The interaction of platinated triplex-forming oligonucleotides with DNA in mammalian cells

triplex-forming oligonucleotides;androgen receptor;prostate cancer;transcription;platinum;cross-link;Biochemistry

Graham, Mindy KimScott, Alan L. ;

Johns Hopkins University

关键词: triplex-forming oligonucleotides;    androgen receptor;    prostate cancer;    transcription;    platinum;    cross-link;    Biochemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/37834/GRAHAM-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

There has been considerable interest in using triplex-forming oligonucleotides (TFOs) to modulate gene expression by targeting homopurine tracts found in the regulatory and transcribed regions of genes. However, because TFOs bind to their DNA targets via hydrogen bonds, they can be displaced by DNA-binding proteins and polymerases, thus reducing their effectiveness as gene regulating agents. This problem can be circumvented by irreversibly anchoring the TFO to its target. This thesis describes studies on platinum-derivatized TFOs (Pt-TFOs) that consist of 2’-O-methylthymidine, 2’-O-methyl-5-methylcytidine and a 3’ and/or 5’ N7-trans-chlorodiammineplatinum(II)-2;;-deoxyguanosine (PtG). The PtG is designed to form a platinum cross-link between the deoxyguanosine of the TFO and a suitably positioned deoxyguanosine in DNA, thus attaching the Pt-TFO to its DNA target. Experiments with model DNA target duplexes showed that: (1) the extent of cross-linking is greatest when the PtG is located on the 3’ end of the TFO and the target deoxyguanosine is on the same strand as the TFO binding site; (2) multiple, contiguous deoxyguanosines in the TFO binding site significantly reduce cross-linking; and (3) a cytosine adjacent to the PtG of the TFO essentially prevents cross-linking. The presence of a 3’- PtG protects the Pt-TFO from degradation by exonucleases found in mammalian serum. A single Pt-TFO cross-linked to the transcribed strand of a DNA reporter plasmid significantly inhibits transcription of the reporter gene in cultured Chinese hamster ovary cells. Experiments with a fluorescein-labeled Pt-TFO demonstrated that the Pt-TFO could be transfected into LAPC-4 cells, a human prostate cancer cell line, using a commercially available polycationic lipid, and that the Pt-TFO localized in the cytoplasm and nuclei of the transfected cells. Pt-TFOs were designed to target four different homopurine tracts in the human androgen receptor (AR) gene.Three of the four Pt-TFOs were found to cross-link to their designated genomic targets in LAPC-4 cells.One of these Pt-TFOs was shown to inhibit AR transcription and knockdown AR protein levels in these cells. Although the levels of knockdown of AR mRNA and protein were modest, the results suggest that Pt-TFOs have potential as agents to control gene expression at the DNA level.