Artificially Evolved Superbinder for Specific Recognition of N⁶-Methyladenine Base Modification in DNA and RNA

N⁶-Methyladenine (m⁶A/6mA) is a functional epigenetic base modification found in RNA and DNA. By selecting one RNA m⁶A reader as a template, we created a series of libraries of 3 × 10⁸ RNA m⁶A reader mutants and developed a yeast surface-display-based evolution approach. Using high-throughput fluorescence-activated cell sorting, we ultimately obtained three evolved 6mA-binding proteins (e6mABPs), which displayed increased affinity for 6mA-containing DNA and reduced affinity for 6mA-free DNA. These e6mABPs are applicable for m⁶A/6mA enrichment and are potentially applied for modulating cell behavior.