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RNA Scaffolds

Volume 1316 of the series Methods in Molecular Biology pp 141-148

Aptazyme-Based Riboswitches and Logic Gates in Mammalian Cells

  • Yoko NomuraAffiliated withDepartment of Biomedical Engineering, University of California, Davis
  • , Yohei YokobayashiAffiliated withDepartment of Biomedical Engineering, University of California, Davis Email author 

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Abstract

This chapter describes a screening strategy to engineer synthetic riboswitches that can chemically regulate gene expression in mammalian cells. Riboswitch libraries are constructed by randomizing the key nucleotides that couple the molecular recognition function of an aptamer with the self-cleavage activity of a ribozyme. The allosteric ribozyme (aptazyme) candidates are cloned in the 3′ untranslated region (UTR) of a reporter gene mRNA. The plasmid-encoded riboswitch candidates are transfected into a mammalian cell line to screen for the desired riboswitch function. Furthermore, multiple aptazymes can be cloned into the 3′ UTR of a desired gene to obtain a logic gate response to multiple chemical signals. This screening strategy complements other methods to engineer robust mammalian riboswitches to control gene expression.

Key words

Riboswitch Aptamer Ribozyme Aptazyme Gene regulation RNA engineering