unbinding of trans-fluorophore is fast, then
the rate-limiting step will be binding of cis-fluorophore from solution. To test
this, we added increasing concentrations of fluorophore (5, 10, and 20 μM).
Increasing the concentration of BI leads to a corresponding increase in the
plateau of fluorescence, suggesting that binding of the fluorophore in solution
is rate limiting, not unbinding of trans-BI. In contrast, adding higher
concentrations of DFHBI-1T has no effect. This suggests that unbinding of
trans-DFHBI is rate limiting. The brightness was computed by measuring the
fluorescence signal in ten cells’ area and subtracting background based on
ontrol cells not expressing Broccoli. Error bars indicate s.e.m. for n=10 cells
per condition.
A concatenated Broccoli tag for imaging single mRNA
We next wanted to determine if these brighter and more
photostable Broccoli-BI complexes would enable imaging of
individual mRNAs in mammalian cells. We developed an RNA
tag comprising multiple consecutive Broccoli aptamers,
analogous to mRNA imaging tags comprising 24-48 MS2
hairpins incorporated into mRNA 3’UTRs, which is currently the
most common approach for single mRNA imaging.[14] In this
approach, mRNAs become fluorescently tagged since the MS2
hairpins bind a MS2 RNA coat protein (MCP) fused to a
fluorescent protein.[14b]
Each Broccoli aptamer was incorporated into F30, an
RNA scaffold that promotes folding of Broccoli (Figure S5a and
S6a).[15] We used “dimeric Broccoli” (dBroccoli), which comprises
two Broccoli aptamers placed end-to-end (Figure S5a and S7a).
Therefore, F30 with one dBroccoli in each arm contains the
equivalent of four Broccoli aptamers (F30-2xdBroccoli) (Figure
S5a).
These two models can be distinguished by measuring if
the plateau in fluorescence increases with increasing levels of
fluorophore in solution.[5b] The plateau occurs when trans-
fluorophore unbinding is balanced by rebinding of cis-
fluorophore. By increasing the fluorophore concentration, the
binding rate is increased in proportion to the concentration of
fluorophore. Therefore, if the plateau in Broccoli fluorescence is
increased with increased fluorophore concentration, it
demonstrates that the binding rate of the cis-fluorophore is rate
limiting. If the plateau is unaffected by the concentration of the
fluorophore, then the trans-fluorophore unbinding is the rate-
limiting step.
Exact sequence repeats can cause recombination or
deletion during plasmid propagation.[16] Additionally, identical
hairpin
sequences
misfold
due
to
“inter-aptamer
hybridization”.[12b] We therefore assembled two separate F30-
2xdBroccoli sequences with a different F30 variant and different
Broccoli aptamers, along with different linkers between each
Broccoli and F30 (Figure S6b, 7 and Tables S2-3).
To test this, we measured the fluorescence of Broccoli in
HEK293T cells cultured with increasing concentrations of
fluorophore. When we tested increasing concentrations of BI, we
observed an increase in the fluorescence plateau with
increasing concentrations of BI (Figure 3d, left). In contrast, we
did not see the change in the plateau with increasing
For each tag tested, we used these two F30-2xdBroccoli
sequences as a minimal unit, which contains 8 Broccoli
aptamers. We then made tags comprising 1, 2, or 3 consecutive
copies of this unit, i.e., (F30-2xdBroccoli)2, (F30-2xdBroccoli)4,
and (F30-2xdBroccoli)6. The fluorescence intensity of the tag
containing 24 Broccoli aptamers was 15.3-fold brighter than a
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10.1002/anie.201914576
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single Broccoli (Figure S5b and S9). A previous study used 64
Spinach repeats but only achieved a 16-fold of fluorescence
increase.[17] The use of the F30 folding scaffold and/or sequence
variants for F30 and Broccoli may allow the fluorescence of the
tag to more linearly correspond to the number of Broccoli
aptamers.[12b]
We next expressed a mCherry mRNA with a 3’UTR
tagged with (F30-2xdBroccoli)6 or (F30-2xdBroccoli)12,
corresponding to 24 or 48 Broccoli aptamers (Figure S10). We
first asked whether the Broccoli-tagged transcripts were full-
length or degraded into Broccoli-containing intermediates. We
performed northern blotting using total cellular RNA 48 h after
transfection with a plasmid expressing the mCherry transcript
tagged with increasing numbers of Broccoli aptamers. In each
case, a single band corresponding to the full-length mRNA was
detected (Figure S11).
We next imaged transcripts in living cells incubated with
10 µM DFHBI-1T or BI. No fluorescent puncta were seen in
COS7 cells treated with DFHBI-1T (Figure S10b). However,
when cells were treated with BI, we observed mobile fluorescent
puncta with transcripts containing (F30-2xdBroccoli)6 or (F30-
2xdBroccoli)12 (24 or 48 Broccoli equivalents) (Figure S10b and
Supplemental Movie 1, 2). These data suggest that BI enables
imaging of Broccoli-tagged mRNAs in mammalian cells.
Broccoli-tagged mRNAs exhibit physiologic localizations in
live cells
We next asked if mRNA containing the concatenated
Broccoli tags exhibit the expected cytoplasmic localization and
mobility normally seen with mRNA in cells. For these
experiments, we used a mCherry mRNA containing a β-actin
3’UTR sequence (Figure 4a), followed by a 24X Broccoli tag
[(F30-2xBroccoli)6]. We used a 3' UTR sequence from the β-
actin transcript since this is commonly used for mRNA imaging
by
MS2-GFP system and confers targeting to neuronal
dendrites.[14b] After the plasmid was transfected in COS7 cells,
mobile puncta were readily detected in the cytosol, with a few
puncta seen in the nucleus (Figure 4b). Similar results were
seen with a 48X Broccoli tag [(F30-2xBroccoli)12] (Figure S12).
Next, we asked if Broccoli-tagged mRNA exhibits
degradation rates normally expected for single mRNAs, which
typically exhibit half-lives of just a few hours.[18] We therefore
added actinomycin D, a transcription inhibitor. We found that
nearly 50% of the puncta were lost at ~2.5 h, indicating a half-life
that is consistent with single mRNA molecules (Figure S13).
We next imaged mRNA prior to and during stress granule
formation induced by arsenite or cold shock. For these
experiments, we used a mCherry mRNA containing a β-actin
3’UTR sequence, followed by
a 24 Broccoli tag [(F30-
2xBroccoli)6]. Prior to the application of arsenite or cold shock,
fluorescent green puncta were observed throughout the cell.
However, upon application of arsenite (500 µM) or cold shock
(10°C), the puncta aggregated into larger foci consistent with
stress granules within 60 min (Figure 4c). These data suggest
that puncta in untreated cells may behave like single mRNAs
that relocalize to granules in response to stress.
As a fourth test, we compared puncta generated using
the Broccoli-BI system to puncta generated with the more
common MS2-GFP system.[14b] Puncta seen when expressing
MS2-tagged mRNA reflect single mRNA.[14a,16,19] To image these
puncta, we co-transfected a plasmid expressing an mCherry
mRNA containing 24X MS2 hairpins, and a plasmid expressing
and GFP-MCP into U2OS cells. As expected, puncta were
readily detectable in the cytoplasm (Figure S14).
We first compared the fluorescent puncta size of mRNAs
tagged with the MS2 tag or the 24X Broccoli tag [(F30-
2xBroccoli)6]. We found that the puncta size of most Broccoli-
tagged mRNAs is similar to the puncta size of MS2-tagged
mRNAs (Figure S15). Therefore, the size of fluorescent puncta
observed using the Broccoli-BI method is consistent with single
mRNAs.
We next used Gaussian analysis of puncta intensity,
which has previously been used to determine if fluorescence
signals represent single mRNAs.[20] We compared the intensity
distribution of puncta, and found both MS2-tagged and Broccoli-
tagged mRNA follow a single Gaussian distribution in puncta
intensity, which again suggests that the puncta are
predominantly single mRNA transcripts in cells (Figure S16).
Figure 4 Imaging and localization of Broccoli-tagged β-actin mRNA in
mammalian cells incubated with BI. (a) Schematic of β-actin mRNA tagged
with six repeated F30-2xdBroccoli units [(F30-2xdBroccoli)6] downstream of a
portion of the 3’UTR of β-actin (shown in gray). (b) The (F30-2xdBroccoli)6-
tagged β-actin mRNA was expressed in COS7 cells and imaged (10 μM BI) (c)
supplemented with arsenite (500 μM, 60 minutes) or subjected to cold shock
(10°C, 30 min) for induction of stress granules. Images were acquired after 1 h
of arsenite treatment or 30 min of cold shock. Exposure times: 500 ms for
FITC filter. Scale bars, 20 μm (Left), 5 μm (Right).
Conclusions
In order to improve the photostability of Broccoli and
Spinach in cells, we computationally screened a library of
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10.1002/anie.201914576
Angewandte Chemie International Edition
RESEARCH ARTICLE
DFHBI derivatives for binding to Spinach, an RNA aptamer that
is highly similar to Broccoli. One molecule, BI, exhibited marked
increases in Broccoli fluorescence in cells. We find that the
mechanism is due to an ability of BI to promote and stabilize
folding of Broccoli at the relatively high (37oC) imaging
temperatures. Broccoli-BI complexes also exhibit improved
photostability, which reflects a reduced ability of BI to undergo
light-induced cis-trans isomerization. We also find that BI is
rapidly ejected when it is isomerized, allowing efficient
replacement with cis-BI and restoration of fluorescence. These
combined properties results in a markedly improved Broccoli
imaging tag, allowing single mRNA to be imaged when tagged
with 24 consecutive Broccoli aptamers in their 3’UTRs.
Like the commonly used 24X MS2 hairpin tag, the 24X
Broccoli tag that can potentially alter the fate of an mRNA in the
cell. Therefore, experiments using RNA imaging tags are best
done by comparing two mRNAs that have the same tag, but
differ by a putative functional element, or differ because of an
experimental treatment that might affect mRNA behavior. Any
result should then be validated with the endogenous transcript.
Although the 24X Broccoli tag is slightly longer than the
24X MS2 tag, Broccoli has the benefit of not recruiting
fluorescent proteins to the mRNA. Additionally, since the
fluorescent proteins usually have nuclear-localization elements,
it is possible that the subsequent recruitment of ~24 nuclear
localization elements to an mRNA might affect its trafficking
behavior.[21] This problem would not be seen when using the
Broccoli tag since no protein is recruited (Table S1). Imaging
with Broccoli should not affect the detection of nuclear mRNAs,
which are usually difficult to detect with the MS2 system due to
nuclear-localized fluorescent proteins (Figure S14).
The unexpected and marked increase of in-cell
fluorescence in BI-treated Broccoli-expressing cell is due to the
ability of BI to stabilize the folded form of Broccoli. Our data
suggests that Broccoli is not well folded in cells at 37°C. BI also
appears to promote folding of Broccoli (see Figure 2e),
suggesting that BI can bind
a
partially folded Broccoli
To the best of our knowledge, this is first time that single
mRNA molecules have been imaged in mammalian cells using a
fluorogenic aptamer and a small fluorophore. We expect that BI
will facilitate the use of Broccoli as an imaging tag for mRNA and
will be generally useful to increase fluorescence signals for
diverse in-cell applications that use Broccoli.
intermediate and convert this Broccoli to the folded form. Thus,
the major effect of BI is to increase the total amount of folded
Broccoli in cells, thus explaining the large increase in
intracellular fluorescence.
The ability of BI to stabilize Broccoli structure is likely due
to the additional contacts that the benzimidazole makes with
Spinach. The improved affinity of BI, compared to DFHBI-1T,
supports the idea that BI makes additional contacts with Spinach
or Broccoli.
The additional contacts of BI with Broccoli likely also
explain the enhanced photostability of Broccoli-BI complexes.
We find that the light-induced isomerization rate is slower, thus
resulting in more fluorescence output before photobleaching.
BI also overcomes an additional problem of DFHBI: the
slow off-rate of the photoisomerized trans-fluorophore. We find
that trans-DFHBI-1T unbinding is slow and rate-limiting for the
restoration of Broccoli fluorescence. In contrast, unbinding of
trans-BI is rapid and not rate limiting. The bulky nature of the
benzimidazole moiety may make the trans-BI unable to be
accommodated in the Broccoli fluorophore binding pocket,
causing its rapid ejection upon photoisomerization. This allows
rebinding of cis-BI to be the rate-limiting step, rather than
unbinding of trans-BI. Since rebinding is relatively rapid
(kon=13,600 M-1 s-1), the Broccoli aptamer does not have long
dark states caused by bound cis-BI, in contast to DFHBI-1T.
BI has other benefits, including small increases in
extinction coefficient and quantum yield relative to DFHBI-1T,
which likely further enhance brightness in cells.
Acknowledgments
We thank W. Zhan, S. Suter and J. Moon for useful comments
and suggestions, R. Wang (Memorial Sloan-Kettering Cancer
Center) for assistance with HRMS. This work was supported by
NIH grant R35 NS111631 (S.R.J.).
Keywords: Fluorescent probes
• Fluorogenic aptamers •
Photostability • RNA folding • Single mRNA imaging
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RESEARCH ARTICLE
TOC:
RESEARCH ARTICLE
Xing Li, Hyaeyeong Kim, Jacob L. Litke,
Jiahui Wu and Samie R. Jaffrey
Page No. – Page No.
Title: Fluorophore-promoted RNA
folding and photostability enable
imaging of single Broccoli-tagged
mRNAs in live mammalian cells
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