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Computational design of sequence-specific DNA-binding proteins.

Cameron J Glasscock1,2,3, Robert J Pecoraro4,5,6, Ryan McHugh4,5

  • 1Department of Biochemistry, University of Washington, Seattle, WA, USA. cjamesglasscock@gmail.com.

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Researchers developed a computational method to design novel DNA-binding proteins (DBPs). These engineered DBPs recognize specific DNA sequences and function in cells for gene regulation and editing applications.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Computational Biology

Background:

  • Sequence-specific DNA-binding proteins (DBPs) are crucial for biological processes and biotechnological applications.
  • Engineering DBPs with novel specificities is essential for advanced applications like genome editing.
  • Computational design of de novo DBPs for arbitrary DNA targets remains a significant challenge.

Purpose of the Study:

  • To develop and validate a computational method for designing small, sequence-specific DNA-binding proteins.
  • To engineer DBPs capable of recognizing specific DNA target sequences with high affinity and specificity.
  • To demonstrate the functionality of designed DBPs in cellular gene regulation and editing.

Main Methods:

  • Utilized a computational approach to design small DBPs targeting specific DNA sequences via major groove interactions.
  • Generated DBP binders for five distinct DNA targets, achieving mid-nanomolar to high-nanomolar affinities.
  • Employed RFdiffusion to achieve higher-order specificity by rigidly positioning binding modules on the DNA double helix.

Main Results:

  • Designed DBPs exhibited specificity matching computational models at up to six base-pair positions.
  • The crystal structure of a designed DBP-target complex confirmed close agreement with the design model.
  • Engineered DBPs successfully repressed and activated transcription in both Escherichia coli and mammalian cells.

Conclusions:

  • The computational method enables the design of novel, sequence-specific DNA-binding proteins.
  • Designed DBPs demonstrate high specificity and functionality in cellular gene regulation and editing.
  • This approach offers a pathway to readily deliverable DBPs for diverse genetic applications.