2616 Biochemistry, Vol. 49, No. 12, 2010 Scheme 1: (A) 1-BD-Modified Oligodeoxynucleotide Number- ing Scheme and (B) Structure and Numbering Scheme for the 1,N2-εdG Adducta aThe imidazole proton of the 1,N2-εdG nucleotide is designated as H2, corresponding to the H8 proton in guanine. followed by further extension and resulting in a bulged 1,N2-εdG lesion in the template, leading to the 50-CATGA-30 -1 deletion product (30). The deletion of dC opposite 1,N2-εdG increases the duplex Tm as compared to that of the complementary modified duplex, and the bulged duplex is more stable than the corres- ponding duplex involving the deletion of dC complementary to an unmodified dG. Structurally, 1,N2-εdG adopts the anti conformation about the glycosyl bond and is accommodated within the DNA helix. We propose that thermal stabilization of this bulged intermediate may be significant with regard to the processing of the 1,N2-εdG lesion into a -1 deletion mutation. MATERIALS AND METHODS Oligodeoxynucleotide Synthesis and Characterization. The oligodeoxynucleotides 50-d(CGCATGGAATCC)-30 and 50-d(GGATTCATGCG)-30, purified by anion-exchange chro- matography, were obtained from the Midland Certified Reagent Co. (Midland, TX). The 1,N2-εdG phosphoramidite (32) was incorporated into 50-d(CGCATXGAATCC)-30 (X = 1,N2-εdG) with an Expedite 8909 DNA synthesizer (PerSeptive Biosystems), using tert-butylphenoxyacetyl-protected cyanoethyl phosphora- midites and the manufacturer’s standard protocols, on a 1 μmol scale. Oligodeoxynucleotide identity was established by MALDI- TOF mass spectrometry: MALDI-TOF MS of 50-d(CGCATX- GAATCC)-30: calcd for [M - H]- m/z 3652.6, found m/z 3653.0. The identity was also established by chromatographic compar- ison of enzymatic hydrolytic products to authentic nucleoside standards by HPLC. HPLC. Analyses were performed using a YMC ODS-AQ column (Waters, Milford, MA) [250 mm 4.6 mm (inside diameter) and 1.5 mL/min for enzymatic digestion gradient 1 and 250 mm 10 mm (inside diameter) and 5 mL/min for purification gradient 2]; solvents were 0.1 M aqueous ammonium formate and acetonitrile. Eluants were detected by UV absor- bance at 260 nm. For HPLC gradient 1: initially 1% acetonitrile, a 15 min linear gradient to 10% acetonitrile, a 5 min linear gradient to 20% acetonitrile, isocratic for 5 min at 20% acetoni- trile, a 3 min linear gradient to 80% acetonitrile, isocratic for 2 min at 80% B, and a 3 min linear gradient to initial conditions. For HPLC gradient 2: initially 1% acetonitrile, a 5 min linear gradient to 8% acetonitrile, a 15 min linear gradient to 11% Shanmugam et al. acetonitrile, a 2 min linear gradient to 80% acetonitrile, isocratic for 1 min at 80% acetonitrile, and a 2 min linear gradient to initial conditions. Capillary Gel Electrophoresis. Analyses utilized a Beckman P/ACE MDQ system (Beckman Instruments, Fullerton, CA; 32 Karat software, version 7.0) monitored at 260 nm using a 31.2 cm 100 μm eCAP capillary with samples applied at 10 kV and run at 9 kV. The capillary was packed with the manu- facturer’s 100-R gel [for single-stranded DNA (ssDNA)] using a Tris-borate buffer system containing 7 M urea. Enzymatic Digestion. Enzymatic hydrolysis was conducted in one step. The oligodeoxynucleotide (0.5 A260 unit) was dissolved in 30 μL of buffer [10 mM Tris-HCl and 10 mM MgCl2 (pH 7.0)] and incubated with DNase I (8 units, Promega), snake venom phosphodiesterase I (0.02 unit, Sigma), and alkaline phosphatase (1.7 units, E. coli; Sigma) at 37 C for 24 h. The mixture was analyzed by reversed phase HPLC gradient 1. Adducted nucleosides were identified by comparison with authentic samples on the basis of retention times, co-injection, and UV spectra. Mass Spectrometry. MALDI-TOF mass spectra (negative ion) of modified oligodeoxynucleotides were obtained on a Voyager Elite DE instrument (Perseptive Biosystems) using a 3-hydroxypicolinic acid matrix containing ammonium hydrogen citrate (7 mg/mL) to suppress multiple sodium and potassium adducts. UV Melting Studies. Samples contained 0.30 A260 unit of duplex oligodeoxynucleotide in 1 mL of buffer containing 10 mM Na2HPO4/NaH2PO4, 0.1 M NaCl, and 5 μM Na2EDTA (pH 7.0) in a 1.0 cm cuvette. The UV absorbance was monitored at 260 nm. A 2.5 C/min temperature gradient was used. The temperature was cycled between 20 and 85 C. The first derivative of the melting curve was used to establish the Tm values. NMR Spectroscopy. Oligodeoxynucleotide concentrations were determined using calculated extinction coefficients at 260 nm (33). The modified and complementary strands were annealed in a 1:1 molar ratio. The duplex was eluted from DNA grade Biogel hydroxyapatite (Bio-Rad Laboratories, Richmond, CA). The duplex was desalted by elution from Sephadex G-25. The sample was lyophilized and prepared in 500 μL of buffer containing 10 mM NaH2PO4, 100 mM NaCl, and 5 μM Na2EDTA (pH 7.0). The duplex concentration was 0.3 mM. Samples for the observation of nonexchangeable protons were prepared in 99.99% D2O. Samples for the observation of exchangeable protons were prepared in a 9:1 H2O/D2O mixture. NMR spectra were recorded at a 1H frequency of 600 MHz. The temperature was 25 C for observation of nonexchangeable protons and 7 C for observation of exchangeable protons. Chemical shifts of the proton resonances were referenced to the water resonance at 4.77 ppm at 25 C and 4.95 ppm at 7 C. For assignment of exchangeable protons, NOESY experiments were conducted using the Watergate pulse sequence (34). The mixing time was 250 ms. For the assignments of nonexchangeable protons and derivation of distance restraints, NOESY spectra were recorded using States-TPPI phase cycling with mixing times of 150, 200, and 250 ms. A relaxation delay of 2.0 s was used. Experiments were recorded with 512 real data points in the t2 dimension and 2K real data points in the t1 dimension. The data in the t2 dimension were zero-filled to give a matrix of 2K 2K real points. Chemical shifts were referenced to water. The NMR data were processed on engineering workstations (Dell, Round Rock, TX) using FELIX2000 (Accelrys, San Diego,