Affinity-enhanced RNA-binding domains as tools to understand RNA recognition

Belén Chaves-Arquero, Katherine M. Collins, Giancarlo Abis, Geoff Kelly, Evangelos Christodoulou, Ian A. Taylor, Andres Ramos

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Understanding how the RNA-binding domains of a protein regulator are used to recognize its RNA targets is a key problem in RNA biology, but RNA-binding domains with very low affinity do not perform well in the methods currently available to characterize protein-RNA interactions. Here, we propose to use conservative mutations that enhance the affinity of RNA-binding domains to overcome this limitation. As a proof of principle, we have designed and validated an affinity-enhanced K-homology (KH) domain mutant of the fragile X syndrome protein FMRP, a key regulator of neuronal development, and used this mutant to determine the domain's sequence preference and to explain FMRP recognition of specific RNA motifs in the cell. Our results validate our concept and our nuclear magnetic resonance (NMR)-based workflow. While effective mutant design requires an understanding of the underlying principles of RNA recognition by the relevant domain type, we expect the method will be used effectively in many RNA-binding domains.

Original languageEnglish
Article number100508
JournalCell Reports Methods
Issue number6
StatePublished - 26 Jun 2023
Externally publishedYes


  • CP: Molecular biology
  • FMRP
  • NMR
  • RNA recognition
  • RNA-binding proteins
  • fragile X
  • method
  • transient protein-RNA interactions


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