Long-range oxidative damage to cytosines in duplex DNA

Abstract

Charge transport (CT) through DNA has been found to occur over long molecular distances in a reaction that is sensitive to intervening structure. The process has been described mechanistically as involving diffusive charge-hopping among low-energy guanine sites. Using a kinetically fast electron hole trap, N(4)-cyclopropylcytosine ((CP)C), here we show that hole migration must involve also the higher-energy pyrimidine bases. In DNA assemblies containing either [Rh(phi)(2)(bpy')](3+) or an anthraquinone derivative, two high-energy photooxidants, appreciable oxidative damage at a distant (CP)C is observed. The damage yield is modulated by lower-energy guanine sites on the same or complementary strand. Significantly, the efficiency in trapping at (CP)C is equivalent to that at N(2)-cyclopropylguanosine ((CP)G). Indeed, even when (CP)G and (CP)C are incorporated as neighboring bases on the same strand, their efficiency of photodecomposition is comparable. Thus, CT is not simply a function of the relative energies of the isolated bases but instead may require orbital mixing among the bases. We propose that charge migration through DNA involves occupation of all of the DNA bases with radical delocalization within transient structure-dependent domains. These delocalized domains may form and break up transiently, facilitating and limiting CT. This dynamic delocalized model for DNA CT accounts for the sensitivity of the process to sequence-dependent DNA structure and provides a basis to reconcile and exploit DNA CT chemistry and physics.

Fig. 1.

Experimental assay to test for hole occupation of pyrimidines during CT through duplex DNA. (Upper) Oxidation of ^CPC leads to rapid decomposition through ring scission and subsequent generation of two characteristic products. (Lower) Two potent photooxidants, Rh and AQ, covalently positioned remote from ^CPC [for Rh through the 5′-sugar and for AQ through the 5′-phosphate (25)] initiate hole injection into DNA. Photooxidation of ^CPC in DNA, detected by HPLC analysis of the enzymatically digested duplexes, requires that the hole first migrate through the lower-energy intervening bases.

Fig. 2.

Long-range oxidative damage to cytosines in duplex DNA. HPLC traces show the oxidative decomposition of ^CPC with increasing irradiation, 0–40 min for Rh-G-1 (Upper Left) and 0 (black) vs. 5 (purple) min for Rh-I-1 (Upper Right). (Insets) Full HPLC traces. Plots of the amount of ^CPC as a function of irradiation time reveals the sequence dependence of ^CPC decomposition with Rh (Lower Left) and AQ (Lower Right). In Lower Left: filled circles, Rh-G-2; open circles, Rh-I-2; filled squares, Rh-G-1; open squares, Rh-I-1. In Lower Right: filled circles, AQ-G-1; open circles, AQ-I-1; filled squares, AQ-I-3. For AQ, note the dramatic increase in efficiency of ^CPC photooxidation when the intervening guanines (AQ-I-1) are replaced with inosines (AQ-I-3). Decomposition of ^CPC is an intraduplex reaction that requires light and photooxidant.

Fig. 3.

Hole distribution on the DNA bridge does not reflect the relative energies of the individual bases. Shown is a plot of the amount of ^CPC (filled circles) or ^CPG (open squares) as a function of irradiation time in AQ-7/Cp-7. These duplexes contain ^CPC and ^CPG at neighboring sites, 5′-^CPC^CPG-3′. Similar results were observed for AQ-8/Cp-8 possessing a 5′-^CPG^CPC-3′ sequence within an otherwise identical duplex.

Fig. 4.

Schematized delocalized domain model for DNA CT. Charge migrates through the DNA bridge by hopping among domains: Extended π-orbitals formed transiently in a manner governed by DNA sequence and dynamics. DNA domains form transiently, irrespective of charge. Base motion causes domains to form and break up, and consequently, migration of charge among domains is conformationally gated. In duplex DNA, hole density is distributed through the domain (high–low, blue–white–red) in a sequence-dependent fashion, as shown, for instance, when an I–C base pair is replaced by a G–C base pair. Here, hole density distribution in the domains is shown schematically by static snapshots, but it will fluctuate in time with the dynamic motion of the DNA bases.