Synthesis and evaluation of a triplex-forming oligonucleotide-pyrrolobenzodiazepine conjugate.
In most cases, unmodified oligonucleotides designed as antigene molecules are incapable of binding to DNA with sufficient stability to prevent gene expression. To stabilize binding to a polypurine tract in the HER-2/neu promoter, a triplex forming oligonucleotide (TFO) was conjugated to a pyrrolo[1,4]benzodiazepine (PBD), desmethyltomaymycin, and site-specific DNA binding was evaluated. An activated ester of the PBD moiety was conjugated by an acylation reaction to a free primary amine on a 50-atom aliphatic linker at the 5' end of the TFO. This long aliphatic linker was designed to provide a bridge from the major groove binding site of the TFO to the minor groove binding site of the PBD. Triplex formation by the resulting TFO-PBD conjugate occurred more slowly and with a nearly 30-fold lower affinity compared to an unconjugated TFO. PBD binding to the triplex target was demonstrated by protection from restriction enzyme digestion, and covalent binding to the exocyclic amino group of guanine was inferred by substituting specific guanines with inosines. Although the binding of the TFO was less efficient, this report demonstrates that in principle, TFOs can be used to direct the binding of a PBD to specific location. Further optimization of TFO-PBD conjugate design, likely involving optimization of the linker and perhaps placing a PBD at both ends of the TFO, will be needed to make gene modification robust.
Zhilina
Zhanna V
ZV
Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724-5024, USA.
Ziemba
Amy J
AJ
Trent
John O
JO
Reed
Michael W
MW
Gorn
Vladimir
V
Zhou
Qun
Q
Duan
Wenhu
W
Hurley
Laurence
L
Ebbinghaus
Scot W
SW
D001569:Benzodiazepines / Q000138:chemical synthesis*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000235:genetics; D011758:Pyrroles / Q000138:chemical synthesis*
D009841:Oligonucleotides; D011758:Pyrroles; C438462:pyrrolo(2,1-c)(1,4)benzodiazepine; C051802:triplex DNA; D001569:Benzodiazepines; D004247:DNA