Abstract
A simple, accessible, and inexpensive assay to quantify the strength of DNA–protein interactions was developed. The assay relies on capturing DNA–protein complexes using an affinity resin that binds tagged, recombinant proteins. Sequential washes with filtration spin cups and centrifugation remove non-specific interactions in a gentle, uniform manner and a final elution isolates specific DNA–protein complexes. SYBR Gold nucleic acid stain is added to the eluted product and the fluorescence intensity accurately quantifies the amount of captured DNA, ultimately illustrating the relative strength of the DNA–protein interaction. The major utility of the assay resides in the versatility and quantitative nature of the SYBR Gold:nucleic acid interaction, eliminating the need for customized or labeled oligos and permitting relatively inexpensive quantification of binding capacity. The assay also employs DNA–protein complex capture by the very common purification tag, 6xHis, but other tags could likely be utilized. Further, SYBR Gold fluorescence is compatible with a wide variety of instruments, including UV transilluminators, a staple to any molecular biology laboratory. This assay was used to compare the binding capacities of different auxin response factor (ARF) transcription factors to various dsDNA targets, including the classical AuxRE motif and several divergent sequences. Results from dose–response assays suggest that different ARF proteins might show distinct comparative affinities for AuxRE variants, emphasizing that specific ARF-AuxRE binding strengths likely contribute to the complex and fine-tuned cellular auxin response.
Key message
A simple yet reliable assay was developed to quantify DNA–protein interactions using virtually any instrument that can measure fluorescence.
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Acknowledgements
The authors thank Prof. Dolf Weijers and his laboratory, Wageningen University, for providing the activator ARF coding sequences and helpful discussions concerning ARF activity. Research grant awards from USDA-NIFA-AFRI (2016-67014-24589), the National Science Foundation/EPSCoR Cooperative Agreement #IIA-1355423, and SD Agricultural Experiment Station (SD00H543-15) are gratefully acknowledged.
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SS performed the experiments, analyzed the data, and wrote the manuscript; BPP performed the experiments, analyzed the data, and edited the manuscript; TL performed the experiments; SC acquired funding, guided the experiments, and edited the manuscript; SSu conceptualized the study, acquired funding, supervised the analysis, and edited the manuscript.
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Schreier, S., Petla, B.P., Lin, T. et al. A simple and sensitive SYBR Gold-based assay to quantify DNA–protein interactions. Plant Mol Biol 101, 499–506 (2019). https://doi.org/10.1007/s11103-019-00922-x
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DOI: https://doi.org/10.1007/s11103-019-00922-x