Charge transport through DNA four-way junctions

Abstract

Long range oxidative damage as a result of charge transport is shown to occur through single crossover junctions assembled from four semi-complementary strands of DNA. When a rhodium complex is tethered to one of the arms of the four-way junction assembly, thereby restricting its intercalation into the pi-stack, photo-induced oxidative damage occurs to varying degrees at all guanine doublets in the assembly, though direct strand scission only occurs at the predicted site of intercalation. In studies where the Mg(2+) concentration was varied, so as to perturb base stacking at the junction, charge transport was found to be enhanced but not to be strongly localized to the arms that preferentially stack on each other. These data suggest that the conformations of four-way junctions can be relatively mobile. Certainly, in four-way junctions charge transport is less discriminate than in the more rigidly stacked DNA double crossover assemblies.

Figure 1

Schematic illustrations of four-way junctions. (A) A folded four-way junction in the presence of magnesium ion, adapted from the crystal structure (45). (B) The non-covalent four-way junction assembly with its strands color coded and labeled with lower case letters. The stacked arms are labeled in Roman numerals. (C) The four-way junction with covalently attached metal complex. The structure of the appended metallointercalator, which is shown schematically in this rendering, is given at the bottom of this figure. (D) Duplex of the same base-stacked sequence as the combined arm I/II from (C) with covalently attached metallointercalator. In all sequences of DNA assemblies used, guanine doublets to be oxidized are shown in red.

Figure 2

Direct photocleavage and piperidine-induced cleavage of four-way junction DNA with non-covalently bound Rh(phi)₂bpy′³⁺ as a function of magnesium concentration. (A) Phosphorimagery after electrophoresis in denaturing 20% polyacrylamide gel with 5′-³²P-end-labeling of strand b of the four-way junction in Figure 1 after photo-irradiation. Lane 1, Maxam–Gilbert A+G; lane 2, C>T sequencing reactions (42); lane 3, photolysis at 313 nm for 10 min; lane 4 photolysis at 365 nm for 2 h followed by piperidine treatment; lane 5, the same assembly without photolysis (dark control, DC) followed by piperidine treatment. Oxidatively sensitive sites are bracketed to the left of the sequencing lanes and the site of the crossover junction is shown by a hollow triangle. The presence or absence of 5 mM Mg(OAc)₂ is indicated above the respective lanes and the arm locations are indicated to the left of each gel. (B) Phosphorimagery of strand c. Lanes as in (A). (C) Phosphorimagery of strand d. Lanes as in (A).

Figure 3

Oxidation of a metallointercalator-bearing duplex representing stacked arms I and II from the four-way junction shown in Figure 1. Lanes are as described in Figure 2.

Figure 4

(A) Long range oxidation of DNA by covalently tethered Rh(phi)₂bpy′³⁺ in four-way junctions as a function of magnesium concentration. The gels are labeled as in Figure 2. Strand b of the covalently tethered four-way junction is shown to be cleaved at the 5′-end on arm I by the covalently attached metal complex upon 313 nm irradiation and long range charge transport is evident upon 365 nm photo-irradiation in the absence and presence of magnesium. (B) Long range oxidation of DNA by covalently tethered Rh(phi)₂bpy′³⁺ in four-way junctions as a function of magnesium concentration. Strand c of the covalently tethered four-way junction is shown to be unaffected by 313 nm irradiation, but long range charge transport is evident upon 365 nm photo-irradiation in the absence and, particularly, in the presence of magnesium.

Figure 5

Schematic illustration of the damage pattern, location and intensity of direct photocleavage and photolysis-induced oxidative damage in four-way junction assemblies by the non-covalent metal complex derived from Figure 4A. Direct photocleavage damage is shown by black arrows, whereas the long range charge transport at 5′-GG-3′ doublets is shown with red arrows. The assemblies are shown with their presumed structural forms, open in the absence of magnesium, and with stacked arms in the presence of magnesium.

Figure 6

Schematic illustration of the damage pattern, location and intensity of direct photocleavage and photolysis-induced oxidative damage in four-way junction assemblies by the covalently tethered metal complex derived from Figure 4B. The relative intensity of damage here is represented qualitatively by the size of arrows.