Figure 1.

Proposed accessibility-based mechanism of antisense hybridization in living cells. (A) Example target region with color-coded local availability (estimated by base-pairing probabilities) is shown in canonical conformation, as would be expected in the native state. In antisense RNAs (asRNA) targeting by the iRS³, the targeted region must unbind from P3 to become single stranded as shown in (B) with a free energy change of –ΔG_Tf. The iRS³ consists of four main elements: a cis-blocking strand (CB, orange), a ribosome binding site (RBS, blue), the sequence encoding green fluorescence protein (GFP, green) and the probe (pink and black) of 8–27 nucleotides targeting a specific region shown in (B). The expected native state of the iRS³ is shown in (C), and it must also unfold to bind the target region as shown in (D) with a free energy change of –ΔG_asf. Finally, the two unfolded structures bind as in (E) with a free energy change of ΔG_asT to stabilize the unfolded iRS³ and allow translation of GFP. Effective asRNA targeting results in a high fluorescence response.