Journal
NUCLEIC ACIDS RESEARCH
Volume 50, Issue 2, Pages 601-616Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkab527
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Funding
- OIST
- KAKENHI from the Japan Society for the Promotion of Science (JSPS) [19K15701, 19H02855]
- Grants-in-Aid for Scientific Research [19K15701, 19H02855] Funding Source: KAKEN
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In this study, a novel library-vs-library in vitro selection strategy was developed to select synthetic and orthogonal RNA-RBP pairs. The selected pairs exhibited picomolar affinity and >4000-fold selectivity.
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 x 10(12) unique RNA sequences and 4.0 x 10(8) 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.
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