Antisense Recognition of the HER-2 mRNA:Effects of Phosphorothioate Substitution and Polyamines on DNA-RNA,RNA-RNA,and DNA-DNA Duplex Stability

摘要

The HER-2 gene is overexpressed in a subset of breast,ovarian,lung,and pancreatic cancers.Antisense oligonucleotides suppress gene expression depending on the stability of the DNA-RNA hybrids formed at the target site.Polyamines,the cellular cations that interact with DNA and RNA,may influence hybrid stability in the cell.Therefore,we studied the ability of natural polyamines (putrescine,spermidine,and spermine) and a series of their structural analogues to stabilize DNA-RNA and RNA-RNA duplexes using melting temperature (T_m) measurements and circular dichroism (CD) spectroscopy.Phosphodiester (PO) and phosphorothioate (PS) oligonucleotides (ODNs) (15 nucleotides,5'-CTCCATGGTGCTCAC-3') targeted to the initiation codon region of the HER-2 mRNA,and complementary RNA and DNA ODNs,were used in this study.The relative order of thermal stability was as follows:RNA-RNA > PO-DNA-RNA > PO-DNA-PO-DNA > PS-DNA-RNA > PS-DNA-PO-DNA > PS-DNA-PS-DNA.The ability of polyamines to stabilize the duplexes improved with the cationicity of the polyamine,with hexamines being more effective than pentamines,which in turn were more effective than tetramines and triamines.However,chemical structural effects were clearly evident with isovalent homologues of spermidine and spermine.CD spectra showed B and A conformations,respectively,for the DNA and RNA helices.DNA-RNA hybrids adopted an intermediate structure between the B and A forms.These data help us to understand the role of endogenous polyamines in DNA-RNA hybrid stabilization,and provide information for designing novel polyamines to facilitate the use of antisense ODNs for controlling HER-2 gene expression.