Identification of ligands for the Tau exona 10 splicing regulatory element RNA by using dynamic combinatorial chemistry

Article abstract of DOI:10.1002/chem.201002065

We describe the use of dynamic combinatorial chemistry (DCC) to identify ligands for the stem-loop structure located at the exona 10-5′-intron junction of Tau pre-mRNA, which is involved in the onset of several tauopathies including frontotemporal dementia with Parkinsonism linked to chromosomea 17 (FTDP-17). A series of ligands that combine the small aminoglycoside neamine and heteroaromatic moieties (azaquinolone and two acridines) have been identified by using DCC. These compounds effectively bind the stem-loop RNA target (the concentration required for 50% RNA response (EC₅₀): 2-58μM), as determined by fluorescence titration experiments. Importantly, most of them are able to stabilize both the wild-type and the +3 and +14 mutated sequences associated with the development of FTDP-17 without producing a significant change in the overall structure of the RNA (as analyzed by circular dichroism (CD) spectroscopy), which is a key factor for recognition by the splicing regulatory machinery. A good correlation has been found between the affinity of the ligands for the target and their ability to stabilize the RNA secondary structure.

Full text of DOI:10.1002/chem.201002065

DOI: 10.1002/chem.201002065
Identification of Ligands for the Tau Exon 10 Splicing Regulatory Element
RNA by Using Dynamic Combinatorial Chemistry
Paula Lꢀpez-Senꢁn,[a] Irene Gꢀmez-Pinto,[b] Anna Grandas,[a] and Vicente Marchꢂn*[a]
Abstract: We describe the use of dy-
namic combinatorial chemistry (DCC)
to identify ligands for the stem-loop
structure located at the exon 10-5’-
intron junction of Tau pre-mRNA,
which is involved in the onset of sever-
al tauopathies including frontotemporal
dementia with Parkinsonism linked to
chromosome 17 (FTDP-17). A series of
ligands that combine the small amino-
glycoside neamine and heteroaromatic
moieties (azaquinolone and two acri-
dines) have been identified by using
DCC. These compounds effectively
bind the stem-loop RNA target (the
concentration required for 50% RNA
response (EC50): 2–58 mm), as deter-
mined by fluorescence titration experi-
ments. Importantly, most of them are
able to stabilize both the wild-type and
the +3 and +14 mutated sequences as-
sociated with the development of
FTDP-17 without producing a signifi-
cant change in the overall structure of
the RNA (as analyzed by circular di-
chroism (CD) spectroscopy), which is a
key factor for recognition by the splic-
ing regulatory machinery. A good cor-
relation has been found between the
affinity of the ligands for the target
and their ability to stabilize the RNA
secondary structure.
Keywords: combinatorial chemis-
try · molecular recognition · RNA
ligands · RNA structures · template
synthesis
Introduction
finity and better specificity could be achieved by combining
two or more small molecules with different affinities and
abilities to recognize some of the secondary structural
motifs present in a particular RNA target.[3] This fragment-
based lead-discovery search approach,[4] in combination with
dynamic combinatorial chemistry (DCC),[5] offers new per-
spectives and opportunities for identifying RNA ligands, as
well as for improving our understanding of RNA recogni-
tion mechanisms.
Over the past few years, DCC has demonstrated its poten-
tial as a tool for the discovery of ligands for biomolecules,
mainly because both the library synthesis and the affinity
screening steps are carried out simultaneously in a single
process.[5] In the field of nucleic acids, DCC has been used
to select a copper(II) coordination complex that binds to an
RNA hairpin,[6] as well as ligands that bind to DNA duplex
or quadruplex structures,[7] and also to stabilize oligonucleo-
tide complexes by covalent linkage of small molecules.[8]
More recently, resin-bound DCC has been used to identify
ligands for RNA structures involved in HIV-1 replication
and in myotonic dystrophy type 1.[9] Typically, most ligands
for DNA and RNA targets that have been identified by
using DCC have high-to-medium affinities (ꢀ2–50 mm).
In the study reported here, we use DCC to identify lig-
RNA plays an essential role in many cellular processes,
from the regulation of gene expression to protein synthesis,
as well as in the progression of viral diseases. This macromo-
lecule is able to adopt more complex three-dimensional
structures than those of DNA; they are similar to those ob-
served in proteins, and the tertiary structure is, as yet, diffi-
cult to predict. Hence, the discovery of selective and, more
importantly, specific ligands for this macromolecule repre-
sents a challenge for medicinal and bioorganic chemistry
and therapy.[1] However, most of the known RNA-binding
ligACHTUNGTRENNUNGands can each bind to several different RNA targets of
unrelated sequence and structure,[2] which compromises
their biological application. We hypothesize that higher af-
[a] P. Lꢀpez-Senꢁn, Prof. A. Grandas, Dr. V. Marchꢂn
Departament de Quꢁmica Orgꢃnica and IBUB
Universitat de Barcelona, Martꢁ i Franquꢄs 1-11
08028 Barcelona (Spain)
Fax : (+34)93-339-7878
E-mail: vmarchan@ub.edu
[b] Dr. I. Gꢀmez-Pinto
Instituto de Quꢁmica-Fꢁsica Rocasolano
CSIC, Serrano 119, 28006 Madrid (Spain)
AHCTUNGTREGaNNUN nds for the Tau exon 10 splicing regulatory element RNA
involved in frontotemporal dementia with parkinsonism
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201002065.
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FULL PAPER
linked to chromosome 17 (FTDP-17).[10] Tau is a microtu-
bule-associated protein that is required for the polymeri-
zation and stability of microtubules, as well as for axonal
transport in neurons. In a normal adult human brain, alter-
native splicing of exons 2, 3, and 10 produces six tau iso-
forms with either three (3R) or four (4R) repeat domains,
with a 4R/3R ratio of approximately 1.[11] However, muta-
tions found in the tau gene of FTDP-17 patients, especially
at the exon 10-5’-intron junction, alter pre-mRNA splicing
and result in an increase in the inclusion of exon 10. The
normal one-to-one isoform balance is thus perturbed, with
the 4R isoform being overproduced in most cases. This
alters the microtubule function and consequently leads to
the development of the tauopathy.[12] The stem-loop struc-
ture located at the exon 10-5’-intron junction seems to be an
important regulatory element in pre-mRNA splicing. Both
in vitro and in vivo experiments have shown that the extent
of exon 10 inclusion is inversely proportional to the stability
of this structure, a stability that is considerably diminished
by mutations (Scheme 1a).[13] Thus, small molecules that se-
Results and Discussion
Construction of the dynamic combinatorial library: First, we
synthesized several building blocks, including known RNA-
binding ligands and some other compounds with the poten-
tial to bind to a particular RNA motif, that could be assem-
bled modularly into new lead ligands for Tau stem-loop tar-
gets (the structures are shown in Scheme 1b).[15] Thiol deri-
vatization was chosen because disulfide exchange is relative-
ly fast in aqueous solutions with pH values near 7–8 and is
fully compatible with RNA. In addition, this approach in-
creases the diversity of the dynamic combinatorial library
(DCL) in comparison with nonsymmetrical bonds, because
the formation of disulfide bridges can afford both homo-
and heterodimers.[16]
Aminoglycoside antibiotics are possibly the most studied
RNA ligands. They are known to be able to discriminate A-
type from B-type duplexes and to have relatively high affini-
ty for RNA structures.[2b,17] However, because natural ami-
noglycosides do not exhibit inherent specificity for biologi-
cal RNA sequences, several
groups have synthesized amino-
glycoside analogues to tune
specificity without compromis-
ing affinity. In order to reduce
the risk of nonspecific electro-
static interactions, we decided
to select a neamine derivative,
Nea, as a building block. This
small aminoglycoside incorpo-
rates rings I and II of neomy-
cin B and still presents a high
charge density. In addition, two
acridine derivatives (Acr1 and
Acr2) that have the ability
either to intercalate in duplex
Scheme 1. a) Sequences and secondary structure of wild-type (wt) and mutated Tau stem-loop target RNAs.
Exonic sequences are shown in capital letters and intronic sequences in lower case, with the +3 and +14
FTDP-17 mutations indicated. Nucleotides involved in base pairs identified previously by NMR spectroscopy
are connected by a dash.[13a] When required, biotin or fluorescein derivatization was performed at the 5’-end.
The ends of the chains were modified with 2’-O-methylribonucleosides to increase stability with RNases (de-
noted by an asterisk). b) Structure and peptide sequences of the building blocks used in the DCC experiments.
regions or to stack with un-
paired nucleobases were also
included in the library of mono-
mers.[18] Although acridines are
also promiscuous molecules,
they have recently been used in
lectively bind to and stabilize this stem-loop structure, and
in particular the mutated variants found in FTDP-17 pa-
tients, would allow the physiological balance between Tau
isoforms to be restored and, consequently, the disease to be
treated.[14] Importantly, most of the ligands identified in this
study by using the DCC approach, in addition to binding
with high-to-medium affinity and stabilizing wild-type (wt)
RNA, are capable of stabilizing the two mutated sequences
(+3 and +14) that cause the highest destabilization of the
stem-loop structure.
the development of RNA ligands with high binding affinity
and good specificity.[19] We also selected a third heteroaro-
matic compound, an azaquinolone derivative, Azq, which
could recognize the bulged adenine of the Tau stem-loop
and provide higher specificity than the aminoglycosides or
intercalators through the formation of complementary hy-
drogen-bond acceptor–donors.[20] Finally, two cationic tripep-
tides containing an aromatic residue, TyrP and TrpP, were
assembled by using solid-phase procedures and were includ-
ed in the library of monomers.
Selection of ligands by using dynamic combinatorial chemis-
try: In order to facilitate the identification of RNA-interact-
ing molecules, DCC experiments were carried out in a solu-
tion phase in the presence of the biotinylated RNA target
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V. Marchꢂn et al.
In subsequent DCC experi-
ments, we included two addi-
tional monomers, Acr2 and
TrpP, both to increase the diver-
sity of the DCL (21 theoretical
compounds may be formed)
and to study the competition
between the two acridines and
the two peptides. As shown in
Figure 2, small amplifications of
Acr1-Nea (60%) and Azq-Nea
(40%) were again observed.
However, other new ligands
were amplified in much higher
proportions, namely, Acr2-Nea
(3600%), Acr2-Acr2 (2300%)
and, to a lesser extent, TyrP-
Figure 1. Schematic representation of a DCC process.
Acr2
(190%),
(140%),
and
TrpP-Acr2
Azq-Acr2
(Figure 1). Previously, UV-monitored thermal-denaturation
experiments and circular dichroism (CD) spectroscopy had
shown that the 5’-end biotin derivatization affects neither
the overall stem-loop structure nor its stability (see the Sup-
porting Information). In addition, the unique monophasic
curve obtained at a higher concentration (25 mm) was further
evidence for the presence of a single intramolecular species
(no transitions were detected at low temperatures) and the
claim that no supramolecular aggregates are formed during
the DCC experiments. After incubation of the thiol-derivat-
ized monomers and the target, the use of streptavidin anch-
ored to magnetic beads allowed the RNA and the interact-
(200%).[23] This result indicates that ligands incorporating
the Acr2 moiety have a higher affinity for the target RNA
than those containing Acr1 or Azq. Importantly, the same
species were amplified when the DCC experiment was car-
ried out in the presence of the biotinylated +3-mutated se-
quence instead of wt RNA (see the Supporting Informa-
tion). This suggests that the binding site is not close to the
mismatched base pair in the +3-mutated sequence.
A crucial parameter in our fragment-based approach is
the selection of the appropriate distance between the build-
ing blocks to allow the simultaneous recognition of two or
more structural motifs in a particular target RNA. This pa-
rameter may be used to tune both the binding affinity and
specificity of the ligand, as found by Tor and co-workers in
some neomycin–acridine conjugates.[24] With this in mind,
we decided to carry out a new DCC experiment with five
monomers: Acr2, TyrP, Azq, Nea, and a neamine derivative
with a shorter spacer between the aminoglycoside core and
the thiol group, Nea2 (Scheme 1b). As expected, the Azq-
Nea, Acr2-Nea, and Acr2-Acr2 ligands were amplified in a
relative ratio similar to that obtained in the previous experi-
ments (see the Supporting Information). Interestingly, the
amplification of the neamine–acridine ligand containing the
shorter linker, Acr2-Nea2, was slightly higher than that of
Acr2-Nea, which suggests a higher affinity.
ing ligACHTUNGTRENNUNGands to be separated easily from the other members
of the DCL.[7] After denaturation of RNA at 908C to re-
lease any bound ligands, MS–HPLC was used to identify
and quantify the compounds. In all cases, a control experi-
ment in the absence of the biotinylated RNA was performed
in parallel to determine the effect of the target on the am-
plification of the ligands.
The first DCC experiment was performed with wt RNA
(25 mm) and the Nea, Acr1, Azq, and TyrP monomers
(100 mm each) in 50 mm tris(hydroxymethyl)aminomethane-
HCl (Tris-HCl) buffer (pH 7.7) containing 100 mm NaCl and
0.1 mm ethylenediaminetetraacetate (EDTA) at room tem-
perature, under an air atmosphere and without stirring.
Comparison with the control experiment (in the absence of
the RNA) indicated clear amplification of two disulfide het-
erodimers, Acr1-Nea (ꢀ100%) and Azq-Nea (ꢀ75%).[21]
Equilibrium was reached within 48 h in both cases, and the
composition of the mixture remained unchanged over the
next 2 days (see the Supporting Information).[22] Further-
more, when the Acr1 monomer was replaced by its disulfide
dimer, Acr1-Acr1 (50 mm), a similar distribution of products
was produced, both in the presence and in the absence of
the RNA. This demonstrated that a true thermodynamic
equilibrium had been reached and that the air-mediated oxi-
dation process is sufficiently slow to allow for equilibration
of the different species.
Binding affinities and specificities of the selected ligands: In
most cases, the selected dimeric ligands were synthesized on
a larger scale by replacing the disulfide linkage with a non-
À
reversible thioether isostere (CH2 S). Quantitative binding
studies were then carried out to determine the binding affin-
ities. Titration experiments were performed by monitoring
the quenching of the fluorescence intensity of fluorescein-
AHCTUNGTREGlNNNU abeled wt RNA as a function of the increase in the concen-
tration of ligand.[25] In all cases, a characteristic dose-depen-
dent saturatable change in fluorescence was observed, which
can be attributed to a slight conformational change in the
RNA target induced by the ligand upon complexation (see
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Tau RNA Ligands
FULL PAPER
Figure 2. Results of DCC experiments involving wt RNA and the Nea, Acr1, Acr2, Azq, TyrP, and TrpP monomers. a) HPLC traces showing the compo-
sition of the DCL in the absence (left) and presence (right) of wt RNA, after 48 h. b) Histograms showing the changes in the DCL composition (left)
and the percentage changes (% amplification) of each species (right) upon introduction of the wt RNA.
the CD results below). EC50 values (the effective ligand con-
centration required for 50% RNA response) of 28.6 mm and
58 mm were obtained for Acr1-Nea and Azq-Nea, respective-
ly, by fitting the data in a dose-response curve. As shown in
Table 1, the overall results are consistent with the DCC am-
spacer between acridine and neamine, Acr2-Nea2, than for
Acr2-Nea.
These results prompted us to evaluate the specificity of
the ligands. Fluorescence binding assays were repeated in
the presence of a 30-fold nucleotide excess of a commercial-
ly available tRNA (Table 1), which is a relevant competitor
because it contains a mixture of both pre- and mature
tRNAs.[9b,24b,25b,26] Interestingly, the specificity (the EC50
value in the presence of the competitor/the EC50 value in
the absence of the competitor) of the ligands was shown to
be highly dependent on the nature of the acridine building
block. (The specificity of Azq-Nea could not be determined
because the emission maximum of fluorescein varied more
than 3 nm at high ligand concentrations.) In the presence of
the competitor, the EC50 values of Acr2-Nea and Acr2-Acr2
for wt RNA were increased by 11-fold and 14-fold, respec-
tively, whereas that of Acr1-Nea was only increased by 4-
fold. Hence, replacement of the Acr2 moiety by Acr1 in the
acridine–neamine ligands increases the specificity. On the
other hand, as shown in Table 1, the specificity ratio for
Acr2-Nea2 was the highest. This result indicates that a
longer spacer confers higher specificity in the Acr2–neamine
ligands. This trend is different from that previously reported
for some acridine–neomycin conjugates with another target,
the HIV-1 RRE RNA.[24b]
Table 1. Binding of the ligands to wt RNA in the absence or presence of
a tRNA competitor.
Ligand
EC50 [mm][a]
EC50 +tRNA [mm][b]
EC50 +tRNA/EC50
neamine
3300
58.0
28.6
5.9
2.1
2.9
n.d.
n.d.
112.2
63.1
47.0
41.0
n.d.
n.d.
3.9
10.7
22.4
14.0
Azq-Nea
Acr1-Nea
Acr2-Nea
Acr2-Nea2
Acr2-Acr2
[a] All fluorescence measurements (0.25 mm RNA) were performed in
10 mm sodium phosphate buffer (pH 6.8), 100 mm NaCl, and 0.1 mm
Na2EDTA. [b] Measured in the presence of a 30-fold nucleotide excess
of a mixture of tRNA (tRNAmix). n.d.: not determined.
plification data because the highest binding affinities are ex-
hibited by the most amplified ligands; this confirms the
power of this methodology for identifying ligands for RNA
targets. Indeed, ligands containing Acr2 showed higher bind-
ing affinities than those containing Azq or Acr1 (EC50 =5.9
and 2.9 mm for Acr2-Nea and Acr2-Acr2, respectively). In-
terestingly, the binding was observed to be approximately
three times stronger for the ligand containing the shorter
These experiments with Tau RNA provide three impor-
tant conclusions, some of which have also been previously
observed in different RNA targets. First, the covalent at-
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V. Marchꢂn et al.
tachment of a heteroaromatic moiety, either azaquinolone
or acridine, causes a substantial increase in the binding af-
finity of the aminoglycoside scaffold (see the neamine entry
in Table 1). Second, ligands containing Acr2 have higher
binding affinities than those containing Acr1 and Azq, and
it seems that a shorter spacer is preferred in the acridine–
neamine ligands with Tau RNA. Third, there is an inverse
correlation between affinity and specificity, because the
higher the affinity, the less specific the ligand seems to be.
as that produced by mitoxantrone, a ligand for Tau RNA re-
cently identified in a high-throughput fluorescence binding
assay.[14a,b,27]
All together, these results show a direct correlation be-
tween the Tm value of the RNA–ligand complex, the level of
amplification observed in the DCC experiments, and the
binding affinities determined by fluorimetry; this is consis-
tent with previous observations in different systems.[7,8] The
most amplified ligands (Acr2-Nea and Acr2-Nea2) have
higher binding affinities and produce a higher degree of sta-
bilization than the least amplified ligands (Azq-Nea and
Acr1-Nea). No stabilization was observed upon incubation
with Acr1-Acr1 because this compound was not templated
by the RNA. As expected, a significant increase in the Tm
value also occurred with Acr2-Acr2. These results indicate
that the 9-(alkylamino)acridine is more stabilizing than that
bearing the 9-carboxamide group. Both the positive charge
of the former (pKa ꢀ5 for Acr1 versus pKa ꢀ8 for Acr2)
and its ability to establish additional stabilizing interactions
may account for this stabilizing effect and the high binding
affinity.[28]
Effect of the selected ligands on the thermal stability of Tau
RNA targets: As previously stated, besides binding with
good affinity and specificity to the Tau RNA targets, effec-
tive ligands for these targets must stabilize them, in particu-
lar the mutated sequences. So, our next objective was to
evaluate the ability, if any, of the most amplified ligands to
produce stability. The impact of the ligands on the thermal
stability of the stem-loop structures (wt and mutated) was
estimated by UV-monitored melting experiments (Table 2).
Table 2. Melting temperature (Tm) values for the complexation of ligands
with target RNAs (1 mm of both the RNA and the ligands in 10 mm
sodium phosphate buffer (pH 6.8), 100 mm NaCl, and 0.1 mm Na2EDTA).
Spectroscopic studies of the complexes formed between Tau
RNA and the selected ligands: With consideration of the
fact that all of the best ligands included a heteroaromatic
moiety, we wanted to gain some insight into the role of this
group upon complexation with RNA. When Acr1-Nea,
Acr2-Nea, and Acr2-Nea2 were titrated with increasing
amounts of wt RNA, a strong hypochromism was observed
in their UV/Vis spectra (30–40% at a 1:1 ratio; Figure 3). In
addition, the 423 and 444 nm bands of the free Acr2-Nea
and Acr2-Nea2 ligands exhibited a 7 and 9 nm redshift, re-
spectively. This shift to higher wavelengths was smaller for
Acr1-Nea (2 nm for the 360 nm band), which is consistent
with previously reported results.[29] Although a similar hypo-
chromism was observed in the case of Azq-Nea, the interfer-
ence of the RNA absorbance near the azaquinolone band
prevented the titration experiment from being completed
(see the Supporting Information). Similar bathochromic ef-
fects were observed in fluorescence titration experiments,
including reduction in the fluorescence intensity of the acri-
dine moieties together with changes in the spectral profiles
(see the Supporting Information). These effects are com-
monly observed when planar heteroaromatic compounds
bind to nucleic acids through an intercalative mechanism or,
at least, when stacking occurs with base-pair nucleobases.
Finally, we used CD spectroscopy to check whether these
ligands might substantially change the overall conformation
of the target RNA structures upon binding. This is a key
issue for the recognition of the ligand-stabilized stem-loop
by the splicing regulatory machinery. Although the +3 and
+14 mutations decrease the thermal stability of the stem-
loop RNAs, they exhibit CD spectra that are very similar to
that of wt RNA. This indicates that the mutations do not
produce a significant change in the structure (see the Sup-
porting Information). When CD spectra were registered in
the presence of the selected ligands, minimal alterations
Ligand
Wild type
Tm [8]
+3 Mutation
+14 Mutation
[a]
[a]
[a]
DTm
Tm [8]
DTm
Tm [8]
DTm
no ligand
neamine
66.4
67.6
67.5
67.1
68.5
68.8
65.4
67.6
–
50.8
51.8
51.9
52.0
53.6
56.5
50.6
53.4
–
54.0
54.4
54.9
54.1
56.0
57.2
53.8
55.4
–
+1.2
+1.1
+0.7
+2.1
+2.4
À1.0
+1.2
+1.0
+1.1
+1.2
+2.8
+5.7
À0.2
+2.6
+0.4
+0.9
+0.1
+2.0
+3.2
À0.2
+1.4
Azq-Nea
Acr1-Nea
Acr2-Nea
Acr2-Nea2
Acr1-Acr1
Acr2-Acr2
[a] DTm =(Tm of the RNA in the presence of ligand)À(Tm of RNA
alone).
In the presence of Azq-Nea or Acr1-Nea, a slight increase
in the Tm value was observed in all RNAs (DTm ꢀ+18C),
with the exception of Acr1-Nea and the +14 mutant, where
no significant shift occurred. This small stabilization, similar
to that obtained with neamine alone, could suggest that the
Azq or Acr1 fragments do not interact with RNA. Howev-
er, the binding affinities (Table 1) and spectroscopic data
(see below) have demonstrated the active participation of
the heteroaromatic moiety in the RNA binding. Replace-
ment of Acr1 by Acr2 in the acridine–neamine ligand
(Acr2-Nea) caused a greater increase in the Tm values for
both the mutated sequences (DTm =+2.8 and +28C for the
+3 and +14 mutants, respectively) and the wt RNA (DTm =
+2.18C). To our surprise, the Tm value of the +3 mutant
was clearly increased (DTm =++5.78C; Table 2) in the pres-
ence of the ligand with the shortest spacer, Acr2-Nea2. The
effect of this ligand on wt RNA and the +14 mutant was
smaller (DTm =++2.4 and 3.28C, respectively) but still higher
than that induced by the ligand with the longest spacer,
Acr2-Nea. It is particularly relevant that the degree of stabi-
lization of the +3 mutant with Acr2-Nea2 is higher than
that produced by neomycin B alone and of the same order
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Tau RNA Ligands
FULL PAPER
small aminoglycoside and a heteroaromatic moiety, are able
to stabilize the +3 and +14 mutated sequences associated
with the development of FTDP-17, as well as the wt RNA.
Furthermore, they bind the RNA target without producing a
significant change in the overall structure of the stem-loop
(as determined by CD spectroscopy), and some of them
could have a preferred binding site near the bulged adenine
of the stem-loop structure, as suggested by the biophysical
experiments. The overall results show a direct correlation
between the affinity of the ligands and their stabilizing prop-
erties. It is important to point out that the most specific li-
gands, such as Acr1-Nea, have a moderate affinity for the
RNA target, whereas those with high affinities (compounds
containing Acr2) are less specific, as inferred from the com-
petition studies performed in the presence of an excess of
tRNA.
The identification of ligands that stabilize the mutated
Tau stem-loop may open the way for the generation of
drugs that restore the physiological balance of Tau isoforms,
which may thus allow the treatment of frontotemporal de-
mentias such as FTDP-17, as well as other neurodegenera-
tive tauopathies including Alzheimerꢆs disease. Moreover,
these ligands may become valuable tools for studying and
understanding the alternative splicing of exon 10 and, for in-
stance, how pre-mRNA secondary structures influence the
regulation of alternative splicing.[12b,30]
Efforts are underway to improve the affinity and especial-
ly the specificity of the most promising ligands, as well as to
elucidate by NMR spectroscopy the structure of their com-
plexes with both wt RNA and mutated sequences, by using
labeled oligoribonucleotides.
Figure 3. UV/Vis titration of a) Acr1-Nea and b) Acr2-Nea ligands
(50 mm) with increasing amounts of wt RNA (0–1 equiv) in a 10 mm
sodium phosphate buffer (pH 6.8) containing 100 mm NaCl and 0.1 mm
Na2EDTA.
Experimental Section
General procedures for DCC experiments: In general, DCC experiments
were repeated at least twice in order to confirm their reproducibility. A
typical experiment includes the following steps:
were observed (a decrease in the ellipticity of the positive
band at 234 nm and a concomitant increase in both the in-
tensity and the wavelength shift of the positive band at
around 267 nm). These results indicate that the overall
stem-loop structure in the Tau RNA target is maintained in
all cases. These data, together with the UV/Vis and fluores-
cence spectroscopy results, suggest that the heteroaromatic
moiety of the ligands might intercalate or stack around the
bulged adenine, as described by Varani and co-workers for
the binding of mitoxantrone to the Tau RNA.[14c]
A) Quantitation of building blocks and target oligoribonucleotides: In
general, building blocks containing thiol were quantified by Ellmanꢆs
test. Procedure: Ellmanꢆs reagent (50 mL; 2 mm dithio-bis-2-nitrobenzoic
acid (DTNB) in 50 mm aqueous NaOAc), aqueous 2m Tris-HCl (100 mL;
pH 8), and H2O (850 mL minus the sample volume) were added into a
1 cm pathlength quartz cuvette and mixed thoroughly. The UV spectrom-
eter was calibrated to zero at 412 nm. The sample was prepared by
adding the appropriate volume (10, 20, 30 mL, etc.) of the thiol. The
sample was mixed well and incubated at room temperature for 2 min.
The absorbance was measured at 412 nm and the concentration was cal-
culated by using the extinction coefficient (e412 =13600mÀ1 cmÀ1). Al-
though the building blocks were dissolved in 0.1% trifluoroacetic acid
(TFA) in H2O to increase their solubility, the pH value of the Ellman so-
lution was not affected.
Conclusion
For the quantitation of acridine derivatives Acr1 and Acr2, the extinction
coefficients of their amino precursors, e343 =1979mÀ1 cmÀ1 and e361
=
In conclusion, we have shown the usefulness of the dynamic
combinatorial chemistry approach to identify ligands that
bind with high-to-medium affinity (EC50 =2–58 mm), as de-
termined by fluorimetry, to the Tau exon 10 splicing regula-
tory element RNA. This confirms the potential of this meth-
odology for developing new RNA-binding compounds. Im-
portantly, most of the selected compounds, which combine a
9910mÀ1 cmÀ1, respectively, were used (determined in 0.1% TFA in H2O).
The concentration of oligoribonucleotides was determined from the
260 nm absorbance value at 258C. The extinction coefficient was calculat-
ed on the basis of dinucleotide frequencies and composition by using the
nearest-neighbor model. The following molar extinction coefficients of ri-
bonucleosides were used for the UV quantitation of oligoribonucleotides:
ei (260 nm): U 9900, A 15400, C 7200, and G 11500 LmolÀ1 cm.
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1951

V. Marchꢂn et al.
B) RNA free exchange experiments: Each building block (24 nmol; in
0.1% TFA in H2O) was combined in an Eppendorf tube and freeze dried
or evaporated in a Speed-Vac. Buffer (240 mL; 50 mm Tris-HCl (pH 7.7),
100 mm NaCl, and 0.1 mm Na2EDTA) was then added, and the mixture
was shaken gently. The DCL mixtures were left to stand at room temper-
ature under air without stirring. After the desired time, an aliquot was
taken (approximately one third), the disulfide exchange was stopped by
addition of a 0.1% TFA solution in water (100 mL; pHꢀ2–3), and the
mixture was subjected to UV–MS–HPLC analysis.
0.58CminÀ1 and measuring the absorbance at 260 nm as a function of
temperature. The reverse denaturation curve (20 to 908C) was then re-
corded. This cooling/heating experiment ensures that the initial state cor-
responds to a thermodynamic equilibrium. In all cases, the two curves
were superimposable, which indicated that the transition was kinetically
reversible. Unless otherwise indicated, the solutions were at a concentra-
tion of 1 mm of both RNA and ligands, in 10 mm sodium phosphate buffer
(pH 6.8), 100 mm NaCl, and 0.1 mm Na2EDTA. The corresponding melt-
ing temperature values (Tm) were determined by using the baseline
method. All experiments were repeated at least three times until coinci-
dent Tm values were obtained. The error in Tm values was Æ0.28C.
B) Circular dichroism: Samples were prepared as described in the UV-
monitored melting experiment section (3 mm of both RNA and ligands, in
10 mm sodium phosphate buffer (pH 6.8), 100 mm NaCl, and 0.1 mm
Na2EDTA). Spectra were recorded on a Jasco J-720 spectropolarimeter
with a thermoregulated cell holder and interfaced with a Neslab RP-100
water bath, at 208C. All CD spectra were baseline subtracted with a sep-
arately acquired buffer spectrum.
In some DCC experiments, the building blocks were incorporated as the
corresponding disulfide homodimers. In those cases, 12 nmol were used
in order to keep the same final concentration of monomer.
C) RNA-templated exchange experiments: Biotinylated RNA (6 nmol; wt
or +3-mutated) was annealed in buffer (240 mL; 50 mm Tris-HCl
(pH 7.7), 100 mm NaCl, and 0.1 mm Na2EDTA) by heating to 908C for
5 min and then slowly cooling to room temperature. After overnight in-
cubation at room temperature, the solutions were stored at 48C. The an-
nealed biotinylated RNA was then added to the Eppendorf tube contain-
ing the evaporated building blocks, and the resulting mixture left to stand
for four days at room temperature under air without stirring. After the
desired time, an aliquot was withdrawn (approximately one third), and
the disulfide exchange was stopped by addition of 0.1% TFA solution in
water (45–70 mL; pHꢀ5–6).
C) UV/Vis titration experiments: A 45–55 mm solution of the ligand (Acr1-
Nea, Acr2-Nea/Nea2, or Azq-Nea) and the corresponding amount of wt
RNA (0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, or 2 equivalents) was prepared in
10 mm sodium phosphate buffer (pH 6.8) containing 100 mm NaCl and
0.1 mm Na2EDTA. The mixture was heated for 5 min to 908C and left to
cool slowly to room temperature. The absorption spectra were recorded
at room temperature. The percentage of absorption quenching was deter-
mined at the following absorption bands: 360 nm (31%) for Acr1-Nea
and 423 (40%) and 444 nm (35%) for Acr2-Nea.
Streptavidin-coated magnetic beads (Biomag Streptavidin, 5 mgmLÀ1 sus-
pension, Qiagen) were used to isolate the biotinylated RNA and the
binding ligands. In all washing procedures, a magnet was used to retain
the beads in the tube while the supernatant was pipetted off. First, the
beads (500 mL of suspension for each DCL aliquot) were separated from
the commercial buffer solution and washed with an acidic buffer (3ꢇ
500 mL of 50 mm Tris-HCl (pH 5.8), 100 mm NaCl, and 0.1 mm
Na2EDTA). DCL aliquots were added to the washed beads and incubat-
ed at room temperature. After 20 min, the beads were retained in the
vessel by using the magnet and the supernatant solution was pipetted off
again. The beads were then treated to remove the noninteracting ligands
and building blocks (3ꢇ200 mL of 50 mm Tris-HCl (pH 5.8), 100 mm
NaCl, and 0.1 mm Na2EDTA). Finally, the beads were washed with a hot
solution of 0.1% TFA in H2O in order to liberate RNA-binding ligands
(3ꢇ200 mL, incubation at 908C for 10 min). The solutions were combined
and evaporated in a Speed-Vac. The final residue was dissolved in 0.1%
TFA in H2O and subjected to UV–MS–HPLC analysis.
D) Fluorescence titration experiments: A solution of the ligand (Acr1-Nea
or Acr2-Nea) and the corresponding amount of wt RNA was prepared in
10 mm sodium phosphate buffer (pH 6.8) containing 100 mm NaCl and
0.1 mm Na2EDTA. The mixture was heated for 5 min to 908C and left to
cool slowly to room temperature. The fluorescence emission spectra were
recorded at room temperature.
E) Fluorescence binding assays: Fluorescence measurements were per-
formed in 1 cm pathlength quartz cells on a QuantaMaster fluorometer
(PTI) at 208C, with an excitation slit width of 4.0 nm and an emission slit
width of 5.2 nm. Upon excitation at 490 nm, the emission spectrum was
recorded over a range between 500 and 550 nm until no changes in the
fluorescence intensity were detected. All binding assays were performed
in 10 mm sodium phosphate buffer (pH 6.8), 100 mm NaCl, and 0.1 mm
Na2EDTA.
D) UV–MS–HPLC analysis: UV–MS–HPLC analysis of DCC libraries
was performed by using a Micromass ZQ mass spectrometer equipped
with an electrospray source and a single quadrupole detector coupled to
a Waters 2695 HPLC instrument (photodiode array detector). The detec-
tion wavelength was set to 260 nm. Elution was performed on a Grace-
Smart C18 column (150ꢇ2.1 mm, 5 mm, flow rate: 0.25 mLminÀ1) with
linear gradients of H2O and ACN; both solvents contained either 0.1%
formic acid or 0.1% formic acid and 0.01% TFA. Typical gradient: 0 to
35% B in 15 min and from 35% to 80% B in 10 min. In some cases,
UV–MS–HPLC analysis was carried out with both elution conditions to
avoid the overlapping of some peaks in order to allow a more accurate
integration.
For each experiment, the fluorescence spectrum of buffer solution
(120 mL) without RNA or ligand was first taken, to be used as the base-
line. After this buffer blank, the spectrum of a 0.25 mm solution of refold-
ed RNA containing fluorescein (120 mL) was recorded, and the baseline
blank was subtracted. Subsequent aliquots of an aqueous ligand solution
(1 mL; increasing in concentration from 0 to 0.75 mm, 0.0005–3000 equiv-
alents, depending on the ligand affinity) were added to the solution con-
taining RNA, and the fluorescence spectrum was recorded after addition
of each aliquot until the fluorescein fluorescence signal at 517 nm
reached saturation (typically 5–10 min). Over the entire range of ligand
concentrations, the emission maxima varied less than 1 nm. The total
volume of the sample never changed more than 20%. The full titration
was repeated in the absence of labeled RNA to correct for the presence
of the ligandꢆs fluorescence. These spectra were subtracted from each
corresponding point of the labeled RNA titrations, and the resulting fluo-
rescence intensity was corrected for dilution (FꢇV/V0).
All peak areas of the HPLC traces were integrated and normalized by
taking into account the extinction coefficient of each compound at the
detection wavelength: e260: Acr1 37090, Acr2 13334, Azq 2930, TyrP 596,
and TrpP 3484mÀ1 cmÀ1. Histograms showing the change in the mol per-
centage for all DCL members at different times were generated in order
to verify that thermodynamic equilibrium had been reached. In addition,
histograms showing the mol percentage changes in each species of the
equilibrium mixture (amplification%) upon introduction of the target
RNA were also generated.
The emission fluorescence at 517 nm was normalized by dividing the dif-
ference between the observed fluorescence, F, and the initial fluores-
cence, F0, by the difference between the final fluorescence, Ff, and the in-
itial fluorescence, F0. This normalized fluorescence intensity was plotted
as a function of the logarithm of the total ligand concentration. Finally,
nonlinear regression with a sigmoidal dose-response curve was performed
with the software package GraphPad Prism 4 (GraphPad Software, San
Diego, CA) to calculate the EC50 values. Experimental errors were less
than or equal to Æ25% of each value.
Evaluation of the interaction between RNA and ligands
A) UV-monitored melting experiments: The impact of the ligands on the
thermal stability of wt and mutated (+3 and +14) stem-loop RNA struc-
tures was estimated by cooling/heating experiments. Samples were placed
in 1 cm pathlength quartz cuvettes in a Jasco V-550 spectrophotometer
equipped with a thermoregulated cell holder. Melting curves were re-
corded by cooling the samples from 90 to 208C at a constant rate of
For competitive experiments, a tRNA from bakerꢆs yeast (Saccharomyces
cerevisiae) was purchased from Sigma. Stock solutions of tRNAmix were
1952
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Chem. Eur. J. 2011, 17, 1946 – 1953

Tau RNA Ligands
FULL PAPER
quantified by using an average extinction coefficient of 9640 cmÀ1 per
base.[24b] The fluorescence binding assays were carried out as described
above with the exception that a 30-fold excess (base) of the tRNAmix was
added to the refolded fluorescein-labeled RNA (or to the buffer for the
titration without target RNA).
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[15] Experimental details are provided in the Supporting Information.
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Acknowledgements
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The authors wish to thank Prof. Carlos Gonzꢂlez for fruitful discussions.
This work was supported by funds from the Spanish Ministerio de Educa-
ciꢀn y Ciencia (grant no. CTQ 2007-68014-01/02), the Generalitat de Cat-
alunya (grant no. 2005SGR-693 and the Xarxa de Referꢄncia de Biotec-
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Acr1-Nea and Azq-Nea was a consequence of the RNA templating.
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Nea, Nea-TyrP, and TyrP-TyrP were eluted very quickly under the
conditions used for the HPLC analysis prevented accurate quantifi-
cation. However, it is noteworthy that close inspection of the MS
traces in the presence of RNA revealed a small amplification of
Nea-Nea and Nea-TyrP.
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Received: July 20, 2010
Published online: January 5, 2011
Chem. Eur. J. 2011, 17, 1946 – 1953
ꢅ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1953