Graphene oxide affinity sample grids for Cyro-EM
Inventors
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Assignees
University of California San Diego UCSD
University of California, San Diego (UCSD)The University of California, San Diego (UCSD) is a leading public research university located in La Jolla, California. Known for its innovative and interdisciplinary approach, UCSD offers a wide range of undergraduate, graduate, and professional programs across various fields. The university is committed to fostering a diverse and inclusive community, promoting sustainability, and driving social mobility through education, research, and public service. UCSD is recognized for its contributions to research and innovation, particularly in areas such as climate science, health innovation, and artificial intelligence.
The University of California, San Diego (UCSD) is a leading public research university located in La Jolla, California. Known for its innovative and interdisciplinary approach, UCSD offers a wide range of undergraduate, graduate, and professional programs across various fields. The university is committed to fostering a diverse and inclusive community, promoting sustainability, and driving social mobility through education, research, and public service. UCSD is recognized for its contributions to research and innovation, particularly in areas such as climate science, health innovation, and artificial intelligence.
Publication Number
US-12360021-B2
Publication Date
2025-07-15
Expiration Date
2039-08-19
Abstract
Herein are innovations that enable facile cryo-EM analysis of diverse samples. Methods of functionalizing sample grids for cryo-EM are described, including methods of creating high quality graphene oxide films on cryo-EM substrates. The cryo-EM sample substrates are functionalized with affinity molecules that efficiently concentrate sample molecules and other specimen types on the grid, away from the air-water interface. Affinity groups include amines and proteins such as tagging system proteins and peptides that can be used to capture diverse sample types with high affinity. Optionally, spacers such as PEG chains are used to place sample particles away from the substrate surface, reducing substrate-induced artifacts.
Core Innovation
The invention presents novel methods, systems, and compositions that enable facile cryo-electron microscopy (cryo-EM) analysis of diverse samples by functionalizing cryo-EM sample grids with high-quality single-layer graphene oxide films. These graphene oxide films are coated on perforated substrates and subsequently functionalized with affinity molecules, including amines, proteins, and peptides, which efficiently concentrate target sample molecules or other specimens on the grid, thereby positioning samples away from the detrimental air-water interface.
The problem addressed by the invention lies in the inefficiency and limitations of conventional cryo-EM sample preparation methods. Traditional grids often expose samples to the air-water interface during the formation of thin vitreous ice films, causing protein denaturation, aggregation, and preferential orientation, which adversely affect image quality and limit high-resolution structural determination. Furthermore, prior art affinity grids either lack general applicability, require high sample concentrations, cause substrate-induced artifacts, or involve laborious target-specific antibody production.
This invention overcomes these barriers by providing a versatile affinity grid system that functionalizes graphene oxide support films with affinity moieties capable of capturing diverse targets through non-specific physiochemical interactions or specific high-affinity binding pairs such as SpyCatcher-SpyTag. The use of intervening spacer molecules, notably polyethylene glycol (PEG) chains, positions samples optimally between the substrate surface and the air-water interface, thereby reducing both substrate-induced and interface-induced artifacts and presenting samples in diverse orientations for rich structural analysis.
Claims Coverage
The patent contains several independent claims that cover functionalized cryo-EM substrates and specific features related to graphene oxide films, affinity moieties, and spacers.
Functionalized cryo-EM substrate with graphene oxide coating
The substrate comprises a plurality of holes, at least 50% of which are covered by a graphene oxide film comprising 1-3 layers of graphene sheets. The graphene oxide film is functionalized with affinity moieties that promote association of target specimens or with functional moieties that facilitate conjugation of affinity moieties.
Diverse substrate materials and coverage parameters
The cryo-EM substrate may comprise amorphous carbon, carbon coated materials, gold, gold coated materials, or silicon nitride. The graphene oxide film coverage includes at least 70% of the hole area or at least 80% of the overall substrate.
Intervening spacers linking affinity moieties to graphene oxide
An intervening spacer conjugates the affinity or functional moiety to the graphene oxide film, with the spacer comprising alkyl chains, polyesters, polyethers, polyalcohols, polyglycols (including PEG), poly(N-isopropylacrylamide), polyacrylamide, poly(acrylic acid), polymethacrylate, or other acrylic polymers.
Affinity moieties comprising binding partners of tagging systems
Affinity moieties include one binding partner of tagging systems that form covalent bonds under suitable conditions. The tagging systems cover SpyCatcher/SpyTag, SnoopCatcher/SnoopTag, HaloTag with chloroalkane ligands, split GFP, DogTag, Isopeptag, SdyTag, biotin-avidin and streptavidin-biotin systems, and polyhistidine tags.
Affinity moieties comprising amines, peptides, antibodies, and nucleic acids
Affinity moieties may be amines, peptides, antibodies or antigen-binding fragments, or nucleic acids that promote specimen association or facilitate conjugation.
Functional moieties facilitating conjugation chemistry
Functional moieties include amine, alkyne, maleimide, NHS, or sulfo-NHS groups that enable further conjugation of affinity moieties onto the graphene oxide film.
The claims comprehensively cover cryo-EM substrates with high-coverage graphene oxide films functionalized by affinity or functional moieties, the incorporation of spacers to optimize specimen positioning, and the use of various affinity groups and tagging systems to capture diverse specimens with high specificity and efficiency.
Stated Advantages
Concentration of sample molecules on the grid allows use of lower specimen concentrations, reducing aggregation and enabling analysis of difficult-to-express or dilute targets.
The use of high-quality single-layer graphene oxide films reduces background and electron scattering compared to amorphous carbon.
Functionalization with specific affinity moieties and spacers preserves sample integrity by preventing protein denaturation and disruption caused by the air-water interface or sample substrate.
The PEG spacers isolate samples from substrate effects and promote diverse particle orientations, enriching structural data.
Functionalized grids can be stored dry at room temperature for long durations, facilitating mass production, affordable shipping, and ready use.
Affinity grids allow 'purification on the grid,' enabling selective capture and enrichment of tagged samples.
The method enables high-resolution structural determination of challenging targets such as human TRAP1 full-length protein, previously limited by conventional methods.
Documented Applications
High-resolution cryo-EM analysis of proteins, including challenging targets such as human mitochondrial Hsp90 (TRAP1).
Structural analysis of diverse biological specimens such as organelles, viruses, cells, DNA origami, and other macromolecules.
Use with tagged proteins employing covalent tagging systems such as SpyCatcher-SpyTag, SnoopCatcher-SnoopTag, and HaloTag for specific capture and structural analysis.
Preparation of cryo-EM specimens that avoid artifacts caused by the air-water interface and substrate interactions.
Concentration and immobilization of DNA-binding proteins by nucleic acid-functionalized grids.
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