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