The SHAPE of tRNA folding and of the 5'-end of the HIV-1 genome

Abstract

Desarrolla un método rápido y exacto para determinar la estructura secundaria del ARN y usarlo para entender el doblamiento de ARN y del extremo 5' del genoma de VIH-1. ARN se dobla en estructuras complejas y difíciles de predecir; durante el proceso de doblamiento algunos nucleótidos se hacen flexibles. En el presente trabajo se identificó una molécula que modifica nucleótidos flexibles. Esta reacción fue ligada a extensión con cebadores y este proceso se llama SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension). Se utilizó SHAPE para demostrar que el doblamiento de ARNt involucra interacciones no jerárquicas. Además, se usó SHAPE para observar el genoma de VIH-1 en cuatro estados diferentes.

Most RNAs function only once they fold to form difficult-to-predict base-paired helices and other structural elements. As an RNA forms a preferred secondary or tertiary structure, a characteristic set of nucleotides becomes constrained by base pairing and higher-order interactions, while unconconstrained positions remain flexible. Determining local nucleotide flexibility as a function of nucleotide position in a folded RNA provides important information that enables the sequence and structure of an RNA to be related to its biological function. I have developed a technology, termed selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), that can be used to interrogate RNA structure under diverse in vitro and in vivo conditions. SHAPE chemistry can be applied to monitor protein binding events and locate promising sites for primer annealing in arbitrary RNA. SHAPE chemistry is based on the discovery that flexible RNA nucleotides preferentially sample conformations that enhance the nucleophilic reactivity of the 2'-hydroxyl group toward electrophiles, such as N-methylisatoic anhydride. Modified sites are detected as stops in an optimized DNA primer extension reaction, followed by sizing of the extension products. SHAPE chemistry scores local flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained residues with a dynamic range of 20-fold or greater. I have applied SHAPE chemistry to observe equilibrium melting of a model tRNA at single nucleotide resolution. I observed that RNA folding is a complex process involving structural rearrangement and the formation of tertiary structure concurrent with secondary structure. Furthermore, I have employed capillary electrophoresis and sophisticated analysis algorithms to create a high-throughput SHAPE (hSHAPE) experiment that can comprehensively interrogate the flexibility of several hundred nucleotides in a single robust experiment. Using hSHAPE, I analyzed the structure of HIV-1 genomic RNA as a function of 4 different biologically relevant states, including infectious viral particles. Despite many thermodynamically plausible structures, the HIV-1 genome exists in a single conformation. I observed the effects of tRNA primer binding, and the effects of nucleocapsid protein on RNA flexibility. hSHAPE chemistry is a promising, scalable approach that can rapidly and accurately analyze the structure of RNA molecules under biologically relevant conditions.