Binding Stoichiometry of an RNA Aptamer and Its Transcription Factor Target

RNA molecules serve informational, structural, and catalytic roles in cells. RNA also offers an interesting raw material for the design or genetic selection of modifiers of gene expression. We have been interested in the possibility that natural and/or artificial RNA ligands might be identified for DNA-binding proteins. With these concepts in mind, our laboratory previously isolated a 31-nucleotide RNA aptamer that specifically binds to human transcription factor NF-κB. This RNA aptamer (α-p50) competitively inhibits DNA binding by NF-κB in vitro. The aptamer may target the DNA-binding groove formed by the junction of the two monomers of NF-κB, perhaps mimicking κB duplex DNA. This model predicts a binding stoichiometry of one RNA aptamer per NF-κB dimer. To test this hypothesis, two complementary biophysical methods were utilized. Both analytical ultracentrifugation and microelectrospray mass spectrometry suggest that 1 mol of α-p50 RNA binds per mole of NF-κB p50 homodimer. Such a result is consistent with the observed ability of the RNA aptamer to block the access of transcription factor NF-κB to its binding site on DNA and highlights the question of how an RNA stem–loop structurally mimics a DNA duplex. This work also demonstrates the successful application of mass spectrometry to characterize noncovalent RNA/protein interactions.