Systematic profiling of single-, double- and triple-halogen substitution effects on the mononucleotide binding of N ⁶ -methyladenosine to human RNA m6A reader YTHDF1

N6-methyladenosine (m6A) is the most plentiful internal modification event in eukaryotic RNAs and regulated dynamically by a variety of its cognate functional partners in a context-dependent manner, namely writers, erasers and readers. The YTHDF1 is a well-documented cytoplasmic m6A reader that can specifically recognize and interact with m6A via its YT521-B homology (YTH) domain. In this study, the three methyl hydrogens of m6A mononucleotide were systematically swapped with four halogens (X = F, Cl, Br and I), totally resulting in the 4 single-, 10 double- and 20 triple-halogen modified counterparts of m6A (i.e. N6-halomethyladenosine, Xm6A). Energetic analyses and affinity assays indicated a moderate binding potency of m6A mononucleotide to YTHDF1 YTH domain, which can be improved by different single-halogen modifications. The electron-correlation calculations revealed that the affinity improvement is primarily donated from formation of additional halogen-π interactions with YTH aromatic cage upon the halogenations. Bromine, but not the bulky iodine, modification reaches the affinity maximum at Brm6A - this can be considered as a compromise between the favorable halogen-π interactions and the unfavorable steric effects caused by halogen modifications. For the same reason, single- and double-modifications generally contribute more favorably to YTH-Xm6A binding than triple-modifications. Further competition assays observed that the m6A mononucleotide and its single-modified Brm6A counterpart can competitively displace a m6A-containing RNA bound in the binding site of YTHDF1 YTH domain in moderate and high potencies, respectively, implying that halogen-modified Xm6A mononucleotides could be exploited as therapeutic strategy by competitively disrupting the cognate intermolecular interaction between YTH domain and RNA m6A.