Development of m6A Readers for Enhanced Recognition In Vitro via Genetic Code Expansion

Abstract The YTH domain was recently demonstrated as a promising tool for recognizing and regulating m6A-modified RNAs. However, the deficiency in binding affinity (micromolar range) and selectivity (off-target binding) for m6A of the wild-type YTH domain limit its sensitivity and accuracy for m6A recognition. Engineering strategies for improving the affinity and selectivity of such epitranscriptomic readers remain lacking. Here, we demonstrate that site-specific introduction of noncanonical tryptophan derivatives within the aromatic m6A-binding pocket of the YTH domain modulates the binding affinity and selectivity. Specifically, we report two YTH variants, with 5-bromotryptophan and 5-hydroxytryptophan incorporated at the W465 of the YTH domain of YTHDF1, which show improved binding affinity and selectivity against m6A-modified RNAs. DFT-based energy decomposition analysis indicates that the binding affinity can be modulated through the substitution group’s effect on the dispersion force in the van der Waals recognition complex, suggesting a critical role of the CH–π interaction in m6A-reader recognition. This work reports new reader domains with improved m6A recognition and demonstrates the potential of m6A reader engineering via genetic code expansion.