Purification of endogenous tDRs by hybridization-based pulldown.

Abstract

As transfer RNAs (tRNAs) are characterized by the existence of a variety of post-transcriptional modifications, transfer RNA-derived RNAs (tDRs) also possess various modifications. Accumulating evidence suggests that these modifications can regulate the biogenesis and the biological functions of tDRs. Therefore, it is important to purify endogenous tDRs for examining the physiological roles of tDRs. Here we present a simple protocol for purification of endogenous tDRs by hybridization-based pulldown. In this method, tDRs of interest are hybridized to biotinylated oligo DNA probes, followed by pulldown using a streptavidin agarose resin. Resin-bound tDR-probe complexes are then isolated by competitive dissociation using excess amount of biotin. After digestion of probes by DNase I, the purified tDRs are obtained. As the pulldown efficiency of this method largely depends on how efficiently tDRs are generated, the yield can be significantly improved by combination with methods for efficient tDR production, such as in lysate RNA digestion method that we previously reported.