Production of pure and functional RNA for in vitro reconstitution experiments

Reconstitution of protein complexes has been a valuable tool to test molecular functions and to interpret in vivo observations. In recent years, a large number of RNA-protein complexes has been identified to regulate gene expression and to be important for a range of cellular functions. In contrast to protein complexes, in vitro analyses of RNA-protein complexes are hampered by the fact that recombinant expression and purification of RNA molecules is more difficult and less well established than for proteins. Here we review the current state of technology available for in vitro experiments with RNAs. We outline the possibilities to produce and purify large amounts of homogenous RNA and to perform the required quality controls. RNA-specific problems such as degradation, 5′ and 3′ end heterogeneity, co-existence of different folding states, and prerequisites for reconstituting RNAs with recombinantly expressed proteins are discussed. Additionally a number of techniques for the characterization of direct and indirect RNA-protein interactions are explained.