Linkers and methods for optical detection and sequencing
Inventors
Lee, Linda G. • Almogy, Gilad • Menchen, Steven
Assignees
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Publication Number
US-11946097-B2
Publication Date
2024-04-02
Expiration Date
Abstract
The present disclosure provides labeling reagents for labeling substrates such as nucleotides, proteins, antibodies, lipids, and cells. The labeling reagents provided herein may comprise fluorescent labels and semi-rigid linkers. Methods for nucleic acid sequencing using materials comprising such labeling reagents are also provided here.
Core Innovation
The invention provides a kit comprising a plurality of linkers for fluorescent labeling reagents and nucleotide/dye conjugates. A linker comprises one or more water soluble groups and two or more ring systems, and includes a non-proteinogenic amino acid comprising a ring system. The linker is coupled to a nucleotide via a nucleobase of the nucleotide, and at least two of the ring systems are connected to each other by no more than two atoms.
The invention addresses water-soluble, semi-rigid, non-proteinogenic amino-acid-based linker components that support optical labeling. Water-solubilizing moieties include pyridinium, imidazolium, quaternary ammonium group, sulfonate, phosphate, alcohol, amine, imine, nitrile, amide, thiol, carboxylic acid, polyether, aldehyde, boronic acid, and boronic ester, and the ring-system rigidity is described as reducing quenching in optical labeling.
The invention further provides cleavable linker components suitable for cleavage and separation of labeled portions, dye removal, or scar formation. Cleavable moieties include azidomethyl group, disulfide bond, hydrocarbyldithiomethyl group, and 2-nitrobenzyloxy group, and disulfide-cleavable linkers are described as generating thiol/hydroxyl scar groups. The document also describes fluorescently labeled nucleotides in sequencing-by-synthesis embodiments and a computer system for interpreting optical signal intensities for sequence read calling.
Claims Coverage
The provided independent claim coverage centers on a kit comprising plural linkers with water-soluble groups, two or more ring systems, nucleobase coupling to a nucleotide, and inclusion of a non-proteinogenic amino acid. Dependent claim refinements further define the permitted water-soluble group classes, ring-to-ring connectivity, and optional cleavable group structures.
Plural linker kit for nucleobase-coupled fluorescent labeling
A kit comprising a plurality of linkers, wherein a linker of said plurality of linkers is coupled to a nucleotide via a nucleobase of said nucleotide.
Water-soluble multi-ring semi-rigid linker
A linker comprising one or more water soluble groups and two or more ring systems.
Constrained ring-system connectivity by no more than two atoms
At least two ring systems of said two or more ring systems are connected to each other by no more than two atoms.
Non-proteinogenic amino acid included in linker
The linker comprises a non-proteinogenic amino acid comprising a ring system of said two or more ring systems.
Direct ring-system connection without an intervening atom
At least two ring systems are directly connected to each other without an intervening atom.
Water-soluble group selection from enumerated categories
The one or more water soluble groups are selected from pyridinium, imidazolium, a quaternary ammonium group, a sulfonate, a phosphate, an alcohol, an amine, an imine, a nitrile, an amide, a thiol, a carboxylic acid, a polyether, an aldehyde, a boronic acid, and a boronic ester.
Kit configured with cleavable group splitting linker into two portions
The kit includes a linker that contains a cleavable group configured to cleave and split the linker into a first portion and a second portion.
Specific cleavable group moieties for linker cleavage
The cleavable group is one of an azidomethyl group, a disulfide bond, a hydrocarbyldithiomethyl group, or a 2-nitrobenzyloxy group.
Overall, the claim coverage centers on a kit of plural nucleobase-coupled linkers where each linker includes one or more water soluble groups and two or more ring systems, with constrained ring-system connectivity and inclusion of a non-proteinogenic amino acid bearing a ring system. Dependent claim coverage further narrows the linker by specifying direct or near-direct ring connectivity, enumerated water-soluble groups, and optional cleavable groups that split the linker into first and second portions.
Stated Advantages
Reducing dye quenching in optical labeling.
Reducing template-copy demands in flow-based sequencing-by-synthesis.
Reducing context-dependent labeling errors and binomial noise when using higher labeling fractions.
Providing a signal that is proportional to incorporated labeled nucleotide number and related to homopolymer length.
Scar thiol/hydroxyl groups are discussed for disulfide-cleavable linkers.
Documented Applications
Fluorescent labeling reagents.
Nucleotide/dye conjugates.
Optical sequencing-by-synthesis using fluorescent/optical labeling reagents and labeled non-terminated nucleotides with polymerase incorporation and optical detection.
Optical fluorescent labeling and sequencing using fluorescently labeled nucleotides and detecting fluorescence during a sequencing process.
Sequencing embodiments using multi-labeled nucleotides where detected signal is proportional to an incorporation number.
Use of cleavable groups to enable dye removal or scar formation, including cleavage between sequencing steps.
Interpreting optical signal intensities for sequence read calling using a computer system.
Evaluation assays including bead-based incorporation and quenching assays for labeled nucleotides and linker performance.
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