Figure 4.

In vitro cleavage assays between the conjugates and the IRES from HCV. Purified 3′-labelled IRES is incubated with either 50 μM free imidazole or increasing concentrations of the naked or the 3′/5′ imidazole-coupled antisense oligonucleotides for one hour at 37°C. Partial RNA sequencing using RNases T1 and U2 under denaturing conditions as well as partial alkaline RNA hydrolysis allows the mapping of the cleavage sites within the IRES RNA sequence. The arrow points to the cleavage site, between G₂₇₁ and C₂₇₂, induced by conjugate 1. The stars point to three additional cleavage sites induced by conjugates 1 and 4, mapped between U₂₉₇ and A₂₉₈ in domain IIIe (*1), between G₁₇₁ and A₁₇₂ between domains IIIa and IIIb (*2) and between G₁₈₁ and A₁₈₂ in domain IIIb (*3). Note that the two highest concentrations of the naked oligonucleotide elicit a weak cleavage between G₁₇₁ and A₁₇₂ (*2).