Details

Autor(en) / Beteiligte

• Fukunaga, Keisuke
• Yokobayashi, Yohei

Titel

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

Ist Teil von

• Nucleic acids research, 2022-01, Vol.50 (2), p.601

Ort / Verlag

England

Erscheinungsjahr

2022

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• RNA-binding proteins (RBPs) and their RNA ligands play many critical roles in gene regulation and RNA processing in cells. They are also useful for various applications in cell biology and synthetic biology. However, re-engineering novel and orthogonal RNA-RBP pairs from natural components remains challenging while such synthetic RNA-RBP pairs could significantly expand the RNA-RBP toolbox for various applications. Here, we report a novel library-vs-library in vitro selection strategy based on Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment (PD-SELEX). Starting with pools of 1.1 × 1012 unique RNA sequences and 4.0 × 108 unique phage-displayed L7Ae-scaffold (LS) proteins, we selected RNA-RBP complexes through a two-step affinity purification process. After six rounds of library-vs-library selection, the selected RNAs and LS proteins were analyzed by next-generation sequencing (NGS). Further deconvolution of the enriched RNA and LS protein sequences revealed two synthetic and orthogonal RNA-RBP pairs that exhibit picomolar affinity and >4000-fold selectivity.