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, Michael M. Seidman

Research output: Contribution to journalArticlepeer-review

54 Scopus citations


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 Mg3+ 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 viva 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.

Original languageEnglish (US)
Pages (from-to)28991-28998
Number of pages8
JournalJournal of Biological Chemistry
Issue number31
StatePublished - Aug 3 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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