Design, synthesis and studies of a high efficacy antisense poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotides

To improve the bioavailability of oligoribonucleotide, our lab has developed a unique method that modifies the oligoribonucleotide with 2, 4-dinitroflurobenzene. The derivatized oligoribonucleotide, poly-2′-O-(2,4-dinitrophenyl)-oligoribonucleotide (poly-DNP-RNA), is resistant to RNase A, B, S, T₁, T₂, and H, and has increased membrane permeability.; In the present study, a high efficacy antisense inhibitor poly-2 ′-O-(2,4-dinitrophenyl)-5′-GGCUGCGUGCCUCCUCACUGG (named antisense poly-DNP RNA-21) has been synthesized by in vitro transcription followed by chemical derivatization. Its base sequence is complementary to that of nt 110 to 130 in RI_α/PKA mRNA which is overexpressed in MCF-7 breast cancer cells. The bioavailable and RNase-resistant antisense poly-DNP RNA-21 was found to inhibit cell growth with IC₅₀ values of 0.05 nM in MCF-7 cells. The control 21-nt RNAs with the same poly-DNP oligonucleotide platform but with scrambled, sense or mismatched base sequence are inactive. Treatment of MCF-7 cells with antisense poly-DNP RNA-21 abolishes both the steady-state concentration of RI_α mRNA and the synthesis of RI_α protein.; At sufficiently high concentration, antisense poly-DNP RNA-21 kills selectively the targeted cancer cells by inducing apoptosis. In subsequent molecular level studies, involvement of two major apoptotic pathways in antisense poly-DNP-RNA-21 induced apoptosis was further explored by western blot assay. Caspase-8 was found to be significantly activated, whereas Bcl-2 protein expression level was only partly regulated.; The observed sequence specificity and extremely low IC₅₀ values of antisense poly-DNP RNA-21 suggest that it is a promising candidate for in vivo testing as an effective anti-cancer agent.