Role of Hydrophobic Effects in the Reaction of a Polynuclear Aromatic Diol Epoxide with Oligodeoxynucleotides in Aqueous Solutions

Abstract

The need for large-scale direct synthesis of stereochemically defined and site-specific benzo[alpha]pyrenediol epoxide-oligodeoxyribonucleotide adducts for detailed NMR and other biochemical and physicochemical studies has necessitated a better understanding of variables that lead to an enhancement of the reaction yields. It is shown that, in aqueous solution, the formation of noncovalent hydrophobic complexes between 7r, 8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[alpha] pyrene (BPDE) and the single-stranded oligonucleotide 5'-d(CCATCGCTACC) precedes the covalent binding reaction of BPDE with the single deoxyguanosine residue. The yield of covalent reaction products (involving reaction of BPDE at the C10 position with the exocyclic amino group of the dG residue) increases with increasing DNA concentration and tends toward saturation at oligonucleotide single-strand concentrations above approximately 3 mM. The addition of NaCl (0.3 M) also tends to enhance the adduct reaction yields. However, organic solvents such as tetrahydrofuran in the reaction mixtures (10-40%) decrease the stabilities of the noncovalent complexes, which in turn leads to reductions in the yields of covalent BPDE-dG oligonucleotide adducts. The efficiencies of formation of hydrophobic complexes were probed by fluorescence and UV absorption techniques using the BPDE tetrol hydrolysis product 7,8,9,10-tetrahydroxytetrahydrobenzo[alpha]pyrene as a model system.

Oxidation of Guanine in G, GG, and GGG Sequence Contexts by Aromatic Pyrenyl Radical Cations and Carbonate Radical Anions: Relationship between Kinetics and Distribution of Alkali-Labile Lesions

“…The fractions of BPT molecules in complexes with the DNA were deduced from an analysis of the fluorescence decay profiles determined by timecorrelated single-photon counting techniques 42 as described by us earlier. 41 In air-equilibrated aqueous solutions, the decay of the BPT fluorescence is close to monoexponential with lifetime, τ 0 = 129 ns (see, Figure S1A in Supporting Information).…”

“…These short lifetimes are attributed to BPT molecules in complexes with double-stranded DNA molecules, and the sum of amplitudes I 1 and I 2 are assumed to be equal to the fractions of BPT molecules (ϕ) bound to the DNA. [41][42][43] The values of ϕ monotonously increase with increasing concentrations of DNA duplexes (see, Figure S2 in Supporting Information); at a duplex concentration ([duplex]) of 100 μM, practically all BPT molecules are bound to DNA and the ϕ values are in the range of 0.8 to 0.9 for a series of six oligonucleotide duplexes (Table S2, Supporting Information). The binding constants, K = (1.4±0.3)× 10 5 M −1 were obtained by fitting the following equation (12) to the experimental data points ( Figure S2).…”

Methylation of Cytosine at C5 in a CpG Sequence Context Causes a Conformational Switch of a Benzo[a]pyrene diol epoxide-N2-guanine Adduct in DNA from a Minor Groove Alignment to Intercalation with Base Displacement

“…63 Caution: benzo[a]pyrene diol epoxides are carcinogenic and mutagenic and should be handled with care, as outlined by National Cancer Institute Guidelines. The modified oligonucleotides were separated from unmodified oligonucleotides, and the modified oligonucleotides containing the single stereoisomeric 10S (+) or 10R (−)-trans-anti- [BP]G adduct (G*, Figure 1) were separated from one another utilizing various reversed-phase HPLC protocols as described in detail elsewhere.…”

“…The modified oligonucleotides were separated from unmodified oligonucleotides, and the modified oligonucleotides containing the single stereoisomeric 10S (+) or 10R (−)-trans-anti- [BP]G adduct (G*, Figure 1) were separated from one another utilizing various reversed-phase HPLC protocols as described in detail elsewhere. 63 The stereo-chemical properties of the modified oligonucleotides were established by enzyme digestion of the modified oligonucleotides and HPLC co-elution of the BPDE-modified base with stereochemically defined trans-anti- [BP]G adduct standards. 64 The stereochemical properties of each mononucleotide adduct were identified by circular dichroism techniques, and by co-elution with the corresponding mononucleotide adduct standards as described earlier for the same B[a]PDE-modified oligonucleotides.…”

Evaluation of the noncovalent binding interactions between polycyclic aromatic hydrocarbon metabolites and human p53 cDNA

“…In our rapid fluorescence displacement assay, the time to measure each PAH metabolite did not exceed five minutes. Therefore, the buffer solution was not suitable and the reaction time was not sufficient to form stable covalent adducts, although covalent adducts could form during the incubation of PAH metabolites and DNA in an aqueous solution at room temperature (Geacintov et al, 1991;Urano et al, 1991;Pirogov et al, 1998). Measurements performed under this situation were done to investigate the predominant noncovalent interaction between the metabolites and DNA.…”