Studies on the mechanism of stabilization of partially phosphorothioated oligonucleotides against nucleolytic degradation.
The use of chimeric oligonucleotides (ODN), in which certain phosphodiester internucleoside linkages are replaced by phosphorothioate (PS) linkages to provide protection against degradation by nucleases, is gaining increasing attention because of their significantly decreased propensity for nonantisense effects as compared with uniformly PS-modified ODN. We have recently reported that partially PS-modified ODN, in which end- capping is used to prevent hydrolysis by exonucleases in combination with PS protection at internal pyrimidine residues which are the major sites of endonuclease degradation, are surprisingly stable in serum. The present study investigates an additional role of the backbone modification in the stabilization of ODN against nucleolytic degradation. We show that the stability of an unmodified ODN in fetal bovine serum is significantly enhanced in the presence of PS-modified ODN. The magnitude of stabilization is strongly dependent on the type and degree of backbone modification. The observed effect is stronger for PS-modified ODN than for methylphosphonate (MP)-modified ODN and increases as the number of PS linkages in the ODN increases. Thus, nuclease stability of partially PS-modified ODN is not only caused by direct prevention of nuclease attack at the phosphate center but is additionally supported by interference of the nucleases with the PS groups of ODN, resulting in decreased degradation. As the degree of many nonantisense effects caused by ODN, such as protein interactions and B cell stimulation, is dependent on the backbone modification, our results may have implications for the use of non-ODN nuclease inhibitors to reduce undesirable side effects.
Uhlmann
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E
Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007-2197, USA.
Ryte
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A
Peyman
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D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004355:Drug Stability; D005092:Exonucleases / Q000378:metabolism; D007700:Kinetics; D016376:Oligonucleotides, Antisense / Q000097:blood / Q000493:pharmacokinetics*; D011743:Pyrimidines; D013873:Thionucleotides; D014709:Vero Cells
D016376:Oligonucleotides, Antisense; D011743:Pyrimidines; D013873:Thionucleotides; D005092:Exonucleases