A Color-Shifting Near-Infrared Fluorescent Aptamer–Fluorophore Module for Live-Cell RNA Imaging

Article abstract of DOI:10.1002/anie.202107250

Fluorescent light-up RNA aptamers (FLAPs) have become promising tools for visualizing RNAs in living cells. Specific binding of FLAPs to their non-fluorescent cognate ligands results in a dramatic fluorescence increase, thereby allowing RNA imaging. Here, we present a color-shifting aptamer-fluorophore system, where the free dye is cyan fluorescent and the aptamer-dye complex is near-infrared (NIR) fluorescent. Unlike other reported FLAPs, this system enables ratiometric RNA imaging. To design the color-shifting system, we synthesized a series of environmentally sensitive benzopyrylium-coumarin hybrid fluorophores which exist in equilibrium between a cyan fluorescent spirocyclic form and a NIR fluorescent zwitterionic form. As an RNA tag, we evolved a 38-nucleotide aptamer that selectively binds the zwitterionic forms with nanomolar affinity. We used this system as a light-up RNA marker to image mRNAs in the NIR region and demonstrated its utility in ratiometric analysis of target RNAs expressed at different levels in single cells.

Full text of DOI:10.1002/anie.202107250

A Journal of the Gesellschaft Deutscher Chemiker
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Chemie
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Accepted Article
Title: A Color-Shifting Near-Infrared Fluorescent Aptamer-Fluorophore
Module for Live-Cell RNA Imaging
Authors: Jingye Zhang, Lu Wang, Andres Jäschke, and Murat Sunbul
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202107250
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10.1002/anie.202107250
Angewandte Chemie International Edition
RESEARCH ARTICLE
A Color-Shifting Near-Infrared Fluorescent Aptamer-Fluorophore
Module for Live-Cell RNA Imaging
Jingye Zhang,[a] Lu Wang,[b] Andres Jäschke*[a] and Murat Sunbul*[a]
[a]
J. Zhang, Prof. Dr. A. Jäschke, Dr. M. Sunbul
Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University
Im Neuenheimer Feld 364, Heidelberg 69120 (Germany)
E-mail: jaeschke@uni-heidelberg.de; msunbul@uni-heidelberg.de
Dr. L. Wang
[b]
Department of Chemical Biology, Max Planck Institute for Medical Research
Jahnstraße 29, Heidelberg 69120 (Germany)
Supporting information for this article is given via a link at the end of the document.
Abstract: Fluorescent light-up RNA aptamers (FLAPs) have become
promising tools for visualizing RNAs in living cells. Specific binding of
FLAPs to their non-fluorescent cognate ligands results in a dramatic
fluorescence increase, thereby allowing RNA imaging. Here, we
present a color-shifting aptamer-fluorophore system, where the free
dye is cyan fluorescent and the aptamer-dye complex is near-infrared
(NIR) fluorescent. Unlike other reported FLAPs, this system enables
ratiometric RNA imaging. To design the color-shifting system, we
synthesized a series of environmentally sensitive benzopyrylium-
coumarin hybrid fluorophores which exist in equilibrium between a
cyan fluorescent spirocyclic form and a NIR fluorescent zwitterionic
form. As an RNA tag, we evolved a 38-nucleotide aptamer that
selectively binds the zwitterionic forms with nanomolar affinity. We
used this system as a light-up RNA marker to image mRNAs in the
NIR region and demonstrated its utility in ratiometric analysis of target
RNAs expressed at different levels in single cells.
milestone towards in vivo RNA imaging because of the lack of
photostable and bright FLAPs that fluoresce in the NIR region.
The fluorogenic nature of FLAPs enables imaging RNAs of
interest (ROIs) in living cells in the presence of their cognate
ligands. However, differences in the cellular uptake of the dye
between different cells, heterogeneous probe distribution within a
single cell, probe instability due to photobleaching, variations in
the cell morphology such as cell thickness and focal plane during
an imaging experiment could cause signal fluctuations and
consequently misinterpretation of data[16]. An effective way to
solve this problem is to use a ratiometric system that enables
imaging both the free dye and the RNA-fluorophore complex
simultaneously. Unfortunately, all FLAPs reported so far are
based on a single-color fluorescence turn-on.
In this work, to address these issues, we report the evolution,
characterization, and application of a novel color-shifting NIR-
fluorescent
aptamer-fluorophore
module
based
on
spirolactamization of fluorophores for RNA imaging in living cells
(Figure 1). The free fluorophore is cyan fluorescent in solution
while the aptamer-bound form fluoresces in the NIR region. We
demonstrate the application of this color-shifting FLAP system as
a genetically encoded tag to visualize mRNAs in the NIR region
using its light-up feature as well as its capability for ratiometric
analysis of RNA expression levels in single cells.
Introduction
RNA labeling methods combined with advanced fluorescence
microscopy enable imaging the complex dynamics of RNA in
living cells with a high temporal and spatial resolution. The
conventional methods to label RNA employ fluorophore-labeled
hybridization probes[1] and molecular beacons[2], yet they suffer
from impermeability and heterogeneous distribution of the
probes[3]. Fluorescent protein-tagged RNA-binding proteins
(RBP) are still the current gold standard for live-cell RNA
imaging[4]. However, these RBP-based tagging systems including
a recently developed CRISPR-Cas system for endogenous RNA
imaging[5] have limitations such as high background fluorescence
and potential alterations in RNA properties due to the bulky tag[3].
The currently emerging field of fluorescent light-up RNA
aptamers (FLAPs) provides powerful tools for live-cell RNA
imaging. Non-covalent binding of cell-permeable, fluorogenic
dyes to their cognate aptamers induces a fluorescence turn-on,
enabling RNA detection with high signal-to-background ratios.
Among literature-reported FLAP systems, the origin of dye
fluorescence quenching can be based on: i) vibrational and
rotational motions (e.g. MG aptamer[6], Spinach[7] and its
relatives[8], Pepper[9]); ii) ground-state complex formation (e.g.
SRB-2[10], DNB[11], Riboglow[12], o-Coral[13] and RhoBAST[14]); iii)
spirolactonization (e.g. SiRA[15]). SiRA was an important
BC binding
aptamer (BeCA)
+ BeCA
- BeCA
Spirocyclic BC
Zwitterionic BC
Cyan fluorescence
Near-infrared fluorescence
Figure 1. Design principle of color-shifting, NIR fluorescent aptamer-fluorophore
module based on benzopyrylium-coumarin (BC) fluorophores. Free BC is in the
cyan fluorescent spirocyclic form, whereas the aptamer (BeCA)-bound BC is in
the NIR fluorescent zwitterionic form.
Results and Discussion
Design of a Color-Shifting Aptamer-Fluorophore Module.
Rhodamine dyes have been widely used for biological imaging
1
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10.1002/anie.202107250
Angewandte Chemie International Edition
RESEARCH ARTICLE
owing to their high quantum yield, excellent photostability, and
good cell permeability[13, 15, 17]. Furthermore, the rational design of
new fluorescent probes that modulate the spirocyclization of
rhodamine dyes is becoming a powerful method for background-
free fluorescence microscopy in vivo[18]. The spirocyclic (lactone
or lactam) form of rhodamine is colorless and non-fluorescent due
to the disruption of the conjugated π-system, whereas the open
form is colored and fluorescent due to the extended π-conjugation.
This fluorescence switching mechanism has also been applied to
NIR-fluorescent silicon rhodamines, enabling precise modulation
of the ring-open and closed forms[18-19]. However, these NIR
fluorescent rhodamine scaffolds can only be used for
fluorescence turn-on probes.
Here, we aimed to design a color-shifting aptamer-
fluorophore module that enables simultaneous imaging of the
RNA-fluorophore complex and the free fluorophore. Moreover, it
is desirable to have the emission of the complex in the NIR region
due to its better live cell compatibility. To this end, we chose an
environmentally sensitive hybrid fluorophore consisting of
benzopyrylium and coumarin moieties (BC) as the core structure
of our probes. Similar to rhodamine dyes, BC fluorophores exist
in an equilibrium between a spirocyclic and a zwitterionic form,
however the closed form is cyan and the open form is NIR-
fluorescent[20]. We envisioned that the color-switching property of
BC fluorophores could be exploited for ratiometric RNA imaging
provided that a high-affinity aptamer binding exclusively to the
open form can be generated. Moreover, the fluorescence light-up
feature of the aptamer can be used for imaging RNAs in NIR
region. The ideal color-shifting BC compound should exist mainly
as a cyan fluorescent spirocyclic form in the unbound state;
however, it should efficiently switch to the NIR fluorescent
zwitterionic form upon binding to the aptamer.
A
BC1: R =
BC1-NH2: R =
BC1-SS-Biotin: R =
BC1-DN: R =
B
C
(N26)
(N26)
Primer binding sites
Primer binding sites
19-nt
40
26-nt 12-nt
26-nt
20-nt
35.3
27.9
30
20
10.5
8
8.7
10
0
6.7
5.6
3.3
2.8
7
1.3
6
0.78
2
0.06
1
3
4
5
9
10 11
SELEX round
Ligand (μM)
RNA (μM)
Washes
4000
100
10
50
25
10
100
6
50
12
Formamide
30
Elution
DTT
In order to increase the chances to evolve an aptamer
binding preferentially to the NIR fluorescent open form, we
planned to use a BC fluorophore that mainly exists in the
zwitterionic form in aqueous solution at physiological pH. This
fluorophore was immobilized on a solid support as bait during the
in vitro evolution of aptamers. After the discovery of the aptamer,
the structure of the BC scaffold was minimally modified in order
to fine tune the open-closed ratio. Finally, the color-shifting
efficiency of the aptamer upon binding to a series of BC analogs
was investigated to find the best aptamer-fluorophore pair for
ratiometric RNA imaging.
Figure 2. In vitro selection of BC-binding aptamers. (A) Chemical structures of
BC1, BC1-NH2, BC1-SS-Biotin and BC1-DN. (B) Design of the partially
structured RNA library. (C) Progress of the in vitro selection monitored by
measuring the fraction of RNA eluted from the BC1-conjugated beads.
reverse-transcribed and PCR-amplified. The obtained DNA was
in vitro transcribed and the enriched RNA pool served as an input
for the next round of selection (Figure S1). After 5 rounds of
SELEX, the fraction of eluted RNA increased to 35%, indicating
the successful selection of BC1-binding aptamers (Figure 2C). In
order to select for high affinity binders, we increased the selection
pressure by gradually decreasing the ligand concentration on the
beads. For this purpose, we synthesized a biotinylated BC1 ligand
containing a linker with a disulfide bond (BC1-SS-Biotin, Figure
2A) and used streptavidin-conjugated beads as solid supports
(Scheme S1). This way, the concentration of BC1-SS-Biotin on
streptavidin-beads could be precisely and easily controlled and
decreased during the course of SELEX. Furthermore, bound RNA
could be specifically eluted with DTT by reducing the disulfide
bond between the BC1 and biotin. Selection stringency was
increased gradually by decreasing the ligand concentration from
4 mM to 10 μM, lowering RNA input concentration from 100 to 10
μM, increasing the washes from 6 to 30 column volumes, and
eluting resin-bound RNA with DTT (Figure 2C). After eleven
iterative rounds, the eluted RNA pool was reverse-transcribed,
PCR-amplified, cloned into plasmids that were subsequently
transformed into bacteria. Single colonies of bacteria were picked
and the plasmids were sequenced by Sanger sequencing.
In vitro Evolution of Aptamers for BC1. We performed SELEX
(Systematic Evolution of Ligands by EXponential enrichment) to
find RNA aptamers binding specifically to the BC zwitterion[21]
.
BC1 was chosen as the ligand for in vitro selection because it
predominantly exists in the NIR fluorescent zwitterionic form[20b]
(Figure 2A). To attach ligands to a solid support for SELEX,
amine-functionalized BC1 (BC1-NH2, Figure 2A) was synthesized
and immobilized on N-Hydroxysuccinimide-activated sepharose
beads (Scheme S1)[15, 20b]
.
We started the selection with an RNA library containing
∼3×1015 different sequences. The sequence of the RNA library
consisted of a 5'-forward primer binding site, a 26-nucleotide (nt)
random region, a 12-nt constant sequence forming a stable stem-
loop, another 26-nt random region and a 3'-reverse primer binding
site[22] (Figure 2B). RNA transcripts were first incubated with a
mock resin to remove sepharose-binding RNA sequences, and
then with sepharose beads functionalized with BC1 (~4 mM on
the resin). After washing the beads, bound RNA was
nonspecifically eluted with formamide solution, precipitated,
Identification of a High Affinity BC1-Binding Aptamer. After
analyzing 60 individual colonies, we identified 52 unique RNA
sequences (Figure S2), all of which were in vitro transcribed for
2
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10.1002/anie.202107250
Angewandte Chemie International Edition
RESEARCH ARTICLE
20
U
A
U
A
U
U
U
U
A
B
C
G
C
A
G
U
C
C
G
G
U
C
A
G
C
60
1000
750
U
C
A
Truncation
Mutation
A
G
150
100
50
BC1-DN
G
G
G
A
KD = 230 ± 50 nM
G
U
G
C
A
U
BC1-DN + BeCA
G
A
A
A
G
G
U
G
G
A
U
G
G
A
G
G
C
C
U
C
10
40
30
G
C
A
20
500
A
G
C
U
G
U
A
A
C
A
C
C
U
G
C
A
G
G
A
C
U
G
A
C
A
A
C
G
C
G
C
G
U
C
G
G
A
C
C
U
C
C
C
G
C
U
U
C
U
U
80
C
G
G
G
U
U
U
U
G
250
0
C
U
A
U
C
BeCA
G
G
G
C
A
C
C
G
C
C
U
A
G
C
(RNA8-2)
1
A
G
G
G
C
G
G
U
C
0
C
A
100
1
38
101
102
103
104
700
750
800
112
Conserved region from library design
Conserved loop evolved through SELEX
Wavelength (nm)
[RNA] (nM)
RNA8
Figure 3. Truncation studies of BC1-binding RNA aptamers. (A) Predicted secondary structure of RNA8 and the minimal BC1-binding aptamer BeCA. The
conserved nucleotides are highlighted in blue or yellow depending on their origin. (B) Fluorescence emission spectra of BC1-DN (100 nM) in the presence or
absence of BeCA (10 μM). (C) KD curve of the BeCA-BC1-DN complex. Fluorescence intensities were recorded after incubating BC1-DN (100 nM) with different
concentrations of the BeCA aptamer (0 – 25 μM). For the fluorescence measurements in panels (B) and (C), an excitation of 652 ± 5 nm and a buffer containing
20 mM Hepes (pH 7.4), 5 mM MgCl2, and 125 mM KCl were used.
screening according to their affinity towards BC1. Since BC1 is
predominantly in the NIR-fluorescent zwitterionic form, binding of
an aptamer to BC1 would not necessarily cause a fluorescence
or spectral change, making BC1 impractical for aptamer
screening. To solve this problem, we synthesized a fluorescence
turn-on probe of BC1 by attaching the contact quencher
dinitroaniline (DN) to yield BC1-DN (Figure 2A and Scheme S2).
DN effectively quenches the fluorescence of BC1 by forming an
intramolecular ground state heterodimer[10]. In the presence of a
BC1-binding aptamer, the fluorophore would interact with the
aptamer rather than the quencher, thereby enhance the
fluorescence. All RNA transcripts were screened based on the
fluorescence intensity enhancement of BC1-DN (100 nM) upon
mixing with aptamers (10 μM) (Figure S2). Out of 52 sequences,
density in the π-conjugated system allows more efficient
nucleophilic attack by the carboxyl group to form the
spirolactone[23]. The second approach was, however, based on
increasing the nucleophilicity of the carboxyl group without
modifying benzopyrylium and coumarin. The conversion of the
carboxyl group to electron-deficient amides has recently been
reported to shift rhodamines’ equilibrium to the spirocyclic
configuration[18a, 20d]. Hence, electron-withdrawing fluorine atoms
(BC 2–4) and electron-deficient amides (BC 5–7) were introduced
to the BC1 scaffold to yield a series of BC analogs (Figure 4 and
Scheme S3). Probes BC 1–7 were synthesized and characterized
BC1
G
A
G
A
A
U
G
G
λabs: n.d./640
λem: n.d./681
D50: 23
8
were determined to be highly active showing >6-fold
U
U
G
C
C
G
A
U
G
C
C
G
C
G
U
A
C
G
C
G
A
U
fluorescence enhancement, 29 were moderately active (3- to 6-
fold), and 15 were less active (<3-fold). After measuring
dissociation constants (KD) between BC1-DN and the highly
active sequences (Figure S3), we discovered that RNA8 showed
the best binding affinity (KD = 920 nΜ) and 10-fold fluorescence
enhancement; therefore, it was selected for further investigation.
Truncation of RNA8 and the stabilization of the terminal
helix by inserting an extra G-C pair yielded a minimal 38-nt RNA8-
2 aptamer, dubbed as BeCA (Figure 3A and Figure S4). BeCA
binds BC1-DN with a KD of 230 nM, and displays a 9.7-fold
fluorescence turn-on (Figure 3B,C). Further truncations or
deletion of the internal hairpin structure caused a significant loss
of fluorogenicity (Figure S4). Sequence alignment and secondary
structure analysis of the aptamers with the highest turn-on values
revealed that sequences GUGG and AGGAA in the main loop are
conserved (Figure 3A, Figure S5A). Additional mutational studies
did not considerably improve the affinity and fluorescence turn-on
of BeCA (Figure S5B).
A
G
U
A
G
C
U
G
Turn-on: 3.9
BeCA
Amidation
BC5
Fluorination
BC2
λabs: 402/644
λem : 475/686
D50: 39
λabs: 419/n.d.
λem: 477/n.d.
D50: >>70
Turn-on: 8.5
Turn-on: 1.6
BC3
BC6
λabs: n.d./638
λem: n.d./681
D50: 30
λabs: 420/652
λem: 478/686
D50: 51
Turn-on: 2.7
Turn-on: 8.2
BC4
BC7
λabs: 413/632
λem: 477/686
D50: 46
λabs: 419/647
λem: 477/686
D50: 62
Fine-Tuning Spirocyclization of BC. The BeCA aptamer was
designed and evolved to selectively bind BC1, which mainly exists
in the zwitterionic form. To obtain a color-shifting ligand for BeCA,
we aimed to increase the propensity of BC1 to form the
corresponding spirocyclic form in the unbound state by chemically
altering its structure. However, these modifications should be
minimal so that the specific interactions between BeCA and BC1
are not negatively affected. For this purpose, we took two different
approaches. The first approach was based on the introduction of
electron-withdrawing fluorine atoms to the aromatic groups in
benzopyrylium and coumarin (Figure 4). Decreasing the electron
Turn-on: 2.1
Turn-on: 2.1
Figure 4. Chemical structures of BC 1–7 and their fluorescence properties. λabs
and λem represented the maximum absorption and emission wavelength (nm)
of the spirocyclic/zwitterionic forms, respectively, in 20 mM Hepes (pH 7.4)
containing 0.1% Triton X-100. n.d. represents not detected. D50 represented the
dielectric constants at which half of the maximum absorbance of zwitterions
were observed in dioxane/water mixture (v/v: 10/90 – 90/10). Turn-on factors
were determined by dividing maximal NIR fluorescence intensities of BC
fluorophores (100 nM) in the presence of BeCA (10 μM) by those of BC
fluorophores (100 nM). For the fluorescence measurements, the BC
fluorophores were excited at their excitation maxima (± 5 nm) in a buffer
containing 20 mM Hepes (pH 7.4), 5 mM MgCl2, and 125 mM KCl.
3
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10.1002/anie.202107250
Angewandte Chemie International Edition
RESEARCH ARTICLE
20a,
23b]
using literature-reported routes[18a,
Information).
(Supporting
A
B
0.06
0.04
0.02
0.00
BC6
BC6 + BeCA
BC6
BC6 + BeCA
1.0
0.5
0.0
We then evaluated how fluorine or amide substitutions can
modulate the equilibrium between spirocylic and zwitterionic
forms by examining the absorbance of BC 1–7 as a function of
the dielectric constant. Using water-dioxane mixtures, we
determined the D50 values, the dielectric constant at which half of
the maximum absorbance of zwitterion was observed (Figure 4
and Figure S6). Water promotes the formation of the zwitterionic
form due to its high dielectric constant (εr = 80.4), whereas
dioxane with a low dielectric constant (εr = 2.2) favors the
spirocyclic form. Rhodamine-based fluorophores with D50 values
around 50 have been shown to be suitable candidates for the
generation of fluorogenic probes for cellular imaging[18a]. BC1
showed a D50 value of 23, indicating that it exists primarily in the
zwitterionic form in aqueous solution. Addition of an electron-
withdrawing fluorine atom to coumarin (BC3) and to
benzopyrylium (BC2) increased the D50 values to 30 and 39,
respectively. This result confirmed that fluorine substitutions
successfully shift the equilibrium towards closed form in aqueous
solution. Furthermore, introduction of two fluorine atoms (BC4),
one for each coumarin and benzopyrylium, elevated the D50 value
to 46, as expected.
Exchanging the carboxylic acid group with an amide group
(BC5) yielded a fluorophore with dramatically increased D50 (≫
70), implying that BC5 is almost completely in the cyan
fluorescent spirolactam form in water. However, the BC
derivatives carrying cyanamide (BC6) and methylsulfonamide
(BC7) have D50 values of 51 and 62, respectively, lower than that
of BC5 due to the decreased nucleophilicity of electron-deficient
amides. Yet, both BC6 and BC7 displayed much higher D50
values than BC1.
Ex
Ex
400 500 600 700 800
Wavelength (nm)
350
450
600
700
Wavelength (nm)
C
D
BC6
BC6 + BeCA
1.0
0.5
0.0
1.0
0.5
0.0
Em
Em
15
100 101 102 103 104
[RNA] (nM)
450
550
700
800
Wavelength (nm)
Figure 5. Photophysical characterization of the BeCA-BC6 complex.
Absorption (A), excitation (B), and emission (C) spectra of BC6 (1 μM) in the
presence and absence of BeCA (25 μM). (D) Plot of the emission ratio
(Fcyan/FNIR) of BC6 (100 nM) as a function of BeCA concentration (0 – 25 μM).
The spectra were measured in a buffer containing 20 mM Hepes (pH 7.4), 5
mM MgCl2, 125 mM KCl, and 0.05% Triton X-100.
fluorescence (휆 = 478 nm) dropped while the NIR fluorescence
(휆 = 684 nm) increased with increasing concentration of BeCA.
The color-shifting process of BC6, from unbound to BeCA-bound
state, produced a dynamic range (cyan/NIR emission ratio
change) as high as 15-fold, substantially higher than the single-
wavelength NIR fluorescence turn-on (Figure 5).
BeCA binds the ligand BC6 with a KD of 220 nM (Figure
S8A), displaying essentially the same affinity as BC1-DN. Notably,
the fluorescence of BeCA-BC6 is independent of magnesium and
potassium ions: 90% of the maximum fluorescence was retained
even in the absence of either of these cations, suggesting that the
folding of BeCA and its complexation with BC1 are independent
of magnesium and potassium (Figure S8B,C). An increase in
temperature from 25 to 37 °C caused a decrease in the
fluorescence of BeCA-BC6 by only 25%, comparable to other
literature-reported, thermally stable aptamer systems[8a, 24] (Figure
S8D).
BeCA as a NIR Light-up Aptamer for RNA Imaging in Live
Cells. First, we analyzed the utility of BeCA-BC6 as a NIR
fluorescence turn-on tag for imaging mRNAs, which generally
possess short half-lives, complex structures and have low
abundance. To this end, eight synonymous copies of BeCA
without any stabilizing scaffold were introduced into the 3'-
untranslated region of the green fluorescent protein (gfp) gene.
Escherichia coli (E. coli) expressing either gfp (control) or gfp-
BeCA8 mRNA were imaged in the presence of BC6 (Figure 6 and
Figure S9). The bright NIR fluorescence emission was detected
predominantly at the poles of bacteria expressing gfp-BeCA8
mRNA, but not in the control cells. The accumulation of mRNA at
the bacterial poles demonstrated by the NIR signal was likely due
to localization of the pET plasmid where mRNA transcription
initiates[24-25]. The average NIR fluorescence intensity at the
bacterial poles of gfp-BeCA8-expressing cells was 6-fold higher
than that of the control bacteria when 200 nM of BC6 was used.
Furthermore, bacteria expressing gfp or gfp-BeCA8 displayed
similar GFP fluorescence intensities, suggesting that the BeCA
Overall, minimal chemical modifications on the BC1 scaffold
enabled fine-tuning of the equilibrium between spirocyclic and
zwitterionic forms as demonstrated by the increased D50 values,
ranging from 23 to >70. As a result, fluorophores BC 4–7 hold
potential to function as color-shifting ligands for the BeCA
aptamer in imaging applications.
Characterization of a Color-Shifting Aptamer-Fluorophore
Module. The ideal color-shifting BC fluorophore for BeCA should
be cyan fluorescent in solution but emit in the NIR region upon
binding to the aptamer. Therefore, the binding of BeCA to the
ideal fluorophore would decrease the cyan fluorescence, increase
the NIR fluorescence, and thereby increase the NIR/cyan ratio.
First, we evaluated whether the chemically modified BC 4–7
analogs bind BeCA and cause a fluorescence increase in NIR
emission and a decrease in cyan fluorescence (Figure 4). Among
these dyes, BC6 showed the highest fluorescence enhancement
of 8.2-fold upon aptamer binding. As indicated by the D50 value,
BC6 displayed its maximum absorbance in the cyan region and
existed mainly in the spirocyclic form (Figure S6). Therefore, BC6
was chosen as the color-shifting ligand for BeCA. Furthermore,
the cyan and NIR fluorescence intensities of BC6 did not
significantly change in the presence of total RNA, supporting the
specific interaction between BC6 and BeCA (Figure S7).
Next, we examined the photophysical features of BC6 in the
presence of BeCA (Figure 5 and Table S1). Titration of BC6 with
the BeCA aptamer led to a decrease in the absorbance peak of
the spirocyclic form (휆 = 420 nm), while an increase in the
absorbance peak of the zwitterionic form (휆 = 665 nm), indicating
the propensity of BC6 to favor the zwitterionic form when bound
to BeCA. Consistent with the absorbance change, the cyan
4
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10.1002/anie.202107250
Angewandte Chemie International Edition
RESEARCH ARTICLE
NIR signal in bacteria expressing BeCA, yet it resulted in elevated
NIR background fluorescence in control bacteria. As expected,
using a higher concentration of BC6 increased the cyan
fluorescence in both BeCA-expressing and control bacteria. Even
in the presence of as low as 200 nM of BC6, both cyan and NIR
channel images of BeCA-expressing cells exhibited excellent
image quality.
gfp
PT7 lacO
gfp
T
PT7 lacO
BeCA8
T
gfp
gfp-BeCA8
GFP
NIR
GFP
NIR
1000
Next, we tested if we can detect variations in cyan and NIR
emission intensities of BeCA-BC6 upon environmental changes
such as exposure to high salt concentration. Upon addition of
ammonium acetate to the BeCA-BC6 complex in vitro, which can
replace mono and divalent cations in the aptamer, we observed
an increase in the cyan fluorescence (1.7-fold) and decrease in
both NIR fluorescence (4.1-fold) and the ratio of NIR/cyan
fluorescence (6.8-fold) intensities. This can be explained by
unfolding of the aptamer followed by the dissociation of the dye
from the aptamer (Figure S12A). Motivated by this result, we
performed a similar experiment with BeCA-expressing live
bacteria that had already been incubated with BC6. After a rapid
treatment of the bacteria with ammonium acetate, confocal
images indicated a fluorescence increase in the cyan channel and
a decrease in both NIR and ratiometric (NIR/cyan) channels as
anticipated (Figure S12B,C). Ratiometric images were obtained
by pixel-to-pixel division of the fluorescence intensities in the NIR
channel by the cyan channel. This experiment allowed us to
visualize the unfolding of the BeCA aptamer in live bacteria
exploiting the ratiometric features of BeCA-BC6.
40
Figure 6. Confocal images of gfp mRNA tagged with BeCA in live bacteria. E.
coli bacteria were transformed with either gfp-BeCA8 or gfp plasmids and
mRNA expression was induced by the addition of IPTG. The cells were
incubated with BC6 (200 nM) for 15 min and imaged. Images in GFP and NIR
channels were acquired using 488 nm and 640 nm lasers. Scale bar, 5 μm.
tag did not significantly affect the transcription and translation
processes.
BeCA was also expressed as synonymous repeats in
mammalian cells and successfully visualized using the
fluorophore BC6 in HEK293T cells (Figure S10). This experiment
demonstrated that BeCA folds correctly in mammalian cells and
could be used to image other target RNAs utilizing its light-up
feature and NIR fluorescence.
BeCA for Ratiometric RNA imaging in Live Bacteria. To
investigate whether the BeCA-BC6 color-shifting aptamer-
fluorophore module could be used for ratiometric imaging of RNAs,
proof-of-principle live-cell imaging experiments were performed.
We expressed BeCA embedded in a tRNA scaffold in E. coli and
imaged bacteria in the presence of BC6 (500 nM) (Figure S11).
BeCA-expressing cells showed a much higher fluorescence
signal in the NIR channel than the tRNA-expressing control cells,
indicating that BeCA selectively binds the BC6 zwitterion and
lights up in the NIR channel. On the other hand, bacteria, whether
expressing BeCA or not, showed similar cyan fluorescence due
to the dynamic equilibrium between the intracellular and
extracellular BC6. Increasing the concentration of BC6 from 500
nM to 1 µM in bacterial imaging did not significantly improve the
We also used the color-shifting capability of BeCA-BC6 for
the analysis of RNA expression levels in living cells. The
transcription of BeCA can be effectively activated by isopropyl-β-
d-thiogalactopyranoside (IPTG)[26]. Cells expressing BeCA were
treated with varying concentrations of IPTG, and imaged in both
cyan and NIR channels simultaneously (Figure 7A). The mean
NIR/cyan ratios of bacteria treated with 20, 50, 100, and 1000 μM
of IPTG were determined to be, respectively, 1.7, 2.4, 3.0 and 3.7
A
B
7
IPTG (μM)
10
20
50
100
1000
5
800
3
1
50
1000
1 0
2 0
5 0
1 0 0
1 0 0 0
[IPTG] (μM)
C
D
10
20
50
100
1000
[IPTG] (μM)
BeCA
0
5
Cyan
a b
NIR
a b
NIR/Cyan
a b
e
e
e
d
d
d
c
c
c
0
Figure 7. Confocal images of live E. coli expressing different levels of BeCA. (A) Bacteria were transformed with plasmids expressing BeCA and the transcription
was induced by addition of IPTG at varying concentrations (10 – 1000 μM). Then, bacteria were incubated with BC6 (200 nM, 15 min) and imaged in M9 medium
containing 2 mM of MgCl2. Images in cyan and NIR channels were acquired using 405 nm and 640 nm lasers, respectively. Scale bar: 5 μm. (B) Quantification of
the mean fluorescence ratio (NIR/cyan) of bacteria expressing BeCA at different IPTG concentrations (10 – 1000 μM). Each dot represents a single cell. Means ±
standard deviation (s.d.) are shown (n = 50 cells from five independent images). Mean of the NIR/cyan ratio for bacteria treated with 10 μM was normalized to 1.
(C) Denaturing polyacrylamide gel analysis of BeCA in total RNAs isolated from bacteria treated with various IPTG concentrations (10 – 1000 μM). (D) Zoom-in
images of the BeCA-expressing cells depicted in panel A. Green arrows indicate the cells a and b, of which BeCA concentration was corrected using ratiometric
analysis. White arrows show transcription heterogeneity at the single-cell level. Transcription efficiency analysis according to the ratiometric images: cell c > cell d
> cell e.
5
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10.1002/anie.202107250
Angewandte Chemie International Edition
RESEARCH ARTICLE
fold higher than that of bacteria treated with 10 μM of IPTG (Figure
7B). These numbers correlate well with in vitro quantification of
BeCA in total RNAs isolated from the bacteria treated with
different concentrations of IPTG (Figure 7C and Figure S13). This
result clearly shows that the ratiometric images obtained by our
color-shifting FLAP system can be utilized to evaluate the
expression levels of RNA transcripts.
We also showed that multiple repeats of BeCA can be
genetically fused to more complex and short-lived mRNAs to
increase the signal-to-background ratio. Combined with the
ratiometric advantages, BeCA-BC6 can be used to precisely
analyze the abundance of target RNAs, which could provide new
insights in gene expression, regulation, developmental plasticity
and disease diagnostics[28]
.
Besides the determination of the intracellular concentration
of a particular ROI at the population (bulk) level, the ratiometric
images obtained from the BeCA-BC6 system could provide more
precise information at the single-cell level. As shown in Figure 7D,
the amounts of RNA transcripts in single bacteria (cells a and b)
do not always correlate well with the NIR fluorescence intensities.
The spirocyclic probes including BC6 have excellent
membrane permeability compared to their zwitterionic
counterparts[20d]. Further, spirocyclic probes are less likely to be
switched to their zwitterionic forms by other cellular components,
thus resulting in less unspecific NIR staining inside the cells. As
demonstrated in this study, aptamer binding can significantly
This might be due to variations in intracellular probe concentration, change their biophysical properties and modulate equilibrium
cell thickness and focal plane. Owing to the dual-fluorescence
nature of BeCA-BC6, we can use the cyan channel to correct for
these discrepancies. Indeed, the ratiometric signals suggest that
cells a and b in Figure 7D had similar levels of intracellular BeCA
transcripts. Extensive heterogeneity of RNA transcription at the
single-cell level in E. coli due to stochasticity in transcriptional
regulation could also be revealed in the ratiometric images
dynamics. In the future, by employing library mutagenesis and
fluorescence activated cell sorting (FACS), it would be possible to
evolve tailored color-shifting fluorophore-aptamer pairs with
various colors, higher brightness, better affinity and improved
thermal stability.
especially at high IPTG concentration (Figure 7D, cells c-e)[27]
.
Acknowledgements
J.Z. was supported by the China Scholarship Council (Grant No.
201606100059). A.J. and M.S. were supported by the Deutsche
Forschungsgemeinschaft (DFG Grant # Ja794/11).
Conclusion
In summary, we have presented the development of a color-
shifting NIR fluorescent aptamer-fluorophore module BeCA-BC6
for live-cell RNA imaging. To achieve that, we exploited the
intramolecular spirocyclization of an environmentally sensitive
hybrid fluorophore, BC. It exists in a dynamic equilibrium between
a cyan-fluorescent, spirocyclic, closed form and a NIR-fluorescent,
zwitterionic, open form. In vitro selection, truncation, and mutation
studies rendered a 38-nt minimal aptamer BeCA, which binds
selectively to the zwitterionic form of BC. By introducing electron-
withdrawing fluorine atoms and electron-deficient amine groups
to BC, we obtained a series of BC analogs with various open-
closed ratios. The best probe BC6 exists primarily in the closed
state with an emission maximum of 478 nm and emits at 684 nm
when bound to BeCA, representing the most NIR-shifted FLAP in
the literature. Thus, BeCA-BC6 is a valuable addition to the RNA
imaging toolbox due to the lack of FLAPs functioning in the NIR
window where cells have much lower absorption (less
phototoxicity), lower auto-fluorescence and deeper penetration.
Moreover, we demonstrated that BC6 showed an emission
ratio change (cyan/NIR) as high as 15-fold upon binding to BeCA.
In live-cell RNA imaging experiments, the cyan fluorescence from
unbound probes reveals the intracellular probe delivery and its
distribution while the NIR fluorescence indicates the RNA location.
BeCA-BC6 is the only aptamer-fluorophore pair which allows
simultaneous imaging of both free fluorophore and the complex.
We used this feature to obtain ratiometric images of bacteria with
different expression levels of BeCA. Ratiometric images, in
contrast to single-color fluorescence images, do not suffer from
problems associated with varying dye uptake, heterogeneous
probe distribution, probe instability, cell morphology and
fluctuations in focal plane. Thus, the dual-color feature of BeCA-
BC6 allowed us to more accurately analyze the expression levels
of RNA transcripts and revealed the transcriptional heterogeneity
at the single-cell level.
Keywords: fluorophores • RNA aptamer • RNA imaging •
ratiometric imaging • SELEX
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RESEARCH ARTICLE
Entry for the Table of Contents
BC binding
aptamer (BeCA)
+ BeCA
- BeCA
Live-cell RNA imaging
Cyan fluorescent spirocyclic BC
Near-infrared fluorescent zwitterionic BC
A color-shifting, near-infrared (NIR) fluorescent aptamer-fluorophore module based on benzopyrylium-coumarin (BC) fluorophores
for ratiometric RNA imaging is reported. Free BC is cyan fluorescent, however its fluorescence emission shifts to NIR upon
complexation with the BC-binding aptamer (BeCA). This system enabled visualization of mRNAs in NIR region and ratiometric
analysis of target RNAs expressed at different levels in single cells.
8
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