Binding affinities of oligonucleotides and PNAs containing phenoxazine and G-clamp cytosine analogues are unusually sequence-dependent

Article abstract of DOI:10.1021/ol701826x

(Chemical Equation Presented) Melting temperatures of DNA duplexes containing the phenoxazine (P) and G-clamp (X) cytosine analogues exhibited a strong and unusual dependence on the nucleoside flanking the modified nucleobase, and the same trend was obser

Full text of DOI:10.1021/ol701826x

ORGANIC
LETTERS
2007
Vol. 9, No. 22
4503-4506
Binding Affinities of Oligonucleotides
and PNAs Containing Phenoxazine and
G-Clamp Cytosine Analogues Are
Unusually Sequence-Dependent
Jose´-Antonio Ortega,† Jose´ Ramo´n Blas,‡ Modesto Orozco,‡ Anna Grandas,†
Enrique Pedroso,*,† and Jordi Robles*,†
Departament de Qu´ımica Orga`nica, UniVersitat de Barcelona, Mart´ı i Franque`s 1-11,
E-08028 Barcelona, Spain, and Departament de Bioqu´ımica i Biologia Molecular,
UniVersitat de Barcelona and Molecular Modeling Unit, IRBB-Parc Cient´ıfic de
Barcelona, Josep Samitier 1-5, E-08028 Barcelona, Spain
epedroso@ub.edu
Received August 8, 2007
ABSTRACT
Melting temperatures of DNA duplexes containing the phenoxazine (P) and G-clamp (X) cytosine analogues exhibited a strong and unusual
dependence on the nucleoside flanking the modified nucleobase, and the same trend was observed in PNA DNA duplexes incorporating X
−
in the PNA chain. Molecular dynamics simulations of the DNA duplexes show that generalized stacking (including secondary interactions of
the ammonium group of X) and hydrogen bonding are good descriptors of the different duplex stabilities.
Numerous biotechnological, biomedical, and material sci-
ences applications of oligonucleotides (ODNs) and analogues
would significantly benefit from enhanced binding affinities
in the formation of secondary structures with their comple-
mentary sequences.1 Nucleobase analogues that maximize
the stacking interactions and increase the number of hydrogen
bonds between base pairs provide a means for enhancing
duplex stability. The tricyclic cytosine analogues phenoxazine
(P) and 9-(2-aminoethoxy)phenoxazine (G-clamp or amino-
G-clamp, X) have emerged as powerful tools to reach this
goal (Figure 1).2 The G-clamp nucleoside phosphoramidite
has been recently made commercially available to allow
introduction in synthetic oligonucleotides.3 The extended π
system of the phenoxazine ring is thought to be responsible
for the increase in the binding affinity, and the G-clamp, in
addition to its enhanced stacking, is believed to simulta-
neously recognize both the Watson-Crick and Hoogsteen
faces of guanine through the formation of four hydrogen
bonds. ODNs containing the phenoxazine and G-clamp
cytosine analogues have shown interesting antisense activities
† Departament de Qu´ımica Orga`nica, Universitat de Barcelona.
‡ Departament de Bioqu´ımica i Biologia Molecular, Universitat de
Barcelona and Molecular Modeling Unit.
(1) (a) Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301,
1884-1886. (b) Pirrung, M. C. Angew. Chem., Int. Ed. 2002, 41, 1276-
1289. (c) Crooke, S. T. Annu. ReV. Med. 2004, 55, 61-95. (d) Manoharan,
M. Curr. Opin. Chem. Biol. 2004, 8, 570-579. (e) Seeman, N. C. Nature
2003, 421, 427-431. (f) Feldkamp, U.; Niemeyer, C. M. Angew. Chem.,
Int. Ed. 2006, 49, 1856-1876.
(2) (a) Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995,
117, 3873-3874. (b) Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998,
120, 8531-8532. (c) Kurchavov, N. A.; Stetsenko, D. A.; Skaptsova, N.
V.; Potapov, V. K.; Sverdlov, E. D. Nucleosides Nucleotides 1997, 16,
1837-1846.
(3) The Glen Report 2006, 18.1, 1-2.
10.1021/ol701826x CCC: $37.00
© 2007 American Chemical Society
Published on Web 10/04/2007

sequence-dependence, although both the CD spectra and
molecular dynamics simulations indicate the formation of a
B-DNA duplex upon hybridization with their complementary
strands.
The synthesis of the phenoxazine- and G-clamp-protected
nucleoside phosphoramidites was performed essentially as
previously described.2 However, in our hands the syntheses
were not at all straightforward, and careful adjustments in
the experimental conditions had to be made to obtain the
desired products and optimize the yields (Supporting Infor-
mation). Phosphoramidites were subsequently used in solid-
phase ODN assembly. It should be mentioned that ODNs
containing the phenoxazine and amino-G-clamp analogues
are light sensitive and start decomposing when exposed to
daylight at room temperature in less than 24 h, but they are
stable enough for large periods when stored lyophilized and
in the freezer. A series of natural and modified 10-mer ODNs
Figure 1. Hydrogen bonding in the base pairs of guanine with the
tricyclic cytosine analogues: (a) phenoxazine and (b) G-clamp.
and 3′-exonuclease resistance.4 Moreover, G-clamp 2′-O-
methyl ODNs have also been shown to inhibit the HIV-1
Tat-TAR interaction more effectively than other ODNs.5 It
is also worth mentioning that tricyclic cytosine analogues
exhibit fluorescent properties and can be exploited as reporter
groups. The high fluorescent quantum yields of the phe-
nothiazine ring are in particular largely insensitive to
interactions with neighboring nucleobases in ODNs or
duplexes.6
Although the binding studies of ODNs incorporating the
phenoxazine analogue showed only moderate increases in
Tm of 3-7 °C, a single incorporation of a G-clamp was
found to increase the Tm of a polypyrimidine DNA decamer
by 18 °C.2 Duplexes containing the 2′-O-methyl-G-clamp
analogue showed Tm increases ranging from 4 to 17 °C,
and the magnitude of the increase was attributed to the
position of the G-clamp within the sequence.5 Phenothiazine-
containing ODNs and peptide nucleic acids (PNAs) also form
more stable duplexes than the unmodified ones, and DNA
duplexes showed an unperturbed B-DNA conformation.6b
Surprisingly, ODNs incorporating another phenoxazine-
derived cytosine analogue, the guanidino-G-clamp, showed
lower melting temperatures (∆Tm ) 6 °C) than the amino-
G-clamp ODNs, although five hydrogen bonds between the
guanidino-G-clamp nucleobase and guanine had been ob-
served by X-ray crystallography.7
5′
having the general sequence GCANCNTACG3′ (where C
denotes either cytosine (C), phenoxazine (P), or G-clamp
(X), and N any nucleoside) were obtained, hybridized to their
complementary strands, and used in UV melting experiments
(Table 1). In the vaste majority of reported sequences, P or
X were flanked by two cytosines. Our sequences were
selected so that either cytosine was combined with a purine
at the 5′ or 3′ side or the two flanking nucleobases N were
identical.
Table 1. Hybridization Properties of Oligonucleotidesa
Tmb
(°C)
∆Tmc
(°C)
∆∆Tmd
(°C)
nucleobase
cytosine (C)
sequence
GCAACATACG
GCAACCTACG
GCACCATACG
GCACCCTACG
GCACCGTACG
GCAGCCTACG
GCAGCGTACG
GCATCTTACG
GCAAPATACG
GCAAPCTACG
GCACPATACG
GCACPCTACG
GCACPGTACG
GCAGPCTACG
GCAGPGTACG
GCATPTTACG
GCAAXATACG
GCAAXCTACG
GCACXATACG
GCACXCTACG
GCACXGTACG
GCAGXCTACG
GCAGXGTACG
GCATXTTACG
46.0
47.5
46.5
51.0
52.5
52.5
54.5
43.5
46.0
47.0
55.0
57.0
58.0
53.5
55.0
47.0
50.0
56.5
62.5
69.0
68.0
66.5
66.0
58.0
phenoxazine (P)
0
-0.5
8.5
6.0
5.5
1.0
0.5
3.5
4.0
The close similarities between the studied sequences
incorporating these cytosine analogs2 prompted us to inves-
tigate whether the thermal stability of the duplexes varied
depending on the sequence context, in particular the residues
flanking the modified nucleoside. In contrast with the original
claim,2b we now report that the binding affinities of ODNs
and PNAs modified with these base analogs exhibit a strong
G-clamp (X)
4.0
9.5
7.5
12.0
10.0
13.0
11.0
11.0
9.0
(4) (a) Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.;
Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3513-
3518. (b) Flanagan, W. M.; Wagner, R. W.; Grant, D.; Lin, K.-Y.; Matteucci,
M. Nat. Biotechnol. 1999, 17, 48-52. (c) Maier, M. A.; Leeds, J. M.;
Ballow, G.; Springer, R. H.; Bharadwaj, R.; Manoharan, M. Biochemistry
2002, 41, 1323-1327.
16.0
18.0
15.5
14.0
11.5
14.5
(5) Holmes, S. C.; Arzumanov, A. A.; Gait, M. J. Nucleic Acids Res.
2003, 31, 2759-2768.
(6) (a) Eldrup, A. B.; Nielsen, B. B.; Haaima, G.; Rasmussen, H.;
Kastrup, J. S.; Christensen, C.; Nielsen, P. E. Eur. J. Org. Chem. 2001,
1781-1790. (b) Engman, K. C.; Sandin, P.; Osborne, S.; Brown, T.; Billeter,
M.; Lincoln, P.; Norde´n, B.; Albinsson, B.; Wilhelmsson, L. M. Nucleic
Acids Res. 2004, 32, 5087-5095.
(7) (a) Wilds, C. J.; Maier, M. A.; Tereshko, V.; Manoharan, M.; Egli,
M. Angew. Chem., Int. Ed. 2002, 41, 115-117. (b) Wilds, C. J.; Maier, M.
A.; Manoharan, M.; Egli. M. HelV. Chim. Acta 2003, 86, 966-978.
a Experiments were performed with ODNs at 5 µM concentration in 5
mM NaH2PO4/Na2HPO4, 1 mM MgCl2, and 140 mM KCl buffer, at pH
7.2. b The error in Tm values was ( 0.5 °C. c Differences between Tm
values of phenoxazine- or G-clamp-modified and unmodified ODNs.
d Differences between Tm values of G-clamp- and phenoxazine-containing
ODNs.
4504
Org. Lett., Vol. 9, No. 22, 2007

Phenoxazine-containing ODNs showed striking sequence-
dependent differences in their ability to stabilize the duplexes
with respect to the unmodified ODNs. In the sequences where
the 5′ flanking nucleobase was a purine (APA, APC, GPG,
and GPC), the phenoxazine nucleobase produced little or no
stabilizing effect (∆Tm ( 1 °C). The most stabilized
duplexes (∆Tm ) 5.5-8.5 °C) were those in which
phenoxazine was flanked by a 5′ cytosine (CPA, CPC, and
CPG), while the TPT duplex was moderately stabilized (∆Tm
) 3.5 °C). These results are in good agreement with those
reported for DNA duplexes incorporating the phenothiazine
analog.6b
As a general trend, G-clamp-containing duplexes were
much more stable than those containing either cytosine or
phenoxazine, thus confirming the outstanding stabilizing
properties of this cytosine analog. However, a noticeable
sequence-dependent effect was again observed that shares
some common trends with phenoxazine-containing duplexes.
The most stabilized duplexes (∆Tm ) 15.5-18 °C) were
those in which the G-clamp was flanked by a 5′ cytosine
(CXA, CXC, and CXG). On the contrary, the lowest
stabilizing effects were observed when the G-clamp was
flanked by a 5′ purine (AXA, AXC, GXG, and GXC), 5′
guanines being significantly more stabilizing (∆Tm ) 11.5-
14 °C) than 5′ adenines (∆Tm ) 4-9 °C). Another clear
effect is that, for a given 5′ residue, the presence of cytosine
at the 3′ side of the G-clamp was more stabilizing than that
of a purine, especially when the 3′ purine was adenine (AXC
vs AXA and CXC vs CXA) and much less when it was
guanine (GXC vs GXG and CXC vs CXG). This behavior
can be attributed to the aminoethoxy arm of the G-clamp,
since it was not observed in phenoxazine-containing du-
plexes, where the differences between these pairs of triplets
were less significant. The consequence for the less stable 5′
purine G-clamp sequences was that the presence of a 3′
cytosine partially counterbalanced the negative impact of the
5′ purine on the binding affinity (AXC vs AXA and GXC
vs GXG).
stabilizing properties of the nucleobase modifications lead
to interesting inversions in the order of stability of certain
pairs of triplets. For example, the GCG triplet gives more
stable duplexes than GCC, but their stabilities are almost
identical when the central C is replaced by X. An inversion
in stability (Tm) is observed for other pairs of triplets: CCG
> CCC but CXC > CXG; ACC > CCA but CXA > AXC;
etc.
∆∆Tm values (Table 1) were calculated to evaluate the
contribution of the appending aminoethoxy arm with respect
to that provided by the phenoxazine ring. These values
clearly reveal that the stabilizing effect of the additional
hydrogen bond formed by the protonated amino group is
higher than that resulting from the stacking interactions of
the phenoxazine ring. Moreover, from the comparison of
∆Tm and ∆∆Tm values it can be inferred that the amino-
ethoxy arm of the G-clamp is the main contributor to the
enhanced binding affinity of the sequences where the
G-clamp is flanked by a 5′ purine.
The melting temperatures of the CCC, CPC, and CXC
duplexes were also determined at different salt concentrations
(70, 140, and 280 mM KCl). As expected, the Tm values
increased with ionic strength because of the decrease in the
repulsion between phosphate groups (Supporting Informa-
tion). However, the increase in Tm values was the same for
the three sequences, irrespective of whether C, P, or X was
present, indicating that the enhanced affinity of the G-clamp-
modified ODNs cannot be attributed to an ionic interaction
between the protonated amino group of X and a phosphate
group of the complementary strand.
It is also worth noting that the CD spectra of the
phenoxazine- and G-clamp-containing duplexes showed the
characteristic profile of a B-form DNA, and did not exhibit
significant differences compared with unmodified duplexes.
In all cases, a positive band centered at 280 nm and a
negative band at 250 nm were observed (Supporting Infor-
mation).
The amino-G-clamp cytosine analog has also been intro-
duced in PNA sequences and reported to produce a dramatic
increase in the stabilities of PNA-DNA and PNA-RNA
duplexes when placed between two cytosines (∆Tm ) 18.2
and 23.7 °C, respectively).8 In order to check whether this
stabilizing effect was maintained in our sequences, we
decided to study two PNAs with the same sequence as the
DNA strands exhibiting the most extreme behavior, those
in which the G-clamp was flanked by two cytosines or two
adenines: H-gcacxctacg-NH2 and H-gcaaxatacg-NH2. PNA
monomers were obtained as previously described with minor
modifications,9 and introduced in PNA sequences using
standard solid-phase procedures. The UV melting curves of
PNA-DNA duplexes showed, again, the remarkable stabiliz-
ing effect of the G-clamp (Supporting Information). The Tms
of the PNA-DNA duplexes formed by H-gcacxctacg-NH2
and H-gcaaxatacg-NH2 were 16 and 8.5 °C, respectively,
Overall, the ∆Tm values shown in Table 1 indicate that
the increase in the binding affinities of oligonucleotides
modified with phenoxazine or G-clamp is far from being
homogeneous. Table 2 depicts the relative order (in paren-
theses) of duplex stabilities of the unmodified and P or X
modified sequences, showing that the non-homogeneous
Table 2. Relative Order of Thermal Stabilities of the Duplexes
(in Parentheses) Formed by the Natural Sequences and the P-
or X-Modified Oligonucleotides
cytosine (C)
phenoxazine (P)
G-clamp (X)
GCG (1)
GCC (2)
CCG (2)
CCC (4)
ACC (5)
CCA (6)
ACA (7)
TCT (8)
GPG (3)
GPC (5)
CPG (1)
CPC (2)
APC (6)
CPA (4)
APA (8)
TPT (6)
GXG (4)
GXC (3)
CXG (2)
CXC (1)
AXC (7)
CXA (5)
AXA (8)
TXT (6)
(8) Rajeev, K. G.; Maier, M. A.; Lesnik, E. A.; Manoharan, M. Org.
Lett. 2002, 4, 4395-4398.
(9) Aus´ın, C.; Ortega, J.-A.; Robles, J.; Grandas, A.; Pedroso, E. Org.
Lett. 2002, 4, 4073-4075.
Org. Lett., Vol. 9, No. 22, 2007
4505

higher than those of the unmodified ones. It is noteworthy
that the difference in ∆Tm between the sequences containing
the cxc and axa triplets was less pronounced than in DNA-
DNA duplexes. We hypothesize that the more flexible
polyamide backbone of PNA, as compared with DNA, allows
to partially offset the lower stabilizing effect when the
G-clamp is flanked by two adenines.
To gain a deeper insight into the reasons underlying the
sequence dependence of the duplex stability of ODNs
containing these cytosine analogues, molecular dynamics
simulations of all 24 duplexes were performed (Supporting
Information).10 All trajectories were stable, sampling regions
close to the canonical B-form and maintaining well the
expected pattern of hydrogen bonds. P or X analogues did
not introduce significant changes in the structure. Both P
and X derivatives made the expected interactions; in
particular, the ammonium-guanine interactions were very
strong in all of the X-duplexes with interaction distances
N-O6 and N-N7 corresponding to those of strong hydrogen
bonds.
The melting temperature is linearly related (r ) 0.94) to
the generalized stacking and hydrogen bond energies in the
central 5 (or 3)-pairs segment (Supporting Information),
showing that these two terms are good descriptors of the
DNA stability. The introduction of the P derivative slightly
increases (around 1 kcal/mol) the hydrogen-bond energy and
clearly improves by 5-10 kcal/mol the stacking energy
(Supporting Information). The sequences where the gains
in stacking are larger are those for which greater increases
in melting temperatures are found (melting temperature and
the gain in melting temperature upon CfP substitution
correlate linearly; r ) 0.8). Clearly, the sequence-dependence
of stacking explains the different gains in stability upon CfP
substitution in different sequences.
The addition of the charged moiety to phenoxazine ring
in X leads to a large gain in hydrogen bonding (between 25
and 30 kcal/mol) and also to an unexpected gain (from 18
to 34 kcal/mol) in generalized stacking (Supporting Informa-
tion), which is largely a consequence of the secondary
interactions of the ammonium group with hydrogen bond
acceptors of bases different from the paired guanine (Figure
2). Note that these secondary interactions (captured in the
generalized stacking term) can be large and are strongly
sequence-dependent, and when combined with the direct
hydrogen bond term are able to reproduce well (r ) 0.93)
the sequence dependent increase in melting upon PfX
substitution, confirming the goodness of the simple descrip-
tors used here.
Figure 2. Snapshot of 5′CX•3′GG tract obtained by molecular
dynamics, showing the secondary interactions of ammonium group
of X with the paired and nonpaired guanines in the opposite strand.
inserted in DNA, many other interactions may be established.
Experimental data and theoretical calculations are in good
agreement and show that ODNs incorporating the tricyclic
cytosine analogues P and X form duplexes with a practically
unperturbed B-DNA conformation. However, unexpected
interactions with neighboring bases give rise to profound and
striking differences in the stability of the resulting duplexes
depending on the sequence context of the modified nucleo-
base. These differences should be taken into account when
designing the incorporation of P and X into ODNs and PNAs
in order to enhance their binding affinities. From our study
it can be concluded, as a general rule, that the substitution
of cytosine by phenoxazine does not enhance duplex stability
if the 5′ flanking nucleobase is a purine, while some
stabilization may be gained if a 5′ cytosine flanks the
phenoxazine ring. When replacing cytosine by the G-clamp,
the highest binding affinities will be obtained if the 5′
nucleobase can be a cytosine. If the 5′ nucleobase has to be
a purine, then guanine is preferred to adenine. The 3′ flanking
nucleobase of the G-clamp also has some effect on duplex
stability and we would recommend cytosine rather than a
purine at this position. Sequences with only one T flanking
P or X were not studied. Although firm predictions cannot
be made, from the comparison of the Tms of CCC and TCT
we would expect duplex stability to slightly decrease when
replacing a single C by a T.
Acknowledgment. This work was supported by funds
from the Ministerio de Educacio´n y Ciencia (grants CTQ2004-
8275-C02-01 and BIO2006-01602), the Generalitat de Cata-
lunya (2005SGR-693 and -286, and CeRBA), and the
Fundacio´ La Caixa.
Supporting Information Available: Details on the
synthesis of phenoxazine and G-clamp building blocks,
ODNs, and PNAs. UV melting profiles, CD spectra, and
theroretical calculations. This material is available free of
charge via the Internet at http://pubs.acs.org.
In summary, atomistic simulations suggest that flat pictures
as those in Figure 1 might be very useful to underline desired
interactions. However, once the modified nucleobase is
(10) Shields, G. C.; Laughton, C. A.; Orozco, M. J. Am. Chem. Soc.
1997, 119, 7463-7469.
OL701826X
4506
Org. Lett., Vol. 9, No. 22, 2007