Research for live cell imaging with fluorescent probes and protein labeling with genetically encoded fluorohydroxybenzoyl-l-lysine

Abstract

Development of imaging technology has enabled visualization of biological molecules in cells with high resolution. Diverse fluorescent tags and small molecules have been developed, and small molecule probes have advantages with relatively small size and photostability. In this study, we have detected mitochondrial peroxynitrite with a benzoindocyanine based probe. Peroxynitrite is a strong oxidizing ROS which can damage cellular components, and its overproduction is implicated in detection of various clinical diseases. We expect our probe to be applied in the detection and screening of peroxynitrite in various physiopathological processes. Expanding genetic code with unnatural amino acids (UAAs) gives new functionalities to proteins. Bio-orthogonal UAAs have been used in biochemical research and protein engineering to provide proximity-enabled protein fusion or site-specific coupling or labeling of fluorescent dyes. Furthermore, recent UAAs have used click-chemistry such as SPAAC and SuFEx that occur orthogonally in biological conditions. We designed and confirmed reactivity of a sulfonyl fluoride specific amino acid, 3-fluoro-4-hydroxybenzoyl-L-lysine (FhbK). We selected evolved Pyrrolysyl-tRNA Synthetase from Methanomethylophilus alvus via Molcular Dynamics study, and FhbK was incorporated into model protein. With its long side chain, incorporated FhbK would have enhanced reactivity with target chemicals. This bio-orthogonal reaction between our novel UAA and chemical dyes having sulfonyl/sulfonate functional groups such as dansyl fluoride, would be particularly useful for imaging and functional studies of proteins in vivo.