Inhibition of transcription by platinated triplex-forming oligonucleotides

摘要

Platinated triplex-forming oligonucleotides (TFOs) consisting of 2'-methoxythymidine and 2'-methoxy- 5-methylcytidine and an N-7 platinated deoxyguanosine (~(Pt)G) at the 5'-(~(Pt)G-TFO), 3'-(TFO-G~(Pt)), or 3'- and 5'-(~(Pt)G-TFO-G~(Pt)) ends of the TFO form mono-(~(Pt)G-TFO and TFO-G~(Pt)) and interstrand (~(Pt)G-TFO-G~(Pt)) cross-links with target DNA as a result of reaction of the PtG with guanines adjacent to the homopurine TFO binding site in the target. The extent of cross-linking is greatest when the ~(Pt)G is located on the 30 end of the TFO and the target guanine is on the same strand as the TFO binding site. Multiple, contiguous deoxyguanosines in the TFO binding site or a cytosine adjacent to the G~(Pt) of the TFO significantly reduce cross-linking. DNA reporter plasmids in which platinated TFOs were cross-linked at a site in the transcribed region between a CMV promoter and a luciferase reporter gene were transfected into Chinese hamster ovary cells, and luciferase expression was compared with that for the corresponding non-cross-linked plasmid. Luciferase expression was inhibited 95 % when TFO-G~(Pt) was bound and cross-linked to the transcribed strand, demonstrating that the cross-linked TFO was able to block transcription elongation. Further inhibition (99 %) was observed in nucleotide excision repair (NER) deficient cells, suggesting that NER may repair this lesion. The 3'-G~(Pt) group of TFO-G~(Pt) protects the TFO from degradation by exonucleases found in mammalian serum. Taken together, these results suggest that platinated TFOs of the type TFO-G~(Pt) may find applications as agents for suppressing DNA transcription and consequently inhibiting gene expression in mammalian cells.