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Melting curves of the four stereoisomers of POPNA with 9
adenine bases po(A )s, in equimolar mixtures with dT are shown
in Fig. 2.‡ All mixtures showed higher melting temperatures (T s)
, 30.5 °C
and 22.7 °C
, respectively. The Job plots showed 1 : 1
9
9
m
than the dA
for trans- -po(A
for trans- -po(A
9
–dT
9
pair, i.e., 34.5 °C for cis-
L
-po(A
9
9 9
)–dT
9
D
9
)–dT
)–dT
9
, 30.1 °C for cis-
9
D-po(A )–dT
L
9
stoichiometry for all hybrids. The temperature range of 5–95%
transition was 18 °C for the cis- -po(A )–dT hybrid. This
temperature range is narrower than that of the Nielsen-type
PNA(A )–dT mixture (26 °C). It must be noted that temperature
dependences of the absorption of po(A )s showed a small jump
L
9
9
9
9
9
around 5 °C, indicating some self-structure formation. The self-
structure at very low temperature does not affect the melting
behavior of the hybrid at higher temperatures (see ESI).
Melting curves of the four po(A
dT XT (X = T, C, A and G) were also measured and the T
collected in Table 1. The A–A and A–G mismatches in the hybrids
of cis- -po(A ) with dT XT lowered the T by ~ 20 °C, while an
A–C mismatch destabilized the duplex by ~ 15 °C. These DT
values are about the same as those observed with Nielsen-type
PNA(A ). The large DT values together with the sharp melting
curves of cis- -POPNA are advantageous for the detection of single
9
)s in equimolar mixtures with
4
4
m
’s are
L
9
4
4
m
Fig. 3 CD spectra of equimolar mixtures of cis-
L
-po(A
9
)–dT
9
, trans-
L-
m
po(A )–dT , cis- -po(A )–dT , trans- -po(A )–dT
9
9
D
9
9
D
9
9
and dA
9
–dT
9
. The
spectra were measured at 5 °C after annealing at 60 °C for 15 min. Other
conditions are the same as in Fig. 2.
9
m
L
mutations in the DNA base sequences.
profiles suggest that the helix senses of all hybrids are the same,
Successful hybridization of all stereoisomers of POPNA is
somewhat surprising, since only limited types of peptides with side-
chain nucleobases have been reported to hybridize with DNAs.
This may be interpreted in terms of flexible main chains that
contain ether linkages. Indeed, three of the four stereoisomers of
despite the opposite main-chain chiralities for the
D- and
L
-
POPNAs. Flexible polyetheramide main chains may adjust their
conformations to the most stable hybrid structure that is close to the
dA –dT right-handed duplex.
9 9
In summary, four stereoisomers of POPNA were synthesized and
hybridized with the complementary DNAs. Of the four stereoi-
po(A
). The trans-
indicating less defined hybrid structure. This may explain the weak
sequence specificity of trans- -po(A ) as found in Table 1. The CD
9
) showed similar CD profiles when hybridized with dT
9
(Fig.
3
L
-po(A )–dT hybrid showed a less intense CD peak,
9
9
somers, the cis-
hybridized with DNA, whereas the trans-
L
-isomer showed the highest stability when
-isomer showed the
L
L
9
lowest stability. The fact that all the stereoisomers of POPNA
hybridized with DNA and the hybrids showed a similar CD pattern
suggests that POPNA chains are flexible and fit their conformations
to essentially the same hybrid structure with the DNA. The cis- -
L
POPNA showed high sequence specificity and a sharper melting
curve than the Nielsen-type PNA, at least for A-rich sequences in
the POPNA.
Notes and references
‡
UV and CD experiments were conducted in aqueous buffer (100 mM
NaCl, 10 mM NaH PO and 0.1 mM EDTA, pH 7.0). Each concentration of
2
4
POPNA and DNA oligomers was 5.0 mM. The UV melting curves were
recorded with cooling the solution at 0.5 °C/0.5 min. Essentially the same
curves were obtained in the heating process. The observed absorbance has
been normalized at 80 °C. The CD spectra were recorded at 5 °C after an
annealing process at 60 °C, 15 min.
1
P. E. Nielsen, M. Egholm, R. M. Berg and O. Buchardt, Science, 1991,
54, 1497; M. Egholm, O. Buchardt, P. E. Nielsen and R. H. Berg, J. Am.
2
Chem. Soc., 1992, 114, 1895; B. Hyrup, M. Egholm, P. E. Nielsen, P.
Wittung, B. Nordén and O. Buchardt, J. Am. Chem. Soc., 1994, 116,
Fig. 2 Temperature dependence of absorption intensity at 260 nm for
equimolar mixtures of cis- -po(A )–dT trans- -po(A )–dT cis-
po(A )–dT , trans- -po(A )–dT and dA –dT . [po(A )] = [dT ] = 5.0
mM in phosphate buffer.
L
9
9
,
L
9
9
,
9
D-
7
964.
9
9
D
9
9
9
9
9
2
3
4
P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Anti-Cancer Drug
Design, 1993, 8, 53; P. E. Nielsen, Bioorg. Med. Chem., 1996, 4, 5; L.
Mologni, P. E. Nielsen and C. Gambacorti-Passerini, Biochem. Biophys.
Res. Commun., 1999, 264, 537.
T. Vilaivan and G. Lowe, J. Am. Chem. Soc., 2002, 124, 9326; R. Schultz,
M. Cantin, C. Roberts, B. Greiner, E. Uhlmann and C. Leuman, Angew.
Chem., Int. Ed., 2000, 39, 1250; C. Garcia-Echeverria, D. Hüsken, C. S.
Chiesi and K.-H. Altmann, Bioorg. Med. Chem. Lett., 1997, 7, 1123.
M. Kuwahara, M. Arimitsu and M. Sisido, J. Am. Chem. Soc., 1999, 121,
256; M. Kuwahara, M. Arimitsu and M. Sisido, Tetrahedron, 1999, 55,
10067; M. Kuwahara, M. Arimitsu, M. Shigeyasu, N. Saeki and M.
Sisido, J. Am. Chem. Soc., 2001, 123, 4653.
Table 1 T
m
s ( °C) of hybrids of various POPNAs with DNAsa
dT
T
4 4
XT
C
A
G
9
po(A )
cis-
L
34.5
30.1
22.7
30.5
19.0
15.5
20.4
16.8
12.9
28.0
20.3
15.5
15.0
13.4
20.8
16.7
cis-
D
trans-
trans-
L
5 M. Shigeyasu, M. Kuwahara, M. Sisido and T. Ishikawa, Chem. Lett.,
2001, 634.
D
a
6 K.-H. Altmann, D. Hüsken, B. Cuenoud and C. Garcia-Echeverria,
Bioorg. Med. Chem. Lett., 2000, 10, 929.
2 4 m
Buffer: 100 mM NaCl, 10 mM NaH PO and 0.1 mM EDTA, pH 7.0. T
evaluated from the midpoint of the transition at [POPNA] = [DNA] = 5.0
mM.
7
T. F. Jenny, J. Horlacher, N. Previsiani and S. A. Benner, Helv. Chim.
Acta, 1993, 76, 248.
C h e m . C o m m u n . , 2 0 0 4 , 1 2 0 8 – 1 2 0 9
1209