Efficient Formation of Site-Specific Protein–DNA Hybrids Using Copper-Free Click Chemistry

Many proteins form dynamic complexes with DNA, RNA, and other proteins, which often involves protein conformational changes that are key to function. Yet, methods to probe these critical dynamics are scarce. Here we combine optical tweezers with fluorescence imaging to simultaneously monitor the conformation of individual proteins and their binding to partner proteins. Central is a protein-DNA coupling strategy, which uses exonuclease digestion and partial re-synthesis to generate DNA overhangs of different lengths, and ligation to oligo-labeled proteins. It provides up to 40 times higher coupling yields than existing protocols and enables new fluorescence-tweezers assays, which require particularly long and strong DNA handles. We demonstrate the approach by detecting the emission of a tethered fluorescent protein and of a molecular chaperone (trigger factor) complexed with its client. We conjecture that our strategy will be an important tool to study conformational dynamics within larger biomolecular complexes.

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“…1a-d and Supplementary Fig. 1a), in line with previously reported efficiencies (just below 20% for a 34 nt anchor) 45 .…”

Section: Resultssupporting

confidence: 92%

“…Coupling of short DNA oligos to proteins. First, we addressed the protein-anchor coupling, which is key to overall efficiency in existing hybridization approaches 45 . Specifically, we interrogated the effect of the anchor length.…”

Section: Resultsmentioning

confidence: 99%

Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA–protein coupling

et al. 2020

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“…However, unwanted by-products of this reaction can be poly-protein constructs and oligo homo-dimers, both of which are not easily separated from the protein-DNA chimera in purification steps following the conjugation reaction. Cysteine residues can also be reacted directly to maleimide-modified DNA oligos, but maleimide has to be supplied in large excess and the potential of maleimide dimerization remains 11,12 . To improve reaction speeds and minimize the excess use of expensive components, Mukhortava and Schlierf 12 developed a two-step protocol, in which cysteines are first functionalised with DBCO-maleimide followed by subsequent conjugation to azide-modified DNA oligos.…”

Section: Introductionmentioning

confidence: 99%

Bioorthogonal protein-DNA conjugation methods for force spectroscopy

Synakewicz

Bauer

Rief

et al. 2019

Sci Rep

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Accurate and stable site-specific attachment of DNA molecules to proteins is a requirement for many single-molecule force spectroscopy techniques. The most commonly used method still relies on maleimide chemistry involving cysteine residues in the protein of interest. Studies have consequently often focused on model proteins that either have no cysteines or with a small number of cysteines that can be deleted so that cysteines can then be introduced at specific sites. However, many proteins, especially in eukaryotes, contain too many cysteine residues to be amenable to this strategy, and therefore there is tremendous need for new and broadly applicable approaches to site-specific conjugation. Here we present bioorthogonal approaches for making DNA-protein conjugates required in force spectroscopy experiments. Unnatural amino acids are introduced site-specifically and conjugated to DNA oligos bearing the respective modifications to undergo either strain-promoted azidealkyne cycloaddition (SPAAC) or inverse-electron-demand Diels-Alder (IE-DA) reactions. We furthermore show that SPAAC is compatible with a previously published peptide-based attachment approach. By expanding the available toolkit to tag-free methods based on bioorthogonal reactions, we hope to enable researchers to interrogate the mechanics of a much broader range of proteins than is currently possible.

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“…DNA conjugates concentration was adjusted to only sparsely cover the beads leading mainly to single-tether formation. The beads were trapped in the foci of a dual beam optical tweezers platform (JPK NanoTracker) (9). Both trapped beads were brought into close proximity for tether formation.…”

Section: Methodsmentioning

confidence: 99%

Structural heterogeneity ofattCintegron recombination sites revealed by optical tweezers

Mukhortava

Pöge

Grieb

et al. 2018

Nucleic Acids Research

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A predominant tool for adaptation in Gram-negative bacteria is the functional genetic platform called integron. Integrons capture and rearrange promoterless gene cassettes in a unique recombination process involving the recognition of folded single-stranded DNA hairpins—so-called attC sites—with a strong preference for the attC bottom strand. While structural elements have been identified to promote this preference, their mechanistic action remains incomplete. Here, we used high-resolution single-molecule optical tweezers (OT) to characterize secondary structures formed by the attC bottom ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${{att}}{{{C}}_{{\rm{bs}}}}$\end{document} ) and top ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${{att}}{{{C}}_{{\rm{ts}}}}$\end{document} ) strands of the paradigmatic attC aadA7 site. We found for both sequences two structures—a straight, canonical hairpin and a kinked hairpin. Remarkably, the recombination-preferred \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${{att}}{{{C}}_{{\rm{bs}}}}$\end{document} predominantly formed the straight hairpin, while the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${{att}}{{{C}}_{{\rm{ts}}}}$\end{document} preferentially adopted the kinked structure, which exposes only one complete recombinase binding box. By a mutational analysis, we identified three bases in the unpaired central spacer, which could invert the preferred conformations and increase the recombination frequency of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${{att}}{{{C}}_{{\rm{ts}}}}$\end{document} in vivo . A bioinformatics screen revealed structural bias toward a straight, canonical hairpin conformation in the bottom strand of many antibiotic resistance cassettes attC sites. Thus, we anticipate that structural fine tuning could be a mechanism in man...

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Efficient Formation of Site-Specific Protein–DNA Hybrids Using Copper-Free Click Chemistry

Cited by 32 publications

References 40 publications

Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA–protein coupling

Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA–protein coupling

Bioorthogonal protein-DNA conjugation methods for force spectroscopy

Structural heterogeneity ofattCintegron recombination sites revealed by optical tweezers

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