Sugar radicals formed by photoexcitation of guanine cation radical in oligonucleotides

Abstract

This work presents evidence that photoexcitation of guanine cation radical (G+*) in dGpdG and DNA-oligonucleotides TGT, TGGT, TGGGT, TTGTT, TTGGTT, TTGGTTGGTT, AGA, and AGGGA in frozen glassy aqueous solutions at low temperatures leads to hole transfer to the sugar phosphate backbone and results in high yields of deoxyribose radicals. In this series of oligonucleotides, we find that G+* on photoexcitation at 143 K leads to the formation of predominantly C5'* and C1'* with small amounts of C3'*. Photoconversion yields of G+* to sugar radicals in oligonucleotides decreased as the overall chain length increased. However, for high molecular weight dsDNA (salmon testes) in frozen aqueous solutions, substantial conversion of G+* to C1'* (only) sugar radical is still found (ca. 50%). Within the cohort of sugar radicals formed, we find a relative increase in the formation of C1'* with length of the oligonucleotide, along with decreases in C3'* and C5'*. For dsDNA in frozen solutions, only the formation of C1'* is found via photoexcitation of G+*, without a significant temperature dependence (77-180 K). Long wavelength visible light (>540 nm) is observed to be about as effective as light under 540 nm for photoconversion of G+* to sugar radicals for short oligonucleotides but gradually loses effectiveness with chain length. This wavelength dependence is attributed to base-to-base hole transfer for wavelengths >540 nm. Base-to-sugar hole transfer is suggested to dominate under 540 nm. These results may have implications for a number of investigations of hole transfer through DNA in which DNA holes are subjected to continuous visible illumination.

Figure 1

Benchmark spectra used for computer analysis. (A) G•⁺ in dGpdG by one-electron oxidation by Cl₂•^– (see Figure 2A also). (B) C5′•, formed via photo-excitation of G•⁺ in 8-D-3′-dGMP. (C) C3′•, produced from G•⁺ in dGuo. (D) C1′•, produced from G•⁺ in TGT. (E) C1′•, produced from G•⁺ in dGpdG. (see supporting information Figure S1 for details regarding Figures 1D and 1E). The three reference markers in this figure and in the other figures in this work represent positions of Fremy's salt resonances (The central marker is at g = 2.0056 and each of three markers is separated from one another by 13.09 G).

Figure 2

(A) ESR spectrum of one-electron oxidized dGpdG (1 mg/ml) by Cl₂•⁻ attack in the presence of K₂S₂O₈ (5 mg/ml) as an electron scavenger in 7.5 M LiCl glass / D₂O. (B) Spectrum found after visible photo-excitation the sample in A for 90 min at 143 K. Nearly complete conversion of G•⁺ to sugar radicals is found. Analyses shows three radicals: C5′• (prominent doublet at the centre), C1′• (prominent quartet) and a very small amount of C3′• visible in the wings. All spectra were recorded at 77 K.

Figure 3

ESR spectra of (A) and (F) of one-electron oxidized TGT (4.5 mg/ml) and TTGTT (4.5 mg/ml) formed by attack of Cl₂•^– in the presence K₂S₂O₈ (8 mg/ml) as an electron scavenger in 7.5 M LiCl glass / D₂O, in annealing to 155 K. These spectra in (A) and (F) are chiefly due to G•⁺. Spectra (B) – (E) and (G) – (I) are obtained after photo-excitation of G•⁺ in the oligonucleotides indicated with visible light at 143 K, showing considerable conversion of G•⁺ to sugar radicals - C5′• (prominent doublet at the centre) as well as to C1′• (prominent quartet). The samples in A and (F) are red-violet due to the absorption of G•⁺ whereas those in (B) – (E) and (G) – (I) are fade in color or are near colorless as conversion to sugar radical occurs. All spectra were recorded at 77 K.

Figure 4

Ice samples of DNA (50 mg/mL in D₂O) with 1 Tl³⁺/10 base pairs were γ-irradiated to 15.4 kGy dose at 77 K and were then annealed to 130 K to remove the •OH signal. (A) ESR spectrum obtained after illumination with the aid of a high pressure Xe lamp (Oriel corporation), at 77 K for 110 min with 380-480 nm band-pass filter. (B) After photo-excitation using a 250 W photoflood tungsten lamp of an identically prepared and handled sample as in A at 143 K for 90 min without any filter. (C) After illumination by the same photoflood lamp of another identically prepared and handled sample as in A and B at 180 K for 90 min. Spectrum (C) also shows outer line component of peroxyl radical which decreases the intensity of the sugar components slightly. All spectra were recorded at 77 K.