Aptazyme-Based Riboswitches and Logic Gates in Mammalian Cells

Methods Mol Biol. 2021:2323:213-220. doi: 10.1007/978-1-0716-1499-0_15.

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.

Keywords: Aptamer; Aptazyme; Gene regulation; RNA engineering; Riboswitch; Ribozyme.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • Allosteric Regulation
  • Animals
  • Aptamers, Nucleotide*
  • Base Sequence
  • Cloning, Molecular / methods
  • Digital Technology / methods*
  • Gene Expression Regulation / genetics*
  • Gene Library
  • Genes, Reporter / genetics*
  • Genetic Engineering / methods*
  • HEK293 Cells
  • Humans
  • Logic*
  • Mammals
  • Plasmids / genetics
  • RNA, Catalytic
  • Riboswitch*
  • Transfection

Substances

  • 3' Untranslated Regions
  • Aptamers, Nucleotide
  • RNA, Catalytic
  • Riboswitch