Biochemistry of HOX Protein DNA Binding
The Stadler Laboratory uses the systematic evolution of ligands by exponential enrichment (SELEX) approach (invented by Larry Gold at the University of Colorado, Boulder) to identify individual HOX protein binding sites. Our adaptation of the SELEX approach, uses purified HOX proteins to identify their preferred binding site from a random mixture of DNA binding sites (see figure below). HOX protein DNA complexes are fractionated on acrylamide gels and the DNA protein complex can be identified by the change in electrophoretic mobility. After 5-6 rounds of site enrichment, the bound DNA sequences are cloned into a plasmid vector and sequenced. To perform this experiment, lab members must learn how to express and purify individual HOX proteins, verify functional folding, and assess DNA binding affinity using quantitative methods.
Determination of the HOXA13 binding site by Selex. (A) Random 16mer DNA. (B) Electrophorhetic mobility enrichment of radiolabeled random 16mer. (C) Sequence alignment of clones selected after 6 successive rounds of binding, electrophorhetic separation, amplification, and rebinding. (D) EnoLogos analysis of nucleotide frequency at each position in 100 purified clones after 6 successive rounds of SELEX.