20] Probing RNA structure and function by circular permutation

Abstract

This chapter describes the way for applying circular permutation (CP) to probe the structure and function of RNA. Five examples are provided that describe the application of CP analysis (CPA) in the analysis of RNA folding, protein–RNA interaction, substrate binding, and catalysis by a bacterial RNase P RNA. These examples illustrate the feasibility of using circular permutation to analyze the structure and function of biologically relevant RNAs. Circular permutation implies covalent linkage of the normal 5´ and 3´ ends of an RNA. Circular RNA can be synthesized by linking the normal 5´ and 3´ ends of RNA with either T4 RNA ligase or T4 DNA ligase and DNA oligonucleotide splint or through the action of a ribozyme. The efficiency and the choice of ligase depend on the structure and the sequence of the 5´ and 3´ ends. T4 RNA ligase is very efficient for RNAs with one unstructured 5´ nucleotide and three unstructured 3´ nucleotides. If the 5´ nucleotide is located in a helix, then the method with T4 DNA ligase should be used. A basic protocol for using T4 RNA ligase to generate circular RNA is described in the chapter; this protocol works well for the circularization of RNAs containing more than 70 nucleotides. A modified protocol using T4 RNA ligase and a DNA template works well for circularization of RNAs containing fewer than 30 nucleotides.