Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3'-terminus and their interaction with DNA within mammalian cells.

5'-[32P]-labelled alkylating decathymidylate [4-(N-2-chloroethyl)N-methylaminobenzyl]-5'-phosphamide derivatives containing cholesterol or phenazinium residues at their 3'-termini were synthesized and used for alkylation of DNA within mammalian cells. The uptake of the cholesterol derivative by the cells and the extent of DNA alkylation are about two orders of magnitude higher than those of a similar alkylating derivative lacking the groups at the 3'-termini. The presence of the phenazinium residue at the 3'-terminus of the oligonucleotide reagent does not improve the reagent uptake by the cells but drastically increases the DNA modification efficiency.

tRNAPhe deprived of 3'-terminal adenosyl residue does not stimulate adenosine aminoacylation catalyzed by phenylalanyl-tRNA synthetase from Escherichia coli.

Phenylalanyl-tRNA synthetase from Escherichia coli does not catalyze the [14C]phenylalanyl residue transfer from phenylalanyl-adenylate to adenosine either in the presence or absence of homologous tRNAPhe and tRNA(-A Phe). When the reaction mixture contained dithiothreitol, radioactive substance was detected having a mobility on HPLC column close to that of aminoacyladenosine. The amount of this product depended on the concentration of dithiothreitol in the mixture.

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.