STUDIES ON THE MECHANISM OF STABILIZATION OF PARTIALLY PHOSPHOROTHIOATED OLIGONUCLEOTIDES AGAINST NUCLEOLYTIC DEGRADATION

The use of chimeric oligonucleotides (ODN), in which certain phosphodi ester internucleoside linkages are replaced by phosphorothioate (PS) l inkages to provide protection against degradation by nucleases, is gai ning increasing attention because of their significantly decreased pro pensity for nonantisense effects as compared with uniformly PS-modifie d ODN, We have recently reported that partially PS-modified ODN, in wh ich endcapping is used to prevent hydrolysis by exonucleases in combin ation with PS protection at internal pyrimidine residues which are the major sites of endonuclease degradation, are surprisingly stable in s erum. The present study investigates an additional role of the backbon e modification in the stabilization of ODN against nucleolytic degrada tion. We show that the stability of an unmodified ODN in fetal bovine serum is significantly enhanced in the presence of PS-modified ODN. Th e magnitude of stabilization is strongly dependent on the type and deg ree of backbone modification, The observed effect is stronger for PS-m odified ODN than for methylphosphonate (MP)-modified ODN and increases as the number of PS linkages in the ODN increases, Thus, nuclease sta bility of partially PS-modified ODN is not only caused by direct preve ntion of nuclease attack at the phosphate center but is additionally s upported by interference of the nucleases with the PS groups of ODN, r esulting in decreased degradation, As the degree of many nonantisense effects caused by ODN, such as protein interactions and B cell stimula tion, is dependent on the backbone modification, our results may have implications for the use of non-ODN nuclease inhibitors to reduce unde sirable side effects.