Book of Abstracts: Albany 2005
Nucleic Acid Third Strand Binding Probes for Targets in the Human MDR-1 Gene
A high level of MDR-1 (multi-drug resistant) gene amplification is associated with a high rate of efflux of anticancer agents from cells, thereby limiting the effectiveness of chemotherapy. Our development of cytogenetic probes for cancer relevant genes is based on the specific recognition of double-stranded homopurine·homopyrimidine DNA target sequences by third strand-binding deoxyoligonucleotides. Two homopurine·homopyrimidine target sequences (15 and 17 bp), located in the promoter region of the human MDR-1 gene, were selected from Genbank for third strand probing in both linear and plasmid target-containing sequences. Homopyrimidine third strands were synthesized with a psoralen moiety at the 5'-end and with 5-methylcytosine residues in place of cytosines. pDR6.5 super-coiled plasmid contains a 6.5kb EcoRI fragment of the MDR-1 gene cloned into a pUC8 vector (gift of Merrill Goldsmith). Probe binding to the target duplex involved incubation and UV-irradiation of the probe with linear duplex or super-coiled plasmid. In the case of the plasmid, the specific fragments of 149 and 173 bp with bound third strand were obtained by restriction. The binding affinity and stability of the triplex formed with the target duplex in both the linear and plasmid cases were determined based upon band-shift in non-denaturing PAGE. The binding affinity was analyzed to determine Kd values. Careful selection of the most favorable conditions for binding experiments (buffer composition, pH, salt concentration and temperature) strongly promoted the thermodynamic stability of triplexes, with Kd values in the 10-7 - 10-9 M range.
Supported by NIH grant CA88547.