Targeted gene knockout by 2'-O-aminoethyl modified triplex forming oligonucleotides.
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 Mg(2+) 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 Mg(2+). 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.
Puri
N
N
NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
Majumdar
A
A
Cuenoud
B
B
Natt
F
F
Martin
P
P
Boyd
A
A
Miller
P S
PS
Seidman
M M
MM
D000577:Amides; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004355:Drug Stability; D005091:Exons; D011564:Furocoumarins; D005821:Genetic Techniques; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007202:Indicators and Reagents; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010756:Phosphoric Acids; D017384:Sequence Deletion
D000577:Amides; D011564:Furocoumarins; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D010756:Phosphoric Acids; C011067:phosphoramidic acid; D007041:Hypoxanthine Phosphoribosyltransferase