RESUMEN
Isatoic anhydride derivatives, including a biotin and a disulfide linker were specifically designed for nucleic acid separation. 2'-OH selective RNA acylation, capture of biotinylated RNA adducts by streptavidin-coated magnetic beads and disulfide chemical cleavage led to isolation of highly enriched RNA samples from an initial 9/1 DNA-RNA mixture. Starting from the parent compound N-methylisatoic anhydride A which was used at 65 °C, we improved the extraction process by designing a new generation of isatoic anhydrides that are able to react under smoother conditions. Among them, a pyridine-based isatoic anhydride derivative 15f was found to be reactive at room temperature, leading to enhance the efficiency and selectivity of the extraction process by significantly reducing DNA side extraction. The extracted and purified RNAs can then be detected by RT-PCR.
Asunto(s)
Biotina/química , Oxazinas/química , Piridinas/química , ARN/aislamiento & purificación , Temperatura , Acilación , Cromatografía Liquida , ADN/química , Ésteres/síntesis química , Ésteres/química , VIH/genética , Espectrometría de Masas , Oxazinas/síntesis química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
An isatoic anhydride derivative conjugated to a biotin and a disulfide linker was specifically designed for the separation of nucleic acids. Starting from a DNA-RNA mixture, a selective 2'-hydroxyl acylation of RNAs followed by capture with streptavidin-coated magnetic beads and cleavage of the disulfide led to elution of RNAs.