Epigenetic CpG duplex marks probed by an evolved DNA reader via a well-tempered conformational plasticity

5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel ‘CpG duplex marks’ could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.