Sensitive Detection of Transcription Factors Using Near-Infrared Fluorescent Solid-Phase Rolling Circle Amplification

Abstract

This study describes a method for analyzing transcription factor (TF) activity, near-infrared fluorescent solid-phase rolling circle amplification (NIRF-sRCA). This method analyzes TF activity in four steps: (i) incubate DNA with protein sample and isolate TF-bound DNA, (ii) hybridize the TF-bound DNA and rolling circle to DNA microarray, (iii) amplify the TF-bound DNA with sRCA that contains biotin-labeled dUTP, and (iv) detect sRCA products by binding of NIRF-labeled streptavidin and NIRF imaging. This method was validated by proof-of-concept detection of purified TF protein and cell nuclear extract. Detection of purified TF protein demonstrated that NIRF-sRCA could quantitatively detect NF-κB p50 protein, and as little as 6.94 ng (∼140 fmol) of this protein was detected. Detection of nuclear extract revealed that NIRF-sRCA could specifically and quantitatively detect NF-κB p50 activity in HeLa cell nuclear extracts, and the activity of this TF in as little as 0.625 μg of nuclear extracts could be detected. Detection of nuclear extract also revealed that NIRF-sRCA could detect the relative activities of multiple TFs in HeLa cell nuclear extracts and the fold induction of multiple TFs in the TNFα-induced HeLa cell nuclear extracts. Therefore, this study provides a new tool for studying TFs.