Affinity Purification of In Vitro Transcribed RNA with Homogeneous Ends Using a 3′-ARiBo Tag

Abstract Common approaches for purification of RNAs synthesized in vitro by the T7 RNA polymerase often denature the RNA and produce RNAs with chemically heterogeneous 5′- and 3′-ends. Thus, native affinity purification strategies that incorporate 5′ and 3′ trimming technologies provide a solution to two main disadvantages that arise from standard approaches for RNA purification. This chapter describes procedures for nondenaturing affinity purification of in vitro transcribed RNA using a 3′-ARiBo tag, which yield RNAs with a homogeneous 3′-end. The applicability of the method to RNAs of different sequences, secondary structures, and sizes (29–614 nucleotides) is described, including suggestions for troubleshooting common problems. In addition, this chapter presents three complementary approaches to producing 5′-homogeneity of the affinity-purified RNA: (1) selection of the starting sequence; (2) Cse3 endoribonuclease cleavage of a 5′-CRISPR tag; or (3) self-cleavage of a 5′-hammerhead ribozyme tag. The additional steps to express and purify the Cse3 endonuclease are detailed. In light of recent results, the advantages and limitations of current approaches to achieve 5′-homogeneity of affinity-purified RNA are discussed, such that one can select a suitable strategy to purify the RNA of interest.