Single-molecule multiplexed profiling of protein–DNA complexes using magnetic tweezers

Epigenetics, such as the dynamic interplay between DNA methylation and demethylation, play diverse roles in critical cellular events. Enzymatic activity at CpG sites, where cytosines are methylated or demethylated, is known to be influenced by the density of CpGs, methylation states, and the flanking sequences of a CpG site. However, how the relevant enzymes are recruited to and recognize their target DNA is less clear. Moreover, although DNA-binding epigenetic enzymes are ideal targets for therapeutic intervention, these targets have been rarely exploited. Single-molecule techniques offer excellent capabilities to probe site-specific protein–DNA interactions and unravel the dynamics. Here, we develop a single-molecule approach that allows multiplexed profiling of protein–DNA complexes using magnetic tweezers. When a DNA hairpin with multiple binding sites is unzipping, strand separation pauses at the positions bound by a protein. We can thus measure site-specific binding probabilities and dissociation time directly. Taking the TET1 CXXC domain as an example, we show that TET1 CXXC binds multiple CpG motifs with various flanking nucleotides or different methylation patterns in an AT-rich DNA. We are able to establish for the first time, at nanometer resolution, that TET1 CXXC prefers G/C flanked CpG motif over C/G, A/T, or T/A flanked ones. CpG methylation strengthens TET1 CXXC recruitment but has little effect on dissociation time. Finally, we demonstrate that TET1 CXXC can distinguish five CpG clusters in a CpG island with crowded binding motifs. We anticipate that the feasibility of single-molecule multiplexed profiling assays will contribute to the understanding of protein–DNA interactions.