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Publication Number
US-11807851-B1
Publication Date
2023-11-07
Expiration Date
Abstract
The present disclosure provides modified substrates such as modified polynucleotides, proteins, antibodies, lipids, and cells and uses thereof. Method of generating the modified substrates, which may comprise cleavage of a cleavable group linking the substrate to a labeling moiety, are also provided herein.
Core Innovation
The invention relates to a composition comprising a compound of Formula (I) or a salt or solvate thereof. In Formula (I), an alkyne is covalently linked to a nucleobase, where R_B comprises a nucleotide, a nucleotide analog, or a nucleoside, and Y is selected from —SH, —S(=O)H, —S(=O)2H, or —S(=O)(OH).
R_A is an optionally substituted 5-12 membered ring system, and R_C is selected from optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, or is absent. The compound further includes broad substituent options for R2, R3, R4, R5, R6, and, where stated, R7, R8, R9, and R10, including ring-forming relationships such as two R3 together forming an optionally substituted 3- to 6-membered heterocycle or carbocycle and two R7 together forming an optionally substituted 3- to 6-membered heterocycle.
The disclosure also describes optical fluorescent labeling reagents for sequencing-by-synthesis, including a dye attached via a semi-rigid, water-soluble cleavable linker to a dGTP analog, and conceptually describes cleavage of cleavable linker groups to separate a fluorescent label from a substrate and generate scarred substrates or scar moiety concepts for sequencing-related use.
Claims Coverage
The consolidated claim coverage centers on one independent claim directed to a composition comprising a Formula (I) compound, or a salt or solvate thereof, with a nucleobase-linked alkyne in R_B, a defined sulfur substituent Y, and broad substituent definitions for the remaining framework. The inventive features reduce to the Formula (I) scaffold, the nucleobase-linked alkyne arrangement, and the enumerated substitution rules for R_A, R_C, and R2–R10.
Formula (I) composition with nucleobase-linked alkyne
A composition comprising a compound of Formula (I) or a salt or solvate thereof, where R_B comprises a nucleotide, a nucleotide analog, or a nucleoside with a nucleobase, and the alkyne of Formula (I) is covalently linked to the nucleobase.
Sulfur substituent Y selection
Y is selected from —SH, —S(=O)H, —S(=O)2H, or —S(=O)(OH).
Optionally substituted ring system R_A
R_A is an optionally substituted 5-12 membered ring system, with substituents on R_A independently selected from R4.
Variable R_C selection or absence
R_C is selected from optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, or is absent.
Substituent framework for R2, R3, R4, R5, and R6
R2 is selected from hydrogen and optionally substituted carbon-based groups including cycloalkyl and heterocycloalkyl options with substituents independently selected from R6, and each R3, R4, R5, and R6 is independently selected from the defined sets, including that two R3 taken together form an optionally substituted 3- to 6-membered heterocycle or carbocycle.
Additional ring-forming relationships
The content also states that two R7 together form an optionally substituted 3- to 6-membered heterocycle, and refers to additional substituent variables R7, R8, R9, and R10 in the broader structural definitions.
Overall, the independent claim coverage is a Formula (I) composition defined by the nucleotide/nucleobase covalent linkage to an alkyne, a defined sulfur-containing Y group, and extensive substituent and ring-forming options across the variable framework.
Stated Advantages
Improves subsequent polymerase extension.
Reduces context effects.
Supports sequencing-by-synthesis.
Quenching between nearby or adjacent dyes is minimized so that signal intensity is proportional to the number of incorporated labeled nucleotides.
A linear relationship where the slope is approximately 1 is stated.
Addresses low labeled-substrate fractions, binomial noise, and context-dependent extension drawbacks.
Documented Applications
Sequencing where fluorescently labeled nucleotides are converted to scarred substrates to support polymerase extension.
Sequencing-by-synthesis.
Optical readout behavior in flow-based sequencing with nucleotide flows, washing, optional chase/cleavage flows, and fluorescence measurement.
Use with terminated and non-terminated nucleotide mixtures, including non-terminated versus terminated/non-terminated mixtures.
Handling homopolymer regions and heteropolymer regions.
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