The Sensitive Side of Protein-dna Interactions

Two groups describe unique methods to sensitively and quantitatively investigate protein-DNA interactions in vitro. A plethora of tools exists to probe protein-DNA interactions, each one with its own strengths and weaknesses. The sensitivity and specificity of the methods, however, are crucial considerations when attempting to draw quantitative conclusions about binding affinity. Two recent papers provide welcome additions to the growing arsenal of methods to quantify protein-DNA binding interactions. Ulf Landegren of Uppsala University and his colleagues were interested in developing alternative, highly sensitive and specific methods for analyzing transcription factor–DNA binding interactions in vitro. Their method of choice was proximity ligation, in which real-time PCR is used as the readout for detecting the interaction between a DNA sequence tagged with an oligonucleotide and a protein recognized by a specific antibody probe tagged with another oligonucleotide sequence (Gustafsdottir et al., 2007). The ligation products are detected by real-time PCR upon hybridization of the probe sequences with a connector oligonucleotide. Charles Cantor and his colleagues at Boston University and SEQUENOM, Inc. were also interested in developing highly sensitive and quantitative in vitro assays to monitor transcription factor–DNA binding interactions. Transcription factors regulate gene expression by binding to DNA at transcription start sites; understanding this process is therefore a fundamental challenge in biology. Cantor and colleagues turned to mass spectrometry to craft a new binding assay. They labeled putative DNA-binding sequences with an oligonucleotide mass tag (OMT), which is a short nucleic acid sequence with a distinct mass. Molecules tagged with OMTs can be easily identified with relatively lowresolution mass spectrometry methods by monitoring the mass alone. Cantor explains, “In a mass spectrometer, each [mass tag] corresponds to a color, like fluorescence... [but unlike fluorescence there is] the potential for [using] probably hundreds of ‘colors’.” Notably, OMTs have dual detection capabilities, in that they can both be amplified by PCR and detected in multiplex by their unique mass. To investigate the binding interactions of the transcription factor NF-κB p50 in multiplex, Cantor and colleagues constructed several putative DNA sequences and tagged each with a unique OMT (Zhang et al., 2007). They also included primer-recognition sequences at 5′ and 3′ ends for PCR amplification. After incubation with the transcription factor, the researchers isolated the binding assemblies from the mixture with a primary antibody recognizing the transcription factor, which was in turn recognized by a secondary antibody attached to a bead. After washing and PCR amplification, the sequences were fed into a mass spectrometer for a quantitative analysis of the binding interactions. Cantor and colleagues demonstrated that 15-plex assays were possible for the purified transcription factor, as well as in HeLa cell nuclear extracts. STEM CELLS