A strategy to enhance the binding affinity of fluorophore-aptamer pairs for RNA tagging with neomycin conjugation

Article abstract of DOI:10.1039/c2cc34498j

Fluorogenic sulforhodamine-neomycin conjugates have been designed and synthesized for RNA tagging. Conjugates were fluorescently activated by binding to RNA aptamers and exhibited greater than 250-400 fold enhancement in binding affinity relative to corresponding unconjugated fluorophores. This journal is The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc34498j

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A strategy to enhance the binding affinity of fluorophore–aptamer pairs
for RNA tagging with neomycin conjugationw
Jongho Jeon,z Kyung Hyun Leez and Jianghong Rao*
Received 22nd June 2012, Accepted 23rd August 2012
DOI: 10.1039/c2cc34498j
Fluorogenic sulforhodamine–neomycin conjugates have been
designed and synthesized for RNA tagging. Conjugates were
fluorescently activated by binding to RNA aptamers and exhibited
greater than 250–400 fold enhancement in binding affinity relative
to corresponding unconjugated fluorophores.
just in the low micromolar range and insufficient for mRNA
imaging in living cells.
In this work, we explored a general strategy to enhance the
binding affinity of fluorophores for aptamers by developing
fluorogenic ASR–neomycin conjugates. Neomycin, an amino-
glycoside known to bind to different RNA structures,6 has
been conjugated to pharmacophores, fluorophores, PNA and
oligonucleotides for specific recognition or targeting of nucleic
acids.7,8 Such conjugates generally displayed higher affinity
and specificity than those of their parent structures. In our
previous study,5 ASR probes specifically bound to a loop
region of Apt10L. Neomycin, however, binds to RNA by
non-specific electrostatic interactions. We hypothesize that
ASR–neomycin conjugates should have an extended binding region
to RNA aptamers and thus display enhanced binding affinity.
Herein we report the synthesis of fluorogenic ASR–neomycin
conjugates via a Click reaction and characterization of their
binding affinity for Apt10 aptamers.
In recent years, increased attention has been paid to the develop-
ment of mRNA tagging methods for the in vitro detection of target
RNA and its imaging in live cells. A number of mRNA visualiza-
tion strategies have been reported, such as molecular beacons,1
and green fluorescent protein (GFP) based systems (e.g. GFP
fusion with the RNA binding domain or GFP aptamer) for living
cell imaging.2 One of the potential concerns with the GFP-based
method is the large size of the formed protein–RNA complex,
which may influence the biochemistry and biology of the target
mRNA under study.
A small fluorogenic molecule-based approach is a promising
method for specific mRNA tagging since small fluorogenic
probes are much smaller and would be amenable to simple
co-incubation with target molecules or cells. They can be washed
away and re-introduced into cells for repetitive imaging. In this
approach, fluorescence of small molecules is initially quenched,
but can be recovered upon binding with a specific RNA aptamer.
So far several fluorescent probe–RNA aptamer pairs have been
developed,3 of which only one (with a Kd of 464 nM) has been
successfully applied to live-cell ribosomal 5S RNA imaging.4
These probes have failed likely due to very low concentrations of
mRNA that normally exist in cells, imposing the requirement for
high fluorophore–aptamer binding affinity. Previously we have
reported aniline substituted sulforhodamine (ASR) analogues
and specific aptamer pairs (Apt10) discovered through a SELEX
(Systematic Evolution of Ligands by EXponential enrichment)
procedure. ASR dyes were fluorescently quenched by a photo-
induced electron transfer (PET) mechanism5 and 50–130 fold
enhancement in the fluorescent signal was achieved after binding
with specific aptamers. However, their binding affinities were
Preparation of azido-neomycin 3 started from a known
N-Boc and O-TIBS (1,3,5-triisopropylbenzene sulfonyl) derivative
of neomycin 2 according to a reported synthetic procedure by
Tor et al. (Scheme 1).8 Substitution with sodium azide followed
by deprotection of Boc groups converted compound 2 to azido
neomycin 3. The fluorophore ASR 4 was obtained from sulfo-
fluorescein.5 ASR 4 was coupled to propargyl amine in order to
introduce an alkyne group that would facilitate its conjugation
with 3. Conjugation between compound 3 and ASR derivative 5
proceeded smoothly using Cu(II)-catalysed click chemistry. Two
fluorogenic conjugates (Scheme 1) have been prepared with a
different length of linker between neomycin and ASR fluoro-
phores to examine the effect of the linker on the binding affinity.
Both 1a and 1b showed very low fluorescence intensity in
aqueous buffer solution. However, their fluorescence intensities
increased ca. 90-fold in 90% glycerol solution, since such a viscous
environment is known to deactivate the PET quenching mechanism
(Fig. S1, ESIw). To determine whether neomycin itself could induce
the fluorescence activation of ASR compounds by non-specific
interactions, various concentrations of neomycin were added
to 1 mM solution of ASR 4 (Fig. S2, ESIw). At low concentra-
tions of neomycin (o1.4 mM), the fluorescence intensity of 4
increased slightly (ca. 1.5 fold), however, this change was
negligible relative to a more than 70–80-fold fluorescence
enhancement when ASR 4 binds to Apt10L (40 mM). Next,
we performed a binding competition experiment using neomycin
Molecular Imaging Program at Stanford (MIPS), Departments of
Radiology and Chemistry, Stanford University, Stanford,
California 94305-5484, USA. E-mail: jrao@stanford.edu;
Fax: +1 650 736 7925; Tel: +1 650 736 8563
w Electronic supplementary information (ESI) available. Experimental
procedures and spectral data for new compounds. See DOI: 10.1039/
c2cc34498j
z These authors contributed equally to this work.
c
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For 1b and Apt10L (Fig. 1b), the plausible binding seems to
have 1.5 : 1 binding stoichiometry, suggesting that the strong
first binding site would be more dominant than the weak
second binding site at 500 nM concentration. As a result, the
neomycin conjugates 1a and 1b have two different binding
sites on the aptamer, which would provide two different
orientations for fluorophore binding. Therefore, Kd and Fmax
for the high affinity binding site (Kd1 and Fmax1) were calcu-
lated by a specific two site-binding equation.9 The fluorescence
titration of Apt10L against compound 5a gave a Kd value of
15.5 ꢀ 1.7 mM and the value of Fmax (maximum fluorescence
fold-enhancement at saturation) as 48 ꢀ 3 (Fig. 2 and Table 1).
The binding affinity (Kd) of Apt10L for compound 5b was
99 ꢀ 33 mM and the value of Fmax was 217 ꢀ 61; the different
affinity observed with 5a and 5b was consistent with our
previous study.5 However, conjugate 1a displayed greatly
enhanced binding affinity of 0.063 ꢀ 0.015 mM (Kd1) for
Apt10L with an Fmax1 of 13.9 ꢀ 0.3. The estimated Kd value
of compound 1b for the high affinity site showed a more than
400-fold enhanced affinity (Kd1 = 0.24 ꢀ 0.03 mM) and Fmax
of 27.9 ꢀ 1.2. While the Fmax values of conjugates 1a and 1b
were lower than those of 5, the observed fluorescent enhance-
ment of neomycin conjugates was higher than those of corres-
ponding parental ASR derivatives (5a and 5b) at low aptamer
concentrations. For example, compounds 5 showed an approxi-
mately 3-fold fluorescence enhancement in the presence of 1 mM
of Apt10L, but conjugates 1a and 1b displayed a >15-fold
fluorescence recovery under the same conditions. These results
further confirm the enhanced affinity of neomycin conjugates
for the target RNA aptamer.
Scheme 1 Synthesis of ASR fluorophore–neomycin conjugates.
as a competitive Apt10L binder. ASR 4 (1 mM) was pre-mixed
with 10 mM of Apt10L, then increasing amounts of neomycin
were added to this mixture and the fluorescence intensity
change was determined (Fig. S3, ESIw). Since the fluorescence
intensity of compound 4 showed little change up to 25 mM of
neomycin, non-specific interactions of neomycin with the
RNA aptamer did not interfere with the specific binding of
the ASR compound.
The binding affinity of neomycin conjugates 1a and 1b to
Apt10L and Apt10M (Fig. S4, ESIw) was determined by
fluorescence titrations. Apt10M is a minimum binding domain
(60-mer) of Apt10L (133-mer). We first determined the Kd
value and fluorescence recovery of compounds 1a and 1b using
Apt10L since this aptamer showed the best binding affinity to
ASR 4 from our previous results.5 The sequence and repre-
sentative secondary structure of Apt10L and its variants are
shown in Fig. S4 (ESIw). Job plot analysis showed that the
highest intensity was observed at the molar ratio of around
0.3 ([RNA]/[RNA + dye]) for Apt10L and 10M with 1a
(Fig. 1a) and for Apt10M with 1b (Fig. 1b), suggesting that
the binding stoichiometry between dye and aptamer is 2 : 1.
Fig. 2 Representative fluorescence titration curves of ASR deriva-
tives 5a and 5b (a) and neomycin conjugates 1a and 1b (b) with
Apt10L. Fluorescence titration curves of conjugates 1a (c) and 1b (d)
with Apt10M variants (M, M1 and M-Lm3) and random RNA.
Table 1 Estimated Kd, Fmax and dynamic linear range (DLR) values
of ASR fluorophores 4, 5a, 5b and neomycin conjugates 1a and 1b
with Apt10L (ND means not determined)
Compound
Kd1 (mM)
Fmax1 (fold)
DLR
1a
1b
5a
5b
4
0.063 ꢀ 0.015
0.24 ꢀ 0.03
15.5 ꢀ 1.7
99.0 ꢀ 33
13.9 ꢀ 0.3
27.9 ꢀ 1.2
48 ꢀ 3.0
11–1000 nM
16–1000 nM
ND
ND
ND
Fig. 1 Job plots to determine the binding stoichiometry between
conjugate 1a with Apt10L and Apt10M (a) and conjugate 1b with
Apt10L and Apt10M (b). FI represents fluorescence intensity. RNA
molar ratio is [RNA]/[RNA + dye].
217 ꢀ 61
135 ꢀ 15.0
39.1 ꢀ 7.6
c
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Table 2 Estimated Kd and Fmax values of conjugates 1a and 1b with
Apt10 variants (M, M1 and M-Lm3) and random RNA (NC means
not calculable)
exploration of the effects of an aminoglycoside (neomycin in
this study) conjugation strategy to enhance the binding affinity
of fluorogenic probes for aptamers. With this approach,
significantly increased binding affinity and high fluorescence
activation were achieved without further screening of RNA
aptamer sequences.
Kd1 for
1a (mM)
Fmax1 for Kd1 for
1a (fold) 1b (mM)
Fmax1 for
1b (fold)
Apt10
M
M1
M-Lm3
0.096 ꢀ 0.026 12.6 ꢀ 1.9 0.063 ꢀ 0.020 11.3 ꢀ 1.7
0.042 ꢀ 0.018 5.6 ꢀ 1.0 NC NC
0.035 ꢀ 0.021 3.7 ꢀ 0.9 0.013 ꢀ 0.011 2.8 ꢀ 0.7
NC NC NC
In summary, we have investigated a bivalent strategy to
enhance the binding affinity of activatable fluorophores for
RNA aptamers through conjugation with neomycin. The
fluorogenic conjugates were obtained by facile Cu(II)-catalysed
click chemistry and displayed a more than 200-fold increase in
binding affinity as compared to unmodified ASR compounds.
Moreover, these conjugates showed specific fluorescence acti-
vation only upon incubation with a selected aptamer. This
strategy may be generally applicable for developing high
affinity fluorophores for specific RNA aptamers.
Random RNA NC
We next examined the specificity of neomycin conjugates for
the following RNA aptamers: Apt10M, its mutants (Apt10M1
and Apt10M-Lm3; structure of each aptamer is shown in
Fig. S4, ESIw) and a random 130-mer RNA containing 90-mer
of randomized sequences (Fig. 2c and d and Table 2). Conjugate
1a showed slightly better Kd and Fmax for Apt10L than Apt10M.
In comparison, conjugate 1b showed a ca. 3.8-fold better Kd
value for Apt10M (0.063 ꢀ 0.020 mM) than for Apt10L (0.24 ꢀ
0.03 mM), suggesting that the long and flexible tetraethylene
linker between the neomycin moiety and the fluorophore was
more suitable for Apt10M than Apt10L. Binding affinities of
1a and 1b for Apt10M were independently measured by a gel
mobility shift assay to compare with the fluorescence assay
and comparable values were obtained: 0.22 ꢀ 0.05 mM for 1a
and 0.17 ꢀ 0.07 mM for 1b (Fig. S5, ESIw).
This work was supported by a Young Investigator Award
from Human Science Frontier Program and a research grant
from NIGMS (1R01GM086196). We thank Dr. Adam
Shuhendler for helpful discussions.
Notes and references
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The estimated maximal fluorescence recovery (Fmax) for
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the fluorescence activation specificity toward aptamers. Both
conjugates showed only ca. 2-fold fluorescence enhancement
with random RNA, suggesting that non-specific binding of the
neomycin group to RNA structures does not significantly affect
the fluorescence activation of the ASR fluorophore. Fluorogenic
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enhancement against selected aptamers (Apt10L and 10M). These
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affinity of the conjugates for Apt10L and Apt10M. They may also
bind some of the aptamer mutants with enhanced binding affinity
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activation is highly specific to the binding of the conjugate to the
aptamer target. Therefore, the neomycin conjugates display both
high binding affinity and high fluorescence turn-on specificity for
the aptamer target.
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The fluorescence dynamic linear ranges of both 1a and 1b
were determined for potential applications as aptamer sensors
(Fig. S6, ESIw and Table 1) and as low as 10 nM of an RNA
aptamer can be detected in solution using a fluorometer. Both
showed no toxicity in mammalian cells at 10 mM (B100 fold
higher than Kd1), suggesting their biocompatibility for cellular
studies (Fig. S7, ESIw).
In previous reports,3,5 repeated SELEX and systematic variant
and mutation studies have been applied to the optimization of
RNA aptamers for improved affinity. This report is the first
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun.