Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs.

Takashi Ohtsuki,Sae Nishimura,Kazunori Watanabe,Masahiko Sisido,Yoshio Kunihiro,Shigeto Kanzaki

doi:10.1038/ncomms12501

Abstract

The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl)methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4 h in neutral aqueous solution, and does not bind to the elongation factor Tu. On irradiation at ∼405 nm at 125 mW cm−2, DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19 s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells.

Highlights

The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues
The caged aatRNA bearing a four-base anticodon was used to phototrigger the translation of a specific mRNA containing the corresponding four-base codon; this caged aa-tRNA containing a photocleavable nitroveratryloxycarbonyl (NVOC) group required ultraviolet irradiation for B30 min for its uncaging, which resulted in a reduction in translation efficiency[12]
For a caged aa-tRNA to serve as an efficient phototrigger for in vitro and in vivo translation, it must possess these properties: (1) before irradiation, the caged aa-tRNA must not deacylate in aqueous solutions; (2) before irradiation, it must not participate in translation due to rejection from either elongation factor (EF)-Tu or the ribosome; (3) it must be uncaged rapidly without any damage to the translation system; and (4) after irradiation, it must decode a specific four-base or nonsense codon

Summary

Introduction

The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. In vitro translation was phototriggered through the decoding of a CGGG four-base codon by using DEACM-Ser-tRNACCCG and streptavidin mRNA-21CGGG (Fig. 4).

Results

Conclusion