Inherent Specificity and Variation Sensitivity as Quantitative Metrics for RBP Binding

RNA-RBP affinity measurements in vitro reveal diverse binding preferences, yet approaches to directly compare motif specificities across RBPs are lacking. Here, we introduce two quantitative metrics: inherent specificity, which measures how selectively an RBP distinguishes its strongest binding motif from all possible motifs, and variation sensitivity, which assesses tolerance to single nucleotide variations within a preferred motif.

Analyzing high-throughput sequencing datasets, we compared these metrics across 100 RBPs in vitro and 27 RBPs in cells, finding a strong correlation between in vitro and cellular measurements for RBPs that bind RNA independently of local structural context. Through RNA binding domain swap CLIP experiments between low-specificity RBM25 and high-specificity HNRNPC, we demonstrated that sequence specificity can be measurably transferred between protein contexts.