RNA affinity tags for purification of RNAs and ribonucleoprotein complexes.
Intrinsic affinity tags are useful tools for the study of macromolecular targets. Although polypeptide affinity tags are routinely used in purification and detection of protein complexes, there has been a relative lack of powerful RNA affinity tags that can be embedded within RNA sequences. Here, the preparation and use of two RNA affinity tags against Sephadex or streptavidin are described. The two tags have different strengths that make them appropriate for slightly different uses. One is a high-affinity ligand for streptavidin that can be specifically eluted by competition with biotin under otherwise native binding conditions. The other tag binds selectively to Sephadex beads, and can be eluted by competition with the soluble dextran that composes Sephadex. When properly placed within another RNA molecule, the tags can be used to effect dramatic purification of RNA or ribonucleoprotein complexes from complex mixtures of cellular RNA.
Srisawat
Chatchawan
C
Department of Biological Chemistry, University of Michigan, 3200 MSRB III, 1150 West Medical Center Drive, Ann Arbor 48109-0606, USA.
Engelke
David R
DR
D020816:Amino Acid Motifs; D001483:Base Sequence; D015152:Blotting, Northern; D004722:Endoribonucleases / Q000378:metabolism; D005821:Genetic Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D012313:RNA / Q000302:isolation & purification* / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism; D043262:Ribonuclease P; D012261:Ribonucleoproteins / Q000302:isolation & purification*; D019809:Streptavidin / Q000737:chemistry
D008024:Ligands; D016337:RNA, Catalytic; D012261:Ribonucleoproteins; D012313:RNA; D019809:Streptavidin; D004722:Endoribonucleases; D043262:Ribonuclease P