RESUMEN
An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.
Asunto(s)
Aminoácidos/química , Ingeniería de Proteínas/métodos , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Secuencia de Bases , Sistema Libre de Células , Codón , ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Variación Genética , Modelos Químicos , Datos de Secuencia Molecular , Plásmidos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Factores de Tiempo , Transcripción Genética , Tirosina/química , Tirosina/farmacología , Proteínas ViralesRESUMEN
A photo-sensitive ribonucleotide of 5-iodo-2-oxo(1H) pyridine (Iy) capable of site-specific incorporation into transcripts was developed. The site-specific Iy incorporation into RNA was achieved by T7 transcription mediated by unnatural base pairing between Iy and its partner, 2-amino-6-(2-thienyl)purine (s). By this specific transcription, Iy was incorporated into an anti(Raf-1) RNA aptamer, which binds to human Raf-1 and inhibits the interaction between Raf-1 and Ras. Protein-dependent photo-dimerization of the aptamer was observed when Iy was located at specific positions in the aptamer, showing that the site-specific incorporation of the photo-sensitive component into RNA achieves highly specific crosslinking. This specific transcription mediated by the unnatural base pair would be a powerful tool for generating high-affinity RNA ligands and for analyzing RNA-RNA and RNA-protein interactions, as well as for constructing RNA-based nanostructures.
Asunto(s)
Emparejamiento Base , ARN Polimerasas Dirigidas por ADN/metabolismo , Nucleótidos/metabolismo , Piridonas/metabolismo , ARN/metabolismo , Bacteriófago T7/enzimología , Bacteriófago T7/genética , Secuencia de Bases , Sitios de Unión , Reactivos de Enlaces Cruzados/metabolismo , Humanos , Enlace de Hidrógeno , Modelos Químicos , Datos de Secuencia Molecular , Estructura Molecular , Nucleótidos/química , Unión Proteica , Piridonas/química , ARN/química , Transcripción Genética , Proteínas ViralesRESUMEN
Site-specific incorporation of a hydrophobic nucleotide analog into RNA, by T7 transcription mediated by unnatural base pairs, was developed. The nucleotide analog, 5-phenylethynyl-3-(beta-D-ribofuranosyl)pyridin-2-one 5-triphosphate (denoted by Ph-yTP), was chemically synthesized and then site-specifically incorporated by T7 RNA polymerase into RNA opposite the pairing partner, 2-amino-6-(2-thienyl)purine (denoted by s) in DNA templates. The introduction of Ph-y into a theophylline-binding RNA aptamer, in which a uridine in the internal loop was replaced by Ph-y, raised the thermal stability of the aptamer. Thus, this unnatural nucleotide analog would be useful for stabilizing RNA tertiary structures and complexes between RNA and other molecules.