Synthesis and properties of cationic oligopeptides with different side chain lengths that bind to RNA duplexes

Article abstract of DOI:10.1016/j.bmc.2013.01.053

A series of artificial peptides bearing cationic functional groups with different side chain lengths were designed, and their ability to increase the thermal stability of nucleic acid duplexes was investigated. The peptides with amino groups selectively increased the stability of RNA/RNA duplexes, and a relationship between the side chain length and the melting temperature (T _m) of the peptide-RNA complexes was observed. On the other hand, while peptides with guanidino groups exhibited a similar tendency with respect to the peptide structure and thermal stability of RNA/RNA duplexes, those with longer side chain lengths, such as l-2-amino-4-guanidinobutyric acid (Agb) or l-arginine (Arg) oligomers, stabilized both RNA/RNA and DNA/DNA duplexes, and those with shorter side chain lengths exhibited a higher ability to selectively stabilize RNA/RNA duplexes. In addition, peptides were designed with different levels of flexibility by introducing glycine (Gly) residues into the l-2-amino-3-guanidinopropionic acid (Agp) oligomers. It was found that insertion of Gly did not affect the thermal stability of the peptide-RNA complexes, but an alternate arrangement of Gly and Agp apparently decreased the thermal stability. Therefore, in the Agp oligomer, consecutive Agp sequences are essential for increasing the stability of RNA/RNA duplexes.

Full text of DOI:10.1016/j.bmc.2013.01.053

Bioorganic & Medicinal Chemistry 21 (2013) 1717–1723
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and properties of cationic oligopeptides with different side
chain lengths that bind to RNA duplexes
⇑
Yusuke Maeda, Rintaro Iwata, Takeshi Wada
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bioscience Building 702, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of artificial peptides bearing cationic functional groups with different side chain lengths were
designed, and their ability to increase the thermal stability of nucleic acid duplexes was investigated.
The peptides with amino groups selectively increased the stability of RNA/RNA duplexes, and a relation-
ship between the side chain length and the melting temperature (Tm) of the peptide–RNA complexes was
observed. On the other hand, while peptides with guanidino groups exhibited a similar tendency with
respect to the peptide structure and thermal stability of RNA/RNA duplexes, those with longer side chain
lengths, such as L-2-amino-4-guanidinobutyric acid (Agb) or L-arginine (Arg) oligomers, stabilized both
RNA/RNA and DNA/DNA duplexes, and those with shorter side chain lengths exhibited a higher ability
to selectively stabilize RNA/RNA duplexes. In addition, peptides were designed with different levels of
Received 4 January 2013
Revised 22 January 2013
Accepted 23 January 2013
Available online 1 February 2013
Keywords:
RNA-binding peptide
Cationic peptide
Artificial peptide
RNAi drug
flexibility by introducing glycine (Gly) residues into the L-2-amino-3-guanidinopropionic acid (Agp) olig-
omers. It was found that insertion of Gly did not affect the thermal stability of the peptide–RNA com-
plexes, but an alternate arrangement of Gly and Agp apparently decreased the thermal stability.
Therefore, in the Agp oligomer, consecutive Agp sequences are essential for increasing the stability of
RNA/RNA duplexes.
DDS
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
creased thermal stability of the dsRNAs also increased their
stability in cells.5 We are thus attempting to develop RNA/RNA du-
In recent years, increased attention has been paid to the devel-
opment of nucleic acid drugs. For example, antisense oligonucleo-
tides and RNA interference drugs (RNAi drugs) are well known
molecular tools for the regulation of gene expression, in which
their mechanisms of action are based on sequence specific interac-
tions.1 The RNAi drugs act on the target mRNA in a sequence selec-
tive manner. Thus, RNAi drugs are attractive because of their high
selectivity for the target and their shorter drug development time.
Therefore, siRNAs have been widely studied for therapeutic appli-
cations; however, such RNA molecules are not sufficiently effective
because of their low-membrane permeability and instability in
cells. To stabilize oligonucleotides against metabolic degradation,
a number of chemical modifications have been proposed,2 and
RNAi drugs generally consist of double stranded RNAs (dsRNAs)
with chemical modifications. A proper modification of an RNA mol-
ecule increases its stability in cells and improves its pharmacoki-
netic properties.3 Another strategy for stabilizing siRNA is the use
of molecules that can non-covalently bind to RNA to protect it from
attack by nucleases. For example, a fusion protein of the peptide
transduction domain-dsRNA binding domain was shown to effec-
tively transport RNAi drugs into primary cells.4 In this case, the in-
plex-binding molecules that are useful as drug delivery systems
(DDSs) for siRNAs.
A wide variety of RNA-binding molecules have been reported,
such as aminoglycosides6 and RNA-binding proteins.7 In paticular,
chemically modified peptides bearing different methylene lengths
in the arginine or lysine residues possess diverse affinities to
RNAs.8 In our previous study,
dideoxy-
a
-(1 ? 4)-linked-2,6-diamino-2,6-
D
-glucopyranose oligomers were synthesized, and their
highly RNA-selective binding ability was demonstrated.9 These re-
sults suggested that the geometry of the cationic groups, particu-
larly the distance between the cationic groups, affects the affinity
and selectivity of the oligomers for nucleic acid duplexes. There-
fore, in this study, we designed a series of cationic oligopeptides
to reveal the relationship between the geometry of the cationic
groups and the affinity of the peptides for nucleic acids. In contrast
to oligosaccharide derivatives, peptides can easily be synthesized,
which is advantageous for obtaining a systematic series of mole-
cules. They can also be readily connected with other functional
groups such as transporter molecules.10 Therefore, RNA duplex-
binding peptides are useful tools for the development of drug
delivery systems (DDSs) for RNAi drugs. The L-arginine (Arg) oligo-
mers designed from the HIV Tat peptide are well known for their
high-membrane permeability11 and the Arg 15mer has been
used as an RNAi transporter by forming a complex with RNA.12
⇑
Corresponding author. Tel.: +81 4 7136 3611.
E-mail address: wada@k.u-tokyo.ac.jp (T. Wada).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.053

1718
Y. Maeda et al. / Bioorg. Med. Chem. 21 (2013) 1717–1723
However, the activity varies with the side chain length,13 and thus
a more detailed study of the relationship between the structure
and activity is important for the development of effective carriers
for RNAi drugs. Thus, to investigate the structural effects on bind-
ing ability, we designed and synthesized a number of cationic oli-
gopeptides bearing different side chain lengths to control the
distance between the cationic functional groups and peptides with
different flexibility by incorporating glycine (Gly).
combination of the functional groups. These peptides were based
on the Agp oligomer and other amino acids such as glycine (Gly),
L-serine (Ser), and L-asparagine (Asn). Glycine was chosen because
of its high flexibility. Ser and Asn were chosen because they are
well known in RNA-binding proteins to form hydrogen bonds with
the phosphate groups of RNA.14
On the basis of molecular mechanics calculations with a GB/SA
water solvation model,15 a cationic oligopeptide 8mer consisting of
Dab can bind to the major groove of an A-type RNA/RNA duplex
12mer, in which all the protonated amino groups of the peptide
form the hydrogen bonds to the phosphate anions of the duplex
(Fig. 2). Therefore, the electrostatic interaction and hydrogen bond-
ing would be important for the binding of cationic oligopeptides to
RNA/RNA duplexes.
2. Results and discussion
2.1. Design of the peptides
A series of cationic oligopeptides was designed (Fig. 1). All pep-
tides contained a unit of N-acetyl-L-tyrosine with two glycine res-
2.2. Melting temperature (Tm) analysis
idues at the N-terminus for UV detection and quantification. In this
study, the 12mer of nucleic acid duplexes was used as a model to
estimate the interaction with peptides bearing four or eight cat-
ionic groups because four pairs of phosphate groups are aligned
on the inward portion of the major groove of the 12mer of A-type
nucleic acid duplexes. First, to compare the effects of the distance
between cationic groups, peptides with different side chain lengths
were designed. Peptides were synthesized with amino groups
The Tm values of the RNA duplexes were measured both in the
absence and presence of an equal amount of peptides. All measure-
ments were performed under-physiological conditions with
10 mM phosphate buffer containing 100 mM NaCl at pH 7.0. Fig-
ure 2 shows the melting temperature enhancements and Table 1
lists the Tm values for the self complementary RNA 12mer
r(CGCGAAUUCGCG)2 in the absence and presence of an equal
amount of peptides with amino groups. In this study, the peptides
were added into the solution of nucleic acid duplexes after anneal-
ing to avoid the aggregation of the peptides at high temperature.
The Tm values were affected by the side chain length, with Dab8
(2) showing the highest Tm value. Comparing the Dab8 (2), Orn8
(3), and Lys8 (4), the Tm value increased as the side chain length
(
L
-2,3-diaminopropionic acid (Dap),
(Dab), -ornithine (Orn), and -lysine (Lys)) and guanidino groups
-2-amino-3-guanidinopropionic acid (Agp), -2-amino-4-guanid-
inobutyric acid (Agb), and -arginine (Arg)). In some molecules,
L-2,4-diaminobutyric acid
L
L
(L
L
L
several glycine units were also inserted into an Agp octamer to
increase the flexibility of the peptides. Peptides with alternate
arrangements were also designed to change the position and
Figure 1. Structures and sequences of cationic peptides.

Y. Maeda et al. / Bioorg. Med. Chem. 21 (2013) 1717–1723
1719
Figure 3. Melting temperature enhancements for the RNA/RNA and DNA/DNA
duplexes at pH 7.0 in the presence of peptides 1–4. Differences in the thermal
melting points (
equimolar amounts of peptide relative to the duplex alone. The dark gray columns
represent Tm for RNA/RNA duplex, and light gray columns represent Tm values
for DNA/DNA duplex.
DTm) are given for the nucleic acid duplexes in the presence of
D
D
Figure 2. Molecular model of L-2,4-diaminobutylic acid (Dab) 8mer binding to A-
type RNA–RNA duplex (12mer).
Table 1
Thermal melting points (Tm in °C) for oligonucleotide duplex (in the absence and
presence of peptides with amino groupsa)b
Peptide
RNA/RNA
D
Tm
DNA/DNA
DTm
None
Dap8
Dab8
Orn8
Lys8
60.7
60.2
74.7
74.2
68.1
48.2
50.8
49.5
51.3
49.7
À0.5
14.0
13.5
7.4
2.6
1.3
3.1
1.5
a
Peptide concentration 4
Buffer (10 mM phosphate buffere for pH 7.0), NaCl (100 mM), together with
l
M.
b
each oligonucleotide strand (4
cate measurements.
lM). Tm values are reported at the means of dupli-
Figure 4. Melting temperature enhancements for the RNA/RNA and DNA/DNA
duplexes at pH 7.0 in the presence of peptides 5–7. Differences in the thermal
melting points (
equal amount of peptide relative to the duplex alone. The dark gray columns
represent Tm for RNA/RNA duplex, and light gray columns represent Tm values
DTm) are given for the nucleic acid duplexes in the presence of an
decreased. These results suggest that the distance between the
amino groups in Dab8 (2) fits to the distance of the opposing phos-
phate groups in the RNA duplexes. This tendency has been previ-
D
D
for DNA/DNA duplex.
ously reported for cationic oligopeptides with D
-amino acids.6 On
the other hand, Dap8 (1) did not stabilize the RNA/RNA duplexes.
This differing behavior may result because the distance of the ami-
no groups in Dap8 is too short to interact with the phosphate
groups in the RNA/RNA duplexes. In fact, none of the peptides with
amino groups increased the thermal stability of the DNA/DNA du-
plex d(CGCGAATTCGCG)2 (Fig. 3). The distance between the phos-
phate groups in the major groove of a DNA/DNA duplex is twice
that in an RNA/RNA duplex. Therefore, none of the peptides were
able to interact with the DNA/DNA duplexes. In the minor groove
of a DNA/DNA duplex, the interstrand phosphate groups are much
closer than those in the major groove. However, the phosphate
groups facing outward of the minor groove are unfavorable to form
hydrogen bonds with the protonated amino groups of cationic
peptides.
Table 2
Thermal melting points (Tm in °C) for oligonucleotide duplex (in the absence and
presence of peptides with guanidino groupsa)b
Peptide
RNA/RNA
D
Tm
DNA/DNA
DTm
None
Agp8
60.7
76.9
75.8
48.2
49.9
48.9
16.2
15.1
1.7
0.7
D-Agp8
73.0
12.3
49.4
1.2
D,L-Agp8
Agb8
Arg8
74.1
73.1
13.4
12.4
53.1
53.7
4.9
5.5
a
Peptide concentration 4
Buffer (10 mM phosphate buffere for pH 7.0), NaCl (100 mM), together with
lM.
b
each oligonucleotide strand (4
cate measurements.
lM). Tm values are reported at the means of dupli-
The peptides with guanidino groups (5, 6, 7) exhibited the same
tendency as the peptides with the amino groups (Fig. 4, Table 2) for
the RNA/RNA duplexes. Peptides with shorter side chain lengths
had higher Tm values. This result indicates that the position of gua-
nidino groups in Agp8 (5) also fits well with the RNA duplex struc-
ture. While the distance between the functional groups is similar in
the peptides with amino and guanidino groups, the peptides with
the guanidino groups exhibited higher Tm values. These different
Tm values can be attributed to the different features of the func-
tional groups. The guanidino group is known to have a stronger
interaction with phosphate groups than amino groups, and thus
the peptides with guanidino groups showed a stronger interaction

1720
Y. Maeda et al. / Bioorg. Med. Chem. 21 (2013) 1717–1723
Table 3
with the nucleic acid duplexes. However, in the case of the DNA/
DNA duplex, the tendency changed because of the differences in
the guanidino and amino functional groups. For the peptides with
guanidine groups, those with longer side chain lengths had higher
Tm values. This result may also be due to the fact that the guanidino
groups strongly interact with the phosphate groups.
The different side chain lengths of the peptides resulted in dif-
ferent thermal stabilities of the nucleic acid duplexes. The duplex
stability may also be affected by the flexibility of the peptides.
Thus, to investigate the effect of incorporation of flexible residues
into the peptide backbone, glycine was inserted into Agp8 (5) to in-
crease the flexibility of the main chain (8, 9, 10). Interestingly, it
was found that an increase in the flexibility of the main chain
did not affect the thermal stability of the peptide–RNA complexes
(Fig. 5, Table 3). These results suggest that the stabilization of the
RNA duplex with Agp8 (5) is mainly attributed to enthalpic factors.
Thermal melting points (Tm in °C) for oligonucleotide duplex (in the absence and
presence of peptides with flexible main chiana)b
Peptide
RNA/RNA
DTm
None
Agp8
60.9
77.5
77.5
78.8
78.6
77.7
77.4
16.6
16.6
17.9
17.7
16.8
16.5
Agp8G1
Agp8G2
Agp8G3
Agp8A2
Agp8P2
a
Peptide concentration 4 lM.
Buffer (10 mM phosphate buffere for pH 7.0), NaCl (100 mM), together with
b
each oligonucleotide strand (4
cate measurements.
lM). Tm values are reported at the means of dupli-
In addition, insertion of L-alanine (Ala) or L-proline (Pro) into Agp8
(5) also did not affect the thermal stability of the RNA duplexes (11,
12). If the peptides invade the major groove, the redundant amino
acid residues will give rise to steric hindrance and decrease the
thermal stability. Therefore, these results suggest that the peptides
interact with the surface of the nucleic acids.
Peptides with alternate arrangements also did not stabilize the
RNA duplexes (Fig. 6, Table 4). Peptides consisting of Agp and Gly
in an alternate arrangement, AgpG (13) did not have any effects
on the RNA/RNA duplex. On the other hand, Agp4 showed an
appreciable stabilization effect for the RNA/RNA duplex. Therefore,
a consecutive Agp sequence is effective for RNA/RNA duplex stabil-
ization. Furthermore, Agp8G3 (10), in which four Agp2 units are
connected, has the same affinity as Agp8; thus, the Agp2 structural
unit is effective for the interaction with RNA/RNA duplexes. In Agp
oligomers with a consecutive sequence, the guanidino groups are
located on both sides of the peptide backbone, and this alignment
is identical to that required for interaction between the guanidino
groups and the phosphates in the major groove of RNA duplexes.
However, peptides with a combination of guanidino and hydroxy
or amide groups (AgpS (14) and AgpN (15), respectively) had no ef-
fects on the thermal stability of RNA duplexes. These results also
suggest that a consecutive arrangement of cationic amino acids is
essential for effective interaction with the phosphates of RNA
duplexes.
Figure 6. Melting temperature enhancements for the RNA/RNA duplexes at pH 7.0
in the presence of peptides 13–15. Differences in the thermal melting points ( Tm
are given for the nucleic acid duplexes in the presence of equimolar amounts of
peptide relative to the duplex alone.
D
)
Table 4
Thermal melting points (Tm in °C) for oligonucleotide duplex (in the absence and
presence of peptides with alternate arrangementsa)b
Peptide
RNA/RNA
D
Tm
None
Agp4
AgpG
AgpS
AgpN
60.3
65.2
62.2
58.9
60.5
The effect of chirality was also examined. The Agp oligomers
consisting of all-L- and all-D-amino acids had similar Tm values,
4.9
1.9
À1.4
0.2
a
Peptide concentration 4 lM.
Buffer (10 mM phosphate buffere for pH 7.0), NaCl (100 mM), together with
b
each oligonucleotide strand (4
cate measurements.
lM). Tm values are reported at the means of dupli-
but the peptides having D/L-alternate arrangements slightly had
lower thermal stability. A decrease in the thermal stability induced
by a heterochiral backbone was also reported for chiral PNA.16
2.3. CD spectroscopy
The structures of the peptides and the RNA–peptide complexes
in solution were analyzed using circular dichroism (CD) spectros-
copy. On the basis of molecular mechanics calculations,15 the ami-
no groups and guanidino groups of the peptides, particularly in
Dap8 (2) and Agp8 (5), can form intramolecular hydrogen bonds
with the amido groups in the main chain. However, in the absence
of nucleic acid duplexes, the spectra of all the peptides indicated
the presence of random coils.17 Therefore, the effect of the second-
ary structures of the peptides was negligible in these cases. The
Figure 5. Melting temperature enhancements for the RNA/RNA duplexes at pH 7.0
in the presence of peptides 1, 8–12. Differences in the thermal melting points ( Tm
are given for the nucleic acid duplexes in the presence of an equal amount of
peptide relative to the duplex alone.
D
)

Y. Maeda et al. / Bioorg. Med. Chem. 21 (2013) 1717–1723
1721
structures of the RNA–peptide complexes were also analyzed. Be-
cause the peptides showed a variety of Tm values depending on
the side chain length and the nature of the cationic functional
groups, there existed not only electrostatic interactions and hydro-
gen bonding, but also structural factors. However, for both the RNA
and DNA duplexes, no appreciable structural changes in the nucleic
acids were observed following the addition of the peptides (Figs. 7
and 8). The CD spectra of the RNA and DNA duplexes in the pres-
ence and absence of peptides were typical for A-type and B-type
helices, respectively.
the exothermic interactions are attributed to the binding of the
peptides to the major groove of RNA/RNA duplexes and the endo-
thermic interactions are related to the interactions with other sites.
3. Materials and methods
3.1. Peptide synthesis
Peptides were synthesized via a conventional solid-phase meth-
od by using the 9-fluorenylmethyloxycarbonyl (Fmoc) strategy.19
The peptide chains were assembled on a Fmoc-NH-SAL-PEG resin
by using Fmoc amino acid derivatives (5 equiv), N,N-diisopropyl-
ethylamine (DIPEA, 10 equiv), and 2-(1H-9-azabenzotriazole-1-
2.4. ITC measurement
Thermodynamic analysis of the peptide–nucleic acid interac-
tions was carried out using isothermal titration calorimetry (ITC)
measurements. The duplex concentration was 2.5 times higher
than that used for the UV melting analyses because the amount
of heat generated during the binding between the nucleic acid du-
plexes and the oligocationic peptides was expected to be insuffi-
cient for calculation of the thermodynamic parameters at the
lower concentration. Figures 9, S8,17 and S917 show the prelimin-
ary results for the ITC titration of the peptides with the self com-
plementary RNA/RNA duplex r(CGCGAAUUCGCG)2 and DNA/DNA
duplex d(CGCGAATTCGCG)2. In addition to electrostatic interac-
tions and hydrogen bonding between the peptides and nucleic
acids, dehydration and dissociation of the phosphates in the buffer
solution were apparently observed. Thus, the interactions were too
complex for calculation of the thermodynamical parameters. How-
ever, it was observed that both of the peptides selectively inter-
acted with the RNA/RNA duplexes. In contrast, only Agb8 (5)
interacted with both the RNA/RNA and DNA/DNA duplexes. This
result may be due to the fact that, in Agb8, the side chain is long
and sufficiently flexible to interact with the phosphate groups of
both the RNA/RNA and DNA/DNA duplexes. In comparison with
the amino-substituted Dap8, the guanidine-substituted Dab8 (5)
had larger endothermic interactions.
yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
(HATU,
5 equiv) in dimethylformamide (DMF) for the coupling, and 25%
piperidine/DMF for the removal of the Fmoc group. After coupling
of the last amino acids, amino groups at the N-termini were pro-
tected with an acetyl (Ac) group using acetic anhydride (10 equiv).
To cleave the peptide from the resin and remove the side chain
protecting groups, the peptide resin was treated with trifluoroace-
tic acid (TFA)-triisopropylsilane-water, (95:2.5:2.5, v/v/v). Peptides
in sat NaHCO3aq (200
the 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine20 (10 equiv
per amino groups) in dioxane (200 l), and stirred overnight at
ll) were added in one portion to a solution of
l
rt, then concentrated in vacuo. To remove the protecting groups
from the guanidino groups, the peptides were treated with TFA–
triisopropylsilane–water (95:2.5:2.5, v/v/v). All peptides were
purified with reverse-phase HPLC (0.05% TFA in water–acetoni-
trile). The peptides were successfully identified by matrix-assisted
laser desorption ionization time-of-flight mass spectrometry
(MALDI-TOF-MS). Compound 1, TOF-MS m/z calcd for [M+Na]+
1048.07; Found 1047.60. Compound 2, TOF-MS m/z calcd for
[M+H]+ 1138.31; Found 1137.22. Compound 3, TOF-MS m/z calcd
for [M+Na]+ 1272.50; Found 1271.56. Compound 4, TOF-MS m/z
calcd for [M+H]+ 1362.73; Found 1361.51. Compound 5, TOF-MS
m/z calcd for [M+H]+ 1362.41; Found 1361.51. Compound 6, TOF-
MS m/z calcd for [M+H]+ 1474.63; Found 1473.77. Compound 7,
TOF-MS m/z calcd for [M+H]+ 1586.84; Found 1585.72. Compound
8, TOF-MS m/z calcd for [M+H]+ 1419.46; Found 1418.53. Com-
pound 9, TOF-MS m/z calcd for [M+Na]+ 1476.52; Found 1476.00.
Compound 10, TOF-MS m/z calcd for [M+H]+ 1533.57; Found
1533.07. Compound 11, TOF-MS m/z calcd for [M+H]+ 1504.57;
Found 1504.21. Compound 12, TOF-MS m/z calcd for [M+H]+
1556.64; Found 1555.68. Compound 13, TOF-MS m/z calcd for
[M+Na]+ 1100.07; Found 1099.25. Compound 14, TOF-MS m/z
Inhibition assays were carried out to clarify the peptide-binding
sites in the RNA duplexes. The peptides were titrated into a solu-
tion of the RNA duplexes in the presence of neomycin, which is
known to bind to the major groove of RNA duplexes.18 The inhibi-
tion of peptide–RNA binding by neomycin was different for the
types of peptides (Fig. S1017). For Dab8 (2), the exothermic
interactions were selectively inhibited, while the endothermic
interactions were still observed. On the other hand, for Agp8 (5),
both of the interactions were inhibited. These results suggest that
Figure 7. CD spectra of the RNA/RNA duplexes in the presence and absence of an equal amount of peptides 1–7 (at 20 °C, pH 7.0, 4 lM each of peptide and duplex).

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Y. Maeda et al. / Bioorg. Med. Chem. 21 (2013) 1717–1723
Figure 8. CD spectra of the DNA/DNA duplexes in the presence and absence of an equal amount of peptides 1–7 (at 20 °C, pH 7.0, 4 lM each of peptide and duplex).
Figure 9. ITC profiles at 25 °C for titration of Dab8 (2) into a solution of RNA/RNA duplex (left) and DNA/DNA duplex (right); each curve is the result of a 2.5
150 M peptide. The duplex concentration was 10 M in a 10 mM phosphate buffer with 100 mM NaCl at pH 7.0; corrected injection heat in the cases of RNA/RNA were
plotted.
ll injection of
l
l
calcd for [M+H]+ 1198.18; Found 1197.67. Compound 15, TOF-MS
m/z calcd for [M+H]+ 1306.29; Found 1305.46.
added after oligonucleotides were annealed. The samples were
prepared as follows. The oligonucleotides were dissolved in a phos-
phate buffer (10 mM) containing NaCl (0.1 M) at pH 7.0. The solu-
3.2. Melting temperature (Tm) analysis
tions of oligonucleotides (4
left for 10 min, and then cooled to 10 °C at a rate of 1 °C/min. The
equal amounts of peptides (final concn: 4 M) were then added to
the solution. The samples were left to equillbrate at the starting
temperature for 30 min, the dissociation of the duplex was ob-
served by heating the solution to 95 °C at a rate of 0.2 °C/min,
and data points were collected at every 0.1 °C.
lM) were first rapidly heated to 95 °C,
Absorbance versus temperature profile measurements were
carried out in quartz cells with a 1 cm path length using an
eight-sample cell changer. The variation in the UV absorbance with
temperature was monitored at 260 nm. The temperature was
scanned from 10 to 95 °C at a rate of 0.2 °C/min. The peptides were
l

Y. Maeda et al. / Bioorg. Med. Chem. 21 (2013) 1717–1723
1723
3.3. CD spectroscopy
Acknowledgments
All CD spectra were recorded at 20 °C. The following instrument
settings were used: resolution, 0.1 nm; sensitivity, 10 mdeg; re-
sponse, 4 s; speed, 10 nm/min; accumulation, 6.
We thank Professor Kohei Tsumoto (University of Tokyo) and
Dr. Satoru Nagatoishi (University of Tokyo) for the ITC measure-
ments and helpful discussions. This work was finally supported
by KAKENHI and CREST, the Japan Science and Technology Agency.
3.4. Conditions for ITC experiments
Supplementary data
The peptides and nucleic acid duplexes were dissolved in a
10 mM phosphate buffer containing 100 mM NaCl at pH 7.0.
Supplementary data (data include CD spectra, UV melting pro-
files, and ITC profiles) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.
053.
The peptide solutions (150
acid duplex solutions (10 M) at 25 °C. Each titration of peptide
solution consisted of a preliminary 0.5 l injection followed by
24 subsequent 1.5 l additions, which were performed over 3 s
lM) were titrated into the nucleic
l
l
l
periods at 120 s intervals. In the inhibition assays, the peptide
solutions were titrated into the nucleic acid solution in the pres-
References and notes
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We have synthesized a series of cationic oligopeptides by
systematically changing the position of the cationic groups. On
the basis of UV-melting analysis, CD spectrometry and ITC mea-
surements, these cationic oligopeptides showed different tenden-
cies for the stabilization of nucleic acid duplexes. Peptides with
amino groups stabilized only RNA duplexes, while peptides with
guanidino groups stabilized both RNA and DNA duplexes. In partic-
ular, Dab8 (2) and Agp8 (5) showed the highest Tm values among
the series of peptides with the same cationic groups but different
side chain length. These results suggest that the distance between
the cationic groups, such as in Dab8 (2) and Agp8 (5), are well fitted
to the distance between the phosphate groups in the major groove
of RNA duplexes. Furthermore, peptides with alternate arrange-
ments and those containing flexible amino acids did not stabilize
the RNA duplexes. These results indicate that at least two consec-
utive sequences of Agp are necessary for effective binding of cat-
ionic oligopeptides to RNA duplexes. Therefore, given their
unique properties, Dab8 (2) and Agp8 (5) will be useful as stabiliz-
ers of dsRNA-based nucleic acid drugs or new materials for their
DDS.
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