title: Construction of semisynthetic DNA-protein conjugates with Phi X174 Gene-A* protein.
abstract: DNA-protein conjugates have frequently been used as versatile molecular tools for a variety of applications in biotechnology to harness synergistic effects of DNA and protein functions. With applications for DNA-protein conjugates growing, easy-to-use and economical methods for the synthesis of DNA-protein conjugates are required. In this study, we developed a method for site-specific labeling of single-stranded DNA (ssDNA) to a recombinant protein of interest (POI) through the Gene-A* protein (Gene-A*) from bacteriophage phi X174, without any chemical modifications of ssDNA. Gene-A* protein is an enzyme that site-selectively cleaves an oligodeoxyribonucleotide (ODN) containing a Gene-A* recognition sequence, at which point a tyrosine residue of Gene-A* is bonded to the 5'-phosphoryl group of the cleavage site via a stable phosphotyrosine linkage. Here, we constructed three kinds of recombinant proteins fused to Gene-A*: N-terminally Gene-A*-fused enhanced green fluorescent protein (EGFP), C-terminally Gene-A*-fused EGFP, and N-terminally Gene-A*-fused firefly luciferase (FLuc). The reaction yields of DNA-protein conjugation catalyzed by the Gene-A* moiety reached 80-90% in the three proteins, and kinetic study revealed that the reaction achieved a steady state after 10 min. Moreover, dot blot analyses were performed to evaluate the hybridization and aptamer-forming ability of ssDNA conjugated to the Gene-A* moiety of a recombinant Gene-A*-FLuc protein. This study demonstrated that a strategy using recombinant proteins fused to Gene-A* could offer a versatile, rapid, easy-to-use, and economical platform for producing DNA-protein conjugates.
authors.affiliation: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology , 4259, Nagatsuta, Midori-ku, Yokohama-shi, 226-8501, Japan.
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Mashimo
authors.forename: Hitomi
authors.initials: H
authors.lastname: Maeda
authors.forename: Masayasu
authors.initials: M
authors.lastname: Mie
authors.forename: Eiry
authors.initials: E
authors.lastname: Kobatake
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism; D010584:Bacteriophage phi X 174 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly; D013917:Thrombin