Reconstruction of tRNAPhe molecules from the fragments by linkage with T-4 RNA ligase in double-stranded regions

Phenylalanine-specific tRNA from yeast was hydrolysed with cobra venom ribonuclease in the double-stranded regions and the fragments isolated. The 'dissected' molecules with nicks in positions 28 and 41 were reconstructed from supplementary fragments and treated with T-4 RNA ligase. A phosphodiester bond between two fragments was formed when the fragment combination (1-28) + (29-76) was used. A strong discrimination in the ligation yield between different nick positions in the same helix is shown.