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Combinatorial Design of a Nanobody that Specifically Targets Structured RNAs

机译：特异性靶向结构RNA的纳米粉底的组合设计

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摘要

Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (approximate to 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs. Most of them adopt a well-defined three-dimensional structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of non-coding RNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA, while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody does not alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof of concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes. (C)2018 Elsevier Ltd. All rights reserved.
机译：转录组测序和分析的最新进展揭示了人类基因组的复杂性。大多数（近似98％）细胞转录物未翻译成蛋白质，代表了巨大的功能性非编码RNA的世界。其中大多数采用明确定义的三维结构来实现其生物功能。然而，目前只有很少的RNA结构，反映了与RNA结晶相关的挑战。然而，这些结构将代表理解非编码RNA的功能及其在细胞中的分子机制的关键步骤。本研究的整体目标是开发一种创新和多功能的工具，以促进结构化RNA（Strnas）的功能研究和结晶。在这项工作中，我们已经设计了能够与低纳米摩尔亲和力特异性结合的骆驼形重链抗体（纳米曲面）的抗体片段，同时可以检测单链DNA / RNA，双链DNA / RNA的无结合或带负电的蛋白质。然而，这种纳米型识别出不同和无关的毒液，该观察结果表明它与这些斯特纳共享的表位结合。最后，我们的数据还表明，纳米体的结合不会改变STRNA的二级结构含量以及在热处理期间的展开/重折叠过程。这项工作构成了一个成功的概念证明，证明纳米级可以被设计成识别相关的RNA相关的表位。 （c）2018年elestvier有限公司保留所有权利。

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  • 来源
    《Journal of Molecular Biology》 |2018年第11期|共19页
  • 作者

    Cawez F.; Duray E.; Hu Y.; Vandenameele J.; Romao E.; Vincke C.; Dumoulin M.; Galleni M.; Muyldermans S.; Vandevenne M.;

  • 作者单位

    Univ Liege Dept Life Sci Ctr Prot Engn Biol Macromol &

    Biochem InBioS B-4000 Sart Tilman Par;

    Univ Liege Dept Life Sci Ctr Prot Engn Biol Macromol &

    Biochem InBioS B-4000 Sart Tilman Par;

    Vrije Univ Brussel Dept Cellular &

    Mol Immunol B-1050 Brussels Belgium;

    Univ Liege Dept Life Sci Ctr Prot Engn Prot Folding &

    Enzymol InBioS B-4000 Sart Tilman Par;

    Vrije Univ Brussel Dept Cellular &

    Mol Immunol B-1050 Brussels Belgium;

    Vrije Univ Brussel Dept Cellular &

    Mol Immunol B-1050 Brussels Belgium;

    Univ Liege Dept Life Sci Ctr Prot Engn Prot Folding &

    Enzymol InBioS B-4000 Sart Tilman Par;

    Univ Liege Dept Life Sci Ctr Prot Engn Biol Macromol &

    Biochem InBioS B-4000 Sart Tilman Par;

    Vrije Univ Brussel Dept Cellular &

    Mol Immunol B-1050 Brussels Belgium;

    Univ Liege Dept Life Sci Ctr Prot Engn Biol Macromol &

    Biochem InBioS B-4000 Sart Tilman Par;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子生物学;
  • 关键词

    nanobody; non-coding RNAs; structured RNAs; RNA/protein interactions; antibody fragment engineering;

    机译：Nanobody;非编码RNA;结构化RNA;RNA /蛋白质相互作用;抗体片段工程;

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