Binding studies on peptide-oligonucleotide complex:: intercalation of tryptophan in GC-rich region of c-myc gene

Transcriptional regulation of the c-myc gene is essential for normal cellular proliferation; differentiation and overexpression of c-myc is associated with several human cancers. C-myc gene, particularly exon 1, which contains the conserved P1 and P2 promoter regions, has been a potential target for the intercalating drugs in chemotherapy. We have chosen a 21-mer GC-rich oligonucleotide sequence starting from 2281 to 2302 of human c-myc gene located 26 base pair upstream of P1 promoter and partially overlapping with the TATA box of P1. In this paper, we have studied the interaction of a tetrapeptide, KWGK-otBut, with duplex of the above 21-mer sequence under low-salt conditions using UV-Vis absorption, UV melting, fluorescence and circular dichroic (CD) spectroscopy. From the fluorescence quenching data, we determined the two binding constants, K-2 (involving only electrostatic interactions) and K-2 (involving intercalation), for the formation of (PN)(1) and (PN)(2) of the two-step mechanism previously established by us. Significant changes were observed in the UV difference absorption spectra and CD spectra of both KWGK and 21-mer duplex upon complex formation even at a very low peptide to nucleotide (P/N) ratios. These spectral changes accompanied by a high value of K-2 (= 5.13) suggest a strong binding of KWGK involving intercalation of the tryptophan in 21-mer duplex. Based on the above data along with changes observed in DeltaH, DeltaS and DeltaG and increase in melting temperature (by about 8 degreesC) of the 21-mer duplex in presence of KWGK, we propose a model for intercalation of tryptophan of in GC-rich region of cm myc gene. Present observations may be explored in understanding the role of intercalation in protein-nucleic acid interactions in c-myc expression and these results could also help in designing oligopeptides or other low molecular weight ligands to modulate gene expression. (C) 2003 Elsevier B.V. All rights reserved.