Site-specific, kinetically inert conjugation of labels and/or carriers to target molecules such as His-tagged proteins via metal complex reagents
Inventors
BENK, Amelie S. • SCHENK, Franziska • WEGNER, Seraphine • Comba, Peter • Spatz, Joachim P.
Assignees
Universität Heidelberg • Max-Planck-Gesellschaft zur Förderung der Wissenschaften eV
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Publication Number
US-12497422-B2
Publication Date
2025-12-16
Expiration Date
Abstract
The present invention relates to means and methods for conjugating/attaching target molecules such as proteins to a label and/or carrier. Specifically, the present invention provides a complex comprising a metal cation coordinating (i) a metal cation ligand being a carbonate selected from CO32− and HCO3— and (ii) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier. This complex can be used for attaching a label and/or a carrier to a target molecule, preferably a protein. The attachment of the label or carrier via the complex of the invention involves the replacement of the metal cation ligand with a coordinating group of the target molecule so that a product complex with the target molecule as primary ligand in the coordination sphere of the metal cation is formed. Accordingly, the present invention also provides for uses and methods involving the attachment of a label and/or carrier to a target molecule. Also provided are the products obtained by the labeling and or carrier-attaching methods of the invention and uses thereof. The invention further relates to methods for producing the complex of the invention and kits comprising the components for producing the complex of the invention.
Core Innovation
The invention relates to a method for attaching a label and/or a carrier to a target molecule by incubating a complex with the target molecule. The complex comprises a metal cation selected from Co3+, Rh3+, and Ir3+, a metal cation ligand being CO3^2−, and a metal cation chelating domain comprising a chelating ligand selected from NTA, IDA, and TED.
The target molecule comprises a protein, peptide, or nucleic acid that can exchange the metal cation ligand in the complex and comprises at least 4 histidine residues or histidine-like residues in a sequence pattern [HnSm]k, with n independently 1 to 4, m independently 0 to 6, and k 2 to 6. The carrier is selected from a polymer, a hydrogel, a microparticle, a nanoparticle, a nanosphere, a microsphere, a bead, and a solid surface.
The description supports cobalt(III) carbonate-mediated immobilization that forms stable [Co(III)(NTA)(His-GFP)] complexes on beads and surfaces. The complex shows improved stability and higher immobilization/labeling efficiencies across different buffer systems and conditions while retaining functionality, including GFP binding and active sortase.
The disclosure further extends the approach to alternative metal-binding domains and metals, including IDA and TALON with Co(III) and Pt(IV) variants, while confirming carbonate coordination. The document reports reduced/elution resistance versus Co(II)-based or nickel controls and chemical inertness against chelators/reducing agents, including mitigation of damage associated with hydrogen peroxide oxidation.
Claims Coverage
Independent claim coverage centers on a method for attaching a label and/or a carrier to a target molecule by incubating a CO3^2−-ligated metal-cation-chelate complex with a target molecule having at least 4 histidine or histidine-like residues in a specified [HnSm]k sequence pattern. The independent claims emphasize the defined metal complex composition, the carbonate ligand, the NTA/IDA/TED chelating domain, the histidine motif target definition, and the broad carrier classes.
Incubating a carbonate-ligated metal complex with a histidine-rich target
Incubating a complex comprising a metal cation selected from Co3+, Rh3+, and Ir3+, a metal cation ligand being CO3^2−, and a metal cation chelating domain comprising a chelating ligand selected from NTA, IDA, and TED, with a target molecule that can exchange the metal cation ligand in the complex.
Target with histidine-rich sequence pattern for metal ligand exchange
The target molecule comprises a protein, peptide, or nucleic acid that can exchange the metal cation ligand in the complex, wherein the target molecule comprises at least 4 histidine residues or histidine-like residues in a sequence [HnSm]k with n independently 1 to 4, m independently 0 to 6, and k 2 to 6.
Attaching a label and/or carrier via defined carrier classes
Attaching a label and/or a carrier by using a carrier selected from a polymer, a hydrogel, a microparticle, a nanoparticle, a nanosphere, a microsphere, a bead, and a solid surface.
The claim coverage is centered on histidine-rich targets undergoing metal-cation ligand exchange with a carbonate-ligated Co3+/Rh3+/Ir3+ complex using NTA, IDA, or TED chelating domains, thereby enabling attachment of a label and/or a broadly defined carrier such as beads and solid surfaces.
Stated Advantages
Improved stability and higher immobilization/labeling efficiencies under stringent wash conditions while retaining functionality.
Reduced/elution resistance versus Co(II)-based or nickel controls.
Chemical inertness against chelators/reducing agents.
Mitigation of damage associated with hydrogen peroxide oxidation.
Documented Applications
Surface immobilization on PEG-passivated nanostructured gold glass using the cobalt(III) carbonate-mediated immobilization approach.
Site-specific biotinylation using Biotin-X-NTA complexes followed by streptavidin capture.
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