Cross-linking nucleotides have been applied for analysis of interactions of nucleic acids with proteins and for improvement of thermal stability of tertiary structures of nucleic acids. Particularly, photo-cross-linking nucleotides have attracted much attention since photo-irradiation can be controlled spatiotemporally without contamination of the biological samples. Because a diazirine moiety has lots of advantages such as high reactivity upon photolysis and sterically less hindrance, many diazirine derivatives have been developed. In this study, we developed novel 2’-O-diazirine-conjugated adenosine (DA) and cytidine (DC) as photo-cross-linking agents for oligonucleotides. The DA and DC phosphoramidite units were synthesized via 2’ alkylation, N-benzoyl protection, dimethoxytritylation, and phosphitylation of corresponding nucleosides. And then, they were used for the solid-phase synthesis of oligodeoxyribonucleotides containing DA or DC in the middle of the sequences (X-ODN), 5’-ccg cgt Xnc gcc g-3’ (X=DA, DC; n=t, a, g, c). First, the UV irradiation conditions for activating the diazirine group were evaluated using reversed-phase HPLC. After UV irradiation at 365 nm (400 mW cm-2) for 30 s, a peak of X-ODN with the diazirine derivatives was completely disappeared and three peaks for the X-ODN with water adducts, self-cross-linking products, and diazo isomers were newly observed. After additional photo-irradiation for 30 min, the peak for the X-ODN with diazo-isomers was almost disappeared. Kinetic studies of the photolysis reactions revealed that the photolysis of the diazirine group was completed within 30 min. Next, we analyzed the photo-cross-linking properties of the full-matched and single base-pair mismatched duplexes. The duple-containing mixtures were irradiated at 365nm (400 mW cm-2) for up to 30 min at 0 °C. Then, the samples were analyzed using 20% denaturing polyacrylamide gel electrophoresis (PAGE). From the results of PAGE, it was revealed that the DA-containing oligodeoxyribonucleotides selectively crosslinked with the oligo-DNAs (and not with the oligo-RNAs). Further investigation of the photo-cross-linking reactions between DA-containing oligodeoxyribonucleotides and non-complementary oligo-DNAs suggests that local structure and stability of the helix are important to enhance the cross-linking efficiency.

Development of photo-cross-linking oligodeoxyribonucleotides with 2’-O-diazirine-conjugated nucleosides

256th National Meeting (2018)

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