Engineering protein and DNA tools for creating DNA-dependent protein switches

Methods Enzymol. 2022:675:1-32. doi: 10.1016/bs.mie.2022.07.002. Epub 2022 Aug 23.


Switchable proteins are capable of changing conformations from inactive (OFF) to active (ON) forms in response to inputs such as ligand binding, pH or temperature change, or light absorption. A particularly powerful class of protein switches, exemplified by the Cas nucleases of CRISPR systems, are activated by binding of specific DNA or RNA sequences. The mechanism by which oligonucleotide binding regulates biological activity is complex and highly specialized in the case of Cas enzymes, but recent advancements in protein and DNA engineering have made it possible to introduce this mode of control into other enzymes. This chapter highlights recent examples of protein switches that combine these two fields of engineering for the purpose of creating biosensors that detect pathogen and other genomic sequences. One protein engineering method-alternate frame folding-has the potential to convert many proteins into ligand-activated switches by inserting a binding protein (input domain) into an enzyme (output domain). The steps for doing so are illustrated using GCN4 as a DNA recognition domain and nanoluciferase as a luminescent reporter that changes color as a result of DNA binding. DNA engineering protocols are included for creating DNA tools (de novo designed hairpins and modified aptamers), that enable the biosensor to be activated by arbitrary DNA/RNA sequences and small molecules/proteins, respectively. These methodologies can be applied to other proteins to gain control of their functions by DNA binding.

Keywords: Alternate frame folding; Aptamer; BRET; Biosensor; GCN4; Luminescence; Nanoluciferase; Toehold-mediated strand displacement.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • DNA / chemistry
  • DNA / genetics
  • Ligands
  • Oligonucleotides
  • Protein Engineering* / methods
  • Proteins* / chemistry
  • Proteins* / genetics


  • Ligands
  • Oligonucleotides
  • Proteins
  • DNA