Aptazyme-Based Riboswitches and Logic Gates in Mammalian Cells
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 wordsRiboswitch Aptamer Ribozyme Aptazyme Gene regulation RNA engineering
We thank Linlin Zhou and Anh Miu for contributions to the development of the screening protocol. This work was supported by National Institutes of Health.