Development of New Method for Molecular Biology Using the Photophore, Diazirine

Abstract

Formation of a covalent bond using photoreactive groups allows one to maintain the complex between the ligand and its binding protein even under denaturing conditions. Thus, the photoreactive groups functioned as the powerful hook in the fishing of specific binding proteins. This is a very useful feature for developing the efficient and sensitive methods in molecular biology. Among the photoreactive groups, carbene-generating phenyldiazirine is a first choice for the application to the methods because the use of phenyldiazirine seems to be promising for achieving efficient crosslinking. This review describes improvements of phenyldiazirine usability and an application of phenyldiazirine to displayed phage screening. We synthesized the photoreactive diazirine units for a solution to preparing photoreactive ligands. Since the photoreactive units can be easily integrated into physiological ligands such as peptides, proteins, DNAs, and sugars by chemoselective reaction, the biochemists, who are not familiar with organic synthesis, can prepare the photoaffinity ligands using their interested ligands. We applied the phenyldiazirine to screening of displayed phages, and investigated the screening efficiency. The phages displayed specific-binding protein were extremely concentrated by using photoreactive peptide bearing diazirine. High efficiency in the screening is due to carry out intensive washing which removes almost all the unspecific binding phages. Moreover, such application overcomes the unavoidable drawback of photoreactive groups, the low efficiency of crosslinking because the isolated genes can be amplified.