Chapter 12 - Analysis of RNA Binding Proteins Using in Vitro Genetics

Abstract

This chapter describes an in vitro genetic system that allows the selection of Ribonucleic acid (RNA) binding proteins that bind tightly to a given RNA target sequence. The system utilizes phage display to physically link RNA binding proteins to their genes. The RNA binding proteins are displayed as fusions outside the filamentous bacteriophage particles and can be trapped on RNA attached to beads. Thus, protein variants with certain RNA-binding characteristics can be selected from a library of mutants through iterative rounds of binding and amplification. Even very rare clones can be retrieved in this way because the selective step occurs in vitro, signifying the complete control over binding conditions. Accessory proteins involved in binding can be added to the binding reaction, so that complex RNA–protein interactions involving numerous components can be studied. The chapter discusses relevant facts about phage morphogenesis, elaborating upon concepts related to efficient display of RNA binding proteins. Binding of displayed RNA binding proteins to RNA is also discussed. The chapter presents application of in vitro genetics to the study of RNA binding proteins. Prospects and future applications are also elaborated.