Assays for affinity profiling of nucleic acid binding proteins

摘要

Methods, compositions and kits are disclosed for assays to determine the binding affinity of DNA-binding proteins or RNA-binding proteins for their corresponding recognition site(s). In particular, assays are disclosed for measuring binding affinities when either the binding protein, or the recognition sequence of the recognition site, or cofactor proteins, contain one or more mutations. The disclosed assays can thus be utilized to measure the effect on transcription factor binding caused by mutations within the recognition site, or mutations within the binding domain of the protein, and to provide binding affinity information that can be correlated with altered gene regulation and expression. The disclosed assays can be personalized to a specific person or organism, with the measured binding affinities based upon an individual's specific binding proteins and recognition sites. Furthermore, embodiments are capable of measuring binding affinities between multiple binding proteins and multiple recognition sites through an entirely in vitro process.

主权项

1. A method for measuring an affinity level of at least one binding protein for at least one recognition site, the method comprising:
hybridizing a target nucleic acid to a capture oligonucleotide; synthesizing a complementary nucleic acid, wherein the complementary nucleic acid extends the capture oligonucleotide and is complementary to the target nucleic acid, and wherein synthesizing the complementary nucleic acid creates a recognition site for a binding protein; introducing the binding protein to the recognition site; and measuring an affinity level of the binding protein for the recognition site.