Single-Molecule Detection of Transcription Factor Binding to DNA in Real Time:  Specificity, Equilibrium, and Kinetic Parameters

Specificity and temporal control of transcriptional machinery are encoded within sequence-specific transcription factors, of which there are thousands in mammalian genomes. Efforts to completely decipher this code will require an understanding of the DNA binding thermodynamic and kinetic properties displayed by each transcription factor, a daunting task given the current methodologies for measuring these interactions. Here, we present a novel methodology to quantify the binding of proteins to target DNA molecules based on single-molecule detection and real-time counting of individual free and bound fluorescently tagged molecules flowing past a detection device. Using this technology, we measured DNA binding by fluorescently tagged domains of four distinct transcription factors, namely, human early growth response protein Egr-1, vertebrate GATA-1, Drosophila GAGA factor, and λ bacteriophage Cro repressor. These proteins represent different structural classes (zinc-finger and helix−turn−helix), quaternary states (monomeric and dimeric), and relative affinities (high, intermediate, and low). Specific binding of each protein to its cognate DNA target was demonstrated at low picomolar concentrations. The equilibrium (Kd) and kinetic (kon and koff) constants governing DNA binding by one of these transcription factors, that of Egr-1, were measured using this approach. Kd values obtained from three different types of saturation titrations were reproducible and consistent, yielding values between 10 and 14 pM that, along with the kinetic constants, agree closely with literature values. Because this methodology offers several significant advantages over other existing approaches, namely, real-time determination, requirement for small amounts of reagents, high reproducibility, exquisite sensitivity, and amenability to high-throughput analysis, it is suitable for characterizing DNA-binding proteins as well as other interacting pairs of molecules that can be fluorescently tagged.