Prototropic equilibria in DNA containing one-electron oxidized GC: intra-duplex vs. duplex to solvent deprotonation

Abstract

By use of ESR and UV-vis spectral studies, this work identifies the protonation states of one-electron oxidized G:C (viz. G•+:C, G(N1-H)•:C(+H+), G(N1-H)•:C, and G(N2-H)•:C) in a DNA oligomer d[TGCGCGCA]2. Benchmark ESR and UV-vis spectra from one electron oxidized 1-Me-dGuo are employed to analyze the spectral data obtained in one-electron oxidized d[TGCGCGCA]2 at various pHs. At pH ≥7, the initial site of deprotonation of one-electron oxidized d[TGCGCGCA]2 to the surrounding solvent is found to be at N1 forming G(N1-H)•:C at 155 K. However, upon annealing to 175 K, the site of deprotonation to the solvent shifts to an equilibrium mixture of G(N1-H)•:C and G(N2-H)•:C. For the first time, the presence of G(N2-H)•:C in a ds DNA-oligomer is shown to be easily distinguished from the other prototropic forms, owing to its readily observable nitrogen hyperfine coupling (Azz(N2)= 16 G). In addition, for the oligomer in H2O, an additional 8 G N2-H proton HFCC is found. This ESR identification is supported by a UV-vis absorption at 630 nm which is characteristic for G(N2-H)• in model compounds and oligomers. We find that the extent of photo-conversion to the C1′ sugar radical (C1′•) in the one-electron oxidized d[TGCGCGCA]2 allows for a clear distinction among the various G:C protonation states which can not be easily distinguished by ESR or UV-vis spectroscopies with this order for the extent of photo-conversion: G•+:C > G(N1-H)•:C(+H+) >> G(N1-H)•:C. We propose that it is the G•+:C form that undergoes deprotonation at the sugar and this requires reprotonation of G within the lifetime of exited state.

Reactions of 5-methylcytosine cation radicals in DNA and model systems: Thermal deprotonation from the 5-methyl group vs. excited state deprotonation from sugar

“…Following our works (Adhikary et al 2010, 2011), one-electron oxidized monomers and one-electron oxidized dsDNA oligomers were photoexcited by using thermoelectrically cooled blue laser (TECBL-20G-405, World Star Tech, Lot 6880, λ = 405 nm, 20 mW) at the range 143 to 148 K. Photoexcitation of samples were also carried out by employing a photoflood lamp (250 W) in the presence of water filter (to cut off the infrared (IR) and UV components of this light) along with a 310 nm cut-off filter at the range 143 to 148 K and a low pressure mercury lamp at 77 K as per our previous works (Adhikary et al 2005, 2008; Sevilla et al 1972). …”

“…Following our ongoing studies with the DNA and RNA monomers (Becker et al 2007, 2010a, 2010b; Adhikary et al 2012, 2013b) and DNA and RNA oligomers (Becker et al 2007, 2010a, 2010b; Adhikary et al 2009, 2010, 2012, 2013a; Khanduri et al 2011), homogeneous solution of each monomer was prepared by dissolving ~ 2 to 3 mg of it in 1 ml of 7.5 M LiCl in D 2 O; whereas to prepare a homogeneous solution of a dsDNA oligomer, ~ 1.5 to 2 mg of it was dissolved in 1 ml of 7.5 M LiCl in D 2 O. K 2 S 2 O 8 (5 to 6 mg/ml) was added to each solution as an electron scavenger to investigate only the formation of the one-electron oxidized species and its subsequent reactions. Following the procedure by Sevilla and co-workers (Sevilla et al 1972), homogeneous solution of 5-Me-2'-dC (0.5 mg/ml) was prepared in 8 M NaClO 4 in D 2 O.…”

“…Our previous work (Adhikary et al 2010) has shown that the 8-mer d[TGCGCGCA] 2 remains double stranded in 7.5 M LiCl in D 2 O till ca. 48°C.…”

“…5). pD of the solutions was adjusted, following our earlier works (Adhikary et al 2009, 2010, 2012, 2013b; Khanduri et al 2011), by quickly adding microliter amounts of 1 M NaOH in D 2 O under ice-cooled conditions. Owing to the high ionic strength (7.5 M LiCl) of these solutions, we were not able to use pH meters for accurate pH measurements.…”

Insight Into the Inhibition of Ribonucleotide Reductases by 2'-chloro-2'-deoxynucleotides and 2'-azido-2'-deoxynucleotides: Biomimetic Studies with Model Substrates

“…Following our works on DNA and RNA-radicals, 79,141,142,148,152,[158][159][160][161][162][163][164][165][166] the anisotropic simulations of the experimentally recorded ESR spectra were carried out by employing the Bruker programs (WIN-EPR and SimFonia). The ESR parameters (e.g., hyperfine coupling constant (HFCC) values, linwidth, etc.)…”