Electron microscopic visualization of tRNA genes with ferritin-avidin: biotin labels

Abstract

A method is described for indirect electron microscopic visualization and mapping of tRNA and other short transcripts hybridized to DNA. This method depends upon the attachment of the electron-dense protein ferritin to the RNA, the binding being mediated by the remarkably strong association of the egg white protein avidin with biotin. Biotin is covalently attached to the 3' end of tRNA using an NH2(CH2)5NH2 bridge. The tRNA-biotin adduct is hybridized to complementary DNA sequences present in a single stranded non-homology loop of a DNA:DNA heteroduplex. Avidin, covalently crosslinked to ferritin, is mixed with the heteroduplex and becomes bound to the hybridized tRNA-biotin. Observation of the DNA:RNA-biotin:avidin-ferritin complex by electron microscopy specifically and accurately reveals the position of the tRNA gene, with a frequency of labeling of approximately 50%.