11] Purification, crystallization, and X-ray diffraction analysis of small ribozymes

Abstract

Publisher Summary Small ribozymes are a family of molecular catalysts composed solely of RNA. This chapter provides an overview of some practical strategies used in the purification, crystallization, and X-ray crystallographic structure determinations of small ribozymes. In studies, two ribozymes has been solved by multiple isomorphous replacement—the hammerhead and leadzyme; they are cited as examples in the chapter. One of the rate-limiting steps in any structure determination is the preparation of crystals suited for X-ray diffraction analysis. For this task, it may be possible to induce crystal growth by selectively modifying the sequence of the target RNA construct. One method involves the incorporation of “sticky-ended’ stems. In this event, the RNA is expected to form a duplex with blunt helical stems. A second strategy incorporates a protein binding site into the ribozyme. This method utilizes a well-characterized protein–RNA interaction placed at an innocuous location in the nucleic acid sequence. A traditional source of RNA for structural studies has been in vitro transcription by T7 RNA polymerase. Subsequently, the RNA is purified to remove failure sequences and N + 1 extensions by use of denaturing polyacrylamide gels. The chapter also describes an alternative approach in which crystallization quality RNA is prepared from solid-phase chemical synthesis using phosphoramidites.