Methods of generating nanoarrays and microarrays
Inventors
Gremyachinskiy, Dmitriy • GALIMIDI, Rachel • Mallick, Parag • Patel, Sujal M.
Assignees
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Publication Number
US-11203612-B2
Publication Date
2021-12-21
Expiration Date
Abstract
The methods described herein provide a means of producing an array of spatially separated proteins. The method relies on covalently attaching each protein of the plurality of proteins to a structured nucleic acid particle (SNAP), and attaching the SNAPs to a solid support.
Core Innovation
The invention provides methods and components for producing spatially separated protein arrays using structured nucleic acid particles (SNAPs). A structured nucleic acid particle is covalently attached to only one protein molecule of interest and comprises a self-hybridized region and nucleic acid origami, including DNA origami SNAPs.
The structured nucleic acid particle carrying the only one protein molecule of interest is attached to an attachment site on a solid support, so that each attachment site is associated with only a single biomolecule via the structured nucleic acid particle. The approach includes controlling spatial separation and array formation using attachment-site geometry and diameter constraints and charged or functionalized surfaces.
The document further describes covalent versus non-covalent SNAP-support attachment, including configurations in which the protein does not contact the solid support. It also describes heterobifunctional crosslinkers, reactive handles, solid support functional group coupling, and strategies for generating attachment sites and protein bioconjugation chemistries.
Claims Coverage
The partial content identifies one independent claim and several dependent refinements. Coverage requires obtaining a structured nucleic acid particle covalently attached to only one protein molecule of interest and attaching that particle to a solid-support attachment site such that the attachment site is attached to the single protein via the structured nucleic acid particle. Dependent claims add geometric constraints, DNA origami, landing-surface and linker positioning, and a photocleavable bond embodiment, for a total of seven inventive features.
Structured nucleic acid particle covalently attached to only one protein of interest and attached to a solid support attachment site
A structured nucleic acid particle is covalently attached to only one protein molecule of interest, where the structured nucleic acid particle comprises a self-hybridized region and comprises nucleic acid origami, and is attached to an attachment site on a solid support so that the attachment site is attached to said only one protein molecule of interest via said structured nucleic acid particle.
Attachment site diameter constrained relative to the SNAP
The diameter of the attachment site is less than the diameter of the structured nucleic acid particle.
Attachment-site array spacing constrained relative to the SNAP
A solid support has an array of attachment sites spaced farther apart than the diameter of the structured nucleic acid particle.
DNA nucleic acid origami SNAP
The nucleic acid origami comprises deoxyribonucleic acid (DNA).
Landing surface and linker position the protein away from the solid support
The nucleic acid origami has a landing surface that contacts an attachment site and uses a linker to position the only one protein molecule of interest away from the solid support relative to the landing surface.
Photocleaveable covalent bond for single-protein attachment
The structured nucleic acid particle includes a photocleaveable bond that covalently attaches only one protein of interest to the attachment site.
Heterobifunctional crosslinker chemistry
Heterobifunctional crosslinkers preserve reactive chemistry such as an NHS-ester for sulfhydryl conjugation through a maleimide moiety, including NHS/amine-to-amine coupling and maleimide/sulfhydryl conjugation.
Coverage centers on a nucleic acid origami-based SNAP that covalently holds only one protein molecule of interest and is attached to solid-support attachment sites. The refinements emphasize single-protein connectivity and geometric or structural constraints that support spatially separated array attachment, including DNA origami, landing-surface and linker positioning, a photocleavable bond embodiment, and heterobifunctional crosslinker chemistry.
Stated Advantages
Enables spatial separation and detection using fluorescently labeled SNAPs and conjugated targets.
Documented Applications
Chip-based click conjugation/detection experiments using fluorescently labeled SNAPs and conjugated targets.
Generation and use of DNA SNAPs, including DNA origami SNAPs, with characterization and validation of size/brightness and imaging.
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