Insights into secondary and tertiary interactions of dengue virus RNA by SHAPE

Abstract

Dengue virus (DENV) is a single-stranded positive-sense RNA virus belonging to the Flaviviridae family. The DENV RNA genome contains multiple cis-acting elements that continue to unravel their essential role in managing viral molecular processes. Attempts have been made to predict the secondary structure of DENV RNA using a variety of computational tools. Nevertheless, a greater degree of accuracy is achieved when these methods are complemented with structure probing experimentation. This chapter outlines detailed methodology for the structural study of DENV subgenomic minigenome RNA by applying highthroughput selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). High-throughput SHAPE combines a novel chemical probing technology with reverse transcription, capillary electrophoresis, and secondary structure prediction software to rapidly and reproducibly determine the structure of RNAs from several hundred to several thousand nucleotides at single-nucleotide resolution. This methodology investigates local structure for all positions in a sequence-independent manner and as such it is particularly useful in predicting RNA secondary and tertiary interactions.