e101 Nucleic Acids Research, 2010, Vol. 38, No. 7 PAGE 2 OF 8 Figure 1. Scheme of the BER pathway. The short-patch sub-pathway is shown. The ﬁlled and open circles represent damaged and undamaged bases, respectively. complementary strand. The obtained 32P-labeled duplex is used as a substrate, and after the incubation with an enzyme, the product is analyzed by denaturing poly- acrylamide gel electrophoresis, followed by detection and quantiﬁcation of the radioactive bands (8,9). However, there are some drawbacks in this type of experiment. In addition to the hazardous nature of the radioisotope, the substrates must be enzymatically labeled through a laborious procedure, and the half-life of 32P is quite short. It is sometimes diﬃcult to accurately determine the molar amount of the labeled oligonucleotide. The enzyme reaction with only a single substrate is monitored in one experiment, and, most importantly, the product must be separated from the intact substrate. In this study, we have developed probes for detecting the excision step of the BER reactions by ﬂuorescence. For this purpose, lesion-containing oligonucleotides bearing a ﬂuorophore and a quencher, which were originally used as molecular beacons (10), were prepared, as shown in Figure 2A and Table 1. While the molecular beacon reports the existence of its target sequence, our hairpin-type BER probe emits ﬂuorescence when the strand is cleaved by a DNA glycosylase/AP lyase. Figure 2. Structures of the oligonucleotide probes (A) and the damaged bases (B) used in this study. The principle is that the ﬂuorophore-labeled 50 fragment of our probe is short enough to dissociate from the quencher strand at the incubation temperature, upon enzymatic cleavage. Destabilization of the duplex upon enzymatic strand scission was used previously to analyze the reaction of a bifunctional enzyme, formamido- pyrimidine glycosylase, but the reaction was monitored by hyperchromicity (11). Various studies using the molecular beacon-type ﬂuorophore/quencher system have been reported thus far. One example related to this study is the use of uracil-containing probes for the analysis of uracil DNA-glycosylase reactions (12,13). The probes contained several uracil bases, and when uracil DNA-glycosylase, which is a monofunctional enzyme, hydrolyzed the glycosidic bonds of the uracil bases, the ﬂuorophore- and quencher-strands were dissociated. This method was used for in vitro (12,13) and in vivo (12,14) analyses. In this study, we incorporated damaged bases induced by free radicals, namely thymine glycol (5,6-dihydro-5,6- dihydroxythymine, Tg) and 5,6-dihydrothymine (DHT), only at a single site, and analyzed the reactions of two bifunctional DNA glycosylases, Escherichia coli Endo III and its human homolog, NTH1. The substrate speciﬁcity was successfully determined by this method. One of the probes was further modiﬁed by incorporating phospho- rothioate linkages, which could prevent non-speciﬁc degradation, and was used to detect the initial step of BER in HeLa cells. MATERIALS AND METHODS Synthesis of oligonucleotide probes Oligonucleotides were synthesized on an Applied Biosystems 3400 DNA synthesizer, using the ultramild phosphoramidites (Glen Research) for the normal bases. The phosphoramidite building blocks for the incorpora- tion of (5R)- and (5S)-Tg were prepared as described previously (15,16). To simplify the synthesis of the (5R)- Tg-containing oligonucleotides, a building block protected with acetyl groups (17) was prepared, as described in the Supplementary Data, and this building block was used for the synthesis of phosphorothioate-containing oligonucleotides. The building block of (5S)-DHT, the phosphoramidites for the attachment of ﬂuorescein and