Fluorescent SSB as a Reagentless Biosensor for Single-Stranded DNA

Abstract

Helicases are an important and much studied group of enzymes that generally couple ATP hydrolysis to the separation of strands of base-paired nucleic acids. Studying their biochemistry at different levels of organization requires assays that measure the progress of the reaction in different ways. One such method makes use of the single-stranded DNA-binding protein (SSB) from Escherichia coli. This is used as a protein framework to produce a "reagentless biosensor," making use of its tight and specific binding of single-stranded DNA. The attachment of a fluorophore to this protein produces a signal in response to that binding. Thus the (G26C)SSB, labeled with a diethylaminocoumarin, gives a ~5-fold fluorescence increase on binding to single-stranded DNA and this can be used to assay the progress of helicase action along double-stranded DNA. A protocol for this is described along with a variant that can be used to follow the unwinding on a single molecule scale.