J. Phys. Chem. B 2001, 105, 6031-6041
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Electron Spin Resonance Study of Electron and Hole Transfer in DNA: Effects of Hydration, Aliphatic Amine Cations, and Histone Proteins Zhongli Cai, Zhenyu Gu, and Michael D. Sevilla* Department of Chemistry, Oakland UniVersity, Rochester, Michigan 48309 ReceiVed: January 29, 2001; In Final Form: April 9, 2001
In this work, we employ electron spin resonance spectroscopy to investigate the effects of hydration, and various cationic complexing agents, such as, aliphatic amine cations and histone proteins, on electron and hole transfer in DNA. Electrons and holes generated by irradiation at 77 K are trapped on DNA and transfer to a randomly interspersed intercalator, mitoxantrone (MX). Monitoring the changes of ESR signals of MX radicals, one electron oxidized guanine (G•+), one-electron reduced cytosine [C(N3)H•], and thymine anion radicals (T•-) with time at 77 K allows for the direct observation of electron and hole transfer. The apparent transfer distance (Da) in bps is derived from the change in radicals with time and is a measure of the total number of bps within the tunneling range. In all solid DNA samples in which tunneling from electrons and holes to an intercalator was investigated, we find that the distance between DNA duplexes is the dominant factor in the degree of transfer observed. In hydrated DNA samples intercalated with MX, the apparent distances and rates of hole and electron transfer to MX decrease as hydration level increases mainly because the distance between DNA duplexes increases with hydration. DNA complexing agents such as poly-lysine, polyethylenimine, nucleohistone, and cationic lipids also reduce the apparent transfer rates by reducing the amount of transfer between duplexes. Transfer rates in DNA complexed with spermine, however, are similar to those in equivalently hydrated MX-DNA. A double layer of cationic lipids is found to nearly isolate DNA duplexes from electron or hole transfer to adjacent duplexes. Our modeling of rates and distances of electron transfer in DNA-complexes allow for estimates of the spacing between DNA duplexes in each complex.
Introduction Owing to the biological significance of DNA damage and repair, electron and hole transfer processes in DNA have attracted considerable experimental and theoretical interest.1-6 Various mechanisms such as single-step tunneling,7-10 multistep incoherent hopping8,11,12 and phonon-like thermally activated hopping13,14 are proposed to explain either long or short range transfer of electrons and holes through DNA. DNA in cellular systems is hydrated to various extents, closely packed with histone proteins in chromatin, or in contact with lipids of the nuclear membrane. There have been previous reports of the effects of hydration level on the free radical yields in irradiated DNA,15-18 as well as the efficiency of hole and electron transfer from the hydration layer to DNA.17,19 However, there are no studies as to the rate of electron and hole transfer through DNA with increasing hydration level or DNA complexed with cationic lipid and proteins. For this reason, a systematic study of the effects of hydration waters, complexing lipids and chromosome structure on the electron and hole transfer along and across DNA duplexes may aid our understanding of processes that are a natural part of radiation damage to living systems.20,21 In our recent efforts, we investigated excess electron transfer for DNA in glasses, ices and solids at low temperatures via ESR.22-24 For DNA in glasses, we reported an overall distance decay constant, β, near 0.9 Å-1 .22 We also found electron transfer between DNA duplexes (ds) was competitive with transfer along the duplex when duplexes approached within ca. 40 Å of each other. These results led to a three-dimensional model that accounts for the electron-transfer both along DNA duplex and across to adjacent DNA duplexes.24 Our most recent work probed into the temperature effects (from 4 to 195 K) on
excess electron and hole transfer through DNA and assigned the ranges of temperature where tunneling, reversible and irreversible protonations, hopping, or recombination make contributions.23 In this work, we employ electron spin resonance (ESR) spectroscopy to investigate the effects of hydration, polymeric and aliphatic amine cations, and nucleosome proteins on electron and hole transfer in γ-irradiated DNA. Specifically, DNA solids intercalated with mitoxantrone (MX) were investigated under these conditions: (1) hydrated to 4-30 waters/nucleotide, (2) complexed with various cationic counterions such as spermine tetra-hydrochloride (SP), poly-L-lysine hydrobromide (PLL), polyethylenimine hydrochloride (PEI), octadecyltrimethylammonium bromide (OCT), and dodecyltrimethyammonium bromide (DOD), and (3) in frozen nucleohistone D2O aqueous solutions. The effects of hydration, polymeric and aliphatic amine cations on electron and hole transfer in γ-irradiated DNA are found to be mainly due to variations in separation distances (Dds) between DNA duplexes or in the case of nucleohistone due to a smaller number of adjacent DNA duplexes (n) than in pure DNA. However, nondistance effects of hydration on the distribution of DNA radicals and their transfer rates are also important. The apparent transfer distance (Da) dependence on separation distances and number of adjacent strands are accounted for by a simple 3-dimensional electron-transfer model.24 Experimental Section Sample Preparation. Preparation of Mitoxantrone-Intercalated DNA Solids Various molar ratios of salmon sperm DNA with MX were prepared by slow addition of ca. 1 mg/mL MX aqueous solution into an aqueous solution of 100 mg/mL DNA
10.1021/jp010358x CCC: $20.00 © 2001 American Chemical Society Published on Web 06/05/2001
6032 J. Phys. Chem. B, Vol. 105, No. 25, 2001 while stirring under nitrogen. The mixture was allowed to sit under nitrogen in the dark for long periods (days to weeks), stirred periodically with a vortex mixer, then freeze-dried when the solutions appeared uniform. Hydrated Samples. About 100 mg of freeze-dried MX-DNA solid or pure DNA was kept for 7 days in a desiccator containing D2O under N2 gas. The hydrated solids were pressed into 4 mm diameter solid plugs with an aluminum press, then kept in desiccators containing saturated LiCl, K2CO3, NaCl, KCl aqueous (D2O) solutions, unsaturated KCl aqueous (D2O) solutions, or D2O under N2 gas for 7 days to attain various hydration levels between 4 and 30 D2O/nucleotide, which were determined by mass measurements. The different solutions control the humidity, thus the level of hydration of DNA. These samples were then placed in liquid nitrogen. SP-(MX)-DNA, PLL-(MX)-DNA and PEI-(MX)-DNA Complex. Aqueous solutions (ca. 50 mg/mL) of spermine tetrahydrochloride, poly-L-lysine hydrobromide, or 5% (W/V) polyethylenimine (pH was adjusted to around 4 with 1 M HCl) were slowly dropped into 25 mM NaCl aqueous solution containing about 10 mg/mL DNA (or MX-DNA) while stirring with a glass rod, as a result, DNA or MX-DNA was precipitated and collected on the rod. In the case of MX-DNA, when the blue color of MX-DNA solution disappeared, precipation was considered complete and the samples of SP-MX-DNA, PLLMX-DNA and PEI-MX-DNA were removed from the glass rod. In the case of pure DNA, equimolar amounts of amine cations to DNA phosphate groups were added. The precipitates were washed with deionized water, excess water removed and pressed into solid plugs. The solid plugs were put in D2O for 2 h to undergo D-H exchange before being placed in liquid nitrogen. OCT-(MX)-DNA and DOD-(MX)-DNA Complex: MX-DNA or pure DNA was dissolved in 50 mM of Sorensen’s phosphate buffer (pH ) 7.5) to obtain a 0.2 mg/mL solution. The OCT(MX)-DNA and DOD-(MX)-DNA complex precipitated while adding 10 mg/mL octadecyltrimethylammonium bromide (OCT) or dodecyltrimethyammonium bromide (DOD) aqueous solution (pH value was adjusted to 7.5 with disodium phosphate) into MX-DNA or DNA solution. The precipitate was collected and washed with deionized water and freeze-dried. “Double-layered” DOD-MX-DNA Complexes: 200 mg DODMX-DNA complex solid was placed into a 50 mL portion of a 20 mg/mL DOD solution, and stirred for 48 h. Disodium phosphate was used to adjust the pH of the DOD solution to 7.5. The precipitate was freeze-dried without washing. The weight increase of the complex suggested sufficient weight gain for approximately a second layer of DOD. (MX)-Nucleohistone and (MX)-DNA in frozen D2O aqueous solution: Aqueous solutions were prepared by addition of 1 mL D2O or D2O aqueous solution of MX to 100 mg calf thymus nucleohistone (39% DNA, 46% protein, 15% unknown)25 or 100 mg salmon sperm DNA. The resulting mixture was allowed to stand in the dark for several days with daily vortex mixing until the solid was dissolved homogeneously. The solution was drawn into a glass tube with inner diameter of 4 mm and frozen in liquid nitrogen; the resultant ice plug was pushed out into liquid nitrogen after warming the glass wall sufficiently. All samples were gamma irradiated for 2.6 kGy (60 min). Irradiation of the DNA solids produced both electron adducts (CD• and T•-) and holes (G•+) within DNA. All preparations were performed in a nitrogen atmosphere. Samples were kept in liquid nitrogen in the dark throughout all experiments.
Cai et al. Methods of Analysis. Electron Spin Resonance. ESR spectra were taken on a Varian Century Series EPR spectrometer operating at X-band with a dual cavity and a 200 mW klystron, with Fremy’s salt (g ) 2.0056, AN ) 13.09 G) as a reference. All ESR spectra were recorded at 77 K within a few minutes after irradiation, and at increasing time intervals thereafter up to 12 days. Benchmark Spectra. Methods of analyses were similar to our previous work.22-24 The benchmark spectra of MX radicals22 (including both one-electron oxidized and reduced MX which appear to have the same ESR spectra) in DNA and G•+, CD•, T•-2c were used in the analysis of each experimental spectrum of hydrated MX-DNA and pure DNA. Linear least-squares fitting of benchmark spectra to experimental spectra is employed to determine the fractional composition of MX radicals, G•+, CD• and T•- in hydrated DNA solids. Hydroxyl radicals were formed in all hydrated samples beyond hydration levels of 8-9 waters/nucleotide19 and small amounts of sugar radicals were also found in hydrated DNA.16 However, both hydroxyl and sugar radicals were ignored in our analyses because they were in low amounts and in the case of the hydroxyl radical the broad ESR spectrum extends well out of the spectral range under investigation. The sugar radicals are estimated to be ca. 10% of the signal and would only have a modest effect on our analyses for the composition of the DNA base radicals and MX radicals. For aliphatic amine cation-MX-DNA complexes, MX-nucleohistone and MX-DNA in frozen D2O aqueous solutions, the benchmark spectra of MX radicals in DNA, as well as the spectra of irradiated DNA complexes (SP-DNA, PLL-DNA, PEI-DNA, OCT-DNA, DOD- DNA, nucleohistone) or DNA were used in the linear least-squares fitting of each experimental ESR spectrum as appropriate. For SP-DNA, PLL-DNA, and PEI-DNA, we did not see radicals from SP, PLL or PEI. For OCT-DNA, DOD- DNA, and nucleohistone, broad background signals were found from OCT, DOD as well as the protein in nucleohistone and were subtracted from the ESR spectra of DNA complexes before our analyses. We assume that electron transfer from the complexes to DNA is fast and that the remaining neutral radicals in the complexes do not participate in electron transfer. Analysis for the Apparent Transfer Distance and Rate. The apparent transfer distances (Da) for overall DNA radicals, holes and electrons are derived from eq 1-3, respectively22,26
Da(MX‚) (t) )
ln(1 - FMX‚(t))
Da(G‚+) (t) ) Da(CD‚ and T‚-) (t) )
2ln(1 - ν) ln(FG‚+ (t)) 2ln(1 - ν) ln(FCD‚ and T‚- (t)) 2ln(1 - ν)
(1)
(2)
(3)
where ν is the mole ratio of MX to DNA bps and Da sums the transfer distances up or down the primary DNA duplex and its near neighbor DNA duplexes within the tunneling range. In effect, 2Da counts the total number of bps on all duplexes within the tunneling range in 3 dimensions and therefore can be related to the tunneling volume. The value of F in each equation comes directly from the fractional composition of the ESR spectra. FMX‚ is the fraction of MX radicals in the overall ESR spectrum; FG+‚ is the fraction of DNA cation radical (G•+) and F(CD‚ and T‚-) is the sum one-electron reduced DNA radicals
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(CD‚ and T‚-). The fraction of MX is to the all DNA radicals after subtraction of background signals due to complexing agents [DOD-MX-DNA, (DOD)2-MX-DNA, OCT-MX-DNA,and nucleohistone]. The fractions of individual DNA radicals are relative to their initial yields in pure hydrated DNA after irradiation. These equations assume complete and random intercalation, a condition we found held at values of V of 1 MX to 30 bp and larger in our earlier work.22 For a tunneling process, the approximate relation for the time dependence of Da, successfully used for tunneling kinetics in glasses26 and in our previous work22 in DNA is
Da(t) ) (1/R) ln(k0t)
(4)
second DNA layer of 6 ds’s at 1.73 x Dds and a third layer of 6 ds’s at 2 x Dds, which would become involved for transfer distances above 1.73 × Dds (which amounts to 37-43 Å, depending on the DNA hydration level.) and 2 × Dds (42-50 Å), respectively. At long times, these additional DNA layers come into play, especially, at the lower hydration where the Dds is the lowest and transfer distances appear longer. With the assumption that the solid samples have hexagonal packing (at least for the first two layers) we modify eq 5 to eqs 6 and 7. When DI(t) > 1.73 Dds
Da(t) ) DI(t) + n(DI(t) - Dds) + n(DI(t) - 1.73Dds)
(6)
When DI(t) > 2 Dds where k0 is the preexponential factor in the rate expression [k ) k0e-βD], R is the apparent value of the distance decay constant β; R is artificially reduced by inter-duplex electron transfer.24 The slope of Da(t) versus ln t, gives 1/R in bp or Å. The apparent transfer rate is therefore, dD/dt ) 1/(Rt). For t in minutes, 1/R is then the apparent transfer rate at 1 min in bp/min or Å /min. Da can be related to the ET distance along a single DNA duplexes (DI) and the inter helical axis distance between duplexes (Dds) according to our previously proposed 3D ET model24
Da(t) ) DI(t) + n(DI(t) - Dds)
(5)
where n is the number of the adjacent DNA duplexes and applies only when DI > Dds. Equation 5 also makes the assumption that the tunneling β is the same up and down the stacked bp’s as across, i.e., inter duplex transfer. In fact, the value of β across duplexes (βT) should be greater than up and down the stacked DNA bases (βπ). We estimate βT ) 1.35 βπ. This could be compensated for by an additional constant before Dds to increase its effective distance. However, correction for the fact that tunneling occurs between sites of spin density introduces an edge to edge correction term (CEdge) which results in an effective reduction in the average across duplex tunneling distances (ca. 5 Å) and compensates for the larger β value for inter ds transfer. Thus, we make the assumption that βπ Dds ) βT(Dds - CEdge) for this work. We also note that the inter duplex path assumed is directly across to adjacent duplexes then up and down these adjacent duplexes. This path is about equivalent to the direct line of sight path from donor to acceptor when the lower β value of the π-way is considered and it simplifies our considerations. Investigations of the conformation of DNA with hydration suggest the A form at low hydrations and the B form for fully hydrated DNA.27 The separation between stacked bp’s is 3.4 Å for both, but due to the off axis twist in the A form the axial lift with each bp is 2.8 Å. We have considered which of these is most appropriate in conversion of bp travel to distance in Å and have concluded that the higher β value for direct transfer again largely compensates for the shorter distance. Thus, 3.4 Å/bp is employed to convert travel distances in bp’s to Å for both A and B forms of DNA. Previous studies on the structure of hydrated Na-DNA film,27 spermine-DNA crystals,28 and cationic liposome-DNA complexes29 all suggest a hexagonal arrangement of DNA packing. For hexagonal packing of the duplexes in solid DNA, n is 6. Therefore, n is taken as 6 for all systems except nucleohistone. For nucleohistone, considering its structure, see Gelbart et al.,30 n is assumed to be 3 for the nucleosome and 6 for so-called linker DNA, we thus estimate an average n of 4.5 for whole nucleohistone. We note that for hexagonal packing there is a
Da(t) ) DI(t) + n(DI(t) - Dds ) + n(DI(t) - 1.73Dds) + n(DI(t) - 2Dds) (7) Molecular Models. Molecular models of SP-DNA, PLLDNA, PEI-DNA, OCT-DNA, and DOD- DNA complexes were produced with Spartan 5.0. The Merck Molecular Force Field (MMFF94) was employed to optimize the molecular geometry of SP-DNA, PLL-DNA, PEI-DNA with the DNA structure frozen and bonds drawn between each pair of positive N in amines and negative P in DNA. Idealized molecular models were produced for OCT-DNA and DOD- DNA complexes. Results Hydrated MX-DNA Solids. The ESR spectra of MX-DNA solids at 7 hydration levels of 4.3, 6.4, 13.3, 14.3, 20.9, 22.3, and 29.8 D2O/nucleotide were followed at increasing time intervals after irradiation, respectively. Linear least-squares fits of benchmark spectra to experimental spectra yield estimates of the fractional composition of MX radical (MX‚), guanine cation radical (G‚+), one electron reduced cytosine (CD‚) and thymine anion radical (T‚-). Figure 1 shows first derivative electron spin resonance spectra found 24 h after γ-irradiation of MX-DNA samples (mole ratio of MX/bp as 1/400) at DNA hydration levels of 4.3, 6.4, 13.3, and 22.3 D2O/nucleotide at 77 K. In addition, the linear least-squares fits of benchmark spectra to each experimental spectrum are shown. The spectra and analyses clearly show that MX radicals decrease in relative amount to the DNA radicals with increased hydration levels of DNA. Thus, the transfer of electrons and holes on DNA to MX decreases substantially with increasing hydration level. There are two possible reasons for this: (1) increased separation between the DNA duplexes with hydration and (2) increased fraction of more stable DNA base radicals CD‚ versus the less stable T‚- (see Supporting Materials). At all hydration levels, the fraction of MX. is found to increase with time after irradiation, while the fractions of G‚+ and the sum of CD‚ and T‚- decrease with time as expected for transfer of holes and electrons to MX. The overall apparent transfer distance (Da) was calculated from the fraction of MX found in MX-DNA solids at various hydration levels. MX captures both holes and electrons and thus its fraction gives the overall transfer of DNA radicals. Figures 2-4 show plots of Da(MX), Da (G‚+), and Da (CD‚ and T‚-) versus natural log time (after irradiation), respectively. A linear dependence of the apparent transfer distance on ln t is clearly observed for all hydration levels in all three figures, suggesting a tunneling process.
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Figure 1. First derivative electron spin resonance spectra found 24 h after γ-irradiation of MX-DNA samples (1 MX:400 bp) at various DNA hydration levels at 77 K. The spectra clearly show that MX radicals decrease in relative amount to the DNA radicals with increased hydration levels of DNA. Thus, the fraction of electrons and holes captured by MX is found to decrease with hydration level. The solid lines are the linear leastsquares fits of benchmark ESR spectra of MX radical, G‚+, CD‚, and T‚- to experimental spectra (dots). The three markers are each separated by 13.09 G. The central marker is at g ) 2.0056.
Figure 2. Plot of the apparent radical transfer distance (Da(MX‚)) vs natural logarithm of time in minutes after irradiation for 1:400 MXDNA solids at various hydration levels. The apparent transfer distance is calculated from the fraction of MX radicals in the overall ESR spectrum using eq 1.
Figure 3. Plot of the apparent hole transfer distance (Da (G‚+)) vs natural logarithm of time in minutes after irradiation for 1:400 MXDNA solids at various hydration levels. The apparent transfer distance is calculated based on the change of the fraction of G‚+, relative to its initial amount formed in pure hydrated DNA after irradiation, using eq 2.
As the hydration level increases from Γ ) 4 to 22 D2O/ nucleotide, both the intercept (Da(1 min)) and the slope (1/R) decrease. Hydration waters up to Γ ) 22 D2O/nucleotide are in an amorphous or glassy state, whereas after 22 D2O/nucleotide a separate ice phase is formed, which steals from the amorphous phase leaving a remaining amorphous hydration level at around 14 D2O/nucleotide with those beyond 14 D2O/nucleotide in the ice phase.15,19 Figure 5 shows the plot of the apparent transfer rate at t ) 1 min of electrons, holes, and overall DNA radicals versus hydration levels as well as versus corresponding interaxial
distances between DNA duplexes (Dds), as estimated from the work of Lee et al.27 Note that the values at 1 min are extrapolated intercepts as experiments were performing at longer time scales. The results show that as hydration increases up to Γ ) 22 D2O/nucleotide, Dds increases and the transfer rate decreases. The plot also clearly shows the near equivalent transfer rates for the hydration levels of 14 and 30 D2O/ nucleotide. Figure 6 shows the apparent transfer distances (t ) 1 min) of electrons, holes and overall DNA radicals after irradiation at 77 K vs hydration levels as well as the corresponding inter-helical axis distance (Dds). As the glassy hydra-
Electron and Hole Transfer in DNA
Figure 4. Plot of the apparent electron-transfer distance (Da (CD‚ and T‚-)) vs natural logarithm of time in minutes after irradiation for MXDNA solids at various hydration levels. The apparent transfer distance is calculated based on the fraction of one-electron reduced DNA bases (sum of CD‚ and T‚-) relative to their initial amount formed in pure hydrated DNA after irradiation, using eq 3.
Figure 5. Plots of the transfer rates of electrons, holes and overall DNA radicals at 77 K vs hydration levels (lower axis) as well as vs the distance between DNA ds’s (upper axis). Values of Dds are estimated from the work of Lee et al.27 The results show that as amorphous (glassy) hydration increases up to Γ ) 22 D2O/nucleotide, Dds increases and transfer rate decreases. At Γ ) 30 D2O/nucleotide, the ice is formed, and leaves the actual amorphous hydration level at around 14 D2O/ nucleotide with the remainder in the ice phase. The plot clearly shows equivalent transfer rates for both hydration levels at 13/14 and 30 D2O/ nucleotide. This result suggests that Dds plays an important role in hydration-dependent hole and electron transfer in DNA.
tion level increases (and consequently Dds increases), the apparent transfer distance decreases. The apparent transfer distances for hydration level of 14 and 30 D2O/nucleotide are equivalent, as for the rates and again result from the formation
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Figure 6. Plots of the apparent transfer distances of electrons (from the decrease in C‚ and T‚-), holes (from the decrease in G•+) and overall DNA radicals (from the increase in MX•) at 1 min after irradiation at 77 K vs hydration levels (lower axis) as well as vs the distance between DNA ds’s (upper axis). The figure clearly shows the transfer distances decrease as the glassy hydration level increases.
of ice phase at Γ ) 30 D2O/nucleotide which reduces the glassy layer to 14.15,19 Amine Cation-MX-DNA Solid Complexes and MXNucleohistone. To further probe the role of Dds on the transfer of DNA radicals in MX-DNA solids, the sodium counterion was replaced by various aliphatic amine cations, e.g., spermine tetrahydrochloride (SP), dodecyltrimethylammonium bromide (DOD), and octadecyltrimethylammonium bromide (OCT), and polymeric amine cations, e.g., poly-L-lysine hydrobromide (PLL), and polyethylenimine hydrochloride (PEI). These species have different molecular sizes and complexing characteristics that allow some control on the separation between the DNA duplexes in MX-DNA samples.28,29,31,32 Figure 7 shows the cross-sectional view of optimized molecular models of SP-DNA, PLL-DNA, and PEI-DNA, and idealized molecular models of DOD-DNA and OCT-DNA complexes. These models show potential geometries and spacing induced by these complexing species in solid samples. The hexagonal packing arrangements of SP-DNA and OCT-DNA are depicted in Figure 8, clearly showing the impact of the molecular size and orientation of the complexing agent on the separation distance between DNA duplexes (Dds).28,29 Nucleohistone consists of nucleosomes (the core particles) connected by short lengths of so-called linker DNA. The structure of nucleohistone can be seen in reference.30 In nucleosomes, a length of DNA containing 146 base pairs wraps 1.75 times around a cylinder-shaped octamer of histone proteins. The radius of octamer is about 50 Å and larger than the tunneling distance, thus the nucleohistone DNA duplexes are partially shielded from each by the histone proteins and provide an example which shows smaller number of adjacent DNA duplexes within the tunneling range but keeps similar separation distance between DNA duplexes. Figure 9 shows the first derivative electron spin resonance spectra found after γ-irradiation of dry DOD-DNA (A), dry OCT-DNA (D), and nucleohistone in frozen D2O aqueous solution (H) at 77 K. All spectra were taken at 77 K. Below
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Figure 7. Minimized molecular models of (A) DNA, (B) SP-DNA, (C) PLL-DNA, and (D) PEI-DNA, and idealized molecular models of (E) DOD-DNA and (F) OCT-DNA.
these spectra are the spectra of irradiated dry DNA (B,E) and irradiated DNA in a frozen aqueous solution (I). The result of subtraction of appropriate amounts (90, 75, and 70% for DODDNA, OCT-DNA, and nucleohistone, respectively) of DNA spectra from that of the complexes are shown in figures C, F, and J. These spectra are from the complexing agents. Figure 9G is that of dry solid OCT, compares well with 9F. Similar subtraction methods show that DOD radicals contribute 25% of the total ESR signal in our samples of (DOD)2-DNA. We did not observe a contribution from SP, PLL and PEI to the overall ESR spectra found for SP-DNA, PLL-DNA and PEIDNA, respectively. We believe this is a result of complete electron transfer to DNA. Some holes formed on the complexes likely also transfer and the remaining holes (probably deprotonated to neutral radicals) are not readily observed. In the analysis of the fraction of MX radicals relative to the sum of MX radicals and DNA base radicals, the background ESR signal (10, 25, 25, and 30% for DOD radicals in DOD-DNA, OCT radicals in OCT-DNA, DOD radicals in (DOD)2-DNA and protein radicals in nucleohistone, respectively) are subtracted from the corresponding ESR spectrum of DNA complexes. In our analyses, we assume that electron transfer from DOD, OCT and protein radicals33 to DNA is fast and that no transfer from the remaining neutral radicals on the complexing species occurs.
Figure 10 shows the plots of the apparent transfer distances of the overall DNA radicals (as measured by the increase in MX•) in fully hydrated solids of MX-DNA, SP-MX-DNA, PLLMX-DNA, and PEI-MX-DNA vs natural logarithm of time in minutes after irradiation. The mole ratio of MX to bp is 1/400 for all four samples. The apparent transfer rate at 1 min (in bps/min) for four complexes are in this order: SP-MX-DNA (3.0 ( 0.4) ) MX-DNA (3.0 ( 0.2) > PEI-MX-DNA (2.1 ( 0.2) ) PLL-MX-DNA (2.0 ( 0.2). As would be expected, the apparent transfer distances at 1 min after irradiation (in bps) are in the same decreasing order: SP-MX-DNA (36 ( 2) > MX-DNA (32 ( 1) > PEI-MX-DNA (30 ( 2) ) PLL-MXDNA (30.5 ( 1). Figure 10 also shows the plot of the apparent transfer distances (measured by fraction of MX radicals) in frozen MXnucleohistone versus natural logarithm of time in minutes after irradiation. The results shown are from three samples at MX:bp loadings of 1:205 to 1:352. Little effect of MX loading on the results is noted. The data for frozen MX-DNA has been presented in Figure 10 of our previous paper24 and is not shown here. The apparent transfer rate and transfer distance at 1 min after irradiation are 1.3 ( 0.1 bps/min and 32 ( 1 bps for nucleohistone, 1.5 ( 0.3 bps/min and 35 ( 2 bps and for DNA,
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Figure 8. Hexagonal arrangements of DNA packing of SP-DNA and OCT-DNA. The figure of OCT-DNA shows an axial down view of a DNA oligomer with one lipid at each phosphate.
Figure 9. First derivative electron spin resonance spectra found after γ-irradiation of dry DOD-DNA (A), dry OCT-DNA (D) and nucleohistone in frozen D2O aqueous solution (H). All spectra were taken at 77 K. Under these spectra are the spectra of irradiated dry DNA (B,E) and irradiated DNA in a frozen aqueous solution (I). The result of subtraction of appropriate amounts of DNA spectra from that of the complexes are shown in figures C(A-0.9B), F(D-0.75E), and J(H-0.7I). Figure 9G is that of dry solid OCT for comparison. The three markers above Figure 9A, 9D, and 9H are each separated by 13.09 G. The central marker is at g ) 2.0056.
respectively. As a consequence of smaller number of adjacent DNA duplexes within the tunneling range, the slightly slower electron transfer in nucleohistone is expected. Figure 11 shows plots of the apparent transfer distances based on the increase in MX ESR signal in freeze-dried solids of MXDNA, DOD-MX-DNA, OCT-MX-DNA, and (DOD)2-MXDNA versus natural logarithm of time in minutes after irradiation. This gives an average of both hole and electron transfer. The MX:bp loadings are 1:107 for OCT-MX-DNA and 1/175 for both DOD-MX-DNA samples. The apparent transfer rates
at 1 min (in bps/min) for four complexes are MX-DNA (3.9 ( 0.2) > DOD-MX-DNA (2.5 ( 0.2) > OCT-MX-DNA (1.9 ( 0.2) > (DOD)2-MX-DNA (1.1 ( 0.1). The apparent transfer distances at 1 min after irradiation (in bps) are MX-DNA (44 ( 2) > DOD-MX-DNA (29 ( 1) > OCT-MX-DNA (24 ( 1) > (DOD)2-MX-DNA (13 ( 1). These results show that the length of the alkyl chain reduces the inter-duplex electron and hole transfer rate. The addition of a second layer of aliphatic amine cations greatly suppresses the transfer of DNA radicals in freeze-dried solids.
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Figure 10. Plots of the apparent transfer distances of the overall DNA radicals in fully hydrated MX-DNA-amine cation complex solids (SPMX-DNA, PLL-MX-DNA and PEI-MX-DNA) and frozen MXnucloehistone D2O aqueous solutions vs natural logarithm of time in minutes after irradiation, showing the effect of spacing between DNA duplexes as altered by SP, PLL, and PEI, and the decreased number of the adjacent DNA ds’s within the tunneling range in nucleohistone. A plot of the apparent transfer distances for DNA radicals in fully hydrated MX-DNA solid are also included for comparison.
Figure 11. Plots of the apparent DNA radical transfer distances measured by the increase in MX radical with time for freeze-dried MXDNA-aliphatic amine cation complexes vs natural logarithm of time in minutes after irradiation. Increasing amounts of lipid between DNA duplexes increases separations and reduces electron transfer between DNA duplexes. Transfer along the strand dominates for DNA samples with a DOD double layer. A plot of the apparent transfer distance for DNA radicals in dry MX-DNA solid are also included for comparison.
Discussion Hydrated MX-DNA Solids. Whether the recombination of G‚+ and T‚- might extensively compete with the hole transfer from G‚+ to MX is a key concern when we derive the hole transfer distance based on the decrease of the fraction of G•+.
Cai et al. The recombination of G‚+ and T‚- leads to neutral base T and G, thus loss of spin signal. However, the total number of spins in irradiated hydrated MX-DNA sample does not show apparent decreases with time. Previous work with hydrated DNA showed decay with time resulting from ion recombinations at high doses at which samples become nearly saturated with radicals.15 The dose used in this work is only 2.6 kGy at which the radicals are far below saturation. On the other hand, MX largely prevents recombination by capture of the intermediates. As shown in Supporting Materials, the fraction of G‚+ remains relatively constant with time in the hydrated pure DNA samples at 2.6 kGy. Other experiments with MX-DNA samples at gamma 13 varied the loading ν from 1/400 to 1/100 MX/bp and gave the same value of Da (1 min) within experimental error although the values of R varied and are likely more sensitive to recombination. Thus, we are confident even though the recombination of G‚+ and T‚- does occur to some extent, it does not compete with the hole transfer from G‚+ to MX sufficiently to affect our values of Da (1 min) reported. At 77 K, the fraction of CD‚, relative to total radicals in MXDNA is not found to be significantly dependent on time after irradiation, in contrast to MX‚, G‚+, and T‚-. Because the conversion of T‚- to CD‚ is observed in pure DNA solids at 77 K, the lack of time dependence of CD‚% may result from similar rates of the electron transfer from CD‚ to MX and from T‚- to CD‚. Thus, the total electron transfer from one electron reduced radicals of DNA (the sum of T‚- to CD‚) to MX is discussed instead of individual T‚- or CD‚. The apparent transfer distance and rates from individual base radicals must be found from experiments with polynucleotides containing only GC or AT base pairs. These studies are presently underway. All measured apparent transfer rates and transfer distances, including that for all DNA radicals combined, as measured by increases in MX., that for holes as measured by the loss of DNA holes (G‚+) and that for electrons as measured by the sum of CD‚ and T‚-, are found to decrease with an increase in the DNA hydration level. The glassy hydration level of DNA affects both the composition of DNA radicals and the distance between DNA duplexes. As shown in Figures 5 and 6, we are confident that Dds plays an important role on the hole and electron transfer in hydrated MX-DNA as suggested in our earlier work24 and that of others.3b,d At this time, it is not certain how the distribution of DNA excess electron radicals between CD‚ and T‚- affects the overall transfer in hydrated MX-DNA. We do know that T‚- is favored at low hydrations and CD‚ is favored at higher hydrations. It is reasonable to suspect the extra stability afforded CD‚ by its protonation (deuteration) would slow the electron transfer from CD‚ and thus favor transfer from T‚-. DI(MX‚), DI (G‚+) and DI (CD‚ and T‚-) are derived from Da’s found for each radical via eq 5 or 6 and are compiled in Table 1. Note that Da sums ET distances on the primary and adjacent duplexes whereas DI estimates the distance of transfer along the primary duplex for each radical species. Although Da’s drop markedly with increasing hydration level, DI’s only slightly decrease with the DNA hydration level. These results are in accord to our explanation for the decreasing apparent transfer distance and rates as hydration level increases: increased separation between the DNA duplexes with hydration is the key factor. Aliphatic and Polymeric Amine Cation-MX-DNA Solid Complexes. Aliphatic and polymeric amine cation-MX-DNA solid complexes were investigated in the dry state (2.5 D2O/ nucleotide) and fully hydrated state (ca. 30 D2O/nucleotide).
Electron and Hole Transfer in DNA
J. Phys. Chem. B, Vol. 105, No. 25, 2001 6039
TABLE 1: Results of Da (1′) and DI(1′) in bps vs DNA Hydration Level for Electron and Hole Transfer to MX in Hydrated MX-DNA Solids CD• and T•- loss Γ(D2O/base) 4.3 6.4 13.3 14.3 20.9 22.3 29.8
G•+ loss
MX• gain
Dds (Å)a
Da (1′)b
DI(1′)c
Da (1′)
DI(1′)
Da (1′)
DI(1′)
20.9 21.4 22.9 23.1 24.6 24.9 23.1
60 ( 5 50 ( 5 42 ( 5 32 ( 4 34 ( 3 36 ( 2 42 ( 5
12.4 ( 11.8 ( 0.7d 11.7 ( 0.7d 10.5 ( 0.6 11.1 ( 0.4 11.4 ( 0.3 11.9 ( 0.7
28 ( 3 27 ( 4 11 ( 7 17 ( 4 8(4 5(4 17 ( 5
9.3 ( 0.4 9.2 ( 0.6 7.4 ( 1 8.2 ( 0.6 7.3 ( 0.6
44 ( 2 39 ( 2 29.5 ( 0.5 27.4 ( 0.5 27.9 ( 0.9 26 ( 2 32 ( 1
11.1 ( 0.3d 10.9 ( 0.3d 10.0 ( 0.1 9.7 ( 0.1 10.2 ( 0.1 10.0 ( 0.3 10.4 ( 0.1
0.7d
8.2 ( 0.7
a
Dds is the inter-duplex center to center separation estimated from the work of Lee et al.27 b Da (1′) is apparent transfer distance at 1 min and is derived from the intercept of a plot of Da (t) vs natural logarithm of time after irradiation, shown in Figure 2 - 4. Da is the sum of transfer distances on all duplexes within the tunneling range. c DI(1′) is the transfer distance on one duplex at 1 min derived by eq 5 assuming n ) 6. d These values for DI(1′) are derived from eq 6 because DI(1′) > 1.73 × Dds the transfer involves a second layer of 6 DNA duplexes.
As shown in Figure 7, the cross-sectional size of the five amine cations increases in the order of spermine tetrahydrochloride (SP) < poly-L-lysine hydrobromide (PLL), polyethylenimine hydrochloride (PEI) < dodecyltrimethyammonium bromide (DOD) < octadecyltrimethylammonium bromide (OCT). The structures shown are suggestive of possible arrangements in the solid state. Those for DOD and OCT are idealized. SP-DNA is reported to have an overall hexagonal packing structure with a single “interhelix” spermine molecule, mediating contacts between neighboring duplexes, and a second “intrahelix” spermine molecule, binding primarily within the minor groove of each of the duplexes in the hexamer. These spermine molecules show a tendency to lie across or between two or three neighboring duplexes, rather than interacting with a single duplex.28 The overall structure with hexagonal symmetry is shown in cross section in Figure 8. For spermine-DNA complexes, our results suggest inter-duplex distances are closer than for extensively hydrated DNA. Figure 7 shows images depicting the increase in separations between DNA duplexes induced by lipid like complexing agents. Obviously, the longer the aliphatic chain, the greater is the expected separation between the DNA duplexes. Recent reports have shown that there is an equilibrium between the columnar hexagonal phase suggested in Figure 8 and a laminar phase of alternating lipid double layer with sheets of DNA.29 Our results with DOD-DNA are consistent only with the columnar hexagonal phase. As shown in Figures 10 and 11, the apparent transfer rate of DNA radicals in hydrated MX-DNA- amine cation complexes decreases in the following order: SP-MX-DNA g MX-DNA > PLL-MX-DNA ) PEI-MX-DNA and in freeze-dry complexes the order of apparent transfer rates is: MX-DNA > DOD-MX-DNA > OCT-MX-DNA. These results clearly support a dependence of the apparent transfer distance on the separation distance between the DNA duplexes and thus explain the effect of amine cations on the electron and hole transfer through DNA. Finally, the addition of a second layer of aliphatic amine cations further suppresses the transfer of DNA radicals to near that found for isolated DNA strands.22 Assuming DI(t) is the same for both MX-DNA and amine cation-MX-DNA solid complexes at the same hydration level, the separation distance between DNA duplexes (Dds) can be roughly estimated by our previously proposed 3D ET model24 with eq 5 or 6. Taking Dds as 20.9 Å for dry DNA (2.5 D2O/ nucleotide) and 23.1 Å for fully hydrated DNA (ca. 30 D2O/ nucleotide), respectively, the Dds for hydrated SP-MX-DNA, PEI-MX-DNA, and PLL-MX-DNA, and dry DOD -MX-DNA, OCT -MX-DNA, and (DOD)2-MX-DNA are derived as 21 ( 2, 24 (1, 25 (1, 28 ( 1, 30 ( 1, and 37 ( 1 Å, respectively.
MX-Nucleohistone. In nucleohistone, the structure and packing on freezing out aqueous solutions keeps the separation distance between DNA adjacent duplexes about that found in hydrated DNA, but the number of adjacent DNA duplexes within the tunneling range are decreased. A structure of nucleohistone can be seen in reference 30. In nucleohistone, a length of DNA containing 146 base pairs wraps 1.75 times around a cylinder-shaped octamer of the four histone proteins H2A, H2B, H3, and H4 with about 25 Å between adjacent duplexes. The nucleohistone DNA duplexes are partially shielded from each by the histone proteins in the core of the structure. On freezing, the nucleosomes are forced in close proximity, placing DNA duplexes on the outside of the nucleosome in juxtaposition. As expected from this structure, see Figure 10, we find slightly slower electron transfer in MXnucleohistone than MX-DNA. This result can be rationalized with the dependence of apparent transfer distance on the smaller average number of adjacent DNA strands, 4.5 rather than 6. The value of n ) 4.5 is justified from a consideration of nucleohistone structure and packing. Using eq 5 and taking Dds as 23.1 Å with n ) 6 for MX-DNA in a D2O ice, the average value of Dds for frozen MX-nucleohistone D2O aqueous solution is found to be 21 ( 3 Å, which is about the separation distance for hydrated DNA and is reasonable considering the nucleosome structure as long as one DNA duplex is forced to be in juxtaposition on freezing. Recently, Lewis et al. reported several definitive works which investigates hole and electron transfer in well-defined systems at ambient temperatures.34 The results yield rates for the excitation driven hole transfer (charge separation) and the subsequent charge recombination event and are shown to be well fit by the Marcus theory. The extrapolated estimate for ko for charge separation is ca. 1 × 1013 s-1 and for recombination ko is ca. 4 × 1012 s-1. Further, they estimate β as 0.7 Å-1 for charge separation and 0.9 Å-1 for recombination. Harriman has also recently reported β values near 1 Å-1 for donor acceptor electron transfer.7 In our work, the radiation process induces charge separation at early times during which some scavenging may occur. However, the dominant transfer in our samples occurs after localization of charge and subsequent tunneling to the intercalator from a number of base pairs away. Our measurements for rates are done long after the initial radiation deposition so that the tunneling we measure is from stabilized ion radicals. These processes are more akin to the recombination event in Lewis et al.’s work. We note that for ambient temperatures other mechanisms involving irreversible protonation reactions of the thymine anion radical would interfere work on long time scales.2a,2c,16,20,23 Our work does involve long times and large distances but the low
6040 J. Phys. Chem. B, Vol. 105, No. 25, 2001 temperatures are used to prevent irreversible protonation and still allow for the observation of fundamental tunneling processes observed at the picosecond time scale at elevated temperatures; however, work at elevated temperatures also includes contributions from activated processes. Conclusions Summary of our Findings. (1) Electron transfer rates and corresponding apparent transfer distances for the transfer from DNA excess electron sites to MX decrease as the hydration level of DNA increases. The dominant effect is clearly the separation distance between DNA duplexes which increases with hydration.27 The dependence of inter-duplex electron transfer on duplex separation has also been reported in our earlier work24 and that of Bernhard and co-workers.3b,d Another effect of the hydration layer is on the free radical composition which at low hydration favors T‚-, and at higher hydration favors the cytosine electron adduct.18 As can be seen in Table 1, the primary distance of travel along the DNA duplex (DI) lengthens somewhat at lower hydration in keeping with the higher amounts of T‚-. (2) Hole transfer rates and corresponding apparent transfer distances for the transfer from G‚+ sites to MX decrease as the hydration level of DNA increases. As with the effect of hydration level on the electron transfer, the increasing separation distance between DNA duplexes with hydration is a key factor; but the effect of DNA hydration level on the prototropic equilibrium within the GC cation radical base pair is likely another factor. The well-known prototropic equilibrium between N-1 on G‚+ and N-3 on C may favor the proton transferred species, ‚G(-H)C(+H) at high hydrations, and aid the stabilization of the hole on guanine.35 This is an interesting possibility that is likely to be amenable to theoretical tests. (3) The apparent ET distances (Da) from the electron adducts (T‚- and CD‚) to MX are found to be nearly twice the hole transfer distances from G‚+ to MX at every hydration level (Table 1). Because Da measures transfer from both the primary and the nearby duplexes a better measure is DI which is the distance of travel along one duplex. We find that the average value of DI for holes is about 72% of that for electron transfer. Clearly, the transfer of excess electrons to MX is more facile than holes at these low temperatures. (4) Replacing sodium counterions with various aliphatic amine cations (lipid like structures) allows for control of the separation of the distance between DNA duplexes. Tunneling is a very sensitive measure of distance and our values of Da yield estimates of these separations between DNA duplexes. With a double layer of lipid the DNA duplexes are nearly isolated so that transfer occurs mainly along one duplex. In effect a lipid double layer provides shielding between the semiconducting DNA duplexes on a distance scale of 37 Å but not true isolation. Previous work with n-alkyl-phthalocyanines has shown that irradiation of the lipid side chains resulted in efficient transfer of charges to the phthalocynanine aromatic groups followed by a charge recombination mainly between adjacent columns of stacked phthalocynanines.36 This system is structurally similar to the DNA-lipid systems under investigation in this work in which we find that inter-DNA duplex recombinations dominate for single lipid layers and intra-DNA duplex recombination dominates for double layers. The value of β was estimated for the phthalocynanine systems to be ca. 1.1 Å-1 at low temperatures and 0.64 Å-1 at higher temperatures.36 Our estimates for β for pure DNA complexed with lipids at low temperatures are also in this range.22-24
Cai et al. (5) Electron transfer in DNA complexed with polyethylenimine, or polylysine shows these agents also separate the DNA strands and hinder electron transfer from one duplex to another. Electron transfer within DNA in nucleohistones is in accord with the structure of the nucleosome in which the histone proteins decrease the number of DNA duplexes in close contact. (6) The fact that electron transfer from adjacent duplexes dominates the ET processes in solid DNA samples and that the tunneling process is very sensitive to distance allows for estimates of the center to center distances between DNA duplexes in the range of 20 to 40 Å. Only when separations between the duplexes are increased beyond 40 Å does the transfer along the primary DNA duplex greatly dominates. Tunneling extent is of course time dependent but the estimate for longer separation ranges than 40 Å would be impractical for most work done with DNA in the solid state. Acknowledgment. This research was supported by the NIH NCI Grant No. RO1 CA45424 and by the Oakland University Research Excellence Fund. Supporting Information Available: As controls for DNA with intercalator, the fractional free radical composition in irradiated pure DNA was investigated at several hydration levels. The ESR spectra of these samples were followed at increasing time intervals after irradiation. The dependence of the fractional composition of various DNA radicals on time and hydration level of DNA is given. This material is available free of charge via Internet at http://pubs.acs.org. References and Notes (1) (a) Meggers, E.; Dussy, A.; Scha¨fer, T.; Giese, B. Chemistry 2000, 6, 485-92. (b) Bixon, M.; Giese, B.; Wessely, S.; Langenbacher, T.; MichelBeyerle, M. E.; Jortner, J. PNAS 1999, 96, 11 713-6. (c) Giese, B.; Wessely, S.; Spormann, M.; Ute Lindemann, S.; Meggers, E.; Michel-Beyerle, M. E. Angew. Chem., Int. Ed. Engl. 1999, 38, 996-998. (d) Meggers, E.; Michel-Beyerle, M. E.; Giese, B. J. Am. Chem. Soc. 1998, 120, 12 950-5. (2) (a) Sevilla, M. D.; Becker, D.; Razskazovskii, Y. Nukleonika 1997, 42, 283-92. (b) Razskazovskii, Y.; Swarts, S. G.; Falcone, J. M.; Taylor, C.; Sevilla, M. D. J. Phys. Chem. B 1997, 101, 1460-7. (c) Yan, M.; Becker, D.; Summerfield, S.; Renke, P.; Sevilla, M. D. J. Phys. Chem. 1992, 96, 1983-9. (3) (a) Debije, M. G.; Bernhard, W. A. J. Phys. Chem. B. 2000, 784551. (b) Debije, M. G.; Bernhard, W. A. Radiat. Res. 1999, 152, 583-9. (c) Debije, M. G.; Milano, M. T.; Bernhard, W. A. Angew. Chem., Int. Ed. 1999, 2752-6. (d) Bernhard, W. A.; Milano, M. T. Radiat. Res. 1999, 151, 39-49. (e) Milano, M. T.; Bernhard, W. A. Radiat. Res. 1999, 152, 196201. (f) Razskazovskiy, Y.; Debije, M. G.; Bernhard, W. A. Radiat. Res. 2000, 153, 436-41. (4) (a) Anderson, R. F.; Wright, G. A. PCCP 1999, 1, 4827-31. (b) Martin, R. F.; Anderson, R. F. Int. J. Rad. Onc. Biol. Phys. 1998, 42, 82731. (c) Anderson, R. F.; Patel, K. B. J. Chem. Soc., Faraday Trans. 1991, 87, 3739-3746. (5) Steenken, S. Bio. Chem. 1997, 378, 1293-7. (6) (a) Nu´n˜ez, M. E.; Barton, J. K. Curr Opin Chem Biol 2000, 4, 199-206. (b) Nu´n˜ez, M. E.; Rajski, S. R.; Barton, J. K. Methods Enzymol 2000, 319, 165-88. (c) Kelley, S. O.; Barton, J. K. Science 1999, 283, 375-81. (d) Wan, C.; Fiebig, T.; Kelley, S. O.; Treadway, C. R.; Barton, J. K.; Zewail, A. H. PNAS 1999, 96, 6014-9. (7) Harriman, A. Angew. Chem., Int. Ed. Engl. 1999, 38, 945-9. (8) Jortner, J.; Bixon, M.; Langenbacher, T.; Michel-Beyerle, M. E. PNAS 1998, 95, 12 759-65. (9) Bixon, M.; Giese, B.; Wessely, S.; Langenbacher, T.; MichelBeyerle, M. E.; Jortner, J. PNAS 1999, 96, 11 713-6. (10) (a) Grozema, F. C.; Berlin, Y. A.; Siebbeles, L. D. A. J. Am. Chem. Soc. 2000, 122, 10 903-9. (b) Grozema, F. C.; Berlin, Y. A.; Siebbeles, L. D. A. Inter. J. Quantum Chem. 1999, 75, 1009-16. (11) Giese, B. Acc. Chem. Res. 2000, 33, 631-6. (12) Giese, B.; Wessely, S.; Spormann, M.; Ute Lindemann, S.; Meggers, E.; Michel-Beyerle, M. E. Angew. Chem., Int. Ed. Engl. 1999, 38, 996998.
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