PNA containing isocytidine nucleobase: Synthesis and recognition of double helical RNA

Article abstract of DOI:10.1016/j.bmcl.2011.01.130

Peptide nucleic acid (PNA1) containing a 5-methylisocytidine (iC) nucleobase has been synthesized. Triple helix formation between PNA1 and RNA hairpins having variable base pairs interacting with iC was studied using isothermal titration calorimetry. The

Full text of DOI:10.1016/j.bmcl.2011.01.130

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2121–2124
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
PNA containing isocytidine nucleobase: Synthesis and recognition of
double helical RNA
⇑
Thomas Zengeya, Ming Li, Eriks Rozners
Department of Chemistry, Binghamton University, The State University of New York, Binghamton, NY 13902, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Peptide nucleic acid (PNA1) containing a 5-methylisocytidine (iC) nucleobase has been synthesized. Tri-
ple helix formation between PNA1 and RNA hairpins having variable base pairs interacting with iC was
studied using isothermal titration calorimetry. The iC nucleobase recognized the proposed target, C-G
inversion in polypurine tract of RNA, with slightly higher affinity than the natural nucleobases, though
the sequence selectivity of recognition was low. Compared to non-modified PNA, PNA1 had lower affinity
for its RNA target.
Received 27 December 2010
Revised 25 January 2011
Accepted 28 January 2011
Available online 1 February 2011
Keywords:
RNA recognition
Ó 2011 Elsevier Ltd. All rights reserved.
Peptide nucleic acid
Triple helix
Modified nucleobases
Isothermal titration calorimetry
Compared to DNA, molecular recognition of double helical RNA
has received little attention. Until relatively recently, RNA was
viewed mostly as a passive messenger in the transfer of genetic
information from DNA to proteins. However, recent discoveries
of the central role that various non-coding RNAs play in gene
expression have revitalized interest in molecular recognition of
double helical RNA. Although RNA is a well-established target of
current antibiotics, designing of new compounds that selectively
recognize RNA has been a challenging task.1
Hydrogen bond mediated base pairing is the key feature of heli-
cal nucleic acids and would be inherently the most effective way of
sequence selective recognition of RNA. Recognition of double heli-
cal DNA via triple helix forming oligonucleotides2 and polyamides3
that form extensive hydrogen bond contacts in the major and min-
or grooves of DNA, respectively, is well established. Compared to
DNA, the wide and shallow minor groove of RNA double helix is
less suited for molecular recognition. Surprisingly and in contrast
to DNA, sequence selective recognition of double helical RNA using
major groove triple helix is little studied.4 The major groove of RNA
is deep and narrow, which may hinder the formation of triple helix.
While DNA does not form triple helix with RNA, modestly stable
RNA triple helices are formed via parallel binding of a pyrimidine
rich RNA third strand to the purine rich strand of a double helix.4
The sequence selectivity derives from recognition of adenosine-
uridine base pairs by uridine (U⁄A–U triplet) and guanosine-cyti-
dine base pairs by protonated cytidine (C⁄G–C triplet) via the
Hoogsteen hydrogen bonding scheme (Fig. 1). Recently, we discov-
ered that peptide nucleic acid (PNA),5 a neutral amide-linked ana-
logue of DNA formed highly stable and sequence selective triple
helices with double stranded RNA.6
A major limitation of triple helical recognition is the require-
ment for a polypurine tract, because only A and G can be recog-
nized by the Hoogsteen hydrogen bonds (Fig. 1). While double
stranded RNA typically does not have long polypurine tracts, it is
common to find stretches of eight and more purine bases inter-
rupted by a couple of pyrimidines in micro RNAs (for structures,
see www.mirbase.org) and other non-coding RNAs. Thus, if the se-
quence range of triple helical recognition could be expanded to
recognize isolated pyrimidine inversions, the approach could be
rendered useful for fundamental biochemical studies and practical
applications.
While extensive work has been done on modified nucleobases
for recognition of C–G and T–A inversion in polypurine tracts of
DNA,2 similar recognition of pyrimidines in RNA is unprecedented.
Herein, we tested a hypothesis that 5-methylisocytosine (iC)
would form a triplet with a C–G base pair in RNA (Fig. 1, iC⁄C–G).
iC was designed by Benner and co-workers7 to engineer unnatural
base pairing schemes as alternatives to Watson–Crick pairing and,
before our study, had not been tried in triple helix context. We
envisioned that iC would recognize cytosine by forming a O4–
N4H hydrogen bond and, potentially, also engaging in an attractive
N3–C5H interaction (Fig. 1). This is similar to the N3–N4H and O2–
C5H hydrogen bonding scheme used for recognition of C–G in DNA
by 5-methyl-2-pyrimidinone8 and derivatives.9 Other designs of C–
G recognition in DNA have included interactions that reach across
⇑
Corresponding author. Tel.: +1 607 777 2441; fax: +1 607 777 4478.
E-mail address: erozners@binghamton.edu (E. Rozners).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.130

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T. Zengeya et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2121–2124
R
O
R
N
R
N
N
4
3
U*A-U
H
C*G-C
iC*C-G
N
H
N
H
O
O
H2N
N
N
H
O
H
H
N H
H
H N
H
O
O
N H
O
N
N
N
N H
N
N
H N
H N
N
N
N
N
N
N
N
R
R
R
N
O
N
N
R
O
N
R
R
O
O
HN H
H NH
O
NH2
O
N
NH2
N
O
N
NH2
N
PNA1
NH
N
O
NH
N
H3N
NH2
N
O
O
N
O
O
O
N
O
O
O
O
O
O
O
N
O
O
O
O
N
N
N
N
N
N
H
N
H
N
N
H
N
H
N
H
NH2
H
NH2
Figure 1. Structures of Hoogsteen U⁄A–U and C⁄G–C and the proposed iC⁄C–G base triplets and PNA (PNA1).
the groove to form hydrogen bonds with G, cationic base substitu-
ents and sugar modifications to stabilize the non-Hoogsteen
triplets.10
We envisioned that late stage alkylation of 5-methylisocytidine
with chloroamide 7 (Scheme 2), following a strategy similar to that
used by Meltzer and co-workers,15 might provide an alternative
route to the desired iC monomer. Chloroamide 7 was obtained by
coupling of amine 4 with chloroacetic acid in the presence of dicy-
clohexylcarbodiimide (DCC).
Although alkylation of 1 with chloroamide 7 gave the expected
product in a relatively low yield, we were able to produce enough
material to proceed with the PNA synthesis and binding studies. As
previously reported,13 cleavage of benzyl ester proceeded unevent-
fully and gave the required Fmoc-PNA-iC(Boc)-OH 9, which was
used in a standard PNA synthesis protocol on an Expedite 8909
DNA synthesizer to prepare PNA1 containing the 5-methylisocyti-
dine nucleobase (see Figs. 1 and 2).
Inspired by our own recent results6 on triple helical recognition
of RNA using PNA, we set out to test if iC installed on a PNA back-
bone (Fig. 1, PNA1) could recognize the C–G inversion in polypu-
rine tract of RNA. While Nielsen and co-workers11 have designed
PNA containing pyridazinone nucleobase for recognition of T–A
and U–A inversions in DNA, to the best of our knowledge, recogni-
tion of C–G inversion by modified PNA had no precedent at the out-
set of our studies.12 We found that the iC nucleobase recognized
the C–G inversion in polypurine tract of RNA with slightly higher
affinity than the natural nucleobases, though the sequence selec-
tivity of recognition was relatively low.
For synthesis of iC PNA monomer we first tried to adopt the
standard PNA chemistry (Scheme 1).13 5-Methylisocytosine14 was
protected with Boc group and alkylated to produce ester 2. The cor-
rect N1 position of alkylation was confirmed by observation of NOE
between H6 and CH2 of the acetate linkage (Scheme 1). The ethyl
ester was cleaved under basic conditions to produce carboxylic
acid 3.
To our surprise, coupling of carboxylic acid 3 with amine 4, fol-
lowing standard procedures,13 resulted in a transfer of the Boc
group on the central amine and gave compound 5 instead of the
expected PNA monomer. A potential explanation for this unex-
pected result could be that the activation of carboxylic acid 3 led
to an intramolecular cyclization and formation of imide 6, which
then could transfer the Boc group to amine 4. Because our goal
was to prepare the iC PNA monomer, we redirected our efforts to
alternative syntheses and the intermediacy of imide 6 remained
a plausible but unconfirmed hypothesis.
The particular sequence of PNA1 allowed convenient testing of
the effect of iC via direct comparison with our previous work that
tested the binding of PNA3–PNA6 (containing a variable nucleo-
base highlighted in bold in Fig. 2) to HRP1–HRP4 (containing a var-
iable base pair).6 The binding affinity and sequence selectivity of
PNA1 was studied using isothermal titration calorimetry (ITC), as
previously described by us.6 ITC directly measures enthalpy of
binding and is one of the best methods to study RNA-ligand inter-
actions.16 ITC has been used to characterize the thermodynamics of
PNA binding to DNA and formation of modified DNA triple heli-
ces.17 Our recent study confirmed that ITC was an excellent meth-
od to study binding of PNA to double helical RNA.6 In our
experimental system, iC in PNA1 was expected to give a matched
triplet with the C–G base pair of HRP3 (see Fig. 1). ITC experiments
(Fig. 3) showed that the affinity of PNA1 for HRP3 was slightly
higher than affinity for HRP1, HRP2 and HRP4, which were ex-
pected to form mismatched triplets with iC (Table 1).
O
O
N
Boc
N
O
4, DCC, HOBt
BrCH2COOEt
71%
N
N
FmocHN
OBn
83%
N
H
NHBoc
H
NHBoc
O
5
O
N
H
NOE 5%
H
1
N
OR
O
O
2 R = Et
3 R = H
H
N
NaOH
>99%
N
O
OtBu
FmocHN
OBn
6
4
Scheme 1. Attempted synthesis of the iC PNA monomer using standard strategy.

T. Zengeya et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2121–2124
2123
O
N
O
N
O
H
N
N
O
FmocHN
OBn
4
N
H
NHBoc
NHBoc
OR
Cl
O
O
1
O
1) Bu4NI
ClCH2COOH
DCC
2) 1, NaH
N
N
FmocHN
FmocHN
OBn
84%
19%
7
8 R = Bn
9 R = H
H2 Pd/C
96%
Scheme 2. Synthesis of the iC PNA monomer using late stage alkylation strategy.
or less. However, we have observed similar phenomenon with mis-
matched complexes in our previous study.6
The binding of PNA1 to HRP3 was confirmed using UV spectros-
copy. The melting curve (Fig. 4) showed two well-resolved transi-
tions. The lower temperature (around 30 °C) transition was
characteristic for melting of triplex to hairpin and single strand
PNA, while the higher temperature (around 90 °C) transition was
due to hairpin melting.6 Analogous experiments using the mis-
matched complexes (HRP1 and HRP2) did not produce clear melting
curves. The complex formation was further confirmed by circular
dichroism spectroscopy (Fig. S1). Overall, the results of UV melting
and circular dichroism experiments were consistent with our previ-
ous observations6 and indicated triple helix formation.
Figure 2. Sequences of RNA hairpins and PNA ligands. Numbering of HRP1–HRP4
and PNA3–PNA6 is retained from Ref. 6.
Comparison of PNA1 with PNAs featuring natural nucleobases
at the variable position (PNA3–PNA6) is shown in Table 2. Affinity
of PNA1 for the matched HRP3 was decreased approximately 50
times compared to PNA3 and PNA4 binding to their complemen-
tary hairpins (HRP1 and HRP2) that featured the matched Hoogs-
teen triplets (highlighted in bold in Table 2). Meanwhile, the
affinities of mismatched combinations involving PNA1 were simi-
lar to that of other mismatches previously studied.6 Thus, the
Analysis of the thermodynamic data in Table 1 revealed that the
binding was driven by favorable entropy that more than compen-
sated for the less favorable enthalpy contribution. The binding or-
der (stoichiometry) indicated that PNA1 formed the expected 1:1
complex with HRP3, a result that is consistent with the proposed
triple helical recognition. We do not have an explanation for why
the binding order of the other mismatched combinations was 0.5
-172.8
-172.9
-173
-173.1
-173.2
-173.3
-173.4
-173.5
0
50
100
150
200
250
Time (min)
Figure 3. ITC titration curve of PNA1 binding to HRP3.
Table 1
Thermodynamic data for binding of PNA1 to RNA hairpinsa
Entry
RNA
Ka
À
D
H (kcal/mol)
À
D
S (cal/mol K)
À
D
G (kcal/mol)
order
1
2
3
4
HRP1
HRP2
HRP3
HRP4
0.51
0.82
1.01
0.77
42.3
27.2
15.5
28.7
116
64
25
7.8
8.1
8.2
8.0
0.5
0.4
1.1
0.3
69
a
Association constants Ka  106 MÀ1 in 100 mM sodium acetate, 1.0 mM EDTA, pH 5.5.

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T. Zengeya et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2121–2124
the challenge of Hoogsteen recognition of pyrimidine nucleobases
1.05
1
having only one hydrogen bonding site.2,8–11 To the best of our
knowledge, our study is the first attempt at overcoming the
requirement for a polypurine tract in RNA triple helices using mod-
ified PNA. Work is in progress in our lab to test other modified
nucleobases that are expected to lead to further improvements in
recognition of C–G inversions in RNA double helices.
PNA1/HRP3
0.95
0.9
Acknowledgment
We thank Binghamton University and NIH (R01 GM071461) for
support of this research.
0.85
0
20
40
60
Temperature
80
100
Supplementary data
Figure 4. UV thermal melting curve of HRP3 (5.25 lM) and PNA1 (18 lM) in
100 mM sodium acetate, 1.0 mM EDTA, pH 5.5.
Supplementary data (experimental procedures, copies of CD
and NMR data, details PNA synthesis and ITC experiments and
data) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmcl.2011.01.130.
Table 2
Sequence specificity of PNA binding to RNA hairpinsa
HRP1a
HRP2a
HRP3a
HRP4a
References and notes
PNA (variable base)
(G–C)
(A–U)
(C–G)
(U–A)
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Chem. Rev. 1997, 97, 1489.
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PNA3 (C)b
PNA4 (T)b
PNA5 (G)b
PNA6 (A)b
0.5
84.0
2.7
1.5
6.0
0.8
0.4
47.0
0.4
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0.02
0.09
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a
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nucleobase to recognize C–G inversions in the polypurine tract of
an RNA double helix, the binding affinity and sequence selectivity
remain modest. In fact, iC is also the best base for recognition of U–
A inversions (Table 2, last column) and may serve as universal base
to be used against pyrimidine inversions instead of the natural
nucleobases. Decreased binding affinity and sequence selectivity,
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G and T–A inversions in polypurine tracts of DNA, and illustrates
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MacKerell, A. D., Jr.; Seidman, M. M. Biochemistry 2010, 49, 7867; (b)
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