A DNA-Assisted Binding Assay for Weak Protein–Protein Interactions

We describe a new method used for quantitating weak interactions between proteins in which the weak interaction is “assisted” by a known DNA–DNA interaction. Oligonucleotides, which are conjugated to proteins of interest, contain short complementary DNA sequences that provide additional binding energy for protein–protein interactions. A stretch of unpaired bases links the protein to the hybridizing DNA sequence to allow formation of both protein–protein and DNA–DNA interactions with minimal structural interference. We validated the DNA-assisted binding method using heterodimerizing coiled-coil proteins. The method was then used to measure the predicted weak interaction between two domains of the Escherichia coli l-arabinose operon regulatory protein AraC. The interaction between domains has the expected magnitude (Kd = 0.37 mM) in the absence of arabinose. Upon addition of arabinose, we detected a weaker and unexpected interaction, which may necessitate modification of the proposed mechanism of AraC. The DNA-assisted binding method may also prove useful in the study of other weak protein–protein interactions.