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J. Biol. Chem., Vol. 276, Issue 31, 28991-28998, August 3, 2001

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Targeted Gene Knockout by 2'-O-Aminoethyl Modified Triplex Forming Oligonucleotides^*

Nitin Puri, Alokes Majumdar, Bernard Cuenoud^§, Francois Natt^¶, Pierre Martin^¶, Andre Boyd, Paul S. Miller, and Michael M. Seidman^**

From the  NIA, National Institutes of Health, Baltimore, Maryland 21224, the ^§ Novartis Horsham Research Centre, Horsham, West Sussex, RH12 4AB United Kingdom, ^¶ Novartis Pharma, Ltd., 4002 Basel, Switzerland, and the  Department of Biochemistry and Molecular Biology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205

Triplex forming oligonucleotides (TFOs) are of interest because of their potential for facile gene targeting. However, the failure of TFOs to bind target 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 triplex 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. 37, 1288-1291). We have prepared psoralen-linked TFOs with varying amounts of the AE-modified residues, and have characterized them in biochemical assays 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 assay was performed in reduced Mg²⁺. Once formed they were also more stable in "physiological" buffer, with the greatest affinity and stability displayed 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 vivo, and showed the highest HPRT gene knockout activity. We conclude that the AE modification can enhance the biological activity of pyrimidine TFOs, but that extensive substitution is deleterious.

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