Strategy to test the predictive value of λ-dynamics for modified RNA and RNA-binding protein interactions. (1) λ-Dynamics and a modified RNA library were used with high-resolution crystal structures of human Pumilio 1 (hPUM1) and human Pumilio 2 (hPUM2) to predict modified RNA–protein binding. (2) Each RNA position in hPUM1 or hPUM2 was changed to a different RNA, and the change in free energy binding was calculated compared to the original RNA–protein structure. See Materials and Methods for more details. Some of the modified RNA modifications tested in silico could be synthesized in vitro. (3) These modified RNAs were tested for RNA binding to probe λ-dynamics prediction accuracy.
