Isolation of biologically active transfer RNA from transfer RNA-DNA hybrids

Abstract

A method for the isolation and purification of specific tRNA-DNA hybrids that avoids the use of ribonuclease is described. This method enables the extraction of biologically active tRNA from its hybrid with DNA. The tRNA-DNA hybrid was prepared by incubating a solution of Escherichia coli DNA denatured by alkali with Escherichia coli tRNA at 70° for 60 min. Excess free tRNA was removed from the hybrid by Sephadex G-200 gel filtration. Further purification of the hybrid was achieved by heating this fraction at 50° for 10 min and adsorption onto nitrocellulose filters. About 80–90% of the tRNA found on the filter was in a true hybrid form with DNA and was resistant to ribonuclease digestion. Extraction of the filters with a solution of low ionic strength followed by deoxyribonuclease digestion yielded tRNA preparations that had amino acid acceptor capacity. The amino acid acceptor capacity of the extracted tRNA in different experiments amounted to 80–90% of that of the initial tRNA. The method is likely to be generally applicable for the determination of the origin and homology of tRNA, in particular in virus-infected systems.