Introduction

Nuclear factor κB (NF-κB) plays an important role in the inducible transcriptional response to pathogenic signals, oxidative stresses, and proinflammatory cytokines (1). NF-κB binds directly to its target genes as a homo- or heterodimer formed with members of the Rel family proteins that include p50, p52, p65/Rel, Rel B, and cRel. In the resting cells, NF-κB is mostly localized at the cytosol in inactive forms bound by the inhibitory IκB proteins (2,3). After stimulation by cytokines, stressors, or pathogenic signals, signal transduction pathways are activated, which lead to the phosphorylation of IκB and result in ubiquitination and degradation of IκB by proteasomes (4). Consequently, NF-κB is released and then translocated to the nucleus, followed by binding to the κB consensus sequence located in the promoter regions of a variety of genes to induce gene expressions (1,5,6). Hence, DNA binding activities of NF-κB reflect cellular responses to various signals. Simple and flexible methods for the detection of DNA binding activities of NF-κB are highly desirable.

A number of techniques have been developed for studying protein-DNA interactions. A commonly used method, electrophoretic mobility shift assay (EMSA) (7,8), detects slower migration of protein-DNA complexes relative to free DNA molecules using a nondenaturing gel electrophoresis. A reporter assay has also been used to detect DNA binding activity (9). The luciferase or β-galactosidase gene is placed under the control of a promoter that contains the consensus sequence recognized by the DNA binding protein. In addition, a chromatin immunoprecipitation (CHIP) assay has been developed for studying protein-DNA interactions (10). However, these assays are laborious, time-consuming, and difficult to apply for high-throughput screening.

Renard et al. (11) established a DNA binding assay on the basis of a modified enzyme-linked immunosorbent assay (ELISA). Recently, methods in relative high-throughput platforms have been developed for studying protein-DNA interactions based on fluorescence resonance energy transfer (FRET) (12,13,14,15). For instance, Lu and his colleagues have described an assay that combines exonuclease III (ExoIII) protection strategy and FRET detection or SYBR® Green I staining method (14,15). In their assay, a double-stranded DNA (dsDNA) probe is designed to contain a pair of FRET fluorophores in the middle and two identical protein binding sites on each side of the FRET fluorophores. The NF-κB protein can protect the probe from ExoIII digestion, which results in a high FRET signal. This binding configuration, however, does not necessarily reflect endogenous DNA binding. Further, protein binding on one side may interfere the binding on the other side, because of the steric effect from complexation with other cotranscription factors likely present in cell extracts.

In this study, we developed a method employing both restriction endonuclease digestion and FRET detection strategy to study NF-κB-DNA interaction. The DNA-FRET probe used is a dsDNA that contains a pair of FRET fluorophores at the same end of the probe and an endonuclease recognition site of the κB DNA consensus sequence. When the transcription factor binds to the dsDNA, it prevents the DNA probe from binding and subsequently being cleaved by the endonuclease, which then results in a high FRET signal. We compared this assay with the commonly used EMSA using purified recombinant NF-κB p50, nuclear extracts, and whole cell lysates. We examined the suitability of this FRET-based assay for high-throughput screening of NFκB activation.

Materials and Methods

FRET Probes

Oligonucleotides were synthesized and high-performance liquid chromatography (HPLC)-purified from Invitrogen (Carlsbad, CA, USA). The sequences of two pairs of NF-κB FRET probes are: (probe 1) 5′-(FAM)AAGTGGGAAATTCCTCT G-3′ 5′-CAGAGGAATTTCCCACTT(TAMRA)-3′; and (probe 2, a mutant probe) 5′-(FAM)AAGTGTTAAATTCCTCTG-3′, 5′-CAGAGGAATTTAACACTT(TAMRA)-3′ (16). The donor [carboxyfluorescein (FAM)] and acceptor [tetramethyl-6-carboxy-rhodamine (TAMRA)] fluorophores are attached to 5′ (dA) and 3′ (dT) via a C6 linker, respectively. The bold sequences represent the NF-κB binding sites, and the underlined sequences are the recognition sites for restriction enzyme ApoI, respectively. To obtain dsDNA-FRET probes, complementary oligonucleotide pairs were mixed at the same molar concentrations of 20 µM in a 100-µL Tris buffer solution (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA). The mixture was heated for 4 min at 92°C and cooled down slowly to 25°C. The formed dsDNA probes were then purified by using native polyacrylamide gel electrophoresis (PAGE).