Selection and characterization of anti-NF-κB p65 RNA aptamers

NF-κB transcription factors include a group of five mammalian proteins that form hetero- or homodimers and regulate hundreds of target genes involved in acute inflammation, HIV-1 transcription activation, and resistance to cancer therapy. We previously used in vitro selection to develop a small RNA aptamer (anti-p50) that binds the DNA-binding domain of NF-κB p50 ₂ with low nanomolar affinity but does not bind NF-κB p65 ₂. Here, we report the in vitro selection of anti-NF-κB p65 RNA aptamers using parallel in vitro selections with either a fully randomized RNA library or a degenerate RNA library based on the primary sequence of the 31-nucleotide anti-p50 RNA aptamer. We report the characterization of these aptamers with respect to NF-κB target specificity, affinity, minimal sequence requirements, secondary structure, and competition with DNA κB sites. These results expand opportunities for artificial inhibition of NF-κB transcription factor dimers containing p65 subunits. Published by Cold Spring Harbor Laboratory Press.