Erroneous results of 3H-thymidine incorporation are related to position of thymidine residues in oligodeoxynucleotides.

: Antisense oligodeoxynucleotides (ODNs) targeted to complementary mRNA sequences have proved to be a powerful approach in assessing the function and the role of unique genes in cell proliferation, differentiation, or transformation. Despite their importance in the development of future therapies, little is known about their fate after uptake by cells. Here, we have examined the contribution of individual nucleotide residues from synthetic nonspecific ODNs on assays commonly used to measure cell proliferation. A dramatic decrease of the 3H-thymidine (3H-T) incorporation was obtained with nonspecific ODNs, while no effect on cell proliferation was observed as assessed by three other techniques. We demonstrate that the presence and position of thymidine in the ODNs directly interfere with the intracellular thymidine pool, leading to faulty data of 3H-T incorporation. As an alternative method, we used 3H-deoxyuridine (3H-dU), which is integrated more efficiently in DNA than thymidine. We observed that 3H-dU incorporation was also decreased. In conclusion, caution should be exercised in the interpretation of the results of 3H-T and 3H-dU incorporation in the presence of oligodeoxynucleotides.