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

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      Nucleic Acids Research
      Oxford University Press

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          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 × 10 12 unique RNA sequences and 4.0 × 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.

          Graphical Abstract

          Graphical Abstract

          Library-vs-library selection of RNA and RNA-binding protein (RBP) resulted in orthogonal RNA–RBP pairs with picomolar affinity and high selectivity.

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          Most cited references73

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          UCSF Chimera--a visualization system for exploratory research and analysis.

          The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/. Copyright 2004 Wiley Periodicals, Inc.
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            Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase

            High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.
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              In vitro selection of RNA molecules that bind specific ligands.

              Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.
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                Author and article information

                Contributors
                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                25 January 2022
                05 July 2021
                05 July 2021
                : 50
                : 2
                : 601-616
                Affiliations
                Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University , Onna, Okinawa 904 0495, Japan
                Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University , Onna, Okinawa 904 0495, Japan
                Author notes
                To whom correspondence should be addressed. Tel: +81 98 982 3396; Email: yohei.yokobayashi@ 123456oist.jp
                Author information
                https://orcid.org/0000-0002-4259-4359
                https://orcid.org/0000-0002-2417-1934
                Article
                gkab527
                10.1093/nar/gkab527
                8789040
                34219162
                051ae2c8-9f1f-46b6-83ef-67f310f40154
                © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 08 June 2021
                : 03 June 2021
                : 19 April 2021
                Page count
                Pages: 16
                Funding
                Funded by: OIST, DOI 10.13039/501100004199;
                Funded by: KAKENHI, DOI 10.13039/501100001691;
                Award ID: 19K15701
                Award ID: 19H02855
                Funded by: Japan Society for the Promotion of Science, DOI 10.13039/501100001691;
                Categories
                AcademicSubjects/SCI00010
                Narese/22
                NAR Breakthrough Article

                Genetics
                Genetics

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