Design of the crosslinking reactions for nucleic acids-binding protein and evaluation of the reactivity

Selective chemical reactions of biomolecules are some of the important tools for investigations by biological studies. We have developed the selective crosslinking reactions to form covalent bonds to DNA or RNA using crosslinking oligonucleotides (CFO) bearing reactive bases. In this study, we designed the cross-linkable 4-amino-6-oxo-2-vinyltriazine derivative with an acyclic linker (acyAOVT) to react with the nucleic acids-binding protein based on our previous results. We hypothesized that the acyAOVT base would form a stable base pair with guanine by three hydrogen bonds at the positions of the vinyl group in the duplex DNA major groove, and the vinyl group can react with the nucleophilic species in the proximity, for example, the cysteine or lysine residue in the nucleic acids-binding protein. The synthesized oligonucleotides bearing the acyAOVT derivative showed a higher reactivity than that of the corresponding pyrimidine derivative without one nitrogen. The duplex containing acyAOVT-guanine (G) formed complexes with Hha1 DNMT even in the presence of 2-mercaptoethanol. We expect that our system will provide a useful tool for the molecular study of nucleic acids-binding proteins.