Figure 1.

Combined modular rational design and in vitro selection for FMN-sensitive allosteric ribozymes. (A) Tripartite construct consisting of a hammerhead ribozyme joined to an FMN-binding aptamer (boxed) via a random-sequence bridge composed of eight nucleotides (N). The three stems that form the unmodified ribozyme are designated I, II and III, and the site of RNA cleavage is indicated by the arrowhead. The randomized bridge serves both as a partial replacement for stem II of the ribozyme and as a flanking stem for the aptamer. The G-C base pair immediately adjacent to the catalytic core is needed for the hammerhead ribozyme to achieve maximal catalytic activity (9, 42). Selection for FMN-inducible (B) and FMN-inhibited (C) allosteric ribozymes gave rise to RNA populations that respond either positively or negatively to the presence of FMN, respectively. The initial RNA pool (G0) and successive RNA populations (G1–G6) are identified.