Purification and Structural Characterization of the Long Noncoding RNAs

Abstract

Long noncoding RNAs (lncRNAs) are now accepted as key players in diverse cellular functions, yet the structure-function relationships of these novel RNAs remain mostly unknown. Homogenous purification of lncRNAs is a necessary first-step for downstream structural studies. The large size of lncRNAs (often more than 1 kb) presents many unique challenges during the purification process. Here, we detail the purification of lncRNAs, including strategies to identify proper folding conditions of the target lncRNA. Next, we discuss two recently developed RNA structure probing techniques, SHAPE-MaP (SHAPE probing followed by mutational profiling) and DMS-MaP (DMS probing followed by mutational profiling). These techniques couple traditional RNA chemical probing methods with next-generation sequencing and allow high-throughput determination of RNA structures. Using the datasets resulting from these orthogonal probing experiments, we lay out the steps to determine and validate the secondary structure of the target lncRNA. Overall, this chapter details an adaptable protocol that can lead to a better understanding of the structure-function relationships of lncRNAs.

Keywords: 1-Methyl-7-nitroisatoic anhydride (1 M7); Chemical probing; DMS (dimethyl sulfate); Mutational profiling; Native purification; SHAPE (Selective 2′-Hydroxyl Acylation analyzed by Primer Extension); lncRNA structure.