Competitive regulation of modular allosteric aptazymes by a small molecule and oligonucleotide effector

Abstract

The hairpin ribozyme can catalyze the cleavage of RNA substrates by employing its conformational flexibility. To form a catalytic complex, the two domains A and B of the hairpin-ribozyme complex must interact with one another in a folding step called docking. We have constructed hairpin ribozyme variants harboring an aptamer sequence that can be allosterically induced by flavin mononucleotide (FMN). Domains A and B are separated by distinct bridge sequences that communicate the formation of the FMN-aptamer complex to domains A and B, facilitating their docking. In the presence of a short oligonucleotide that is complementary to the aptamer, catalytic activity of the ribozyme is completely abolished, due to the formation of an extended conformer that cannot perform catalysis. However, in the presence of the small molecule effector FMN, the inhibitory effect of the oligonucleotide is competitively neutralized and the ribozyme is activated 150-fold. We thus have established a new principle for the regulation of ribozyme catalysis in which two regulatory factors (an oligonucleotide and a small molecule) that switch the ribozyme's activity in opposite directions compete for the same binding site in the aptamer domain.

Redox‐Active Riboswitching: Allosteric Regulation of Ribozyme Activity by Ligand‐Shape Control

“…[4] The 11-nucleotide bulge segment (nucleotides 8-13 and 24-28 in Figure 1 b) has been used before for the construction of FMN-dependent hammerhead aptazymes [5] and for competitive regulation of modular allosteric aptazymes by a small molecule and an oligonucleotide effector. [6] Furthermore, a natural RNA fold within mRNA has been discovered that specifically interacts with FMN to regulate the expression of bacterial genes involved in the biosynthesis and transport of riboflavin and FMN. [7] We have engineered two variants of hairpin aptazymes, HPAR2 and HPAR5 (Figure 1).…”

Ligand‐dependent ribozymes

“…In vitro selection and mixed approaches proved to be very successful because they allowed the generation of a broad variety of aptazymes active in vitro using different types of actuator platforms (e.g., HHR, hairpin, HDV, X motif, group I intron, and artificial ribozymes) . An interesting application of this approach was described by Sen and coworkers who used an in vitro selected aptamer specific for binding one but not the other of two isomers of a photoconvertible compound for the construction of an allosteric HHR whose catalysis is controlled by irradiation with visible versus ultraviolet light …”

Reversible Photo-regulation of a Hammerhead Ribozyme Using a Diffusible Effector

“…30,31 Rate enhancements of ∼900-fold, as measured here, have typically been observed only in cases where both rational design and in vitro selection have been used in conjunction to create allosteric ribozymes. 32 The UG-dihydropyrene ribozyme, however, demonstrates that modular rational design alone may contribute to sizable rate enhancements. Likely, a very effective structural stabilization of the communication module is enabled by the binding of the BDHP-PEG ligand to the ribozyme aptamer domain.…”