In vitro selection of random RNA fragments to identify protein-binding sites within large RNAs

Abstract

In vitro selection experiments have various goals depending on the composition of the initial pool and the selection method applied. We developed an in vitro selection variant that is useful for the identification of minimal RNA binding sites for proteins within large RNAs. A pool of randomly fragmented RNA is constructed from a large RNA, which is the natural binding partner for a protein. Such a pool contains all the potential binding sites and is therefore used as starting material for affinity selection. A successful in vitro selection with the purified protein will identify the protein's natural RNA target site. The method has been developed for ribosomal systems and is a general approach providing a basis for the functional and structural characterization of large ribonucleoprotein particles. © 2008 Humana Press.