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Chem Biol Drug Des. 2015 Nov;86(5):1242-52. doi: 10.1111/cbdd.12592. Epub 2015 Jun 23.

Changing blue fluorescent protein to green fluorescent protein using chemical RNA editing as a novel strategy in genetic restoration.

Author information

1
School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan.

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.

KEYWORDS:

CVU oligodeoxynucleotide; antisense oligodeoxyribonucleotide; chemical RNA editing; deamination; photochemical reaction

PMID:
26031895
DOI:
10.1111/cbdd.12592
[Indexed for MEDLINE]

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