The binding behavior of N⁶‐Methyladenosine (m6A) to human RNA m6A reader pediatric YTHDF3 can be rationally altered by halogen modification of m6A's N⁶‐methyl group: An integrated in silico‐in vitro (iSiV) approach

N 6 ‐adenosine methylation, or N 6 ‐methyladenosine (m6A), is the most prevalent and reversible modification event in mammalian messenger and noncoding RNAs, which can be dynamically regulated by writers, erasers, and readers in a context‐dependent manner. The human YTHDF3 is a widely documented m6A reader that recognizes and interacts with m6A through its YTH domain, where the N 6 ‐methyl group of m6A is tightly captured by an aromatic cage defined by the YTH tryptophan residue triad Trp438–Trp492–Trp497. Considering that the aromatic cage has only a limited size that can accommodate only small atoms and moieties, we herein attempted to investigate the substitution effects of halogen modification of m6A's N 6 ‐methyl group (N 6 HCH 3 ) on YTHDF3 YTH recognition and binding. The N 6 ‐methyl group possesses three chemically equivalent hydrogens; each of which can be substituted by four halogens (X 1 , X 2 and X 3 = F, Cl, Br and I). An integrated in silico‐in vitro ( iSiV ) strategy was employed to examine the structural, energetic, and affinity effects of halogen modification on the binding of m6A mononucleotide to YTHDF3 YTH domain by systematically substituting one or more of the three N 6 ‐methyl hydrogens of m6A with four halogens, totally resulting in 34 N 6 ‐halomethyladenosine analogs (xm6A), including 4 single‐, 10 double‐, and 20 triple‐substituted xm6A. It is revealed that the halogen modification can create favorable halogen–π interactions with the π‐electron‐rich aromatic cage, thus conferring affinity and specificity to the binding of xm6A to YTHDF3 YTH domain. However, multiple substitutions with bulky halogen atoms such as I would cause unfavorable steric overlaps and clashes against the small aromatic cage, thus considerably impairing the binding potency of the resulting xm6A. The native m6A mononucleotide binds to YTH with a moderate affinity, which can be modestly or considerably improved by different single‐halogen substitutions. The single Br‐substitution and double Cl‐Br‐substitution were determined as the best candidates to improve xm6A binding affinity, which are good compromises between the favorable halogen–π interactions and unfavorable steric effects eliciting from the substitutions.