The RNA recognition motif (RRM) is the most common RNA binding domain across eukaryotic proteins. It is therefore of great value to engineer its specificity to target RNAs of arbitrary sequence. This was recently achieved for the RRM in Rbfox protein, where four mutations R118D, E147R, N151S, and E152T were designed to target the precursor to the oncogenic miRNA 21. Here, we used a variety of molecular dynamics-based approaches to predict specific interactions at the binding interface. Overall, we have run approximately 50 microseconds of enhanced sampling and plain molecular dynamics simulations on the engineered complex as well as on the wild-type Rbfox·pre-miRNA 20b from which the mutated systems were designed. Comparison with the available NMR data on the wild type molecules (protein, RNA, and their complex) served to establish the accuracy of the calculations. Free energy calculations suggest that further improvements in affinity and selectivity are achieved by the S151T replacement.

Computational Biomedicine Institute for Advanced Simulation IAS 5 and Institute of Neuroscience and Medicine INM 9 Forschungszentrum Jülich Jülich Germany

Department of Chemistry University of Washington Seattle Washington United States of America

Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic

JARA HPC Jülich Supercomputing Centre Forschungszentrum Jülich GmbH Jülich Germany

Regional Centre of Advanced Technologies and Materials Department of Physical Chemistry Faculty of Science Palacky University Olomouc Olomouc Czech Republic

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In Vitro Evolution Reveals Noncationic Protein-RNA Interaction Mediated by Metal Ions