2024 AIChE Annual Meeting
Engineering Binding Affinity of Yth to m6A-RNA Leveraging Yeast Surface Display and Next-Generation Sequencing for Comprehensive Mutant Library Analysis
Engineered RNA-binding proteins (RBPs) show promise for diverse applications in research and therapeutic development. RBPs can be used to track and alter RNA localization in the cell. Harnessing the capabilities of RNA-binding proteins (RBPs) can enhance the analysis and characterization of RNA modification profiles. Moreover, RBPs can be used to regulate translation through influencing alternative splicing and inhibiting RNA function [1]. Modulating the interaction between YTH-domain containing proteins and methylated RNA ligands could advance the development of research tools in biotechnology. The YTH domain specifically binds methylated RNA ligands and is conserved across the five N6-methyladenosine (m6A) reader proteins in the human proteome. Both holo and apo structures of YTH have been solved through x-ray crystallography. Notably, tryptophan residues contained in the hydrophobic binding pocket have been shown to be important in binding m6A via an “aromatic cage,” quantitatively evidenced by alanine scanning [2]. N6-methyladenosine (m6A) is the most prevalent internal modification in RNA across eukaryotic organisms. Consequently, the modification plays a significant role in various disease pathologies, particularly in cancer. Increased m6A modifications can promote the stability of oncogenic mRNA, driving tumor progression. Accordingly, to illustrate a possible application of engineered RBPs, characterizing m6A modifications in the transcriptome could serve as a biomarker for tracking cancer progression. Prior studies have determined that YTH has a 10-fold higher affinity to m6A modified RNA (KD≈0.18µM) as compared to unmodified RNA (KD≈2.8µM) [3]. Further affinity maturation of the YTH-m6A interaction through directed evolution of the YTH structure could produce a novel protein with nanomolar affinity better suited for the development of biological detection tools. Here, yeast surface display is utilized to characterize the binding affinity and specificity of the YTH domain to m6A modified RNA. Subsequently, fluorescence-activated cell sorting will allow for selection of high affinity, highly specific binders from mutant libraries. With this, multiple rounds of directed evolution can be performed. Mutant libraries generated through error-prone PCR are to be sequenced using nanopore sequencing to robustly analyze mutations tested.
References: [1] Chen, Y., & Varani, G. (2013). Engineering RNA-binding proteins for biology. The FEBS Journal, 280(16), 3734–3754. https://doi.org/10.1111/febs.12375 [2] Miller, L., et al. (2023). Characterization of epitranscriptome reader proteins experimentally and in silico: Current knowledge and future perspectives beyond the YTH domain. Computational and Structural Biotechnology Journal, 21, 3541–3556. https://doi.org/10.1016/j.csbj.2023.06.018 [3] Wang, X., et al. (2014). m6A-dependent regulation of messenger RNA stability. Nature, 505(7481), 117–120. https://doi.org/10.1038/nature12730