Inhibition of c-myc expression in cells by targeting an RNA-protein interaction using antisense oligonucleotides.

Antisense oligodeoxynucleotides (ODNs) are designed to bind to and inhibit a target mRNA. We used a novel approach for the design of ODNs to the c- myc mRNA using protein binding sites as targets for ODN action. Our strategy was to identify ODNs that could interfere with the coding region determinant-binding protein (CRD-BP), a protein that binds to the CRD region of the c- myc mRNA. Using an in vitro gel shift assay, we show that ODN molecules can occlude the CRD-BP from the mRNA. The best ODN, CRD-ODN4, was able to inhibit RNA binding of the CRD-BP by 75%. This effect was sequence-specific and concentration dependent. K562 cells treated with a 2′- O -methyl derivative of CRD-ODN4 showed a concentration-dependent decrease in both c- myc mRNA and protein levels, with a maximal 65% inhibition of protein expression at 200 nM CRD-ODN4. In contrast, a 2′- O -methyl ODN derivative targeting the translation initiation codon (antimyc-aug) reduced c- myc protein but actually increased mRNA levels, an effect resulting at least partly from stabilization of the c- myc mRNA. CRD-ODN4 treatment did not alter the c- myc mRNA half-life. CRD-ODN4 was more effective in inhibiting K562 cell growth than antimyc-aug, reducing cell number by ≈70% after 48 h of exposure to 750 nM. The correlation between ODN effects on RNA-protein interactions in vitro and those observed in cells supports the hypothesis that CRD-ODN4 inhibits the interaction between the CRD-BP and the c- myc mRNA and that disrupting this RNA-protein interaction reduces c- myc expression in cells.