We describe a fast and simple one-step affinity-purification
method for the isolation of specific RNA-binding proteins.
An in vitro-transcribed hybrid RNA consisting of an aptamer
sequence with high binding specificity to the antibiotic
streptomycin and a putative protein-binding RNA sequence
is incubated with crude extract. After complex formation,
the sample is applied to an affinity column containing
streptomycin immobilized to Sepharose. The binding of the
in vitro-assembled RNA–protein complex to streptomycin-Sepharose
is mediated by the aptamer RNA and the specifically bound
proteins are recovered from the affinity matrix by elution
with the antibiotic. Employing two well-characterized RNA–protein
interactions, we tested the performance of this new method.
The spliceosomal U1A protein and the bacteriophage MS2
coat protein could be isolated via their appropriate RNA
motif containing hybrid RNA from crude yeast extracts in
high yield and purity after only one round of affinity
purification. As the purification principle is independent
of the extract source, this new affinity chromatography
strategy that makes use of an in vitro-selected antibiotic-binding
RNA as a tag, “StreptoTag,” should be applicable
to extracts from other organisms as well. Therefore, we
propose StreptoTag to be a versatile tool for the isolation
of unknown RNA-binding proteins.