Polyamide amino acids: A new class of RNA ligands

Article abstract of DOI:10.1039/b815324h

This communication introduces a new class of promising RNA ligands, named polyamide amino acids (PAA), which are able to bind a targeted bulged stem-loop RNA fragment (HIV-1 TAR RNA) with micromolar affinities and with specificity comparative to dsDNA and tRNA; both the affinity and the specificity of PAA for TAR depend on their length and on the nature of the amino acid residues. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b815324h

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COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Polyamide amino acids: a new class of RNA ligandsw
´
Vanessa Bonnard, Stephane Azoulay, Audrey Di Giorgio and Nadia Patino*
Received (in Cambridge, UK) 2nd September 2008, Accepted 11th February 2009
First published as an Advance Article on the web 6th March 2009
DOI: 10.1039/b815324h
This communication introduces a new class of promising
RNA ligands, named polyamide amino acids (PAA), which are
able to bind a targeted bulged stem-loop RNA fragment
(HIV-1 TAR RNA) with micromolar affinities and with
specificity comparative to dsDNA and tRNA; both the affinity
and the specificity of PAA for TAR depend on their length and
on the nature of the amino acid residues.
Fig. 1 (A) Structures of synthesized PAA. (B) Secondary structures
Unlike regular double helix DNA, single-stranded RNA has
the ability to fold into well-defined tertiary structures, which
are often involved in biological functions via specific
protein–RNA or RNA–RNA recognition. Synthetic molecules
which are able to interact with high affinity and specificity with
RNA structures, and thus to inhibit their function, constitute
very attractive tools for molecular biology and medicine.1
In this context, we designed a new family of compounds
named ‘‘polyamide amino acids’’ (PAA), constituted by an
[oligo-N-(2-aminoethyl)glycine] backbone, onto which a-amino
acid residues were bound (Fig. 1A). This oligomeric backbone,
also found in peptide nucleic acids (PNA), could constitute a
convenient scaffold for building new RNA ligands. As a mimic
of the RNA sugar-phosphate moiety, it has enabled PNA to
hybridize to complementary RNA with high affinity.2 Most
likely as complementary nucleic bases, amino acid residues are
also prime RNA recognition motifs, as RNA–protein interactions
play a central role in many biological processes. Therefore,
PAA could become a new family of specific RNA ligands,
recognizing their RNA target through both electrostatic
and non-electrostatic interactions (H-bonding, p-stacking,
Van der Waals), which could involve a-amino groups, amino
acid side chains and backbone amide bonds of PAA, with the
phosphates and nucleic bases of RNA.
of TAR RNA and of bulge-truncated TAR RNA fragments.
we selected the HIV-1 TAR hairpin fragment (Fig. 1B), often
taken as a model RNA target to identify RNA ligands. The
two basic residues (K and R) were introduced for promoting
electrostatic interactions and/or hydrogen bonds. Moreover,
among the wide variety of identified TAR synthetic ligands,
peptides and peptidomimetics containing these basic amino
acid residues were shown to bind tightly and specifically to
TAR.4 The aromatic and hydrophobic residues were selected
for favoring p-stacking and van der Waals interactions.
Supported synthesis of tetramers 1–8 was carried out on a
b-alanine functionalized MBHA-LL resin, starting from fully
N-protected PAA monomers 9–12, and repeating a three-step
procedure (Scheme 1A): (i) on-resin condensation of the
selected PAA monomer, (ii) capping of the unreacted amino
groups, and (iii) Boc cleavage by acidolysis. After N-acetylation
of the last PAA residue and cleavage from the resin, crude
PAA 1–8 were obtained in high yields (495%) and high
HPLC purity (from 80 to 90%). PAA 1–8 were purified by
semi-preparative HPLC and fully characterized by HRMS,
1H and 13C NMR spectroscopy. Concerning the synthesis of
the N-protected PAA monomer building blocks 9–12, these
synthons were prepared using
a
two-step procedure
The specificity of RNA–ligand interactions is typically
assumed to be the result of non-electrostatic interactions,
while electrostatic interactions are responsible for non-specific
aspects.3 Thus, finding the proper balance between these two
types of interactions is a key element to identify specific
ligands. On these bases and as a preliminary semi-empirical
study, we designed hetero- (1–4) and homo- (5–8) tetra-PAA
(Fig. 1C) incorporating four natural amino acids, i.e. arginine
(R), lysine (K), phenylalanine (F) and alanine (A), and
evaluated their potential to bind an RNA bulged stem-loop
fragment with affinity and specificity. For this binding study,
(Scheme 1B) consisting of the condensation of the N-Z
protected amino acid residues onto the N-Boc methyl or
Laboratoire de Chimie des Mole´cules Bioactives et des Aroˆmes, UMR
´
6001 CNRS UNSA, Institut de Chimie de Nice, Universite de Nice
Sophia Antipolis, 06108 Nice Cedex, France. E-mail: patino@unice.fr;
Fax: (þ33) 4 92 07 61 51; Tel: (þ33) 4 92 07 61 46
w Electronic supplementary information (ESI) available: PAA synth-
esis and characterization, CD and fluorescence assays experimental
sections. See DOI: 10.1039/b815324h
Scheme 1 (A) Solid-phase synthesis of tetra-PAA. (B) Synthesis of
PAA monomers.
ꢀc
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Table 1 Dissociation constants for PAA–RNA interactions in the absence or in the presence of nucleic acid competitorsa
Kd (TAR) (mM)
without competitor
K0d (TAR) (mM)
K00 (TAR) (mM)
Specificity TAR
vs. TARabd
d
PAA sequence
with dsDNAb
Kd/K0d
with tRNA mixc
Kd/K00
Kd (TARab) (mM)
d
FRKA (1)
RFAK (2)
KARF (3)
AKFR (4)
KKKK (5)
RRRR (6)
AAAA (7)
0.75 ꢁ 0.05
1.8 ꢁ 0.2
2.1 ꢁ 0.3
1.2 ꢁ 0.2
1.2 ꢁ 0.4
0.08 ꢁ 0.02
41000
0.86 ꢁ 0.09
1.7 ꢁ 0.2
3.0 ꢁ 0.3
2.2 ꢁ 0.2
2.6 ꢁ 0.3
0.10 ꢁ 0.02
nde
1.1
1
1.6 ꢁ 0.4
3.3 ꢁ 0.6
2.4 ꢁ 0.2
2.9 ꢁ 0.9
9.7 ꢁ 0.5
1.8 ꢁ 0.9
nd
2.1
1.8
1.1
2.4
8
1.0 ꢁ 0.3
0.9 ꢁ 0.7
2.2 ꢁ 0.7
3.4 ꢁ 0.8
5.2 ꢁ 1.3
0.22 ꢁ 0.05
41000
1.3
0.5
1.0
2.8
4.3
2.8
nd
1.4
1.8
2.2
1.3
nd
22.5
nd
a
All standard fluorescence measurements were performed in buffer A (20 mM HEPES (pH 7.4 at 25 1C), 20 mM NaCl, 140 mM KCl and 3 mM
MgCl2). Measured in the presence of a 100-fold nucleotide excess of a 15-mer duplex DNA. Measured in the presence of a 100-fold nucleotide
b
c
excess of a mixture of natural tRNA (tRNAmix). Determined as Kd (TARab)/Kd (TAR) ratio. Not determined.
d
e
allyl ester backbone 13a–b, then carboxyl deprotection
(e.g. saponification for methyl esters (- 9–11) or reaction
with catalytic Pd0(PPh3)4 for the allyl ester (- 12)).
100-fold nucleotide excess of a 15-mer DNA duplex (Table 1).
For all tested compounds, only a slight affinity decrease was
observed (maximum 2.2-fold for PAA 5), demonstrating a
high TAR RNA vs. dsDNA specificity of PAA, even in the
case of the highly charged PAA 5. The PAA 1–6 specificity for
TAR RNA was also assessed by measuring binding affinities
(K00d) in the presence of a 100-fold nucleotide excess as a
mixture of natural tRNA (Table 1). As expected, the octa-
cationic PAA 5 and 6 were less specific than the hexa-cationic
PAA 1–4. Indeed, in the presence of tRNA, the affinities of
PAA 5–6 for TAR decreased 8- and 22-fold, respectively, while
those of PAA 1–4 were only slightly affected (Table 1). The
diminution in the competitive effect of tRNA as the number of
amines decreases likely reflects a decrease in the non-specific
electrostatic binding energy. However, this energy loss may be
compensated for by a binding energy increase resulting from
non-ionic interactions, as illustrated in the cases of PAA 6 and
We first studied by circular dichroism (CD) the possible
PAA binding to the TAR fragment, as the interaction of TAR
RNA with small ligands has been reported to induce TAR
conformational changes, liable to be visualized by this
method.5 The CD spectra of PAA 1–8 alone did not reveal
any of the characteristic shapes of secondary peptide structure,
suggesting that PAA are either unstructured or adopt a
non-canonical structure. In the presence of PAA 1–8, a dose
dependant modification of the molecular ellipticity intensity in
the CD spectrum of TAR was observed (see ESIw). These
modifications consisted of a decrease in the 265 nm band
intensity (indicating a change in base stacking), and a
concomitant increase in the negative Cotton effect at 208 nm
(indicating a change in helicity). The magnitude of these
changes depended on the overall positive charges of the
PAA (see CD spectra of RRRR 6 vs. AAAA 7 in ESIw), but
it was not strictly correlated with the number of amines on the
PAA (hetero-PAA 1–4 vs. KKKK 5).
PAA 4, which display similar K00 values.
d
We also investigated the interaction of tetra-PAA 1–6 with a
bulge-truncated TAR fragment (TARab; Fig. 1B) (Table 1).
Octa-cationic PAA 5 and 6 displayed specificity for TAR
comparative to TARab, their affinity for TAR being approxi-
mately 4- and 3-fold higher than for TARab, respectively.
Concerning hetero-PAA 1–4, their specificity was shown to be
sequence-dependent. Indeed, PAA 4 was more specific for
TAR than for TARab (3-fold) while conversely, PAA 2 was
found to be 2-fold more specific for TARab than for TAR.
Moreover, we compared the affinity and specificity of tetra-
PAA 1, 2 and 6 for TAR RNA with those of truncated
derivatives (tri-PAA 10, 20 and 60) (Table 2).
These results prompted us to investigate the binding of
tetra-PAA 1–8 to TAR RNA by monitoring the fluorescence
change of a fluorescently-labelled (Alexa 488) TAR fragment.
This method, broadly used to study interactions between RNA
and small ligands,6 allowed us to determine dissociation
constants (Kd) (Table 1) and thermodynamic parameters
(Table 3) of PAA–TAR complexes. All curves fitted well a
1 : 1 stoichiometry model, except for FFFF 8, which seems to
follow a 2 : 1 (ligand : RNA) behavior, although the final
stoichiometry could not be unambiguously deduced. Apparent
dissociation constants ranged from 80 nM to 2 mM, octa-
cationic RRRR 6 and tetra-cationic AAAA 7 displaying the
highest and the lowest affinities, respectively, highlighting the
importance of electrostatic interactions for binding to TAR
RNA. However, affinities were not strictly correlated with the
number of cationic charges. Indeed, hexa-cationic hetero-PAA
1–4 and octa-cationic KKKK 5 bound to TAR with similar
affinities. Moreover, octa-cationic RRRR 6 displayed a Kd
value one order of magnitude lower than that of octa-cationic
KKKK 5. Altogether, these results indicate that the formation
of the PAA–TAR complexes probably results both from
electrostatic and non-electrostatic interactions.
As expected, RNA affinity increased when going from the
tri- to the tetra-PAA, either by adding an A or R residue.
However, this affinity gain was not strictly correlated with the
Table 2 Comparison of tri- and tetra-PAA
Affinity gain (tri-tetra)
Binding specificitya
TAR vs. TARab
TAR
TARab
PAA
FRK (10)
FRKA (1)
FAK (20)
RFAK (2)
RRR (60)
RRRR (6)
ꢂ 9
ꢂ 13
1.9
1.3
0.9
0.5
0.5
2.8
ꢂ 38
ꢂ 70
ꢂ 33
ꢂ 175
To estimate the TAR RNA vs. dsDNA specificity of PAA
1–6, binding affinities (K0d) were measured in the presence of a
a
Measured by Kd (TARab)/Kd (TAR).
ꢀc
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Table 3 Thermodynamic parameters for PAA–TAR associations
TDS1nel
DG1el ¼ TDS1elc
a
a
PAA sequence DG1 (kJ molꢃ1
)
DH1 (kJ molꢃ1
)
TDS1 (kJ molꢃ1
)
TDS1/DH1 DG1nelb (kJ molꢃ1
)
(kJ molꢃ1
)
(kJ molꢃ1
)
FRKA (1)
RFAK (2)
KARF (3)
AKFR (4)
KKKK (5)
RRRR (6)
AAAA (7)
ꢃ35.1 ꢁ 0.2
ꢃ32.6 ꢁ 0.3
ꢃ32.2 ꢁ 0.5
ꢃ33.9 ꢁ 0.6
ꢃ33.9 ꢁ 1.1
ꢃ40.6 ꢁ 1.1
ꢃ14.6 ꢁ 0.2
ꢃ70.3 ꢁ 1.9
ꢃ70.7 ꢁ 5.6
ꢃ69.9 ꢁ 2.7
ꢃ76.6 ꢁ 1.4
ꢃ59.0 ꢁ 1.1
ꢃ77.0 ꢁ 0.9
ꢃ85.4 ꢁ 2.4
ꢃ34.7 ꢁ 0.4
ꢃ38.1 ꢁ 1.1
ꢃ37.7 ꢁ 2.7
ꢃ42.7 ꢁ 1.4
ꢃ25.1 ꢁ 5.8
ꢃ36.4 ꢁ 5.0
ꢃ70.8 ꢁ 20.4
0.50
0.54
0.54
0.56
0.43
0.46
0.79
ꢃ26.1 ꢁ 0.9 (74%) ꢃ44.2 ꢁ 2.8
ꢃ26.2 ꢁ 0.9 (80%) ꢃ44.5 ꢁ 6.5
ꢃ25.8 ꢁ 0.5 (80%) ꢃ44.1 ꢁ 3.6
ꢃ28.8 ꢁ 1.0 (85%) ꢃ47.8 ꢁ 2.4
ꢃ9.0 ꢁ 1.1
ꢃ6.4 ꢁ 1.2
ꢃ6.4 ꢁ 1.0
ꢃ5.1 ꢁ 1.6
ꢃ19.7 ꢁ 1.3 (58%) ꢃ39.3 ꢁ 2.4 ꢃ14.2 ꢁ 2.4
ꢃ24.1 ꢁ 2.8 (58%) ꢃ52.9 ꢁ 3.7 ꢃ16.5 ꢁ 3.9
ꢃ8.4 ꢁ 0.2 (57%) ꢃ77.0 ꢁ 2.6
ꢃ6.2 ꢁ 0.4
b
a
Determined by temperature effect experiments using the DG1T ¼ DH1Tr þ DCP (T ꢃ Tr) ꢃ TDS1Tr ꢃ TDCP ln(T/Tr) (see ESIw). Determined by
salt effect experiments using the equation log (Kd) ¼ log (Knel) ꢃ ZC log [KCl] (see ESIw). In brackets: percentage of non-electrostatic interactions
c
(DG1nel/DG1). DG1el ¼ DG1 ꢃ DG1nel ¼ TDS1el.
number of cationic charges, since for example, adding a
PAA(R) monomer to PAA 20 led to a 38-fold affinity increase
for TAR, while adding the same residue to PAA 60 resulted in
a 175-fold increase. Concerning its impact on TAR vs. TARab
(nearly 6-fold), whereas adding the same residue to 20 led to a
2-fold decrease in specificity. Lastly, addition of a PAA(A)
monomer to 10 led to a slight decrease in the specificity for
TAR vs. TARab. Altogether, these results unambiguously
show that affinity and specificity for a RNA target may be
modulated by the length and the nature of the PAA sequence.
In order to have further insight into the binding mode of
the PAA–TAR interactions, DH1 and DS1 thermodynamic
parameters associated with the formation of the complexes
were determined from DG1 versus temperature curves (T1 from
278 to 308 K) (Table 3). Non-electrostatic (DG1nel, DH1nel
(E DH1), DS1nel) and electrostatic (DG1el, DH1el (E 0), DS1el)
components were obtained by examining the dependency of the
dissociation constants on the ionic strength of the solution.7 In
any case, the Gibbs energy (DG1 ¼ DG1el þ DG1nel) reflects a
balance of one unfavorable and two favorable contributions. The
unfavorable contribution (TDS1nel) mainly stems from the
entropic cost of bimolecular complex formation. This factor is
overwhelmed by two favorable contributions, which stem from
the polyelectrolyte effect (DG1el ¼ ꢃTDS1el) and non-covalent
ligand–RNA interactions (DH1nel), the latter providing the
predominant driving force for complex formation. As expected,
the non-electrostatic contribution of the total binding
(DG1nel/DG1) was lower for octa-cationic PAA 5 and 6 (o60%)
than for hexa-cationic PAA 1–4, for which the non-electrostatic
part clearly dominates the binding (from 74 to 85%).
Concerning the lowest charged AAAA 7, the significant
favorable enthalpic factor (DH1 ¼ ꢃ85 kJ molꢃ1) was
strongly balanced by a highly unfavorable entropic factor
(TDS1 ¼ ꢃ70 kJ molꢃ1; TDS1/DH1 ¼ 0.79), leading to the
highest DG1 value of the series (ꢃ14 kJ molꢃ1). So, even if the
electrostatic part (DG1el) of the total Gibbs energy for PAA 7
was 2-fold lower than for the highest charged PAA 5 and 6
of PAA to behave as specific RNA ligands, via electrostatic
and non-electrostatic interactions. Both the affinity and the
specificity of PAA for an RNA target may be modulated by
their length and by the nature of the amino acid residues.
Further work will focus on the preparation of PAA libraries
containing a wide variety of natural or non-natural amino
acids in order to identify ligands capable of binding with high
affinity and specificity to biologically relevant RNA targets.
This research was supported by ANRS, Sidaction, CG 06,
FRM and CAMPL. V. Bonnard is a recipient of a MENRT
PhD fellowship. We thank E. Margeat for helpful discussions
and for his assistance with the BIOEQS program.
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(ꢃ6.2, ꢃ14.2 and ꢃ16.5 kJ molꢃ1
, respectively), the
DG1nel/DG1 ratio was the same for the three compounds.
In conclusion, considering that only four different amino
acid residues were selected for this preliminary study, our
results are very promising, as they demonstrate the potential
ꢀc
This journal is The Royal Society of Chemistry 2009
2304 | Chem. Commun., 2009, 2302–2304