## 3 Results
### Crosslinking Reactions to DNA Methyl Transferase  
We chose cytosine-5-DNA methyl transferase (DNMT) as a model DNA binding protein. DNMT performs the methylation of cytosines for epigenetic modification of the genome that is involved in regulating many cellular processes [6]. In addition, aberrant methylation can cause many diseases, such as cancer [22; 23; 24]. The efficient inhibition of DNMT offers the possibility of interfering with the methylation process, which may allow control of the epigenetic regulation of cells and treat various cancers [25]. The mechanism of the cytosine-5 methylation with DNMT is illustrated in Figure 6A. A thiol of the Cys residue in the active site of DNMT acts as a nucleophile and attacks the C6 position of the cytosine to form an enzyme-linked intermediate. The resulting nucleophilic C5 position of the cytosine is then methylated by S-adenosyl-l-methionine (SAM). Subsequent abstraction of the proton at the 5 position and b-elimination provides the 5-substituted pyrimidine and active enzyme (Figure 6A) [26]. Based on this mechanism, the modified bases forming a covalent bond to DNMT were reported as irreversible inhibitors [27; 28; 29; 30; 31]. We expected that _acy_AOVT (**2**) replacement of the cytosine methylated by DNMT can react with the Cys residue in this enzyme (Figure 6B). In our investigation, we used the bacterial DNMT, i.e., the commercially-available Hha1 DNMT-1 from _Haemophilus haemolyticus_. Hha1 DNMT-1 methylated the internal 2\({}^{\prime}\)-deoxycytidine in the target sequence 5\({}^{\prime}\)-G\(\underline{\text{C}}\)GC-3\({}^{\prime}\), in which AOVT was substituted in place of the internal 2\({}^{\prime}\)-dC. We synthesized **ODN6**, which contained AOVT in the Hha1 DNMT-1 recognition sequence and evaluated the crosslink reactivity to DNA. The base selectivity was similar, but the reactivity to the target DNA was significantly lower compared to that of the previous sequence (Figure S12). We selected DNA2 containing methyl cytosine as a target DNA because Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA. The methylation conditions were determined using the duplex DNA between DNA**2** and DNA**3** contained cytosine instead of acyclic AOVT and the mono-methylated product in DNA**3** was confirmed by MALDI-TOF MS. The duplex DNA (**ODN6** and **DNA2**) was incubated with Hha1 DNMT-1 in the reaction buffer containing 5 mM 2-mercaptoethanol at 37 \({}^{\circ}\)C in the determined conditions. The reaction mixture was analyzed by sodium dodecyl sulfate \(-15\%\) polyacrylamide gel electrophoresis (SDS\(-\)PAGE) and visualized with  
Figure 5: Comparative thermal denaturing analysis of duplexes **ODN7**/**DNA1** or **RNA1**. \(T_{\text{m}}\) values were measured in 50 mM MES buffer (pH 7.0) containing 100 mM NaCl. Figure 6: **(A)** Plausible mechanism for methylation by cytosine-5 methyltransferases (DNMTs) with SAM as a cofactor (**B**) Possible mechanism for covalent trapping with AOVT (**2**) derivative. silver staining. The slower mobility band (*) was observed around 51 kD and not observed with the non-reactive duplex DNA and Hha1 DNMT-1 (Figure 7A). We performed the reaction using the 5' FAM-labelled **ODN6** and analyzed by SDS-PAGE. The slower mobility band stained silver matched the fluorescent band in the reaction using the 5' FAM-labelled **ODN6** (Figure 7B) and this band was derived from the complex between Hha1 DNMT-1 and the duplex DNA. The quantification of the products was next performed by using the FlamingoTM Gel staining in-gel fluorescence analysis. We confirmed the linear correlation between the protein amount and the fluorescence intensity. Thus, the complex between the protein and ODN can be quantified by measuring the fluorescence intensity on the gel (Figure 7C).