In vitro selection of a 3′ terminal short protector that stabilizes transcripts to improve the translation efficiency in a wheat germ extract

Ogawa, Atsushi; Kutsuna, Akane; Takamatsu, Masashi; Okuzono, Tatsuya

doi:10.1016/j.bmcl.2019.06.058

Cited by 5 publications

(3 citation statements)

References 16 publications

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“…36 The sequences of these synthetic nucleic acids are described in Supporting Information. The sequence of a synthetic plasmid, pEU-nLuc-Venus encoding the NanoLuc gene, 37 has been reported elesewhere 21 and that of another synthetic plasmid, pTheo5-PSIV-IRES encoding the PSIV (Plautia stali intestine virus) IRES, 38 is described in Supporting Information.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

In Vitro Selection of RNA Aptamers Binding to Nanosized DNA for Constructing Artificial Riboswitches

Ogawa

Itoh

2020

ACS Synth. Biol.

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We here designed an in vitro selection scheme for obtaining an aptamer with which to rationally construct an artificial riboswitch as its component part. In fact, a nanosized DNA-binding aptamer obtained through this scheme allowed us to easily and successfully create eukaryotic riboswitches that upregulate internal ribosome entry site-mediated translation in response to the ligand (nanosized DNA) in wheat germ extract, a eukaryotic cell-free expression system. The induction ratio of the best riboswitch ligand-dose-dependently increased to 21 at 300 μM ligand. This switching efficiency is much higher than that of the same type of riboswitch with a widely used theophylline-binding aptamer, which was in vitro selected without considering its utility for constructing riboswitches. The selection scheme described here would facilitate obtaining various ligand/aptamer pairs suitable for constructing artificial riboswitches, which could serve as elements of synthetic gene circuits in synthetic biology.

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Section: ■ Results and Discussionmentioning

confidence: 99%

In Vitro Selection of RNA Aptamers Binding to Nanosized DNA for Constructing Artificial Riboswitches

Ogawa

Itoh

2020

ACS Synth. Biol.

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“…The sequences of primers and a template DNA for PCR are described in the Supporting Information. A simple stem-loop structure and an in vitro -selected terminal protector were added to the 5′ and 3′ terminus of Broccoli, respectively, to inhibit its degradation in WGE …”

Section: Methodsmentioning

confidence: 99%

“…A simple stem-loop structure and an in vitroselected terminal protector were added to the 5′ and 3′ terminus of Broccoli, respectively, to inhibit its degradation in WGE. 22 In vitro transcription was conducted with a T7-Scribe Standard RNA IVT Kit (CellScript) according to the manufacturer's protocol. Broccoli-encapsulating and Broccolifree SGUVs were prepared as described above (under the optimized conditions) except that the droplet included 2 μM Broccoli and the exterior included 100 μM DFHBI.…”

Section: T H I S C O N T E N T Imentioning

confidence: 99%

Preparation of a Millimeter-Sized Supergiant Liposome That Allows for Efficient, Eukaryotic Cell-Free Translation in the Interior by Spontaneous Emulsion Transfer

Takahashi

Ogawa

2020

ACS Synth. Biol.

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We sought to prepare millimeter-sized supergiant unilamellar vesicles (SGUVs) by spontaneous emulsion transfer for efficient, eukaryotic cell-free translation in the interior. Although the conventional protocols require that a considerably high concentration of sucrose be encapsulated into the SGUVs for their efficient formation, such high amounts of sucrose severely inhibited cell-free translation based on wheat germ extract (WGE). We thus optimized the preparation conditions to permit SGUV formation at a much lower concentration of sucrose that has almost no effect on WGE translation. Under the optimized conditions, we successfully prepared WGE translation system-encapsulating SGUVs that allow for protein synthesis with a high efficiency comparable to that outside a liposome. The optimization also resulted in a high rate of successful SGUV formation (>90%) and a decent stability of the formed SGUVs (>60 min). These SGUVs are expected to serve as research tools in cell-free synthetic biology and as foundations for artificial cell-based biosensors.

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Mutation of the start codon to enhance Cripavirus internal ribosome entry site-mediated translation in a wheat germ extract

Ogawa

Takamatsu

2019

Bioorganic & Medicinal Chemistry Letters

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In vitro selection of a 3′ terminal short protector that stabilizes transcripts to improve the translation efficiency in a wheat germ extract

Cited by 5 publications

References 16 publications

In Vitro Selection of RNA Aptamers Binding to Nanosized DNA for Constructing Artificial Riboswitches

In Vitro Selection of RNA Aptamers Binding to Nanosized DNA for Constructing Artificial Riboswitches

Preparation of a Millimeter-Sized Supergiant Liposome That Allows for Efficient, Eukaryotic Cell-Free Translation in the Interior by Spontaneous Emulsion Transfer

Mutation of the start codon to enhance Cripavirus internal ribosome entry site-mediated translation in a wheat germ extract

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