preparation of oligonucleotides were purchased and utilized as recommended by the instrument’s manufacturer. Given that only thioated oligodeoxyribonucleotides were prepared, the iodine oxidation step of the synthesis cycle was replaced with a sulfurization step employing 0.05 M 3H-1,2-benzo- dithiol-3-one 1,1-dioxide in MeCN, as recommended in the literature (33,34). The sulfurization step was performed before the capping step, and the reaction time for these steps was 120 and 60 s, respectively. The dedimethoxytritylation step of the synthesis cycle was effected over a period of 60 s with a freshly prepared solution of 3% trichloroacetic acid (w/v) in dichloromethane. Oligonucleotide deprotection and purification The synthesis column containing the 50-O-dimethoxytritylated oligonucleotide was placed into a stainless steel pressure ves- sel and exposed to pressurized ammonia (10 bar at 25 C) for 12 h (35). Upon removal of residual ammonia from the pres- sure container, the 50-O-DMTr-oligonucleotide was eluted off the column with 40% MeCN in 0.1 M triethylammonium acetate (TEAA, pH 7.0) (1 ml). The puriﬁcation of each oligonucleotide was accomplished by reverse phase high per- formance liquid chromatography (RP-HPLC) employing a UV detection system and a semi-preparative 5 mm Supelcosil LC-18-S column (10 mm · 25 cm). An elution gradient for the puriﬁcation of 50-O-DMTr-CpG ODN fma1555 or 50-O- DMTr-ODN fma1556 was optimized as follows: starting from 5% MeCN in 0.1 M TEAA (pH 7.0), 1.5% MeCN/min was pumped at a ﬂow rate of 3 ml/min for 30 min. A different elution gradient was however used for the puriﬁcation of 50-O- DMTr-CpG ODN 1555; thus starting from 0.1 M TEAA (pH 7.0), 1% MeCN/min was pumped at a ﬂow rate of 3 ml/ min for 40 min. The product peaks were collected and the eluates were evaporated using a stream of air without heating. The residue was dissolved in 80% acetic acid (1 ml) and the solution was left standing at ambient temperature for 30 min. The acidic solution was also evaporated through the use of a stream of air without a heat source. Each oligo- nucleotide was then dissolved in a solution of 40% MeCN in 0.1 M TEAA (pH 7.0) (1 ml) and puriﬁed by RP-HPLC using the same conditions (column and elution gradient) as those employed for the puriﬁcation of the respective 50-O- DMTr-CpG ODN fma1555, 50-O-DMTr-ODN fma1556 and 50-O-DMTr-CpG ODN 1555. The pooling and evaporation of eluates containing each puriﬁed 50-O-deprotected oligonuc- leotide was performed in a manner similar to that described for the respective 50-O-DMTr-oligonucleotides. After recon- stitution of each puriﬁed oligonucleotide in ddH2O (1 ml), its concentration was determined by UV spectrophotometry at 260 nm. The recovered yields of CpG ODN fma1555 and ODN fma1556 were 65 and 68 OD260 units, respectively, whereas the recovered yield of CPG ODN 1555 was 95 OD260 units. Each oligonucleotide solution was stored frozen at 20 C. Oligonucleotide characterization RP-HPLC analysis of puriﬁed CpG ODN fma1555 was per- formed using an analytical 5 mm Supelcosil LC-18S column (4.6 mm · 25 cm) according to the following conditions: starting from 0.1 M TEAA (pH 7.0), a linear gradient of Nucleic Acids Research, 2005, Vol. 33, No. 11 3553 Figure 1. RP-HPLC profiles of d(GPS(FMA)CPS(FMA)TPS(FMA)APS(FMA)GPS(FMA)- APS(FMA)CPS(FMA)GPS(FMA)TPS(FMATPS(FMA)APS(FMA)GPS(FMA)CPS(FMA)- GPS(FMA)T) [CpG ODN fma1555]. Magenta line: Chromatographic profile of purified CpG ODN fma1555. Brown line: Chromatographic profile of purified CpG ODN fma1555 that was heated in 1· PBS (pH 7.2) for 626 h at 37 C. Turquoise line: Chromatographic profile of purified CpG ODN fma1555 that was heated in 1· PBS (pH 7.2) at 37 C for 73 h. RP-HPLC analyses were performed using a 5 mm Supelcosil LC-18S column (4.6 mm · 25 cm) accord- ing to the following conditions: starting from 0.1 M TEAA (pH 7.0), a linear gradient of 1% MeCN/min is pumped at a flow rate of 1 ml/min for 40 min. Peak heights of each profile are normalized to the highest peak, which is set to one arbitrary unit. 1% MeCN/min was pumped at a ﬂow rate of 1 ml/min for 40 min. An RP-HPLC proﬁle of the puriﬁed oligonucleotide is shown in Figure 1. Samples of puriﬁed CpG ODN fma1555, CpG ODN 1555 and ODN 1556 were analyzed employing a Waters 1525m binary pump equipped with a Waters 2777 sample manager, which was connected online to an orthogonal Electrospray Ionization Time of Flight (ESI-TOF) mass spectrometer (Micromass LCT Premier, Waters, Milford, ME). The mobile phase for direct introduction of the sample was composed of 0.2% formic acid (40%) and acetonitrile (60%). MS chroma- tograms were acquired in the positive ion mode using an ESI-MS capillary voltage of 3.5 kV, a sample cone voltage of 60 V, and an MCP detector voltage of 2200 V. Desolvation gas ﬂow rate was maintained at 400 l/h, cone gas ﬂow rate at 50 l/h. Desolvation temperature and source temperature were set to 150 and 80 C, respectively. The acquisition range was set at m/z 50–2000. The 2.1 s scan cycle consisted of a 2 s acquisition time and a 0.1 s interscan delay. Instrument cal- ibration was performed routinely in positive ion mode prior to MS experiments by direct infusion of sodium formate in 2- propanol:water (9:1 v/v) at 10 ml/min. Leu-enkephalin was used as a lock mass and MassLynx was the operating software for the MS system. Raw summed spectra were deconvoluted using the MaxEnt1 software. To further characterize CpG ODN fma1555 and ODN fma1556, the puriﬁed oligonucleotides (1 OD260 unit each) were dissolved in 1· Phosphate-buffered saline (PBS, pH 7.2) (0.5 ml) and placed in a heat block, pre-heated to 37 – 2 C, to thermolytically cleave the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group over a period of 626 h. Each fully deprotected oligonucleotide (0.25 OD260 unit) was ana- lyzed by PAGE using a denaturing 20% polyacrylamide–7 M urea gel (40 cm · 20 cm · 0.75 mm), which was prepared as described by Maniatis et al. (36). Electrophoresis was carried out at 350 V until the bromophenol blue dye of the loading buffer traveled 80% of the length of the gel. The gel was then stained by soaking in a solution of Stains-all, as reported elsewhere (10). A photograph of the gel is shown in Figure 2.