e100 Nucleic Acids Research, 2007, Vol. 35, No. 15 PAGE 4 OF 6 Figure 3. The major products of 50-phosphorylated dsDNA identiﬁed by ESI–FT ICR MS. Identiﬁed fragments are indicated in dark gray. Theoretical masses were calculated assuming that CTD hydrolyzes a phosphodiester bond at 50-side of a phosphate. Figure 4. Identiﬁcation of the precise structure of the reaction products. (A) There are several potential structures corresponding to a single mass. Theoretical masses (Mt) are indicated to the right of each DNA fragment. (B) 50-modiﬁcation of the substrate enables the precise structure of the corresponding product peak to be determined. For example, if CTD hydrolyzes a phosphodiester bond at the 50-side of a phosphate, only one kind of 12-mer fragment (whose theoretical mass is highlighted) will retain a hydroxyl moiety at the 50-end. (C) Comparison of the deconvoluted mass spectra of products generated from 50-phosphorylated (a) and hydroxylated (b) substrates. Arrows indicates the shifted masses. Diﬀerences in the masses are almost identical to the diﬀerence in the mass of the phosphoryl and hydroxyl groups. exonuclease activity. Gel electrophoretic analysis also ruled out the possibility of 50 to 30 exonuclease activity of CTD because there was no observable accumulation of short fragments during the incision of 50-labeled substrates (Figure 5). We then tested the oligonucleotide analog where the nucleotide at the 30-terminal end was replaced with LNA, which contains an extra 20-O, 40-C-methylene bridge on the ribose ring. It had been reported that this substrate shows a tolerance to exonuclease activity (23,24). Although the 30–50 exonuclease activity of KOD poly- merase was aﬀected by the replacement of the 30-terminal end, the replacement did not aﬀect the nuclease activity of CTD (Figure 6). Taken together, electrophoretic analysis also demonstrated that CTD does not possess an exonuclease activity. Thus, we have conﬁrmed the validity of the ESI–FT ICR mass spectrometric analysis. The results in this study showed that ESI–FT ICR MS can precisely analyze the digestion pattern of a non- sequence-speciﬁc endonuclease. In comparison, the ana- lysis of a sequence- or structure-speciﬁc endonuclease activity should be relatively straightforward using this methodology. The information about substrate speciﬁcity