Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure

Article abstract of DOI:10.1016/j.bmcl.2017.11.004

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC₅) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions.

Full text of DOI:10.1016/j.bmcl.2017.11.004

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
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Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability
of DNA (dC5) i-motif structure
⇑
Chandrasekhar Reddy Gade, Nagendra K. Sharma
School of Chemical Sciences, National Institute of Science Education and Research (NISER), HBNI-Mumbai, Bhubaneswar, Jatni 752050, Odisha, India
a r t i c l e i n f o
a b s t r a c t
Article history:
This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical
studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC5)
under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation
of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich
sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions.
Ó 2017 Elsevier Ltd. All rights reserved.
Received 9 August 2017
Revised 1 November 2017
Accepted 2 November 2017
Available online xxxx
Keywords:
Hybrid i-motif
Aep-PNA
DNA
Ion-ion interaction
Enhanced stability
Since after discovery of DNA duplex structure, DNA are also
known to form triplex, tetraplex, and other many more DNA poly-
morphs.1 Importantly, DNA forms two type of tetraplex structures
from the repeated G-/C-rich sequences: (i) the G-quadruplex (G4),
which encompasses of stacked guanine tetrads stabilized by mono-
valent cations (Na+/K+); and (ii) the i-motif, a cytosine-rich struc-
ture comprising of two hemi-protonated parallel duplexes
intercalated in an antiparallel orientation (Fig. 1).2,3 The role of
those DNA tetraplexes structures are reportedly considered in
the maintenance of telomere and gene regulation. Recently, the
occurrence of i-motif are noticed in regulatory regions of the
human genome such as centromeres, telomeres, and oncogene
promoter regions.4 These structures are also considered as poten-
tial materials for nanotechnology research.5,6
Structurally, the DNA i-motif is reportedly a compact structure
with short base-pairing distances (ꢀ3.1 Å), helical twist (ꢀ12–16°)
between adjacent C:C+H base pairs and close sugar-sugar contacts,
CAHAO interactions (Fig. 1).3 However, the repulsive interactions
also exist between the charged C-imino protons and phosphates
groups of DNA strands, which intend the destabilization of that i-
motif structure. In vitro, the formation of DNA i-motif structures
are occurred only under acidic conditions, near acidic pH range
(3.5–5.5) and the maximum stability are noticed at pH equal to
the pKa of the cytosine N-3. Though DNA i-motifs are stable at neu-
tral pH conditions in vivo presumably due to other cellular factors
such as negative super helicity, cellular proteins and molecular
crowding conditions.7 Recently, the formation of DNA i-motif are
noticed in vitro, at neutral pH conditions, in presence of metal ions
(Cu2+/Ag+).8 Furthermore, the stable i-motif structures have been
achieved by sugar/phosphate backbone modified DNA analogues
which are following-Thio-phosphoramidates,9 RNA,10 Locked
nucleic acid (LNA),11 and 20-fluoro DNA analogue.12
Nevertheless the stabilization of DNA i-motifs are also explored
by hybridization with similar C-rich sequence of DNA analogues,
and with the sequence specific DNA binding small synthetic
molecules.13 In repertoire of structurally backbone modified DNA
analogues, Peptide nucleic acid (PNA), consisting aminoethylglyci-
nate (aeg) backbone, has emerged as a potential DNA analogue
because of its promisable binding affinities with DNA duplex/tri-
plex structures.14 The C-rich sequence (TC5/TC8) of aeg-PNAs are
also explored in the formation of stable i-motif.15,16 Moreover
modified PNA as Alanyl-PNA, alanine backbone instead of aeg,
are reportedly known the formation of the stable i-motif from its
TC8 sequence.17 Further, aeg-PNA are successfully employed for
the formation of hybrid DNA (or RNA) i-motif structure which
are more stable than respective DNA (or RNA) i-motif.18,19 The sta-
bilization of PNA:DNA (or PNA:RNA) hybrid i-motif are presumably
considered because the hybridized aeg-PNA strand diminish the
ion-ion repulsive interaction between negatively charged DNA’s
phosphate backbone in that hybrid i-motif.
These results inspired us to design the hybrid DNA i-motif from
the positively charged backbone containing DNA or PNA analogue
which could enhance the stability of DNA i-motif structure near
⇑
Corresponding author.
E-mail address: nagendra@niser.ac.in (N.K. Sharma).
https://doi.org/10.1016/j.bmcl.2017.11.004
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Gade C.R., Sharma N.K. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.11.004

2
C.R. Gade, N.K. Sharma / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
H
(100 mM, pH4.5) at 10 °C (Fig. 3). The CD spectra of DNA i-motif
N
N
O
H
H
O
structure gives maxima (ꢀ296 nm) and minima (ꢀ261 nm) respec-
tively. We observed similar CD spectrum with control DNA dC5. We
were unable to observe the characteristic CD signal of i-motif with
alone aep-PNA (pC5) even at low pH (4.5). The CD spectrum of
aep-PNA pC5 did not match with the CD of control DNA (dC5).
The CD spectra of pC5, however, give only minima at ꢀ263 nm
under that pH (4.5) condition. This CD signature resembled to
the self-duplex type of secondary structure. This self-duplex possi-
bly forms from the PNA pC5 and cytosine protonated (N3-atom of
cytosine) PNA pC5 (pC+-H)5 by hydrogen bonding. The repulsive
interactions between positively charged prolyl amine of pC5 PNA
backbone might prevent the formation of stable tetraplex struc-
ture. Interestingly, the CD spectrum of hybrid dC5:pC5 (1:1)
showed characteristic CD signature with the consisting maxima
(ꢀ300 nm) and minima (ꢀ261 nm) like control dC5.Importantly,
the marginal red shift (ꢀ4.0 nm) was observed in the CD maxima
of hybrid sample. The comparative CD spectral analyses strongly
support the hybridization of pC5 with dC5 which lead to the
formation of hybrid i-motif pC5:dC5 structure.
O
N
acidic pH
O
N
C
N
N
H
O
H
DNA-Cn (dCn)
C+H---C
DNA i-motif
Fig. 1. DNA cytosine (dC), hydrogen bonding in C+HꢁC, and i-motif structure.
aep-PNA
Hybrid PNA:DNA i-motif
DNA
O
H
N
O
C
Low pH
O
P
O
O-
O
+
O
C
HN
pC5
dC5
(ion-ion interactions)
Hydrogen bonding (C+H----C )
To find the appropriate pH conditions for the formation of
hybrid i-motif, we recorded the pH dependent CD spectra of dC5/
hybrid dC5:pC5 (1:1) (Fig. 3). The characteristic CD signal of control
i-motif forming DNA dC5 are diminished with increasing pH values
(Fig. 4A). The similar pH dependent CD signals depletion are
observed with hybrid (pC5:dC5) (Fig. 4B). These results indicate
that the hybrid pC5:dC5 (1:1) also forms DNA i-motif type of struc-
ture, as like control, only at low pH range (4.0–6.0).
Further the stability of hybrid i-motif structure was examined
by the temperature dependent CD-spectra analyses. We recorded
the temperature dependent CD spectra of annealed hybrid pC5:
dC5 (1:1) and control dC5 at acidic pH 4.5. The characteristic i-motif
CD signals (maxima & minima) of hybrid structure were signifi-
cantly depleted in cooperative manner with increasing the temper-
ature as like control dC5 (Supplementary Materials). The stability of
DNA structures (duplex, triplex and tetraplex) are measured in
term of their Tm (Thermal melting) values, which are extracted
from respective sigmoidal UV/CD-melting profiles.25 The CD ther-
mal melting profiles of hybrid pC5:dC5, at wavelength 299 nm, is
appeared as negative sigmoidal as like control dC5. Such melting
profiles are characteristic for DNA i-motif structures. Thus, hybrid
pC5:dC5 also forms DNA i-motif type of structure and their profiles
indicate that hybrid i-motif structure is more stable than lone DNA.
Fig. 2. Proposed hybrid i-motif formation.
acidic conditions, possibly due to ion-ion attractive interaction.
Since aminoethylprolyl (aep) modified PNA analogues, conforma-
tionally constrained chiral PNA, is reported and found that
aep-PNA is strongly stabilized hybrid DNA duplexes/triplexes structure
with complementary DNA.20–23 The G-rich sequence of aep-PNA
are also explored in the formation of stable G-quadruplex.24 Thus
we planned to employ aep-PNA, cytosine rich sequence, to explore
the formation of stable hybrid DNA i-motif structure by introduc-
ing ion-ion attractive interactions between amino backbone of
aep-PNA and phosphate backbone of DNA (Fig. 2). Herein, we
describe the synthesis and biophysical studies of aep-PNA cytosine
pentamer (pC5) for hybrid DNA i-motif structure with DNA dC5
by CD/UV/NMR/ESI-MS techniques.
Results and discussion
We began with the synthesis of N4-acetyl aep-PNA cytosine
(aep-PNA-C) monomer from L-4-Hydroxyproline by following the
reported procedure (see in Suppl. Materials).21 This monomer
was employed for the synthesis of aep-PNA C5 (pC5), designed i-
motif forming sequence, by solid support peptide synthesis meth-
ods using MBHA resin (Scheme 1). After cleavage from the resin,
oligopeptide pC5 was isolated by gel filtration (Sephadex G-15),
and then purified by HPLC. The purified pC5 was characterized by
ESI-Mass studies (see in Supplementary Materials). This PNA pC5
was used to prepare the hybrid DNA:PNAi-motif structure with
DNA-C5 (dC5) and performed a comparative biophysical studies
such as CD/UV/NMR/ESI-Mass studies of DNA:PNA hybrid i-motif
structure. The DNA (dC5) was purchased and directly used to per-
form biophysical experiments.
CD studies. We recorded the CD spectra of the annealed PNA pC5,
DNA dC5, and hybrid pC5:dC5 (1:1) samples in sodium acetate buffer
Fig. 3. CD-Spectra of pC5 (45.0 mM)/dC5 (45.0 mM)/hybrid dC5:pC5 (1:1), 22.5 mM
each.at pH 4.5 (10 °C).
Scheme 1. Synthesis of aep-C-monomer and aep-PNA-C5.
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C.R. Gade, N.K. Sharma / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Fig. 4. pH dependent CD spectra of dC5(45.0 mM) (A); and hybrid pC5:dC5, 22.5 mM
each (B).
Fig. 5. Thermal UV-melting profiles at different pH at k295nm: (A) DNA dC5; (45.0
mM) (B) DNA dC5: PNA pC5 hybrid (1:1), 22.5 mM each.
Thermal UV-studies
The temperature dependent UV studies are one of the inexpen-
sive and accurate methods to determine Tm of DNA structures. DNA
duplexes/tetraplex structures have characteristic sigmoidal melt-
ing curves at different wavelengths. The positive sigmoidal melting
profiles at 260 nm are known for duplexes/triplexes/tetraplexes
while the negative sigmoidal melting profile at wavelength 300
nm are characteristic for DNA tetraplexes (G-quadruplex and i-
motif).25 The first derivative curve of those sigmoidal curves give
accurate Tm of specific DNA structures. Pleasantly, we performed
the UV melting experiments for annealed pC5/dC5/hybrid pC5:dC5
(1:1) sample at k295nm under acidic conditions (pH 4.0–6.0), and
then plotted their respective thermal melting profiles (Fig. 5 &
SI). At pH 4.5, the melting profiles of dC5 is appeared as negative
sigmoidal curves (at k295nm) which are characteristic for the denat-
uration of i-motif structure (Fig. 5A). The similar melting profiles,
negative sigmoidal curves, are also observed with dC5 under other
pH conditions: pH 5.0, 5.5, and 6.0. The melting profiles of hybrid
dC5:pC5 at different pH conditions (pH 4.5–6.0) are depicted in
Fig. 5B which also exhibit i-motif’s characteristic negative sig-
moidal curve (at k295nm) under acidic conditions. In contrast, the
melting profiles of pC5, at pH range 4.5–6.5, exhibit positive sig-
moidal types curve (at k295nm) (Supplementary Materials) which
is non-characteristic for i-motif structure. The positive sigmoidal
curves for lone PNA pC5 are appeared under acidic pH conditions
possibly because of the self-duplex formation from protonated
and non-protonated cytosine of pC5 C+H:C) via hydrogen bonding.
However, we could not observe the sigmoidal curve (at k295nm
)
with dC5/pC5/hybrid dC5:pC5 at neutral pH 7. Importantly, the
UV-melting profiles of hybrid dC5:pC5 and control dC5 are charac-
teristically similar to that of DNA i-motif structure.
Thus aep-PNA (pC5) is hybridized with DNA (dC5) and formed
hybrid i-motif structure as like control DNA (dC5) under acidic con-
ditions. The stability of those hybrid i-motifs was compared with
control DNA (dC5) i-motif by DNA denaturation temperature (Tm)
value. We, then, extracted the Tm values of hybrid (dC5:pC5) and
dC5 under different pH conditions from their respecting UV-melt-
ing profiles (see Table 1). At pH 4.5, the Tm value of hybrid (dC5:
pC5) i-motif is remarkably higher than control dC5 with tempera-
ture difference (
D
Tm) ꢀ15.0 °C (Table 1, Entry 1). This difference
is almost 2.5 folds higher than that of reported Tm (ꢀ6–7.0 °C) of
the unmodified DNA:PNA hybrid i-motif structure.19
At other different pH conditions, the
DTms of hybrid dC5:pC5
and control dC5 are ꢀ18.0 °C, ꢀ9.0 °C, and ꢀ13.0 °C at respective
pHs 5.0, 5.5 and 6.0 (Table 1, Entry 2–4). Importantly, more than
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C.R. Gade, N.K. Sharma / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
1H NMR signal of that proton is characteristically appeared at d
Table 1
15.0–16.00 (ppm) in DNA i-motif structure.26 Herein, we recorded
the 1H NMR spectrum of annealed hybrid pC5:dC5 (1:1) and control
annealed-dC5 under acidic pH (4.5) conditions at temperature 25
°C. The 1H NMR spectrum of imino-regions of hybrid dC5:pC5 and
control dC5 are depicted in Fig. 6A & B respectively. The 1H NMR
spectrum of both control dC5 and hybrid dC5:pC5 exhibit promi-
nent signals at d 15.0–16.00 ppm which are characteristic peaks
for DNA i-motif structure. However no characteristic 1H NMR sig-
nal (d 15.0–16.00) are noticed in the NMR spectra of annealed-
pC5 (data not shown). These NMR results strongly support the
hybrid DNA:aep-PNAi-motif formation as like control DNA (dC5)
i-motif structure.
pH dependent UV-Tm/DTmof dC5/Hybrid dC5:pC5.
Entry
pH
Tm (°C) of dC5
Tm (°C) of dC5:pC5(1:1)
D
T (°C)*
1
2
3
4
4.5
5.0
5.5
6.0
46.73
36.13
31.26
22.66
62.00
54.06
40.11
35.82
15.27
17.93
8.85
13.16
*
D
Tm is difference in Tm of dC5 and dC5:pC5 (1:1).
one transitions in UV-melting profile of hybrid at pH 5.5 are
observed. It may appeared due to other complexes such as hybrid
triplex and duplex structure. These UV-melting studies strongly
support the formation of stable hybrid DNA:aep-PNAi-motif struc-
ture under acidic conditions at least by 9.0 °C. In contrast, the melt-
ing profiles of annealed-pC5 at different acidic pH range (4.5–6.0)
did not match with characteristic i-motif structure.
ESI-MS studies
Further, to confirm the above hybrid i-motif structure, we
recorded the mass spectra of control dC5 and hybrid pC5:dC5
(1:1) sample before/after annealing under acidic (pH 4.5) condi-
tions. The mass spectra are provided in Supplementary Materials,
while the selective mass spectra of annealed- hybrid (pC5:dC5) are
depicted in Fig. 7. Further, the prominent mass peaks of dC5/pC5/
hybrid (dC5:pC5), are extracted from their respective mass spec-
trum and summarized in Table 2. The mass spectra of un-annealed
dC5 or pC5 showed mass peaks of respective monomeric unit as Mp
(molecular mass of pC5) and Md (molecular mass of dC5) (Table 2,
Entry 1 & 2). The selected region of mass spectra of annealed
dC5/pC5 are provided in Supplementary Materials. Fig. 7, the mass
spectra of annealed hybrid dC5:pC5 (1:1) exhibit prominent mass
peaks at 1104.98 (m/z) which matches to mass of hemi-protonated
hybrid tetraplex dC5:pC5 (1:1) (Table 2, Entry 3). We also observed
quintuple charged sodium adducts of hybrid tetraplexes (1104.98
+ ꢀ4.4) with mass difference of ꢀ4.4 units (Supplementary
Materials). These mass data confirm the formation of hybrid
NMR studies
We further examined i-motif formation by hybrid pC5:dC5
i-motif structure under acidic conditions by 1H NMR studies and
compared with the control dC5i-motif structure. Since imino proton
of protonated cytosine (N3-H ) is involved in hydrogen bonding
with N3 of non-protonated cytosine in DNA i-motif structure, and
Table 2
ESI-Mass analysis of pC5/dC5/hybrid dC5:pC5.
Entry
pC5/hybrid pC5:dC5
(calculated Mass)
Mass (Observed)*
1
dC5
1382.05 (MdꢁH)ꢁ*
(Md) 1383.2760
pC5(Mp) 1375.6900
Annealed-pC5:dC5
(1:1); (pH 4.5)
2
3
1376.71 (Mp+H)+
1104.98
(2Mp+2Md+10H)5+
;
1036.80 (2Md+Mp+5H)4+
;
;
1382.39,(2Md+Mp+5H)3+
1384.75 (2Md+5H)2+
Fig. 6. 1H NMR of hydrogen bonded imino N-H of protonated cytosine at pH 4.5 at
25 °C (700 MHz):(A) DNA dC5(300 mM); (B) hybrid dC5:pC5 (1:1), each 200 mM.
*
Md: Molecular mass of DNA dC5; Mp: Molecular mass of pC5.
Fig. 7. ESI-MS of hybrid i-motif dC5:pC5 (1:1).
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C.R. Gade, N.K. Sharma / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
pC5:pC5i-motif structure. The mass peak of annealed hybrid
A. Supplementary data
dC5:pC5 also exhibit at m/z 1036.80 and 1382.39 which match to
the mass of protonated hybrid triplex as 2dC5:pC5 (Table 2, Entry
3). The appearance of mass peak of hybrid trimer is probably
occurred due to the separation of one pC5 strand from hybrid tet-
raplex. In literature, formation of triplex from DNA
i-motif structure are also noticed during nano-ESI mass analy-
sis.16Another mass peak is also prominent at 1384.75 which match
to mass of protonated self-duplex dC5-dimer (Table 2, Entry 3).
These mass analyses confirmed the formation of hybrid pC5:dC5
(1:1) i-motif structure under acidic conditions along with few
other species (hybrid triplex & DNA self-duplex). We recorded
the mass spectrum of annealed-dC5 with negative mode ESI
method but observed only mass peak of monomeric dC5 and its
ions along with sodium salts (Supplementary Materials). Presum-
ably the i-motif structure of lone dC5 are not stable under that
conditions.
In summary, we have successfully completed the synthesis of
aep-PNA-C5 (pC5) and accomplished the comparative biophysical
studies by CD/UV/NMR/ESI-Mass. The repeated C-rich sequence
of modified PNA, aep-PNA, forms hybrid DNA:aep-PNA (1:1) i-
motif and enhanced the stability of that i-motif structure. The
remarkable stability of that hybrid i-motif is most probably due
to additional strong ion-ion attractive interactions between tertiary
amine backbone of aep-PNA and phosphate backbone of DNA under
near acidic condition. Importantly, the formation of i-motif by only
aep-PNA (pC5) is not observed under acidic conditions. Hence this
type of hybrid i-motif analogues could be potential candidate for
the regulation of specific gene expressions, and other applications
related to i-motif.
Synthetic procedures for the aep-C monomer, the NMR and
HRMS of all the compounds also provided. Synthetic procedures
for aep-C5 pentamer, HPLC chromatogram and mass spectrum are
provided. Temperature dependent CD spectra of DNA and hybrid
i-motif, UV-Vis, spectral data for pC5, are provided.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2017.11.004.
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Acknowledgment
This paper is dedicated to Prof. K. N. Ganesh for his 64th
birthday.
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