## 3 Results
### Synthesis of the Oligonucleotides Containing acy_AOVT and Evaluation of the Crosslinking Reactivity  
We synthesized two kinds of **ODNs1**, **2** by a DNA synthesizer using phosphoramidite **10** (Scheme 2). The synthesized ODNs were treated with 45 mM K\({}_{2}\)CO\({}_{3}\)/MeOH containing 10 mM 1-octanethiol to cleave them from the resin. After the DMTr-ON purification by reverse-phase (RP) HPLC, detritylation was carried out in an aqueous 10% AcOH solution at room temperature for 30 min. The obtained ODNs were further purified by RP-HPLC and were characterized by a MALDI-TOF MS analysis. The sulfide group of ODNs**1**, **2** was oxidized with magnesium monoperoxyphthalate (MMPP) and treated with aqueous 10% AcOH to give ODNs5 and **6** according to the reported procedure [20]. The characterization of the ODNs was performed by MALDI-TOF MS.  
**Scheme 2. **_Reagents and conditions_: (**a**) DNA synthesizer; (**b**) 45 mM K\({}_{2}\)CO\({}_{3}\)-MeOH, 10 mM 1-octanethiol, rt, 4 h; (ii) 10% AcOH, rt, 30 min. ; (**c**) 1 mM MMPP (2 eq) in carbonate buffer (pH 10), rt, 1 h; (**d**) 5% aq. AcOH, rt, 2 h.  
The crosslinking reactions using the reactive ODN5 to initially the complementary target DNA1 or RNA1 labelled with fluorescein at the 5\({}^{\prime}\) end were investigated under neutral conditions. The reaction mixture was analyzed by 20% polyacrylamide gel electrophoresis (PAGE) containing 7 M urea and the yields of the cross-linked product were calculated based on the fluorescent intensity of each band observed on the gels. Figure 3 shows a comparison of the reactivity of ODN5 toward the different bases at the target site of the DNA1 or RNA1. ODN5 showed relatively high crosslinking reactivity to dC, dT, and dG, except for dA in the DNA. On the other hand, the crosslinked product was observed in high yields with rC and rU, and no significant products were observed with rG and rA in the RNA. The comparison of the reactivity to DNA or RNA with acyAOV **2**, 4-amino-6-oxo-2-vinyl pyrimidine having an acyclic linker (**11:** acyAOVP) and Et-AOVT **1**, is shown in Figure 4. By comparison, acyAOVT **2** and acyAOVP **11** showed a significant difference in the reaction rate and the base selectivity to the target DNA and RNA (Figure 4A,B). The reaction rate with _acy_AOVT **2** was faster than that of _acy_AOVP **11**. The higher reaction rate with _acy_AOVT **2** might be attributed to the electron-withdrawing effect of the triazine increasing the reactivity of the 6-vinyl group of _acy_AOVT **2** than that of the pyrimidine-type _acy_AOVP **11**. Compared to the target base selectivity in **DNA1** with previously reported the _acy_AOVP **11**[21], AOVT **1** and **2** showed a drastically increased reactivity with the cytosine. On the other hand, the crosslinking yields to **RNA1** with AOVT **1** and **2** were significant higher to cytosine and uracil, and relatively lower to guanine than that of AOVP **11**. The reaction rate and selectivity to the pyrimidine bases (C, T and U) with _acy_AOVT **2** was comparable to that of Et-AOVT **1**. The reactivity to adenine (A) in DNA and RNA with **2** was significantly lower than that of Et-AOVT **1** (Figure 4A,C). Next, we carried out a thermal denaturing study of the duplex containing the non-reactive _acy_AOVT. The **ODN7** bearing the non-reactive _acy_AOVT (SMe) was prepared by the addition of NaSMe to the vinyl derivative under weak acidic conditions for avoiding the decomposition of _acy_AOVT to guanylurea (Scheme 3). Figure 3: Denaturing gel electrophoresis of crosslink reaction to target DNA1 and RNA1 with **ODN5**. The reaction was performed in 50 mM MES-buffer (pH 7.0) containing 100 mM NaCl, **ODN5** (5 \(\upmu\)M) and cDNAs or cRNAs (2 \(\upmu\)M) at 37 \({}^{\circ}\)C.  
The melting temperature (\(Tm\)) of the **ODN7/DNA1** or **RNA1** is summarized in Figure 5. The \(Tm\) values of the **ODN7/DNA1** or **RNA1** were observed to be lower than that of the unmodified natural duplex (\(T_{\rm m}=67\)\({}^{\circ}\)C).