The development of bioactive triple helix-forming oligonucleotides.
We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in mammalian cells. We have described psoralen-conjugated TFOs containing 2'-O-methyl (2'OMe) and 2'-O-aminoethoxy (AE) ribose substitutions. TFOs with a cluster of 3-4 AE residues, with all other sugars as 2'OMe, were bioactive in a gene knockout assay in mammalian cells. In contrast, TFOs with one or two clustered, or three dispersed, AE residues were inactive. Thermal stability analysis of the triplexes indicated that there were only incremental differences between the active and inactive TFOs. However the active and inactive TFOs could be distinguished by their association kinetics. The bioactive TFOs showed markedly greater on-rates than the inactive TFOs. It appears that the on-rate is a better predictor of TFO bioactivity than thermal stability. Our data are consistent with a model in which a cluster of 3-4 AE residues stabilizes the nucleation event that precedes formation of a complete triplex. It is likely that triplexes in cells are much less stable than triplexes in vitro probably as a result of elution by chromatin-associated translocases and helicases. Consequently the biologic assay will favor TFOs that can bind and rebind genomic targets quickly.
Seidman
Michael M
MM
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA. seidmanm@grc.nia.nih.gov
Puri
Nitin
N
Majumdar
Alokes
A
Cuenoud
Bernard
B
Miller
Paul S
PS
Alam
Rowshon
R
D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D018390:Gene Targeting; D006801:Humans; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides / Q000737:chemistry*
D002843:Chromatin; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D004247:DNA