Changing Blue Fluorescent Protein to Green Fluorescent Protein Using Chemical RNA Editing as a Novel Strategy in Genetic Restoration
Changing Blue Fluorescent Protein to Green Fluorescent Protein Using Chemical RNA Editing as a Novel Strategy in Genetic Restoration
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使用化学 RNA 编辑将蓝色荧光蛋白变为绿色荧光蛋白作为遗传恢复的新策略
DOI：
10.1111/cbdd.12592
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发表时间：
2015
影响因子：
3
通讯作者：
Hitoshi Suzuki and Toshifumi Tsukahara
中科院分区：
文献类型：
--
作者：
Luyen T. Vu;Thanh T. K. Nguyen;Shafiul Alam;Takashi Sakamoto;Kenzo Fujimoto;Hitoshi Suzuki and Toshifumi Tsukahara
利用BFP（蓝色荧光蛋白）基因的胞嘧啶到GFP（绿色荧光蛋白）基因199位尿苷的转变作为模型，我们成功地控制了光化学RNA编辑，实现了胞苷（C）到尿苷（U）的定点脱氨基。以含5′-羧基乙烯基-2 ′-脱氧尿苷（CVU）的寡脱氧核苷酸（ODNs）为靶，以单链100-nt BFP DNA和体外转录的全长BFP mRNA为靶，进行可逆性光连接。使用UV（366 nm）照射进行与响应性ODN的光交联，随后进行热处理，并通过光裂解（UV，312 nm）切割交联的核苷酸。使用限制性片段长度多态性（RFLP）和荧光测量分析产物。Western blotting和荧光分析结果表明，体外翻译蛋白是由定点RNA编辑后的mRNA合成的。我们使用RFLP检测到大量的靶碱基取代片段，并使用荧光光谱法观察到过渡GFP信号的高度可重复光谱，这表明蛋白质稳定性。ODNc恢复了约10%的C至U转变。因此，我们成功地使用非酶定点脱氨进行体外遗传修饰。在不久的将来，将进行包括培养细胞和模型动物在内的体内研究，以治疗遗传疾病。
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 (CVU) were used for reversible photoligation, and single‐stranded 100‐nt BFP DNA andin 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 thatin 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 restorationin vitro. In the near future,in vivostudies that include cultured cells and model animals will be conducted to treat genetic disorders.
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影响因子：
16.6
作者：
Nishikura K
通讯作者：
Nishikura K
Masayuki Hayashi、Isao Saito：通过可逆 DNA 光连接将胞嘧啶位点特异性转变为尿嘧啶。
DOI：
--
发表时间：
2006
期刊：
Chemical Communications 30
影响因子：
--
作者：
Kenzo;Fujimoto;Shigeo;Matsuda;Yoshinaga;Yoshimura;Takashi;Matsumura
通讯作者：
Matsumura
影响因子：
11.4
作者：
Farajollahi, Sanaz;Maas, Stefan
通讯作者：
Maas, Stefan
影响因子：
16.8
作者：
通讯作者：
--
使用 T 淋巴细胞培养物对 Lesch-Nyhan 综合征突变 (hprtMontreal) 进行分子分析
影响因子：
5.3
作者：
T. Skopek;L. Recio;D. Simpson;L. Dallaire;S. Melançon;H. Ogier;J. O'neill;M. Falta;J. Nicklas;R. Albertini
通讯作者：
R. Albertini