Simple identification of two causes of noise in an aptazyme system by monitoring cell-free transcription.

Ichihashi, Norikazu; Kobori, Shungo; Yomo, Tetsuya

Ichihashi, Norikazu; Kobori, Shungo; Yomo, Tetsuya.

Afiliación

Ichihashi N; Graduate School of Information Science and Technology, Osaka University, Suita, Osaka, Japan; Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Suita, Osaka, Japan. Electronic address: ichihashi@ist.osaka-u.ac.jp.
Kobori S; Graduate School of Information Science and Technology, Osaka University, Suita, Osaka, Japan.
Yomo T; Graduate School of Information Science and Technology, Osaka University, Suita, Osaka, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan; Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Suita, Osaka, Japan; Earth-Life Science Institute, Tokyo Institute of Technology, Meguro, Tokyo, Japan.

Methods Enzymol ; 550: 93-107, 2015.

Article en En | MEDLINE | ID: mdl-25605382

RESUMEN

Aptazymes are artificially synthesized ribozymes that catalyze reactions in response to ligand binding. Certain types of aptazymes can be utilized as RNA-based regulators of gene expression. These aptazymes contain a sequestered ribosome-binding site (rbs) and release the rbs through self-cleavage in response to ligand binding, inducing the expression of the downstream gene. One of the most important properties of aptazymes as gene expression regulators is their signal-to-noise ratio (S/N ratio), the ratio of target expression in the presence of ligand to that in the absence of ligand. One strategy to improve the S/N ratio is to decrease the noise (expression in the absence of ligand) due to leaky translation without rbs release or ligand-independent rbs release. In this chapter, we describe an easy method to identify the main cause of noise using a cell-free reconstituted transcription-translation system, an ideal platform for the quantitative understanding of biochemical reactions because researchers can strictly control the experimental conditions and the concentrations of all components. This knowledge would be useful for designing aptazymes with high S/N ratios.

Asunto(s)

Sistema Libre de Células/metabolismo; ARN Catalítico/metabolismo; Transcripción Genética/genética; Modelos Teóricos

Palabras clave

Aptazyme; Cell-free translation system; In vitro; Mathematical model; Pure system; Signal-to-noise ratio

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transcripción Genética / Sistema Libre de Células / ARN Catalítico Tipo de estudio: Diagnostic_studies / Etiology_studies Idioma: En Revista: Methods Enzymol Año: 2015 Tipo del documento: Article Pais de publicación: Estados Unidos

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