A Multicolor Large Stokes Shift Fluorogen-Activating RNA Aptamer with Cationic Chromophores

Article abstract of DOI:10.1002/chem.201805882

Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4-hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogues that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functionalization at the C2 position of the imidazolone, this strategy yielded DMHBO⁺, which binds to the Chili aptamer with a low-nanomolar K_D. Because of its highly red-shifted fluorescence emission at 592 nm, the Chili–DMHBO⁺ complex is an ideal fluorescence donor for F?rster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems.

Full text of DOI:10.1002/chem.201805882

A Journal of
Accepted Article
Title: A multicolor large Stokes shift fluorogen-activating RNA aptamer
with cationic chromophores
Authors: Christian Steinmetzger, Navaneethan Palanisamy, Kiran R.
Gore, and Claudia Höbartner
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.201805882
Link to VoR: http://dx.doi.org/10.1002/chem.201805882
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A multicolor large Stokes shift fluorogen-activating RNA aptamer
with cationic chromophores
Christian Steinmetzger,[a] Navaneethan Palanisamy,[b]# Kiran R. Gore[c]§ and Claudia Höbartner*[a,b,c]
Abstract: Large Stokes shift (LSS) fluorescent proteins (FPs) exploit
excited state proton transfer pathways to enable fluorescence
emission from the phenolate intermediate of their internal
4-hydroxybenzylidene imidazolone (HBI) chromophore. An RNA
aptamer named Chili mimics LSS FPs by inducing highly Stokes-
shifted emission from several new green and red HBI analogs that are
non-fluorescent when free in solution. The ligands are bound by the
RNA in their protonated phenol form and feature a cationic aromatic
side chain for increased RNA affinity and reduced magnesium
dependence. In combination with oxidative functionalization at the C2
position of the imidazolone, this strategy yielded DMHBO+, which
binds to the Chili aptamer with a low-nanomolar KD. Because of its
highly red-shifted fluorescence emission at 592 nm, the Chili–
DMHBO+ complex is an ideal fluorescence donor for Förster
resonance energy transfer (FRET) to the rhodamine dye Atto 590 and
will therefore find applications in FRET-based analytical RNA systems.
to an RNA aptamer prevents non-radiative deactivation of the
excited state, thereby restoring the intrinsic fluorescence.
The majority of known fluorogenic aptamers is characterized
by a small or moderate difference between their excitation and
emission maxima. However, fluoromodules with large Stokes
shifts display significant advantages, such as reduced
reabsorption of the emitted light,[12] convenient use as FRET
donors for far-red fluorescence reporters,[13] and the ability to
produce multiple emission colors by simultaneous excitation of
different fluorophores at a single wavelength.[14] Large energy
differences between absorption and emission peaks can result
from intramolecular charge transfer (ICT), or from inter- or
intramolecular excited state proton transfer (ESPT), among other
mechanisms.[12] Fluorescent proteins that exhibit large apparent
Stokes shifts (termed LSS FPs) have been engineered to exploit
ESPT from the aromatic OH group of the HBI chromophore to a
neighboring amino acid of the barrel scaffold. Prominent
members of this family are the orange/red emitting proteins
LSSmOrange[14] and LSSmKate,[15] which are excited at 437 and
460 nm and emit at 572 and 605 nm, respectively. Similarly large
Stokes shifts and fluorescence emission in the 600 nm region
have not been reported for fluorogenic RNA aptamers. In this
study, we aim to mimic LSS green and red fluorescent proteins by
capitalizing on fluorogen-activating RNA aptamers that bind the
protonated form of HBI analogs and enable ESPT and emission
from an excited deprotonated intermediate.
Fluorogen-activating RNA aptamers (FLAPs) have emerged
as powerful tools for tagging and visualizing RNA in vitro and in
vivo, and, among various other applications in biochemistry and
synthetic biology, they are used as components of metabolite and
protein sensors.[1] These artificial functional RNAs form specific,
non-covalent complexes with conditionally fluorescent chromo-
phores that are non-emissive free in solution, but show strongly
enhanced fluorescence emission in the bound state. Early
examples, including the Malachite green aptamer[2] and the
sulforhodamine aptamer,[3] are currently receiving renewed
attention for further optimization and reselection,[4,5] and new
chromophore-binding aptamers, such as cyanine dye-activating
Dir2s[6] and Mango RNA aptamers,[7,8] continue to be developed.
A prominent class of fluorogenic aptamers known as mimics of
fluorescent proteins, named Spinach,[9] Broccoli,[10] and Corn,[11]
were evolved to bind analogs of the GFP chromophore 4-
hydroxybenzylidene imidazolone (HBI). Synthetic HBI derivatives
are non-fluorescent in aqueous solution, but non-covalent binding
Spinach, Broccoli and Corn are members of the HBI-binding
aptamer family that activate derivatives of 3,5-difluoro-HBI
(DFHBI), which are almost completely deprotonated at physio-
logical pH.[9] Here, we capitalized on 3,5-dimethoxy-substituted
HBI (DMHBI) derivatives that have a higher pKa and can be
excited at the absorption maximum of the phenol form. We first
examined the 13-2 aptamer, which was originally reported to
activate the fluorescence of DMHBI, showing an emission at 529
nm upon excitation at 398 nm.[9] Based on the analysis of the
predicted secondary structure,[16] we introduced an extra-stable
UUCG tetraloop into 13-2min, with the intention to stabilize an
apical hairpin and to assist folding of the ligand binding pocket.
This modified 52-nt RNA aptamer (Figure 1) showed approx. 2-
fold enhanced fluorescence of bound DMHBI compared to the
original 60-nt RNA, and the fraction of RNA that folded into a
ligand binding-competent conformation was significantly
enhanced (Figure S1). The 52-nt RNA resulting from the
optimization of 13-2min was used for all further experiments
discussed in this manuscript, and – in line with the “vegetable
nomenclature” – was named Chili because of its special
properties to activate green, yellow and red chromophores.
[a]
[b]
C. Steinmetzger, Prof. Dr. C. Höbartner
Institute of Organic Chemistry, University of Würzburg
Am Hubland, 97074 Würzburg (Germany)
E-mail: claudia.hoebartner@uni-wuerzburg.de
N. Palanisamy, Prof. Dr. C. Höbartner
International Max Planck Research School Molecular Biology,
University of Göttingen (Germany)
#
present address: BIOSS Center for Biological Signaling Studies,
University of Freiburg (Germany)
[c]
Dr. K.R. Gore, Prof. Dr. C. Höbartner
Center for Nanoscale Microscopy and Molecular Physiology of the
Brain (CNMPB), Göttingen, (Germany)
§
present address: Department of Chemistry, University of Mumbai
(India)
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201805882
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complex with the Chili RNA. The DMHBI+ derivative carries a
trimethylammonium-substituted phenyl ring at N3, which may
provide additional stacking interactions with nucleobases, and the
cationic substituent may assist electrostatic interactions with the
negatively charged RNA backbone. These features should
enforce a rigid fluorophore conformation and stabilize the position
of the fluorophore at the binding site. The other two new DMHBI
analogs were extended at the C2 position: DMHBI–Imi mimics
Figure 1. a) Structures and names of the HBI derivatives investigated in this
work. b) Sequence of 52-nt Chili RNA and gel image of Tb3+-footprinting,
comparing unfolded and folded RNA in the absence and presence of DMHBI.
Full gel image in Figure S3. The flexible loop region is indicated in red.
The high guanine content of the Chili RNA sequence suggests
that it contains guanine quartets in the ligand binding site. This
hypothesis is supported by the finding that DMHBI fluorescence
is strongly dependent on the K+ concentration with an apparent
KD of 12 mM and a Hill coefficient of 2.5 (Figure S2), suggesting
the involvement of a two-tiered G quadruplex.[17,18] This feature is
reminiscent of other HBI-binding aptamers, including Spinach and
Corn, for which the crystal structures show highly unusual
quadruplex topologies,[18] as well as Mango aptamers, which also
contain quadruplexes as ligand-binding structural elements.[19]
Despite the overall similarity of the 2D secondary structure
schemes of Chili and Spinach/Broccoli (Figure S1), their
sequences are significantly different. In the absence of a 3D
structural model, it remains unknown which guanines of Chili are
involved in putative G quartets. However, it is interesting to note
that, compared to Spinach, Chili has additional nucleotides that
are expected to form larger loops extruding from a putative
quadruplex core. This is supported by structural probing data that
clearly identify the region of nt 34–38 in the folded and ligand-
bound state as more susceptible to backbone hydrolysis
compared to the unfolded state, i.e., in the absence of ligand and
Mg2+ (Figure 1b and Figure S3).
Figure 2. a) Normalized excitation and emission spectra (dashed and solid lines,
resp.) of DMHBI (blue), DMHBI+ (green), DMHBI-Imi (yellow), and DMHBO+
(orange) bound to Chili RNA. b) Integrated fluorescence intensity of each dye in
the presence (+) or absence (–) of the Chili RNA at pH 7.5. c) Titration with Chili
RNA reveals higher affinity for all three new chromophores compared to DMHBI
(KD listed in Table 1, experimental details given in supporting information).
the chromophore of the protein Kaede, which contains a histidine-
derived arylvinyl substituent.[21] Other red fluorescent proteins,
such as DsRed and its relatives, carry acylimine substituents
instead of arylvinyl groups at C2.[22] In combination with the
trimethylammoniumphenyl side chain described above, the oxime
in DMHBO+ was chosen as a hydrolytically stable acylimine mimic.
The three new chromophores were synthesized starting from
4-hydroxy-3,5-dimethoxybenzaldehyde (Scheme S1). DMHBI+
was obtained in three steps, starting with the condensation with
4-(dimethylamino)aniline. The imine was incorporated into the
HBI scaffold in a 1,3-dipolar cycloaddition reaction with an imidate
ylide, which was prepared from ethyl acetimidate and methyl
glycinate.[23] The aromatic dimethylamino group was alkylated
with methyl iodide to obtain DMHBI+. To generate DMHBO+, the
cycloaddition product was oxidized with selenium dioxide and the
resulting aldehyde was reacted with hydroxylamine, followed by
alkylation with methyl iodide. The Kaede analog DMHBI-Imi was
generated from DMHBI via a scandium triflate-catalyzed Aldol
condensation with imidazole-4-carboxaldehyde.[24]
The presence of the flexible bulge in Chili suggested an
opportunity to modulate the fluorescence properties of the RNA-
chromophore complexes by ligand engineering. Candidate sites
for modification are the positions N1 and C2 of the imidazolone,
similar to variations observed in fluorescent proteins, originating
from the primary protein sequences. In analogy, the DFHBI ligand
was earlier modified at these two sites, resulting in DFHBI-1T as
a brighter Spinach ligand[20] and DFHO as the ligand responsible
for orange emission in complex with the Corn aptamer.[11]
The fluorescence properties of the four dyes and their binding
and activation by Chili were investigated. Chili–DMHBI and Chili–
DMHBI+ showed excitation maxima near 400 nm and emission
maxima near 540 nm (Figure 2a), demonstrating that the
Here, we designed and synthesized three new DMHBI ligands
(Figure 1) and investigated their fluorescence properties in
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Me3N+Ph substituent at N3 has little influence on the conjugated
-electron system of DMHBI. However, a more than sevenfold
increase in fluorescence enhancement was observed compared
to the parent DMHBI (Figure 2b), suggesting that the cationic side
chain prevents non-radiative deactivation of the excited state
more effectively. The other two chromophores were expected to
yield red-shifted fluorescence emission. Indeed, Chili–DMHBO+
showed bathochromic shifts of the excitation (455 nm) and
emission maxima (592 nm), exceeding other RFP mimics like the
Corn (545 nm) and Red Broccoli (582 nm) systems.[11] Excitation
of Chili–DMHBI-Imi at 463 nm resulted in a bimodal emission
profile with maxima at 545 and 594 nm. The fluorescence
intensities of Chili–DMHBO+ and Chili–DMHBI-Imi were
comparable within 20% to the Chili–DMHBI complex, which is
also reflected in their comparable fluorescence quantum yields
(Table 1). Importantly, none of the DMHBI derivatives showed
unspecific fluorescence activation, as demonstrated by control
experiments with E.coli tRNA, other non-cognate RNA aptamers
(including Spinach), and DNA quadruplexes (Figures S4, S5).
at 456 nm (Figure 3b, S9). This suggests that the Chili RNA
exclusively binds the DMHBO+ chromophore in its protonated
state. The bathochromic shift of 20 nm compared to the free
phenol likely originates from a combination of -stacking and
electrostatic interactions.[26] Excitation of the protonated
chromophore–RNA complex is followed by fast deprotonation,
which results in the large apparent Stokes shift of 136 nm. These
results suggest that a functional group of the RNA near the phenol
acts as a proton shuttle. The identity of this functional group on a
nucleobase or in the ribose-phosphate backbone remains to be
identified.
Binding affinity and magnesium dependence are of crucial
importance for the application of fluorogenic RNA aptamers. In
the absence of Mg2+, the fluorescence intensity of the Chili
complexes with DMHBI and DMHBI-Imi was reduced to approx.
50% of the value obtained with 5 mM Mg2+. In contrast, the
cationic ligands DMHBI+ and DMHBO+ retained approx. 80% of
their maximum intensity even in the absence of Mg2+ (Figure S6),
suggesting that the positively charged side chain partially
compensated for the reduced availability of divalent metal ions.
Other recently reported systems, in which arylvinyl-substituted
HBI analogs were shown to be activated by parallel-stranded DNA
quadruplexes, required 50 mM Mg2+.[25]
Figure 3. a) Absorbance vs pH titration of DMHBO+ gives a pKa of 6.9. b) The
UV-VIS spectra of DMHBO+ in aqueous buffer at pH 7.5 (dashed line) and
bound to the Chili aptamer (solid line). c) Time course of the fluorescence
emission at 592 nm after mixing Chili and DMHBO+ under pseudo-first order
conditions follows a biexponential model. d) The apparent rate constants from
c) plotted versus DMHBO+ concentration.
The dissociation constants of the new FLAP complexes were
determined by titrating each fluorophore with increasing amounts
of RNA (Figure 2c, Table 1). We found a KD of 570 nM for DMHBI
binding to Chili RNA, similar to the original 13-2 aptamer.[9] For
the brighter Chili–DMHBI+ complex an almost 10-fold stronger
binding was observed with a KD of 63 nM. The DMHBI-Imi
chromophore was bound with similar affinity (KD of 71 nM),
suggesting that the bulky imidazolylvinyl substituent at C2 was
also well-accommodated. Interestingly, the cationic DMHBO+
chromophore displayed the tightest binding with a KD of 12 nM,
suggesting synergistic effects of the C2 and N3 substituents.
Such low-nanomolar affinities have been observed for the Mango-
TO1-biotin fluoromodules,[7,8] but could not be reached by
previous iterations of HBI-binding aptamers. The increase in
binding affinity and fluorescence enhancement by the Me3N+Ph
substituent is specific for DMHBI derivatives in complex with Chili.
Analogous modification of DFHBI and DFHO (Scheme S2)
completely eliminated binding and fluorescence activation by
Spinach and Broccoli (Figure S5).
Next, we studied the kinetics of DMHBO+ activation by the Chili
aptamer. The time course of fluorescence emission at 592 nm
after mixing Chili and DMHBO+ under pseudo-first order reaction
conditions followed a biexponential model with two apparent rate
constants (Figure 3c, d), of which only one was linearly dependent
on the dye concentration. This can be interpreted as a fast binding
of the chromophore to a partially preorganized binding site,
followed by a slow reorganization of peripheral residues.
Prolonged incubation of the RNA with K+- and Mg2+-containing
buffer prior to the addition of DMHBO+ had no influence on the
observed association kinetics (Figure S7). Therefore, folding of
the putative quadruplex is not rate-limiting and the slow refolding
of the binding site likely involves loop nucleotides. Overall, the
Chili LSS FLAP shows a much faster fluorescence turn-on
compared to the slow maturation times of LSS red fluorescent
proteins, which are in the range of hours.[14]
In consequence of this distinctive performance, the properties
of DMHBO+ and its complex with Chili were further investigated.
The pKa of the free chromophore was determined as 6.9 (Figure
3a), which results in approx. 80% deprotonation at pH 7.5. and
the appearance of the phenol (at 436 nm) and phenolate (at 547
nm) absorbance bands. Interestingly, the RNA-bound
chromophore exhibited a single absorption band with a maximum
The tight binding and strong fluorescence enhancement of
Chili–DMHBO+ can be used to visualize RNA in native and
denaturing polyacrylamide gels (Figure S13, S15). Moreover, the
Chili–DMHBO+ complex serves as a fluorescence donor for
FRET-based analytical systems, as demonstrated with an Atto
590-labeled Chili RNA aptamer (Figure 4) that carries the
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10.1002/chem.201805882
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rhodamine dye at its 3’-end.[27] Upon binding of DMHBO+ and
excitation at 456 nm, fluorescence emission from Atto 590 was
observed, which can be rationalized by the strong overlap of the
DMHBO+ emission and Atto 590 absorption bands (Figure S14).
The large Stokes shift of Chili–DMHBO+ rules out direct excitation
of the acceptor, as shown in the absence of DMHBO+ (Figure S14).
Furthermore, the FRET effect allowed monitoring of DNA-
catalyzed site-specific cleavage of the Atto 590-labeled extension
of the aptamer. Upon addition of a 10-23 deoxyribozyme with
appropriate binding arms,[28] the DMHBO+ emission intensity
increased, while Atto 590 intensity decreased concomitantly
(Figure 4b). The time-dependent change in the ratio of
fluorescence signals (I590/I620) (Figure 4c) correlates well with the
gel-based analysis (Figure 4d). The polyacrylamide gel was
stained with DMHBO+ and imaged with green and blue epi
illumination (i.e., green: excitation of Atto 590; blue: excitation of
DMHBO+). The false-color overlay confirms the presence of both
chromophores in the full-length construct and identifies the single-
labeled cleavage products (Figure 4d, S15). This proof-of-
principle experiment suggests that the Chili–DMHBO+-Atto 590
FRET pair can be used for ratiometric RNA sensors and multicolor
fluorescence experiments.
the three-dimensional structure of the binding site to comprehend
the structural basis of the proton relay in the RNA responsible for
the large apparent Stokes shift, and to enable engineering of even
brighter LSS aptamer variants for applications in vitro and in cells.
Acknowledgements
This work was supported by the European Research Council
(ERC consolidator grant No. 682586), by the Cluster of
Excellence and DFG Research Center Nanoscale Microscopy
and Molecular Physiology of the Brain, University of Göttingen,
and by the University of Würzburg.
Table 1. Photophysical data of Chili aptamer complexes[a] in comparison to
selected fluorescent proteins.
Fluorophore
Ex/Em[b] (nm)
Stokes
shift (nm)
Quantum
yield[c]
KD (µM)
DMHBI
400/537
137
0.08
0.40
0.08
0.10
0.57
DMHBI+
DMHBI-Imi
DMHBO+
413/542
129
0.063
0.071
0.012
463/545,594[d]
456/592
82,131
136
GFP[22]
475/508
572/582
437/572
460/605
33
0.79
0.33
0.45
0.17
-
-
-
-
Kaede[21]
10
LSSmOrange[14]
LSSmKate2[15]
135
145
[a] in 40 mM HEPES pH 7.5, 125 mM KCl, 5 mM MgCl2 [b] Excitation and
emission maxima. [c] relative quantum yield using Coumarin 153 as
standard. [d] Two maxima of the bimodal emission spectrum.
Keywords: RNA aptamer • fluorescence • large Stokes shift •
fluorescent protein • fluorescence resonance energy transfer
Figure 4. a) A Chili aptamer-based FRET sensor monitors DNA-catalyzed RNA
cleavage. Donor: DMHBO+ (orange), acceptor: Atto 590 (red). b) Fluorescence
emission spectra of Atto 590-labeled Chili–DMHBO+ before addition of DNA
enzyme (red curve). Upon addition of 10-23 DNA enzyme, spectra were taken
every 6 sec (every 20th spectrum shown up to 20 min). c) Kinetics of RNA
cleavage monitored by a decrease in acceptor emission (red) and an increase
in donor emission (orange). d) Denaturing PAGE imaged with green and blue
epi illumination after staining with a 1 µM solution of DMHBO+. Time points: 0.25,
0.5, 1, 2, 5, 10, 20, 40, 60 min. Experimental conditions for c) and d): 0.5 µM
RNA, 5 µM DNA enzyme, 40 mM HEPES pH 7.5, 125 mM KCl, 5 mM MgCl2.
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10.1002/chem.201805882
Chemistry - A European Journal
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
“My shadow side so amplified.” (Red
C. Steinmetzger, N. Palanisamy,
Hot Chili Peppers)
K.R. Gore, C. Höbartner*
A large Stokes shift fluorogen-
activating RNA aptamer (LSS FLAP)
named “Chili” strongly amplifies
green or red fluorescence emission
of otherwise non-fluorescent, tightly
bound cationic ligands upon
Page No. – Page No.
A multicolor large Stokes shift
fluorogen-activating RNA aptamer
with cationic chromophores
excitation with blue light. Chili allows
visualization of RNA and can be
used in FRET sensors.
This article is protected by copyright. All rights reserved.