RESUMEN
We have rationally constructed a novel regulation-type of artificial riboswitch that ligand-dose dependently upregulates translation initiation mediated by a 3' cap-independent translation element (3' CITE) with no major hybridization switches in a plant expression system (wheat germ extract).
Asunto(s)
Regiones no Traducidas 3' , Regulación de la Expresión Génica de las Plantas , Células Germinativas/metabolismo , Ligandos , Biosíntesis de Proteínas , Riboswitch , Triticum/genética , Triticum/metabolismo , Sistema Libre de Células , Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes ReporterosRESUMEN
We have developed a novel type of biofunction-assisted, signal-turn-on sensor for simply and homogenously detecting DNA. This sensor system is composed of two types of in vitro-transcribed label-free RNAs (a 3' premature amber suppressor tRNA probe and an amber-mutated mRNA encoding a reporter protein), RNase H, and a wheat germ extract (WGE). A target DNA induces the 3' end maturation of the tRNA probe, which is enhanced by RNase H and leads to the expression of a full-length reporter protein through amber suppression in WGE, while there is almost no expression without the target due to the inactivity of the premature probe. Therefore, the target can be readily detected with the activity of the translated reporter. The catalytic reuse of the target with the help of RNase H in addition to various bioprocesses in WGE enables this sensor system to exhibit relatively high selectivity and sensitivity.
Asunto(s)
ADN/análisis , Sondas Moleculares/metabolismo , Extractos Vegetales/química , ARN de Transferencia/metabolismo , Ribonucleasa H/metabolismo , Triticum/química , Biocatálisis , ADN/metabolismo , Humanos , Extractos Vegetales/metabolismo , Triticum/metabolismoRESUMEN
Correction for 'Biofunction-assisted aptasensors based on ligand-dependent 3' processing of a suppressor tRNA in a wheat germ extract' by Atsushi Ogawa et al., Org. Biomol. Chem., 2015, 13, 6681-6685.
RESUMEN
We have developed a novel type of biofunction-assisted aptasensor that harnesses ligand-dependent 3' processing of a premature amber suppressor tRNA and the subsequent amber suppression of a reporter gene in a wheat germ extract.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Extractos Vegetales/genética , ARN de Transferencia/química , Teofilina/análisis , Triticum/genética , Vasodilatadores/análisis , Aptámeros de Nucleótidos/genética , Secuencia de Bases , Genes Reporteros , Extractos Vegetales/química , ARN de Transferencia/genética , Supresión Genética , Triticum/químicaRESUMEN
High-quality wheat germ extract (hqWGE) is very useful for the high-yield production of various types of protein. The most important key to high productivity is the design of mRNA templates. Although the design has been refined for straightforward and efficient translation in hqWGE, there is still room for improvement in untranslated regions (UTRs), especially the 3' UTR length, because a long, cumbersome 3' UTR is commonly used for translation enhancement. Here we examined some short viral 3' cap-independent translation enhancers (3' CITEs) to identify effective ones for efficient translation in hqWGE. We then combined the most effective 3' CITE and a 5' enhancer to further increase the translation efficiency. mRNA with the optimal short 3' and 5' UTRs, both of whose length was less than 150 nt, exhibited a productivity of 1.4 mg/mL in prolonged large-scale protein synthesis in hqWGE, which was comparable to that of control mRNA with a commonly-used long 3' UTR (â¼1200 nt).
