Targeted gene knockout by 2 '-O-aminoethyl modified triplex forming oligonucleotides

Triplex forming oligonucleotides (TFOs) are of interest because of their po tential for facile gene targeting. However, the failure of TFOs to bind tar get sequences at physiological pH and Mg2+ concentration has limited their biological applications. Recently, pyrimidine TFOs with 2 ' -O-aminoethyl ( AE) substitutions were shown to have enhanced kinetics and stability of tri plex formation (Cuenoud, B., Casset, F., Husken, D., Natt, F., Wolf, R. M., Altmann, K. H., Martin, P., and Moser H. E. (1998) Angew. Chem. Int, Ed. 3 7,1288-1291). We have prepared psoralen-linked TFOs with varying amounts of the AE-modified residues, and have characterized them in biochemical assay s in vitro, and in stability and HPRT gene knockout assays in vivo. The AE TFOs showed higher affinity for the target in vitro than a TFO with uniform 2 ' -OMe substitution, with relatively little loss of affinity when the as say was performed in reduced Mg2+. Once formed they were also more stable i n "physiological" buffer, with the greatest affinity and stability displaye d by the TFO with all but one residue in the AE format. However, TFOs with lesser amounts of the AE modification formed the most stable triplexes in v ivo, and showed the highest HPRT gene knockout activity. We conclude that t he AE modification can enhance the biological activity of pyrimidine TFOs, but that extensive substitution is deleterious.