PAGE 3 OF 8 Designation Fl-RTg Fl-STg Cy3-STg Cy5-DHT Fl-T aRef. 24. Table 1. Oligonucleotide probes used for the E. coli Endo III and human NTH1 reactions Base (X) (5R)-Tg (5S)-Tg (5S)-Tg DHT T Fluorophore (Y) Fluorescein Fluorescein Cy3 Cy5 Fluorescein Quencher (Z) Dabcyl Dabcyl BHQ2 BHQ2 Dabcyl Excitation Wavelength (nm) Emission Wavelength (nm) Quenching Eﬃciencya (%) 494 494 547 646 494 520 520 563 662 520 91 91 93 96 91 Nucleic Acids Research, 2010, Vol. 38, No. 7 e101 cyanine dyes (Cy3TM and Cy5TM), the controlled-pore glass (CPG) for the 30-end modiﬁcation with 4-(dimethyla- mino)azobenzene (Dabcyl), and the Beaucage sulfurizing reagent were purchased from Glen Research. BHQ-2 CPG was purchased from Biosearch Technologies. Chain assembly and deprotection were performed according to the manufacturers’ recommendations. Cleavage from the support and removal of the alkali-labile protecting groups were simultaneously performed with ammonia water at room temperature for 2 h. The tert- butyldimethylsilyl group for the protection of the Tg was removed with triethylamine trihydroﬂuoride, as described previously (9). After deprotection, the oligonucleotides were analyzed and puriﬁed on a Gilson HPLC system, using a Waters mBondasphere C18 5 mm 300 A˚ column (3.9 150 mm) with a linear gradient of acetonitrile in 0.1 M triethylammonium acetate at pH 7.0. A Waters 2996 photodiode array detector was used for analysis. Quantiﬁcation of oligonucleotides Aliquots of oligonucleotides were incubated with DNase I (50 U; Takara) in a buﬀer (30 ml), containing 40 mM Tris– HCl (pH 7.5), 8 mM MgCl2 and 5 mM dithiothreitol (DTT), at 37 C for 24 h. Then, a buﬀer (5 ml), containing 0.3 M ammonium acetate (pH 4.6), 2.8 M NaCl and 10 mM ZnSO4, water (10 ml), and S1 nuclease (5 ml, 800 U; Takara) were added, and the mixture was incubated again at 37 C for 24 h. This mixture was diluted with water (2.5 ml), and a buﬀer (14 ml), containing 0.5 M Tris–HCl (pH 9.0) and 10 mM MgCl2 and alkaline phosphatase (3.5 ml, 14 U; Takara) were added. After an incubation at 37 C for 2 h, the products were analyzed by high-performance liquid chromatography (HPLC) using an Inertsil ODS-2 column (4.6 150 mm; GL Sciences) with a linear gradient of acetonitrile (from 0 to 10% for 20 min) in 0.1 M triethylammonium acetate at pH 7.0. The molar concentrations of the original solutions were calculated by comparing the peak areas with those obtained from an unmodiﬁed oligonucleotide with a known molarity. Analysis of the Endo III reaction by gel electrophoresis Escherichia coli Endo III was purchased from New England Biolabs. Sample solutions (250 ml) contained each oligonucleotide (37.5 pmol) and E. coli Endo III (the amounts are shown in the legend to Supplementary Figure S3), in a buﬀer consisting of 20 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.1 mg/ml bovine serum albumin (BSA). After an incubation at 37 C for 30 min, followed by heating to 95 C for 5 min, the mixtures were evaporated on a SpeedVac concentrator, and the residues were dissolved in water (10 ml). Each sample was mixed with 90% formamide (10 ml) containing bromophenol blue, heated to 95 C for 3 min and loaded onto a 20% polyacrylamide gel containing 7.5 M urea, 89 mM Tris borate (pH 8.3) and 2 mM EDTA. Electrophoresis was performed at a constant power of 35 W. The bands were detected on a FujiFilm FLA-7000 Bio-imaging system at Osaka University Radioisotope Research Center, using the Y520 and O580 ﬁlters for ﬂuorescein and Cy3, respectively. Measurement of ﬂuorescence intensity Human NTH1 was prepared as described previously (18). Sample solutions (250 ml) contained each oligonucleotide (12.5 pmol) and the enzyme (the amounts are shown in Figures 3, S4 and S5) in a buﬀer consisting of 20 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.1 mg/ml BSA. After an incubation at 37 C for 30 min, the ﬂuorescence intensity was measured at 37 C on a JASCO FP-6500 spectroﬂuorometer equipped with an EHC 573 temperature controller. The excitation and emission wavelengths for each ﬂuorophore are listed in Table 1. The excitation and emission bandwidths for the measurement of ﬂuorescein were 3 nm, and those for the cyanine dyes were 5 nm. The measurement was repeated three times, and the data were averaged. The 100% cleavage was deﬁned as the ﬂuorescence intensity obtained for 50 nM solutions of Y-ACGCGA, in which Y represents a ﬂuorophore, at 37 C in the above- mentioned buﬀer. Determination of kinetic parameters Fl-RTg and Fl-STg (2–75 and 0.3–10 nM, respectively) were incubated with E. coli Endo III (101 pM) at 37 C, in a buﬀer (250 ml) consisting of 20 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.1 mg/ml BSA. The ﬂuorescence intensity was measured at 1-min inter- vals, in the same way as described above, and using these velocity data, kinetic parameters were calculated from Lineweaver–Burk plots.