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

ABSTRACT: 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.

SUBMITTER: Fukunaga K

PROVIDER: S-EPMC8789040 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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

Fukunaga Keisuke K Yokobayashi Yohei Y

Nucleic acids research 20220101 2

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 P ...[more]

PMID: 34219162

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Project description:Directed evolution is a powerful tool that can bypass gaps in our understanding of the sequence-function relationship of proteins and still isolate variants with desired activities, properties, and substrate specificities. The rise of directed evolution platforms for polymerase engineering has accelerated the isolation of xenobiotic nucleic acid (XNA) synthetases and reverse transcriptases capable of processing a wide array of unnatural XNAs which have numerous therapeutic and biotechnological applications. Still, the current generation of XNA polymerases functions with significantly lower efficiency than the natural counterparts and retains a significant level of DNA polymerase activity which limits their in vivo applications. Although directed evolution approaches are continuously being developed and implemented to improve XNA polymerase engineering, the field lacks an in-depth analysis of the effect of selection parameters, library construction biases and sampling biases. Focusing on the directed evolution pipeline for DNA and XNA polymerase engineering, this work sets out a method for understanding the impact of selection conditions on selection success and efficiency. We also explore the influence of selection conditions on fidelity at the population and individual mutant level. Additionally, we explore the sequencing coverage requirements in directed evolution experiments, which differ from genome assembly and other -omics approaches. This analysis allowed us to identify the sequencing coverage threshold for the accurate and precise identification of significantly enriched mutants. Overall, this study introduces a robust methodology for optimizing selection protocols, which effectively streamlines selection processes by employing small libraries and cost-effective NGS sequencing. It provides valuable insights into critical considerations, thereby enhancing the overall effectiveness and efficiency of directed evolution strategies applicable to enzymes other than the ones considered here.

Dataset Information

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

Publications

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

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