Combinatorial selection and binding of phosphorothioate aptamers targeting human NF-κB RelA(p65) and p50

David J. King, Suzanne E. Bassett, Xin Li, Susan A. Fennewald, Norbert K. Herzog, Bruce A. Luxon, Robert Shope, David G. Gorenstein

Research output: Contribution to journalArticle

57 Scopus citations

Abstract

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-κB proteins RelA(p65) and p50, duplex thioaptamers have been selected that demonstrate high-affinity, competitive binding with the duplex 22-mer binding site, IgκB. 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 Igκ was determined to bind each p65 and p50 homodimer with a 1:1 stoichiometry and with affinities, determined by global analysis, Kd = 4.8 ± 0.2 nM for p65 and Kd = 0.8 ± 0.2 nM for p50. A global analysis tool for competitive NF-κB/Igκ 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-κB site as the known promoter element IgκB (Kd = 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.

Original languageEnglish (US)
Pages (from-to)9696-9706
Number of pages11
JournalBiochemistry
Volume41
Issue number30
DOIs
StatePublished - Jul 30 2002

ASJC Scopus subject areas

  • Biochemistry

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    King, D. J., Bassett, S. E., Li, X., Fennewald, S. A., Herzog, N. K., Luxon, B. A., Shope, R., & Gorenstein, D. G. (2002). Combinatorial selection and binding of phosphorothioate aptamers targeting human NF-κB RelA(p65) and p50. Biochemistry, 41(30), 9696-9706. https://doi.org/10.1021/bi020220k