Combinatorial selection and binding of phosphorothioate aptamers targeting human NF-kappa B RelA(p65) and p50.
Previously, we reported the in vitro combinatorial selection of phosphorothioate aptamers or "thioaptamers" targeting the transcription factor NF-IL6. Using the same approach and purified recombinant human NF-kappa B proteins RelA(p65) and p50, duplex thioaptamers have been selected that demonstrate high-affinity, competitive binding with the duplex 22-mer binding site, Ig kappa B. Binding energetics of RelA(p65) and p50 homodimers were studied using a quantitative electrophoretic mobility shift assay or EMSA. As a reference system for competitive aptamer binding, the duplex 22-mer phosphoryl binding site known as Ig kappa was determined to bind each p65 and p50 homodimer with a 1:1 stoichiometry and with affinities, determined by global analysis, K(d) = 4.8 +/- 0.2 nM for p65 and K(d) = 0.8 +/- 0.2 nM for p50. A global analysis tool for competitive NF-kappa B/Ig kappa binding was developed and utilized to measure the affinity of thioaptamers selected by both p65 and p50. The competition results indicate that the thioaptamers bind and compete for the same NF-kappa B site as the known promoter element Ig kappa B (K(d) = 78.9 +/- 1.9 nM for a p65-selected aptamer and 19.6 +/- 1.3 nM for a p50-selected thioaptamer). Qualitative gel shift binding experiments with p50 also demonstrate that the nature of enhanced affinity and specificity can be attributed to the presence of sulfur. Collectively, these results demonstrate the feasibility of the thioaptamer in vitro combinatorial selection technology as a method for producing specific, high-affinity ligands to proteins.
Department of Human Biological Chemistry and Genetics and Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas 77555, USA.
David J
DJ
King
Suzanne E
SE
Bassett
Xin
X
Li
Susan A
SA
Fennewald
Norbert K
NK
Herzog
Bruce A
BA
Luxon
Robert
R
Shope
David G
DG
Gorenstein
D001483:Base Sequence; D020650:Combinatorial Chemistry Techniques; D017931:DNA Primers; D006801:Humans; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding
D017931:DNA Primers; D016328:NF-kappa B; D010710:Phosphates; C083120:phosphorodithioic acid