RESUMEN
Methods for rapidly assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are critical to our ability to manipulate and interface with biology. We describe a framework for engineering RNA devices from preexisting aptamers that exhibit ligand-responsive ribozyme tertiary interactions. Our methodology utilizes cell sorting, high-throughput sequencing and statistical data analyses to enable parallel measurements of the activities of hundreds of thousands of sequences from RNA device libraries in the absence and presence of ligands. Our tertiary-interaction RNA devices performed better in terms of gene silencing, activation ratio and ligand sensitivity than optimized RNA devices that rely on secondary-structure changes. We applied our method to build biosensors for diverse ligands and determine consensus sequences that enable ligand-responsive tertiary interactions. These methods advance our ability to develop broadly applicable genetic tools and to elucidate the underlying sequence-structure-function relationships that empower rational design of complex biomolecules.
Asunto(s)
Aptámeros de Nucleótidos/química , Ingeniería Genética/métodos , Riboswitch/genética , Aptámeros de Nucleótidos/metabolismo , Técnicas Biosensibles , Interpretación Estadística de Datos , Citometría de Flujo/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Biblioteca de Genes , Proteínas Fluorescentes Verdes/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ligandos , Nepovirus/genética , ARN Catalítico/química , Resonancia por Plasmón de Superficie , Teofilina/metabolismo , Teofilina/farmacologíaRESUMEN
SELEX was used to create an RNA aptamer targeted to protein tyrosine phosphatase 1B (PTP1B), an enzyme implicated in type 2 diabetes, breast cancer and obesity. We found an aptamer that strongly inhibits PTP1B in vitro with a Ki of less than 600 pM. This slow-binding, high-affinity inhibitor is also highly selective, with no detectable effect on most other tested phosphatases and approximately 300:1 selectivity over the closely related TC-PTP. Through controlled synthesis of truncated variants of the aptamer, we isolated shorter forms that inhibit PTP1B. We also investigated various single-nucleotide modifications to probe their effects on the aptamer's secondary structure and inhibition properties. This family of aptamers represents an exciting option for the development of lead nucleotide-based compounds in combating several human cancers and metabolic diseases.