DNA methylation, nucleic acid structure, and rett mutations tune MeCP2 binding affinity and cooperativity.

Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-associated factor whose dysfunction causes Rett syndrome. Although MeCP2 preferentially binds methylated DNA, its affinity for methylated substrates is only ∼ threefold higher than for unmethylated DNA, raising the question of how MeCP2 selectively recognizes its targets. Here, we quantify binding of full-length WT MeCP2 and Rett-associated variants (R106W, T158M, R270X, and R306C) to nucleic acid substrates that vary in length, secondary structure, methylation pattern, CpG symmetry, and strand composition. WT and mutant MeCP2 preferentially bind double-stranded DNA but also interact with single- and double-stranded DNA and RNA with nanomolar affinity. Binding to single-stranded targets is largely driven by the formation of local duplex structures, whereas 5-methylcytosine provides stabilizing contacts and enhances MeCP2 affinity when canonical duplex geometry is absent. Rett-associated mutations segregate into mechanistic classes: mutations within the methyl-CpG-binding domain (R106W and T158M) weaken methylation-dependent recognition and reduce binding affinity, whereas C-terminal mutations (R270X and R306C) preserve high-affinity binding but diminish cooperative interactions consistent with impaired higher-order bridging. These findings identify MeCP2 as a methyl-sensitive nucleic acid binder whose interactions are shaped by local nucleic acid topology and modulated by cytosine methylation, providing a mechanistic framework for understanding how Rett-associated mutations disrupt chromatin regulation.