Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes

a1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA


Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using “allosteric selection,” a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3′,5′-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062–1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This “affinity maturation” effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches.

(Received November 24 2000)
(Revised December 21 2000)
(Accepted January 9 2001)

Key Words: aptamer; catalytic RNA; effector; hammerhead; in vitro selection; molecular recognition.

c1 Reprint requests to: Ronald R. Breaker, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA; e-mail: ronald.breaker@yale.edu.
p1 Present address: Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, Nebraska 68178, USA.
p2 Present address: Sankyo Co., Ltd. 2-58, Hiromachi 1-Chrome, Shinagawa-Ku, Tokyo 140-8710, Japan.