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<p>Sequence-specific DNA-binding proteins (DBPs) have critical roles in biology and biotechnology and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize short specific target sequences through interactions with bases in the major groove and use this method to generate binders for five distinct DNA targets with mid-nanomolar to high-nanomolar affinities. The individual binding modules have specificity closely matching the computational models at as many as six base-pair positions and higher-order specificity can be achieved by rigidly positioning the binders along the DNA double helix using RFdiffusion. The crystal structure of a designed DBP–target site complex is in close agreement with the design model and the designed DBPs function in both <i>Escherichia coli</i> and mammalian cells to repress and activate transcription of neighboring genes. Our method provides a route to small and, hence, readily deliverable sequence-specific DBPs for gene regulation and editing.</p>

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Computational design of sequence-specific DNA-binding proteins

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年9月12日

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  • Cameron J. Glasscock,
  • Robert J. Pecoraro,
  • Ryan McHugh,
  • Lindsey A. Doyle,
  • Wei Chen,
  • Olivier Boivin,
  • Beau Lonnquist,
  • Emily Na,
  • Yuliya Politanska,
  • Hugh K. Haddox,
  • David Cox,
  • Christoffer Norn,
  • Brian Coventry,
  • Inna Goreshnik,
  • Dionne Vafeados,
  • Gyu Rie Lee,
  • Raluca Gordân,
  • Barry L. Stoddard,
  • Frank DiMaio,
  • David Baker

摘要

Sequence-specific DNA-binding proteins (DBPs) have critical roles in biology and biotechnology and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize short specific target sequences through interactions with bases in the major groove and use this method to generate binders for five distinct DNA targets with mid-nanomolar to high-nanomolar affinities. The individual binding modules have specificity closely matching the computational models at as many as six base-pair positions and higher-order specificity can be achieved by rigidly positioning the binders along the DNA double helix using RFdiffusion. The crystal structure of a designed DBP–target site complex is in close agreement with the design model and the designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method provides a route to small and, hence, readily deliverable sequence-specific DBPs for gene regulation and editing.