title: 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.
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Di Tomasso
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Alix
authors.initials: A
authors.lastname: Salvail-Lacoste
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bouvette
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: James G
authors.initials: JG
authors.lastname: Omichinski
authors.affiliation: Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, Quebec, Canada. Electronic address: pascale.legault@umontreal.ca.
authors.forename: Pascale
authors.initials: P
authors.lastname: Legault
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001408:Bacillus anthracis / Q000737:chemistry / Q000235:genetics; D001419:Bacteria / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017123:Bacteriophage T7 / Q000378:metabolism; D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods*; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D016337:RNA, Catalytic; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: ARiBo tag; Affinity purification of RNA; CRISPR; CRISPR-specific nuclease; Hammerhead ribozyme; Lambda N protein; Lambda boxB RNA; Riboswitch; T7 RNA polymerase; glmS ribozyme