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Bioconjug Chem. 2009 Aug 19;20(8):1444-8. doi: 10.1021/bc900137y. Epub 2009 Jul 2.

Plate capture assay of fluorescent oligonucleotide duplex reporter-transcription factor complexes.

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  • 1Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

Abstract

We previously developed prototype oligodeoxyribonucleotide (ODN) duplex fluorescence energy transfer (FRET) reporters for optical sensing of NF-κB transcription factor. We report here a plate-binding assay designed for optimizing the above reporters. Nitrilotriacetate-bearing plates were prepared by using sequential (1) aminosilylation; (2) carboxylation; (3) coupling of Nα,Nα-bis(carboxymethyl)-L-lysine or, alternatively, by replacing steps 1 and 2 by treating the glass with 3-(triethoxysilyl)propylsuccinic anhydride. FRET reporters were obtained by covalent linking of Cy5.5 (fluorescence donor) and IRdye800CW (fluorescence acceptor) to complementary ODN strands encoding a high-affinity p50 binding site. Recombinant 6 × His tagged NF-κB p50 was used for immobilizing the protein on glass plates via linked NTA-Ni(II) groups. Imaging and quantification of the fluorescence intensity in the wells was performed in two channels (700 and 800 nm) using a near-infrared scanning device with microscopic resolution. The fluorescence intensity of the ODN duplex reporter was detectible in the plates at the concentration of 5 pM. NF-κB p50-ODN reporter interaction was studied by measuring the ratio of 700 nm (donor) to 800 nm (acceptor) fluorescence intensities. Using the plate assay, we were able to measure p50-mediated interference with FRET at low density of plate binding.

PMID:
19572630
[PubMed - indexed for MEDLINE]
PMCID:
PMC2889241
Free PMC Article
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