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bioRxiv · 10.1101/2025.06.06.658291

Quantitative Assessment of Protein-DNA Interactions via SYBR Green Fluorescence

Wang, J.·Wang, Z.·Wang, P.·Sun, S.·Li, X.·Tian, Z.·Xu, R.·Shi, Y.·Wang, Y.
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Abstract

Protein-DNA interactions are crucial for cellular processes, but current quantification methods lack sensitivity. We developed the Real-time PCR-based DNA Binding Assay (RP-DBA) to detect and quantify these interactions. The target protein, expressed as a Strep-tag II fusion, is purified and incubated with double-stranded DNA probes containing 4 bp 3 overhangs. Protein-DNA complexes are immobilized on Strep-Tactin beads, washed, and eluted. A complementary single-stranded DNA amplification arm is added, extended by Taq polymerase, and quantified via qPCR with SYBR Green. RP-DBA enables real-time kinetic analysis and is 4- to 10-fold more sensitive than EMSA, depending on amplification arm length (30-90 bp). Its simplicity, speed, accuracy, and high-throughput potential make it a valuable tool for advancing molecular biology research.

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Wang, J., Wang, Z., Wang, P., Sun, S., Li, X., Tian, Z., Xu, R., Shi, Y., Wang, Y.. 2025-06-07. Quantitative Assessment of Protein-DNA Interactions via SYBR Green Fluorescence. https://doi.org/10.1101/2025.06.06.658291

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