Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Triplex-Forming Oligonucleotides (TFO-s) as Probes for Promoter Region of Cancer Relevant Human mdr1 Gene
The over-expression of human mdr1 (multidrug resistant) gene leads to intensive efflux of cytotoxic anticancer drugs out of malignant cells and aggressive tumor behavior. Rational mdr1 gene targeting by TFO-s within the promoter region represents a perspective way to evaluate and regulate mdr1 gene expression. Gene targeting is based on the highly sequence - specific recognition of oligopurine-oligopyrimidine DNA-duplex tract by synthetic oligopyrimidine third strand.
Two target tracts (15 and 17 base pairs) for in vitro binding assay have been chosen from Genbank database and synthesized. Both sequences were located within the promoter region of human mdr1 gene. TFO-s were synthesized as third-strand probes with a psoralen moiety at the 5?-terminus and 5mC residues in place of cytosines. The degree of local triplex formation by each TFO-probe with corresponding target duplex was assessed based upon band shift and intensity data in non-denaturing PAGE. The probes? binding ability was analyzed to determine apparent dissociation constant (Kd) values. High affinity TFO binding makes the designed probes highly suitable for ex/in vivo applications.
1. The developed TFO-probes could be exploited for cytogenetic quantitative detection of valuable TISH-technology (third strand in situ hybridization). This is very sensitive ex vivo procedure for timely clinical diagnosis of MDR-phenomenon.
2. The same oligopurine-oligopyrimidine stretches in gene promoter region serve as target sites also for transcription factors. TFO?s competitive binding leads to blocking of transcription initiation. Thus the over-expression of human mdr1 gene can be artificially down-regulated by TFO-s at the transcriptional level in vivo (?antigene? therapy).
Acknowledgements This work was performed at the Department of Molecular Biology (Prof. J.R. Fresco) of Princeton University, New Jersey, USA. Supported by NIH grant CA88547.
Inst. of Fine Organic Chemistry