Plate capture assay of fluorescent oligonucleotide duplex reporter-transcription factor complexes.
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.
Zhang
Surong
S
Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Bogdanov
Alexei
A
D001665:Binding Sites; D002232:Carbocyanines / Q000737:chemistry*; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D017930:Genes, Reporter; D016328:NF-kappa B / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*
C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D009571:Nitrilotriacetic Acid