Results and Discussion
- Peptide nucleic acid–DNA duplexes: Long range hole migration from an internally linked anthraquinone

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The synthetic details and characterization of these compounds and the oligomers described below are reported elsewhere (unpublished work).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quenching are consistent with an intercalated conformation of the AQ and are reported in detail elsewhere (unpublished work).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, 3336). 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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