Computational design of sequence-specific DNA-binding proteins

Cameron J Glasscock^#^{1

2

3}, Robert J Pecoraro^#^{4

5

6}, Ryan McHugh^#^{4

5}, Lindsey A Doyle⁷, Wei Chen^{4

5}, Olivier Boivin^{8

9}, Beau Lonnquist^{4

5

10}, Emily Na^{4

5}, Yuliya Politanska^{4

5}, Hugh K Haddox⁶, David Cox^{11

12}, Christoffer Norn^{4

5

13}, Brian Coventry^{4

5

14}, Inna Goreshnik^{4

5}, Dionne Vafeados^{4

5}, Gyu Rie Lee^{4

5

14}, Raluca Gordân^{9

15

16

17

18}, Barry L Stoddard⁷, Frank DiMaio^{4

5}, David Baker^{19

20

21}

Affiliations

¹ Department of Biochemistry, University of Washington, Seattle, WA, USA. cjamesglasscock@gmail.com.
² Institute for Protein Design, University of Washington, Seattle, WA, USA. cjamesglasscock@gmail.com.
³ Department of BioSciences, Rice University, Houston, TX, USA. cjamesglasscock@gmail.com.
⁴ Department of Biochemistry, University of Washington, Seattle, WA, USA.
⁵ Institute for Protein Design, University of Washington, Seattle, WA, USA.
⁶ Department of Physics, University of Washington, Seattle, WA, USA.
⁷ Division of Basic Sciences, Fred Hutchinson Cancer Center, Seattle, WA, USA.
⁸ Program in Genetics and Genomics, Duke University, Durham, NC, USA.
⁹ Center for Advanced Genomic Technologies, Duke University, Durham, NC, USA.
¹⁰ Department of Bioengineering, University of Washington, Seattle, WA, USA.
¹¹ Department of Biochemistry, Stanford University School of Medicine, Palo Alto, CA, USA.
¹² Department of Medicine, Division of Hematology, Stanford University, Stanford, CA, USA.
¹³ BioInnovation Institute, Copenhagen, Denmark.
¹⁴ Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA.
¹⁵ Department of Computer Science, Duke University, Durham, NC, USA.
¹⁶ Department of Genomics and Computational Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
¹⁷ Department of Biostatics and Bioinformatics, Duke University, Durham, NC, USA.
¹⁸ Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA.
¹⁹ Department of Biochemistry, University of Washington, Seattle, WA, USA. dabaker@uw.edu.
²⁰ Institute for Protein Design, University of Washington, Seattle, WA, USA. dabaker@uw.edu.
²¹ Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA. dabaker@uw.edu.

^# Contributed equally.

PMID: 40940539
DOI: 10.1038/s41594-025-01669-4

Computational design of sequence-specific DNA-binding proteins

Cameron J Glasscock et al. Nat Struct Mol Biol. 2025.

. 2025 Sep 12.

doi: 10.1038/s41594-025-01669-4. Online ahead of print.

Authors

Affiliations

¹ Department of Biochemistry, University of Washington, Seattle, WA, USA. cjamesglasscock@gmail.com.
² Institute for Protein Design, University of Washington, Seattle, WA, USA. cjamesglasscock@gmail.com.
³ Department of BioSciences, Rice University, Houston, TX, USA. cjamesglasscock@gmail.com.
⁴ Department of Biochemistry, University of Washington, Seattle, WA, USA.
⁵ Institute for Protein Design, University of Washington, Seattle, WA, USA.
⁶ Department of Physics, University of Washington, Seattle, WA, USA.
⁷ Division of Basic Sciences, Fred Hutchinson Cancer Center, Seattle, WA, USA.
⁸ Program in Genetics and Genomics, Duke University, Durham, NC, USA.
⁹ Center for Advanced Genomic Technologies, Duke University, Durham, NC, USA.
¹⁰ Department of Bioengineering, University of Washington, Seattle, WA, USA.
¹¹ Department of Biochemistry, Stanford University School of Medicine, Palo Alto, CA, USA.
¹² Department of Medicine, Division of Hematology, Stanford University, Stanford, CA, USA.
¹³ BioInnovation Institute, Copenhagen, Denmark.
¹⁴ Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA.
¹⁵ Department of Computer Science, Duke University, Durham, NC, USA.
¹⁶ Department of Genomics and Computational Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
¹⁷ Department of Biostatics and Bioinformatics, Duke University, Durham, NC, USA.
¹⁸ Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA.
¹⁹ Department of Biochemistry, University of Washington, Seattle, WA, USA. dabaker@uw.edu.
²⁰ Institute for Protein Design, University of Washington, Seattle, WA, USA. dabaker@uw.edu.
²¹ Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA. dabaker@uw.edu.

^# Contributed equally.

PMID: 40940539
DOI: 10.1038/s41594-025-01669-4

Abstract

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.

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Conflict of interest statement

Competing interests: C.G., R.P., R.M., C.N., F.D., D.C., B.C., H.H., G-R.L. and D.B. are coinventors on a provisional patent application that incorporates discoveries described in this paper. The other authors declare no competing interests.

Update of

Computational design of sequence-specific DNA-binding proteins.
Glasscock CJ, Pecoraro R, McHugh R, Doyle LA, Chen W, Boivin O, Lonnquist B, Na E, Politanska Y, Haddox HK, Cox D, Norn C, Coventry B, Goreshnik I, Vafeados D, Lee GR, Gordan R, Stoddard BL, DiMaio F, Baker D. Glasscock CJ, et al. bioRxiv [Preprint]. 2023 Sep 21:2023.09.20.558720. doi: 10.1101/2023.09.20.558720. bioRxiv. 2023. Update in: Nat Struct Mol Biol. 2025 Sep 12. doi: 10.1038/s41594-025-01669-4. PMID: 37790440 Free PMC article. Updated. Preprint.

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Computational design of sequence-specific DNA-binding proteins

Affiliations

Computational design of sequence-specific DNA-binding proteins

Authors

Affiliations

Abstract

Conflict of interest statement

Update of

References

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