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Sequence-Directed Covalent Protein-DNA Linkages in a Single Step Using HUH-Tags
Klaus N Lovendahl1, Amanda N Hayward1, Wendy R Gordon1
1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Journal of the American Chemical Society
|May 9, 2017
Summary
We developed HUH-tags to covalently link proteins and DNA. These tags react specifically with DNA sequences, enabling applications in gene editing, single-molecule studies, and cellular imaging.
Area of Science:
- Biochemistry
- Molecular Biology
- Chemical Biology
Background:
- Covalent protein-DNA linkages are crucial for various biological processes and biotechnological applications.
- Existing methods for protein-DNA conjugation often require modified DNA or lack specificity.
- Novel strategies are needed for efficient and specific protein-DNA conjugation in biological systems.
Purpose of the Study:
- To develop a robust and versatile method for covalently linking proteins and DNA.
- To identify and characterize HUH-endonuclease domains as fusion partners (HUH-tags) for specific DNA sequence recognition.
- To demonstrate the utility of HUH-tags in gene editing, single-molecule studies, and cellular imaging.
Main Methods:
- Engineering HUH-endonuclease domains as fusion proteins (HUH-tags).
- Testing HUH-tag reactivity with unmodified single-stranded DNA sequences.
- Identifying orthogonal HUH-tags that recognize distinct DNA sequences.
- Integrating HUH-tags into Cas9-mediated gene editing systems.
- Constructing doubly DNA-tethered proteins for single-molecule experiments.
- Applying HUH-tags for cellular imaging in live and fixed cells.
Main Results:
- HUH-tags demonstrate robust covalent linkage with specific, unmodified single-stranded DNA sequences.
- Five distinct HUH-tags were identified, exhibiting orthogonal reactivity with different DNA sequences.
- HUH-tags were successfully employed as fusion partners in Cas9 gene editing.
- Doubly DNA-tethered proteins were constructed for advanced single-molecule studies.
- The method was validated for cellular imaging applications in both live and fixed cells.
Conclusions:
- HUH-tags provide a versatile and robust platform for covalent protein-DNA conjugation.
- The orthogonality of identified HUH-tags expands their utility in complex biological systems.
- HUH-tag technology offers significant potential for advancing gene editing, single-molecule biophysics, and live-cell imaging.
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