USE OF PARTIALLY PHOSPHOROTHIOATED ANTISENSE OLIGODEOXYNUCLEOTIDES FOR SEQUENCE-DEPENDENT MODULATION OF HEMATOPOIESIS IN CULTURE

To distinguish between sequence-dependent effects and non-specific cyt otoxicity of phosphorothioate antisense oligonucleotides (AS-oligos), we introduced AS-oligos blocking expression of 2Hs, the Home sapiens c ell division controller cdc, kinase, its hematopoietically expressed h omolog CHED, and the acetylcholine-hydrolyzing enzyme butyrylcholinest erase (BCHE) into primary murine bone marrow (BM) culture. Antisense o ligonucleotides were fully phosphorothioated (Ts) or prepared with thr ee phosphorothioate groups at their 3' termini (S3). Each of these oli gos could cause reductions in colony counts either as a result of its sequence-dependent biological capacity or due to sequence-independent cytotoxicity. The Ts and S3 forms of the matching sense oligo, S-BCHE, served for comparison. The S3 forms of AS-2Hs, AS-BCHE, and S-BCHE ca used more limited drops in colony counts than their Ts counterparts, r eflecting lower cytotoxicity. When incubated with electroblotted BM pr oteins, Ts but not S3 oligos intensively labeled two protein bands. Mo reover, S-end P-32-labeled (Ts) S-BCHE labeled nuclear proteins in sit u in small, mitotic cells, suggesting correlation between oligo-protei n interactions and the sequence-independent cytotoxicity of Ts AS-olig os. Extension of the apparently nontoxic AS-CHED by two adenosine resi dues at the 3' end, creating a potential for intramolecular hydrogen b ond formation, resulted in increased toxicity. These findings recommen d the use of nonlooped, partially phosphorothioated oligos for the mod ulation of hematopoiesis.