Directed evolution of orthogonal RNA–RBP pairs through library-vs-library in vitro selection

Keisuke Fukunaga, and Yohei Yokobayashi

Nucleic Acids Research, gkab527, doi:10.1093/nar/gkab527

In cells, there are many RNA binding proteins (RBPs) that specifically bind to compact RNA motifs to regulate gene expression and to assemble ribonucleoprotein (RNP) complexes. RBPs are also useful for visualizing cellular RNAs and building synthetic gene circuits. However, re-engineering an RBP to recognize non-natural RNA motifs but not the natural RNA target remains a challenging task while such orthogonal RNA-RBP pairs can be useful for various applications. The authors generated large pools of RBP mutants displayed on T7 phage particles and RNA mutants, and they executed sequential selection and amplification cycles (PD-SELEX) to select for novel RNA-RBP pairs. After six rounds of selection, the authors identified two orthogonal RNA-RBP pairs that exhibit picomolar dissociation constants with high selectivity.

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