RNA affinity tags for the rapid purification and investigation of RNAs and RNA-protein complexes.
Isolation of ribonucleoprotein particles from living cells and cell lysates has allowed the identification of both simple bimolecular interactions and the members of large, extended complexes. A number of different strategies have been devised to isolate these complexes by using affinity purification methods that are specific for the RNA rather than the protein components of these complexes. We describe the use of two such RNA affinity tags: small RNAs that bind with high affinity and specificity to either Sephadex beads or streptavidin affinity resins and can be eluted under mild, native conditions that retain intact complexes. The tags can be inserted into appropriate locations in genes encoding the RNA components, and ribonucleoproteins can be assembled either in vivo or in vitro before affinity isolation. Strategies toward the design and production of these tagged RNA sequences are discussed, and the purification procedure is outlined.
Walker
Scott C
SC
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
Scott
Felicia H
FH
Srisawat
Chatchawan
C
Engelke
David R
DR
D002846:Chromatography, Affinity / Q000379:methods*; D003911:Dextrans / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry
D003911:Dextrans; D012261:Ribonucleoproteins; D012313:RNA; D019809:Streptavidin; C025614:sephadex