Engineering preferences of hairpin PNA binding to complementary DNA: Effect of N7G in aeg/aep PNA backbone

Article abstract of DOI:10.1081/NCN-100002516

AegPNA and aepPNA monomeric units bearing the N7-guanine nucleobase as a substitute for C⁺ have been demonstrated to bind to a GC base-pair of a duplex in a pH-independent manner when placed in the third strand. The aepPNA backbone exerts a pre

Full text of DOI:10.1081/NCN-100002516

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ENGINEERING PREFERENCES OF HAIRPIN PNA BINDING
TO COMPLEMENTARY DNA: EFFECT OF N7G IN aeg/
aep PNA BACKBONE
V. A. Kumar a , M. D'Costa a & K. N. Ganesh a
a Division of Organic Chemistry (Synthesis) , National Chemical Laboratory , Pune,
Maharashtra, India
Published online: 07 Feb 2007.
To cite this article: V. A. Kumar , M. D'Costa & K. N. Ganesh (2001) ENGINEERING PREFERENCES OF HAIRPIN PNA BINDING
TO COMPLEMENTARY DNA: EFFECT OF N7G IN aeg/aep PNA BACKBONE, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7,
1187-1191, DOI: 10.1081/NCN-100002516
To link to this article: http://dx.doi.org/10.1081/NCN-100002516
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1187–1191 (2001)
ENGINEERING PREFERENCES OF HAIRPIN PNA
BINDING TO COMPLEMENTARY DNA: EFFECT
OF N7G IN aeg/aep PNA BACKBONE
V. A. Kumar,∗ M. D’Costa, and K. N. Ganesh
Division of Organic Chemistry (Synthesis),
National Chemical Laboratory, Pune, Maharashtra, India
ABSTRACT
AegPNA and aepPNA monomeric units bearing the N7-guanine nucleobase as a
substitute for C+ have been demonstrated to bind to a GC base-pair of a duplex
in a pH-independent manner when placed in the third strand. The aepPNA
backbone exerts a preference for binding in the antiparallel Hoogsteen mode
over the parallel Hoogsteen mode.
Aminoethylglycyl (aeg) Peptide Nucleic Acids (PNAs) are a class of DNA
mimics that are neutral and achiral and bind to complementary DNA/RNA
sequences with high affinity and sequence specificity (1). Homopyrimidine PNA2:
DNA triplex formation is pH-dependent due to the requirement of N3-protonation
of cytosine for the Hoogsteen (HG) mode of recognition. PNA, with no configura-
tional and conformational constraints, accommodates both Watson-Crick (WC) &
HG binding modes in either antiparallel or parallel orientations (2). Aminoethylpro-
lyl (aep)PNA recently reported by us (3) is a chiral positively charged PNA analogue
and oligothymine aepPNAs show excellent binding properties with complemen-
tary DNA sequences. Chiral aepPNA is homomorphous with DNA and may induce
orientational preferences in binding to DNA sequences. Mixed polypyrimidine
sequences using this chiral backbone are therefore expected to display orientation-
selective binding to DNA.
∗Corresponding author.
1187
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C
Copyright 2001 by Marcel Dekker, Inc.
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KUMAR, D’COSTA, AND GANESH
Figure 1. Hydrogen bonding schemes.
In this communication, we describe the synthesis of triplex forming mixed
polypyrimidine sequences using the aepPNA units at predetermined positions and
study of their effect on directing the orientation selectivity. N7G is known to
mimic C+ in its hydrogen bonding pattern (4) in the HG mode (Fig. 1) and hence,
aeg/aepPNA-N7G monomeric units are introduced to circumvent the requirement
of acidic pH for stable triplex formation.
The aegPNA-N7G ester 2 was synthesized by alkylation of N2-isobutyryl-
guanine GiBu with the ethyl N-Boc-aminoethyl-N-chloroacetyl glycinate in
presence of K2CO3. The protected monomer units, (2S, 4S) aepPNA-N7GiBu 6b
and (2S, 4S) aepPNA-CCBz 6a were synthesized similarly, by alkylation of GiBu
and CCBz with the common intermediate 5. The compound 5 was prepared by
mesylation of the known compound 4 (3) (Scheme 1). The products were purified
by column chromatography and characterized (5) by 1H, 13C and mass spectrome-
try. The ester hydrolysis in all cases was effected with 1N NaOH in aq. MeOH to get
the monomer synthons 3, 7a & 7b. The PNA sequences (PNA 9–11) incorporating
these new monomers are listed in Scheme 2. The other aegPNA monomers, N-Boc-
ꢀ-aminohexanoic acid and protected lysine units were synthesized according to the
literature procedures (6). The PNA oligomers synthesized by standard protocols
(6) were purified by FPLC and characterized by MALDI-TOF mass spectrometry
(5).
The designed control bisPNA sequence (PNA 8) with aegPNA-C units in
both arms binds to both DNA 12 and DNA 13 sequences without discrimination
as studied by UV melting experiments (ꢁTm = 0◦C, Table 1). A higher Tm was
observed at acidic pH in both the cases (ꢁTm ∼7◦C) indicating equally efficient
HG and WC modes of recognition in either orientation (Scheme 2) of the target
strand. Replacement of aegPNA-C in one of the arms by aegPNA-N7G (PNA 9)
allows pH-independent triplex formation with the target DNA strands 12 & 13,
again, without any orientation selectivity (ꢁTm = 2◦C). This may be possible, as
N7G is capable of recognizing a GC base pair in HG mode with either parallel or
antiparallel orientation of the third strand.
In contrast to the aegPNA backbone, introduction of aepPNA-C units in this
sequence (PNA 10) led to a stronger binding with DNA 13 (Tm = 48◦C) as com-
pared to DNA 12 (Tm = 37.5◦C) at neutral pH exhibiting a significant bias in

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HAIRPIN PNA BINDING TO COMPLEMENTARY DNA
1189
Scheme 1a.
Scheme 1b.
the directionality of binding (ꢁTm ∼ 10◦C). This selectivity was less pronounced
(ꢁTm ∼ 5◦C) at acidic pH, which can be explained on the basis of the differential
protonation of cytosine N3 in aegPNA-C and aepPNA-C units in the HG strand.
The aepPNA backbone being protonated even at physiological pH, N3-protonation
of cytosine in aepPNA-C is probably less pronounced at pH 5.8 and is reflected
Scheme 2. PNA Oligomer Sequences.

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KUMAR, D’COSTA, AND GANESH
Table 1.
DNA
PNA
DNA 12
DNA 13
Tm (◦C)
Tm (◦C)
aegPNA-C
aegPNA-N7G
aepPNA-C
PNA 8
PNA 9
PNA 10
PNA 11
45 (52.7)
45 (47)
37.5 (47)
42 (43)
45 (52)
47 (45)
48 (52)
51 (49)
aepPNA-N7G
Figures in parentheses denote the Tm at 5.8 pH. Tm studies were per-
formed in 0.01 M sodium phosphate buffer, pH 7.4 with a heating rate
of 0.5◦C per min. The Tm values were obtained from the peaks in the
first derivative plots of % hyperchromicity at 260 nm Vs temperature
and are accurate to 1◦C. Each experiment was repeated at least twice.
in the corresponding Tm values. This result indicates that the presence of chiral
aepPNA units stabilizes the PNA:DNA complex when the HG strand containing
these units is in the antiparallel orientation with respect to the DNA central strand.
The aepPNA-N7G units in this sequence (PNA 11) confirm the selectivity for the
antiparallel HG mode of binding of the third strand (ꢁTm ∼9◦C). The aegPNA-C
units of this sequence (PNA 11) are consequently in the WC mode of binding and
hence, no change was observed in Tm at lower pH (ꢁTm 1◦C).
In summary, the introduction of aepPNA units in the backbone of triplex
forming PNA oligomers strongly influences the recognition of DNA in an orien-
tation selective manner. The triplex formation is pH-dependent when aepPNA-C
units are used, while aepPNA-N7G units permit triplex formation at physiolo-
gical pH.
ACKNOWLEDGMENTS
MD thanks CSIR, New Delhi for a fellowship. VAK and KNG acknowledge
Department of Biotechnology, New Delhi, for a research grant.
REFERENCES
1. Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624–630.
2. Egholm, M.; Christensen, L.; Dueholm, L. D.; Buchardt, O.; Coull, J.; Nielsen, P. E.
Nucleic Acids Res. 1995, 23, 217–222.
3. D’Costa, M.; Kumar, V. A.; Ganesh, K. N. Org. Lett. 1999, 1, 1513–1516.
4. (a) Brunar, H.; Dervan, P. B. Nucleic Acids Res. 1996, 24, 1987–1991. (b) Hunziker,
J.; Priestley, E. S.; Brunar, H.; Dervan, P. B. J. Amer. Chem. Soc. 1995, 117, 2661–
2662.
5. Characteristic signals: 2: 1H NMR (CDCl3) δ: 10.1 (s, 1H, G-NH), 7.9 (s, 1H,
G-H8). 13C NMR δ: 167.2 (G-CH2-CO), 162.9 (G-C2), 156.4 (G-C4), 148.0 (G-C6),

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HAIRPIN PNA BINDING TO COMPLEMENTARY DNA
1191
1
145.2 (G-C8), 112.2 (G-C5). 6b: H NMR (CDCl3) δ: 12.1 (br s, 1H, G-NH),
8.4 (s, 1H, G-H8), 5.6 (m, 1H, H4). 13C NMR δ: 156.4 (G-C2), 156.0 (G-C4), 147.5
(G-C6), 142.3 (G-C8), 111.5 (G-C5), 59.5 (C4). MALDI-TOF: PNA 8: [M]obs
4499.75, [M]calc = 4495.64; PNA 9: [M]obs = 4580.88, [M]calc = 4575.69.
=
6. Nielsen, P. E.; Egholm, M. Eds. in Peptide Nucleic Acids: Protocols and Applications
1999, Horizon Scientific Press, Norfolk, UK.

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