The Structure of PNA Conjugates.
PNA oligomers were synthesized from N-Boc protected (2-aminoethyl)glycine monomers (25). The AQ-containing monomers are shown in Fig. 2.
In AQ1, the anthraquinone group is linked to the peptide backbone by a
single amide bond whereas for AQ2 there is an intervening glycine group
between the quinone and backbone. A third monomer (Ac, used in control
experiments) has an acetyl group bound to the backbone nitrogen atom of
the (2-aminoethyl)glycine. The synthetic details and characterization
of these compounds and the oligomers described below are reported
elsewhere (unpublished work).
The PNA–AQ monomers were incorporated into 19-base oligomers (Fig. 3).
The quinones are at the central position of PNA-1, equidistant from two
CC sequences (sites B and C), with a third CC sequence (site A) two
bases beyond site B. Hybridization of the PNA-1 with its complementary
DNA-1Z oligomers gives duplexes with three GG sites to act as traps of
a migrating radical cation. Placement of the quinone at the center of
the duplex permits study of both the distance and directional
dependence of hole migration.
A model of a PNA–DNA duplex containing an intercalated, covalently linked AQ1 group was built in sybyl 6.0 (Tripos Associates, St. Louis) using the coordinates for a PNA–DNA structure determined using NMR spectroscopy by Erikkson
and Nielsen (27).
One of the internal base pairs of the duplex was removed by excision of
the PNA and DNA bases. The AQ carboxamide was then linked to the PNA
backbone in place of the nucleobase while the DNA base was replaced by
a hydrogen atom to create an abasic site. Even though this structure
was not subjected to energy minimization, it clearly showed that there
is space within the helix to accommodate the intercalated AQ group.
This result and other experiments including thermal denaturation and
phosphorescence quenching are consistent with an intercalated
conformation of the AQ and are reported in detail elsewhere
(unpublished work).
Light Causes Long Range DNA Damage.
The irradiation of AQs intercalated in duplex DNA gives efficient, piperidine-requiring strand cleavage selectively at GG
sites by an electron transfer mechanism (16, 17). The base sequence in PNA-1/DNA-1X was specifically designed to probe for the corresponding reaction in PNA/DNA hybrids
and to examine its mechanism. DNA-1X was labeled at its 5′ terminus with 32P by standard methods (28)
and hybridized with the complementary PNA oligomers. Irradiation of the
PNA-1(AQ1)/DNA-1X hybrid at 350 nm lead to piperidine-requiring DNA
strand cleavage at the three GG sites (Fig. 4, lane 8). Cleavage at the 3′-G is favored for site A whereas the 5′-G is favored at site B (Fig. 3);
cleavage occurs with equal efficiency at each G of site C. DNA strand
cleavage also is observed at the abasic residue directly opposite the
AQ. The pattern of cleavage bands is identical for the PNA-1(AQ2)
hybrid (Fig. 4, lane 12) although the efficiency is lower. Control experiments with PNA-1(Ac), which lacks the quinone group, show essentially
no cleavage of the DNA strand at any site (Fig. 4, lanes 1–4).
Additional control experiments show that cleavage of the
DNA strand results from an intramolecular reaction initiated by
excitation of the quinone in the hybrid duplex. In the photocleavage
experiment described above, the PNA and labeled DNA were hybridized in
the presence of a stoichiometric amount of unlabeled DNA-1X (relative
to PNA-1). This ensures that all of the PNA will be hybridized during
the experiment, with some fraction of the hybrids containing labeled
DNA-1X strands. Hybridization of PNA-1(AQ1) with labeled DNA-1X and
with a 10-fold excess of unlabeled DNA-1X strongly inhibits cleavage of
the labeled DNA because the fraction of PNA-1(AQ1) that is hybridized
to labeled DNA-1X is 10-fold lower. However, hybridization in the
presence of a stoichiometric amount of unlabeled complementary DNA and
a 9-fold excess of noncomplementary single-stranded DNA has no effect
on the cleavage of the labeled DNA. (The presence of a stoichiometric
amount of complementary, unlabeled DNA in the latter case ensures that
the PNA is hybridized while maintaining the concentration of
unhybridized DNA.) The lack of inhibition in this experiment
demonstrates that cleavage is not mediated by a freely diffusing
intermediate because the excess of single-stranded, unlabeled DNA
should effectively inhibit such a process. Finally, cleavage is still
observed when excess unlabeled DNA-1X is added after hybridization with
labeled DNA, demonstrating the kinetic stability of the PNA(AQ)–DNA
hybrids studied in these experiments.
Selective cleavage of DNA at guanine has been observed in reactions initiated by 1-electron oxidation and by reaction of singlet
oxygen (1O2) (29). The G-selective cleavage of the PNA–DNA hybrid cannot be caused by freely diffusing 1O2, but a recent report raises the possibility of one-dimensional intramolecular diffusion of 1O2 in a groove of duplex DNA (30),
and this could account for the long range cleavage that is observed.
However, this path is unlikely in the present case because the lifetime
of the requisite AQ triplet is shortened by electron transfer
quenching. Additional control experiments were performed that compare
the cleavage of PNA-1(AQ1)–DNA hybrids caused by irradiation of
methylene blue, a known 1O2 generator (31), with direct irradiation of the quinone in the hybrid duplex. Inspection of Fig. 5 shows cleavage of DNA upon irradiation of methylene blue that is enhanced significantly when D2O is substituted for H2O (compare Fig. 5, lanes 4 and 2), proving that the cleavage in this case is caused by reaction with 1O2 generated by excited methylene blue (32). No effect of D2O is seen for the quinone-initiated cleavage (data not shown). It is important to note that the cleavage pattern due to the
reaction of 1O2 is significantly different from that seen for irradiation of the quinone. In particular, densitometric analysis indicates
that the ratio of cleavage of the 5′-G to the 3′-G at site A is 1.6 for 1O2 and 0.7 for the AQ-initiated reaction. Clearly, the long range G-selective cleavage reaction of the PNA–DNA hybrid duplex
cannot involve 1O2 but must occur by electron transfer to generate a base radical cation and long distance migration of the hole as has been
previously proposed for anthraquinones in duplex DNA.
Mechanistic interpretation of reactions with extraordinarily low quantum yields is risky because minor structural isomers
or impurities can confound the analysis. The quantum yields for cleavage at the GG step (ΦGG) by photonucleases range from 1.4% for an intercalated AQ derivative (with a 10-fold preference for cleavage at the 5′-G)
(16) to 0.000005% reported for a Rh(phi)2DMB+3 complex linked covalently to a 5′ terminus of duplex DNA and presumed to be intercalated (14). We determined ΦGG in a PNA–DNA hybrid duplex by an HPLC technique.
The PNA-2(AQ1)/DNA-3 hybrid duplex (Fig. 3) was designed for measurement of ΦGG.
Cleavage at the 5′-G gives, after 5′-dephosphorylation, 5′-GAAT-3′, and
cleavage at the 3′-G gives 5′-AAT-3′. These oligonucleotides are
separated easily and quantified by reversed phase HPLC. In
air-saturated solution, the ΦGG
for PNA-2(AQ1)/DNA-3 is 0.17%. The ratio of 5′ to 3′ G cleavage is 1:3,
a preference opposite to that seen in duplex DNA. Clearly, the
efficiency of radical cation generation, migration, and conversion to a
piperidine-cleavable lesion is relatively high in the
quinone-containing hybrid PNA–DNA duplex.
The
3′-G at site A of the PNA-1(AQ1)/DNA-1X hybrid is more than 25 Å from
the AQ group, and the short linkage between the quinone and the PNA
backbone prohibits direct contact between the donor (G) and acceptor
(AQ), yet cleavage is observed at this and other remote sites within
the duplex. One model for long range oxidative damage involves
instantaneous delocalization of the radical cation, i.e., the electron
is transferred directly from the GG site to the excited state
photosensitizer. Selective GG cleavage in such a process would reflect
a higher radical cation density at these most easily oxidized sites. An
alternative mechanism postulates oxidation of a base at a distinct
position (e.g., adjacent to the photosensitizer) followed by migration
of the radical cation by sequential electron transfers, i.e.,
hole-hopping. In this model, the hole will be distributed among the
various low oxidation potential sites only if the rate of hopping is
faster than the rate of irreversible chemical reaction (e.g., addition
of water or O2 to the radical cation) at a particular site. The data presented thus far cannot distinguish between these two mechanistic
models.
A Deep Trap Reveals the Mechanism.
Introduction
of a low oxidation potential trap in the hybrid duplex allows a clear
test of the mechanism for migration of oxidative damage. In particular,
the central position of the AQ acceptor between two GG reaction sites
(as opposed to the linkage of the photosensitizer to the duplex
terminus) allows the placement of a trap so that instantaneous
delocalization of the hole into the trap can be distinguished from its
arrival at the trap by a series of hops. The delocalization model
predicts inhibition of cleavage at all GG sites by the trap because the
duplex is considered to be one continuous orbital system. Instantaneous
connection of the hole and the trap decreases the likelihood of
reaction at all other sites. On the other hand, migration by the
hopping mechanism will exhibit a distinct directional preference for
cleavage inhibition because the hole cannot “know” of the trap’s
existence until it hops into it.
The
ideal trap is a modified base that does not distort the structure of
the PNA–DNA duplex and that has an oxidation potential significantly
below that of the GG sequence. We selected the guanine derivative
7,8-dihydro-8-oxoguanine (8-OxoG; Fig. 3) as the trap on this basis. This modified base is often detected as a byproduct of oxidative damage (14, 20, 23, 31, 33–36). Although structural information is not available for PNA-containing duplexes, Williams and coworkers (37)
recently determined the structure of a DNA duplex having an 8-OxoG
substitution by x-ray crystallography and found that the oxidized base
caused little perturbation. We found that substitution of an 8-OxoG for
G on DNA in a PNA/DNA hybrid lowers the melting temperature of the
duplex only 1°C. Foote and Sheu (36) report that the oxidation potential of 8-OxoG is 0.4–0.5 V below that of guanosine (as their t-butyldimethylsilyl-protected nucleoside derivatives). Considering that the difference in oxidation potential between G and
A is only ≈0.1 V (38), 8-OxoG should provide a deep trap for holes in the PNA/DNA hybrid.
Photoinduced cleavage of the PNA-1(AQ1)/DNA-1T hybrid yields a cleavage pattern nearly identical to that observed for DNA-1X
(Fig. 6, lane 4; compare with Fig. 4,
lane 8). DNA-2 is analogous to DNA-1T except that 8-OxoG is substituted
for G at site B. Irradiation of the PNA-1(AQ1)/DNA-2 hybrid shows
significantly enhanced cleavage at site B, but the more dramatic effect
is observed at site A, where cleavage is almost completely inhibited
(lane 10). The 8-OxoG acts as a barrier to cleavage at this distal GG
site. It is important to note that 8-OxoG substitution has no effect on
cleavage at site C, which is located in the opposite direction from the
trap. Clearly, the hole does not sense the presence of the 8-OxoG trap
when the selection of migration direction occurs. Consequently, the
hole cannot be in electronic contact with the trap as is required by
the instantaneous delocalization model. These findings provide evidence
that radical cations in a PNA–DNA duplex migrate by a discrete hopping
mechanism.
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