title: Changing blue fluorescent protein to green fluorescent protein using chemical RNA editing as a novel strategy in genetic restoration.
abstract: Using the transition from cytosine of BFP (blue fluorescent protein) gene to uridine of GFP (green fluorescent protein) gene at position 199 as a model, we successfully controlled photochemical RNA editing to effect site-directed deamination of cytidine (C) to uridine (U). Oligodeoxynucleotides (ODNs) containing 5'-carboxyvinyl-2'-deoxyuridine ((CV) U) were used for reversible photoligation, and single-stranded 100-nt BFP DNA and in vitro-transcribed full-length BFP mRNA were the targets. Photo-cross-linking with the responsive ODNs was performed using UV (366 nm) irradiation, which was followed by heat treatment, and the cross-linked nucleotide was cleaved through photosplitting (UV, 312 nm). The products were analyzed using restriction fragment length polymorphism (RFLP) and fluorescence measurements. Western blotting and fluorescence-analysis results revealed that in vitro-translated proteins were synthesized from mRNAs after site-directed RNA editing. We detected substantial amounts of the target-base-substituted fragment using RFLP and observed highly reproducible spectra of the transition-GFP signal using fluorescence spectroscopy, which indicated protein stability. ODNc restored approximately 10% of the C-to-U transition. Thus, we successfully used non-enzymatic site-directed deamination for genetic restoration in vitro. In the near future, in vivo studies that include cultured cells and model animals will be conducted to treat genetic disorders.
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan.
authors.forename: Luyen T
authors.initials: LT
authors.lastname: Vu
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan.
authors.forename: Thanh T K
authors.initials: TT
authors.lastname: Nguyen
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan.
authors.forename: Shafiul
authors.initials: S
authors.lastname: Alam
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan.
authors.forename: Kenzo
authors.initials: K
authors.lastname: Fujimoto
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Tsukahara
mesh_terms: D001483:Base Sequence; D003562:Cytidine / Q000737:chemistry / Q000235:genetics*; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry / Q000235:genetics; D017393:RNA Editing; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D014466:Ultraviolet Rays; D014529:Uridine / Q000737:chemistry / Q000235:genetics*
chemical_list: C520201:5-carboxyvinyl-2'-deoxyuridine; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D003562:Cytidine; D003857:Deoxyuridine; D014529:Uridine
keywords: CVU oligodeoxynucleotide; antisense oligodeoxyribonucleotide; chemical RNA editing; deamination; photochemical reaction