Towards a circular bis-peptide nucleic acid

Article abstract of DOI:10.1081/NCN-120022727

En route to a circular bis-PNA molecule, we have synthesized and characterized the DNA binding of several "clamp"-type bis-PNAs. In order to incorporate charge into a circular PNA, a new linker based on the achiral 2-aminoethylglycine has been used.

Full text of DOI:10.1081/NCN-120022727

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Towards a Circular bis -Peptide Nucleic Acid
a b
a
R. H. E. Hudson
a
& J. Tse
Department of Chemistry , The University of Western Ontario , London, Ontario, Canada
Department of Chemistry , The University of Western Ontario , Room 209, N6A 5B7,
b
London, Ontario, Canada
Published online: 31 Aug 2006.
To cite this article: R. H. E. Hudson & J. Tse (2003) Towards a Circular bis -Peptide Nucleic Acid, Nucleosides, Nucleotides
and Nucleic Acids, 22:5-8, 1023-1027, DOI: 10.1081/NCN-120022727
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1023–1027, 2003
Towards a Circular bis-Peptide Nucleic Acid
*
R. H. E. Hudson and J. Tse
Department of Chemistry, The University of Western Ontario, London,
Ontario, Canada
ABSTRACT
En route to a circular bis-PNA molecule, we have synthesized and characterized
the DNA binding of several ‘‘clamp’’-type bis-PNAs. In order to incorporate
charge into a circular PNA, a new linker based on the achiral 2-aminoethylglycine
has been used.
Various strategies have evolved in DNA chemistry to facilitate the formation of
[1]
triple helices. In this vein, Nielsen and coworkers originally developed peptide
[
2]
nucleic acid (PNA) as a triplex-forming ligand. However, it was discovered that
pyrimidine motif triple-helices tend to form by a strand invasion mechanism at
A-rich target sequences to yield PNA(Y):DNA(R):PNA(Y) structures. To favour this
interaction, ‘‘clamp’’-type bis-PNA molecules in which the PNA binding domains are
[
3]
covalently linked at one end only have been reported. Our aim is to synthesize a cir-
cular bis-PNA molecule in which the PNA binding domains are covalently linked at
both ends, Sch. 1. Such a molecule is hypothesized to be even more efficient at for-
ming triple helices than ‘‘clamp’’-type bis-PNA molecules by analogy to DNA-based
systems. Combined with internal modifications, especially cationic groups, this type
of molecule may perform better than clamp-type bis-PNA under physiological ionic
*
Correspondence: R. H. E. Hudson, Department of Chemistry, The University of Western
Ontario, Room 209, N6A 5B7 London, Ontario, Canada; Fax: þ1519 661 3022; E-mail:
rhhudson@uwo.ca.
1
023
DOI: 10.1081/NCN-120022727
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1024
Hudson and Tse
Scheme 1. Cartoon representation of the proposed biomolecular triplex formation by a
circular PNA.
conditions. Study of the circular PNA will also allow us to investigate the contemp-
orary thoughts on the mechanism of strand displacement triplex formation.
Peptide nucleic acid is a nucleic acid mimic based on a 2-aminoethylglycine
polyamide with pendant nucleobase (B ¼ Ade, Gua, Thy, Cyt, etc.) groups as shown
in Fig. 1. Homopyrimidine PNA can form triple-helices of the composi-
tion PNA(Y):DNA(R):PNA(Y) at A-rich target sequences. If the target exists as a
duplex, these triplexes will occur by strand invasion, under appropriate conditions.
The formation of these structures is favoured by covalently linking the PNA binding
domains together creating molecules known as bis-PNA. Several ‘linker’ structures
have been used to connect the PNA domains that vary in length and composition
[
3]
e.g., the 27-atom tris(8-amino-3,5-dioxaoctanoic acid), 23-atom based on lysine
[4]
[4]
and 6-aminohexanoic acid, 24-atom linker based on a trioxa-amino acid and
[
5]
an 18-atom hexapeptide linkage.
[
5]
Starting with the available x-ray crystallographic structure, we have designed
a novel linker that is based on the backbone monomer of PNA, Sch. 2. Prelimin-
ary molecular modeling results using MM þ force field suggest that the energy of
a linker having a composition of aeg-gly-aeg-gly-aeg (aeg ¼ N-2-aminoethylglycine,
gly ¼glycine) will be similar to those reported earlier. This linker offers the opportu-
nity of studying the effect of placing positive charges in the linker region, without the
introduction of chiral centers. Importantly, the linker has enough length and confor-
mational flexibility to loop out of the way of a DNA strand that is longer than the
bis-PNA molecule.
0
0
The sequence chosen for the DNA target strand is 5 -AAGGAAA-3 that is
amenable to triplex formation. For future comparison to an authentic closed circular
bis-PNA, we have synthesized the sequences listed in Table 1 which include the
components for a termolecular triplex and two structural isomers of a bimolecular
Figure 1. Generic structure of PNA.

Circular bis-Peptide Nucleic Acid
1025
Scheme 2. Linker synthesis and composition. Notes: Coordinates for bis-PNA complexed
[
8]
with DNA have been adapted from Ref. . DNA strand rendered in space filling mode, bis-
PNA rendered in wireframe (PNA sequences) and space filling (peptidic linker).
Table 1. Sequences used.
Number
PNA sequences
a
1
H
H
2
N-lys-TTCCTTT-lys-CO
N-lys-TTTCCTT-lys-CO
2
H
H
b
2
2
2
c
3
H N-TCTCTTT-(egl) -TTTCTCT-lys-NH
2
2
2
3
2
2
c
4
H
H
N-TTCCTTT-(egl)
3
-TTTCCTT-lys-NH
c
5
N-TCTCTTT-aeg-gly-aeg-gly-aeg-TTTCTCT-lys-NH
2
2
c
6
H N-TTCCTTT-aeg-gly-aeg-gly-aeg-TTTCCTT-lys-NH
2
d
7
H
H
2
N-CTT-aeg-gly-aeg-gly-aeg-TTCCTTT- aeg-gly-aeg-gly-aeg-TTTC-CO
N-CTTT-aeg-gly-aeg-gly-aeg-TTTCCTT- aeg-gly-aeg-gly-aeg-TTC-CO
2
H
H
d
8
2
2
DNA sequences
0
0
0
0
0
0
0
0
0
9
5 -CGCAAGGAAACGC-3
5 -CGCAAAGGAACGC-3
0
0
0
0
0
1
1
1
9
1
1
1
0
1
2
X
0X
1X
2X
5 -CGCAGAGAAACGC-3
5 -CGCAAAGAGACGC-3
e
5 -CGCAAGXAAACGC-3
5 -CGCAAAXGAACGC-3
e
e
e
0
0
5 -CGCAGAXAAACGC-3
5 -CGCAAAXAGACGC-3
a
Hoogsteen complementary strand.
b
Watson-Crick complementary strand.
Clamp bis-PNA.
c
d
Pre-circular bis-PNA.
e
DNA mismatch sequences (X ¼ A, C, T). PNA sequences were synthesized by standard man-
ual synthesis utilizing the BOC=CBz strategy and their identity confirmed by MALDI-MS
analysis. Definitions: aeg ¼ 2-aminoethylglycine; egl ¼ 8-amino-3,6-dioxaoctanoic acid.

1026
Hudson and Tse
triplex namely the clamp and pre-circular bis-PNAs. Hybridization studies with fully
matched DNA (Table 2) and target sequences containing a single mismatch for com-
0
0
plexes with either a 5 - or 3 -linker crossover indicate excellent sequence discrimina-
tion (data not shown). In general, a lower buffer pH favors both duplex and triplex
formation, presumably based on a higher net charge on the PNA components and
protonation of the cytosine nucleobase (for triplex formation). Two sequences and
two linkers were examined in the context of a clamp-type bis-PNA, that was pre-
[
3]
viously reported by Nielsen and coworkers termed Nielsen sequence (NS), Nielsen
linker (NL) and our sequence (HS) and linker (HL). Each PNA domain has the same
composition but slightly different sequence. Within the experimental error, there is
no sequence effect (cf. NS, NL complex 3:12 and HS, NL complex 4:10) nor a sub-
0
0
stantial effect for 3 -crossover vs. 5 -crossover. These data also show that the aeg-gly
HL) linker is comparable to the established egl (NL) linker, offering clearly better
(
stability only under acidic conditions. This suggests quaternarizing the secondary
amino group may offer a significant stabilizing effect even at neutral pH. The stability
of the complex formed by the pre-circular ligand is somewhat less stable than the
Table 2. Thermal denaturation studies.
ꢀ
Tm, C
Complex
composition
Binding mode
pH ¼ 5
58.2
59.5
50.2
47.4
61.0, 49.0 (b)
60.0, 49.5 (b)
pH ¼ 7
44.4
45.3
35.0
30.7
46.5, 31.0 (b)
46.6, 35.8 (b)
Linear
1
1
2
2
1
1
:9
:10
:9
:10
:2:9
:2:10
parallel duplex
antiparallel duplex
antiparallel duplex
parallel duplex
triplex
triplex
Clamp
0
0
0
0
0
0
0
0
3
4
5
6
3
4
5
6
:12
:10
:12
:10
:11
:9
5 -crossover NS, NL
5 -crossover HS, NL
68.0
67.0
73.5
72.0
69.0
71.5
74.5
78.0
55.5
53.5
54.5
53.5
54.6
55.6
56.0
57.5
5 -crossover NS, HL
5 -crossover HS, HL
3 -crossover NS, NL
3 -crossover HS, NL
:11
:9
3 -crossover NS, HL
3 -crossover HS, HL
Pre-circular
7
8
7
8
:9
:9
:10
:10
W=C break
64.0
55.0
60.0
58.0
47.0
36.5
46.0
36.2
Hoogsteen break
Hoogsteen break
W=C break
Note: All thermal denaturation measurements were done in the following buffer: 10 mM
4
ꢁ
þ
PO
by the first derivative method (± 0.5 C). b ¼ biphasic.
3
, 100 mM Na , 0.1 mM EDTA, pH 7. pH adjusted with HCl. Tm values were estimated
ꢀ

Circular bis-Peptide Nucleic Acid
1027
clamp-type bis-PNAs as judged by their respective Tm values. This is probably due
to the break in one of the binding domains. However, the thermal stability of these
complexes, in the best cases, are comparable to that of the termolecular triplex but
exhibit a monophasic melting transition suggesting that both PNA domains are
binding in a cooperative manner.
We are currently pursuing carbodiimide-mediated ligation of the pre-circular
[
6,7]
bis-PNA assembled on a DNA template,
course.
and will present these results in due
ACKNOWLEDGMENTS
The authors wish to thank NSERC (Canada) for support of this work (RHEH).
NSERC and the Ontario Graduate Scholarship Program are thanked for support
(JT). We are also grateful for support from the University of Western Ontario
through the Academic Development Fund.
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tion of DNA by strand displacement with a thymine-substituted polyamide.
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1
.
.
2
3
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.
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8