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Artificial Riboswitches

Volume 1111 of the series Methods in Molecular Biology pp 221-235

Date:

Dual-Selection for Evolution of In Vivo Functional Aptazymes as Riboswitch Parts

  • Jonathan A. GolerAffiliated withUniversity of CaliforniaU.S. Department of Energy Joint BioEnergy Institute
  • , James M. CarothersAffiliated withUniversity of CaliforniaU.S. Department of Energy Joint BioEnergy Institute
  • , Jay D. KeaslingAffiliated withUniversity of CaliforniaU.S. Department of Energy Joint BioEnergy Institute

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Abstract

Both synthetic biology and metabolic engineering are aided by the development of genetic control parts. One class of riboswitch parts that has great potential for sensing and regulation of protein levels is aptamer-coupled ribozymes (aptazymes). These devices are comprised of an aptamer domain selected to bind a particular ligand, a ribozyme domain, and a communication module that regulates the ribozyme activity based on the state of the aptamer. We describe a broadly applicable method for coupling a novel, newly selected aptamer to a ribozyme to generate functional aptazymes via in vitro and in vivo selection. To illustrate this approach, we describe experimental procedures for selecting aptazymes assembled from aptamers that bind p-amino-phenylalanine and a hammerhead ribozyme. Because this method uses selection, it does not rely on sequence-specific design and thus should be generalizable for the generation of in vivo operational aptazymes that respond to any targeted molecules.

Key words

In vitro selection RNA aptamer Ribozyme Aptazyme Riboswitch Synthetic biology