EFFECT OF DERIVATIZATION OF RIBOPHOSPHATE BACKBONE AND TERMINAL RIBOPHOSPHATE GROUPS IN OLIGORIBONUCLEOTIDES AN THEIR STABILITY AND INTERACTION WITH EUKARYOTIC CELLS

Various derivatives of oligoribonucleotides were synthesized by the H- phosphonate method. Different modifications of the ribophosphate backb one were designed in order to protect the derivatives against nucleoly tic enzymes present in the biological media. These modifications inclu de coupling of fluorescein moiety to 3'-terminal ribose, 2'-O-methylat ion of ribose, introduction of phosphorothioate internucleotide bonds throughout the molecule, replacement of the two last 3'-terminal phosp hodiester bonds by phosphoroamidates and coupling of the last 3'-termi nal nucleotide via the 3'-3'-phosphodiester bond. All modifications we re tested for their effect on the stability of the derivatives against phosphodiesterase from snake venom and nucleases of the cell culture media. 2'-O-methylated oligoribonucleotides containing either terminal 3'-3'-linkage or two 3'-terminal phosphoroamidate internucleotide bon ds appeared to be the most stable under the most severe conditions use d. The results demonstrate a possibility to use protected oligoribonuc leotide derivatives for experiments in vivo when the use of deoxy-anal ogues might be ineffective. The uptake of 2'-O-methylated derivatives and their 5'-cholesterol conjugates (coupled via a disulfide bond) by human carcinoma cells did not differ from that of the corresponding ol igodeoxyribonucleotides. 85% of the bound derivatives were found in th e membrane-cytosolic fraction, while only 15% were found in the nuclea r fraction. The oligonucleotide moiety of 2'-O-methyloligoribonucleoti de-cholesterol conjugate was not translocated through the cellular mem brane. After cleavage of the linkage between cholesterol and oligonucl eotide by dithiothreitol the major portion of the oligonucleotide moie ty was released into the media. The derivatives, as well as their 5'-c holesterol conjugates, which entered the cells, were stable and protec ted from action of dithiothreitol dissolved in culture media. These re sults demonstrate an endocytosis mechanism of penetration as observed in similar experiments using oligodeoxyribonucleotides.