RESUMEN
Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.
Asunto(s)
Empalme Alternativo/genética , Regulación de la Expresión Génica/genética , Ingeniería Genética/métodos , ARN/genética , Factores de Transcripción/genética , Secuencias de Aminoácidos/genética , Animales , Línea Celular , Biología Computacional , Secuencia de Consenso/genética , Exones/genética , Biblioteca de Genes , Genes Reporteros/genética , Humanos , Intrones/genética , Mutagénesis Sitio-Dirigida/métodos , Dominios Proteicos/genética , Isoformas de Proteínas/genética , ARN/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Emergent RNA technologies employ sequence and structural information to perform a diversity of biological functions. Synthetic RNA molecules have been developed for a wide array of applications, including genetic regulation, environmental sensing, and diagnostics devices. Recent advances in chemical synthesis and computational design of RNA have enhanced our ability to program novel functions and expand upon current biomedical applications for therapeutics and diagnostics. In this review, we highlight recent advances in synthetic RNA devices that have been engineered for biomedical systems, while addressing the current limitations and challenges of translating these engineered functional RNAs to clinical applications.