[go: up one dir, main page]

WO2025221895A1 - Échafaudages polymères solubles dans l'eau pour marquage de colorant - Google Patents

Échafaudages polymères solubles dans l'eau pour marquage de colorant

Info

Publication number
WO2025221895A1
WO2025221895A1 PCT/US2025/024973 US2025024973W WO2025221895A1 WO 2025221895 A1 WO2025221895 A1 WO 2025221895A1 US 2025024973 W US2025024973 W US 2025024973W WO 2025221895 A1 WO2025221895 A1 WO 2025221895A1
Authority
WO
WIPO (PCT)
Prior art keywords
type
substituted
nucleotide
unsubstituted
unlabeled
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/024973
Other languages
English (en)
Inventor
Nam Trong NGUYEN
Jacob George CORRIE
Iuliana Petruta MARIA
Wayne N. GEORGE
Andrew A. Brown
Xavier VON HATTEN
Xiaolin Wu
Oliver MILLER
Dale WEEKES
Madushani Dharmarwardana
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Illumina Inc
Original Assignee
Illumina Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Illumina Inc filed Critical Illumina Inc
Publication of WO2025221895A1 publication Critical patent/WO2025221895A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B69/00—Dyes not provided for by a single group of this subclass
    • C09B69/10—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds
    • C09B69/105—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds containing a methine or polymethine dye

Definitions

  • the present disclosure generally relates to compositions, kits, methods and systems for nucleic acid sequencing applications.
  • Nucleic acid sequencing methodology has evolved significantly from the chemical degradation methods used by Maxam and Gilbert and the strand elongation methods used by Sanger. Today several sequencing methodologies are in use which allow for the parallel processing of thousands of nucleic acids all in a single sequencing run. The instrumentation that performs such methods is typically large and expensive since the current methods typically rely on large amounts of expensive reagents and multiple sets of optic filters to record nucleic acid incorporation into sequencing reactions.
  • One aspect of the present disclosure relates to a water soluble polymer scaffold, comprising: a plurality of first functional groups that are capable of reacting specifically with a plurality of detectable labels to form covalent bonding; a plurality of unsubstituted or substituted polyacrylamide spacer units; one or more second functional groups that is capable of reacting specifically with a biomolecule to form covalent bonding; and optionally one or more reactive oxygen species (ROS) quencher moieties.
  • ROS reactive oxygen species
  • Another aspect of the present disclosure relates to a labeling reagent, comprising a plurality of detectable labels covalently attached to the water soluble polymer scaffold in accordance with the present disclosure, through the plurality of first functional groups of the water soluble polymer scaffold.
  • Another aspect of the present disclosure relates to a method of determining the sequences of a plurality of different target polynucleotides, comprising: (a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b) contacting the solid support with an incorporation mixture comprising DNA polymerase and one more of four different types of nucleotides under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides; wherein each of the four types of nucleotides comprises a 3′ blocking group; and the incorporation mixture comprises a first type of unlabeled nucleotide having a first reactive moiety covalently attached to the first type of unlabeled nucleotide; (a)
  • kits for sequencing application comprising: an incorporation mixture composition comprising one or more of four different types of nucleotides each comprising a 3′ blocking group, wherein a first type of unlabeled nucleotide having a first reactive moiety covalently attached to the first type of unlabeled nucleotide; and a first labeling reagent in accordance with the present disclosure, comprising a plurality of first detectable labels, wherein the one or more second functional groups of the first labeling reagent is capable of reacting specifically with the first reactive moiety of the first type of unlabeled nucleotides to form covalent bonding.
  • FIGs.1A and 1B schematically illustrate exemplary structures of water soluble polymer scaffolds according to certain embodiments of the present disclosure.
  • FIG. 2A plots fluorescence intensity maxima of three different poly(acrylamide)-based water soluble polymer scaffolds as a function of UV absorbance maxima.
  • FIG.2B plots brightness per dye moiety and brightness per scaffold relative to brightness of a single dye molecule.
  • FIG.3A is a scatter plot of sequencing by synthesis (SBS) runs using standard SBS reagents with labeled nucleotides.
  • FIGs. 3B and 3C are SBS scatter plots using a post incorporation labeling method with either a labeling reagent comprising a single dye, or a multi- dye labeling reagent containing a water soluble copolymer scaffold according to certain embodiments of the present disclosure.
  • FIG.4A is a scatter plot of sequencing by synthesis (SBS) runs using standard SBS reagents with labeled nucleotides.
  • FIGS. 4B and 4C are SBS scatter plots using a post incorporation labeling method with either a labeling reagent comprising a single dye, or a multi- dye labeling reagent containing a water soluble block copolymer scaffold according to certain embodiments of the present disclosure.
  • DETAILED DESCRIPTION [0016] The present disclosure provides next-generation sequencing compositions, methods, kits, and systems. Certain disclosure relates to polymer scaffolds prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization to produce polymer scaffolds with functionalities for multi-dye attachments.
  • the labeled polymer scaffolds can be used in post- incorporation labeling (PIL) sequencing by synthesis methods as described herein.
  • PIL post- incorporation labeling
  • the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.”
  • the term “comprising” means that the process includes at least the recited steps but may include additional steps.
  • the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.
  • An array can include different probe molecules that are each located at a different addressable location on a substrate.
  • an array can include separate substrates each bearing a different probe molecule, wherein the different probe molecules can be identified according to the locations of the substrates on a surface to which the substrates are attached or according to the locations of the substrates in a liquid.
  • Exemplary arrays in which separate substrates are located on a surface include, without limitation, those including beads in wells as described, for example, in U.S. Patent No.6,355,431 B1, US 2002/0102578 and PCT Publication No. WO 00/63437.
  • Exemplary formats that can be used in the invention to distinguish beads in a liquid array for example, using a microfluidic device, such as a fluorescent activated cell sorter (FACS), are described, for example, in US Pat. No. 6,524,793. Further examples of arrays that can be used in the invention include, without limitation, those described in U.S. Pat Nos.
  • FACS fluorescent activated cell sorter
  • covalently attached or “covalently bonded” refers to the forming of a chemical bonding that is characterized by the sharing of pairs of electrons between atoms.
  • a covalently attached polymer coating refers to a polymer coating that forms chemical bonds with a functionalized surface of a substrate, as compared to attachment to the surface via other means, for example, adhesion or electrostatic interaction. It will be appreciated that polymers that are attached covalently to a surface can also be bonded via means in addition to covalent attachment.
  • non-covalent interactions differs from a covalent bond in that it does not involve the sharing of electrons, but rather involves more dispersed variations of electromagnetic interactions between molecules or within a molecule.
  • Non-covalent interactions can be generally classified into four categories, electrostatic, ⁇ -effects, van der Waals forces, and hydrophobic effects.
  • electrostatic interactions include ionic interactions, hydrogen bonding (a specific type of dipole-dipole interaction), halogen bonding, etc.
  • Van der Walls forces are a subset of electrostatic interaction involving permanent or induced dipoles or multipoles.
  • ⁇ -effects can be broken down into numerous categories, including (but not limited to) ⁇ - ⁇ interactions, cation- ⁇ & anion- ⁇ interactions, and polar- ⁇ interactions.
  • ⁇ -effects are associated with the interactions of molecules with the ⁇ -orbitals of a molecular system, such as benzene.
  • the hydrophobic effect is the tendency of nonpolar substances to aggregate in aqueous solution and exclude water molecules.
  • Non-covalent interactions can be both intermolecular and intramolecular.
  • Non-covalent interactions can be both intermolecular and intramolecular.
  • certain radical naming conventions can include either a mono-radical or a di-radical, depending on the context.
  • a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a di-radical.
  • a substituent identified as alkyl that requires two points of attachment includes di-radicals such as –CH2–, –CH2CH2–, –CH2CH(CH3)CH2–, and the like.
  • halogen or “halo,” as used herein, means any one of the radio-stable atoms of column 7 of the Periodic Table of the Elements, e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.
  • C a to C b refers to the number of carbon atoms in an alkyl, alkenyl or alkynyl group, or the number of ring atoms of a cycloalkyl or aryl group. That is, the alkyl, the alkenyl, the alkynyl, the ring of the cycloalkyl, and ring of the aryl can contain from “a” to “b,” inclusive, carbon atoms.
  • a “C 1 to C 4 alkyl” group refers to all alkyl groups having from 1 to 4 carbons, that is, CH 3 -, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and (CH3)3C-;
  • a C3 to C 4 cycloalkyl group refers to all cycloalkyl groups having from 3 to 4 carbon atoms, that is, cyclopropyl and cyclobutyl.
  • a “4 to 6 membered heterocyclyl” group refers to all heterocyclyl groups with 4 to 6 total ring atoms, for example, azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, morpholine, and the like. If no “a” and “b” are designated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl, or aryl group, the broadest range described in these definitions is to be assumed.
  • the term “C1-C6” includes C1, C2, C3, C4, C5 and C 6 , and a range defined by any of the two numbers .
  • C 1 -C 6 alkyl includes C 1 , C 2 , C3, C4, C5 and C6 alkyl, C2-C6 alkyl, C1-C3 alkyl, etc.
  • C2-C6 alkenyl includes C2, C3, C4, C 5 and C 6 alkenyl, C 2 -C 5 alkenyl, C 3 -C 4 alkenyl, etc.
  • C 2 -C 6 alkynyl includes C 2 , C 3 , C 4 , C 5 and C6 alkynyl, C2-C5 alkynyl, C3-C4 alkynyl, etc.
  • C3-C8 cycloalkyl each includes hydrocarbon ring containing 3, 4, 5, 6, 7 and 8 carbon atoms, or a range defined by any of the two numbers, such as C3-C7 cycloalkyl or C5-C6 cycloalkyl.
  • alkyl refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds).
  • the alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated).
  • the alkyl group may also be a medium size alkyl having 1 to 9 carbon atoms.
  • the alkyl group could also be a lower alkyl having 1 to 6 carbon atoms.
  • the alkyl group may be designated as “C1-C4 alkyl” or similar designations.
  • C1-C6 alkyl indicates that there are one to six carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t- butyl.
  • alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.
  • alkoxy refers to the formula –OR wherein R is an alkyl as is defined above, such as “C1-C9 alkoxy,” including but not limited to methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy, and the like.
  • alkenyl refers to a straight or branched hydrocarbon chain containing one or more double bonds.
  • the alkenyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term “alkenyl” where no numerical range is designated.
  • the alkenyl group may also be a medium size alkenyl having 2 to 9 carbon atoms.
  • the alkenyl group could also be a lower alkenyl having 2 to 6 carbon atoms.
  • the alkenyl group may be designated as “C 2- C 6 alkenyl” or similar designations.
  • C 2- C 6 alkenyl indicates that there are two to six carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1- yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl- ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2,-dienyl, and buta-1,2-dien-4-yl.
  • Typical alkenyl groups include, but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and the like.
  • aromatic refers to a ring or ring system having a conjugated pi electron system and includes both carbocyclic aromatic (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine).
  • carbocyclic aromatic e.g., phenyl
  • heterocyclic aromatic groups e.g., pyridine
  • the term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of atoms) groups provided that the entire ring system is aromatic.
  • aryl refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) containing only carbon in the ring backbone. When the aryl is a ring system, every ring in the system is aromatic.
  • the aryl group may have 6 to 18 carbon atoms, although the present definition also covers the occurrence of the term “aryl” where no numerical range is designated. In some embodiments, the aryl group has 6 to 10 carbon atoms.
  • the aryl group may be designated as “C 6 -C 10 aryl,” “C 6 or C 10 aryl,” or similar designations.
  • aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.
  • An “aralkyl” or “arylalkyl” is an aryl group connected, as a substituent, via an alkylene group, such as “C7-14 aralkyl” and the like, including but not limited to benzyl, 2- phenylethyl, 3-phenylpropyl, and naphthylalkyl.
  • the alkylene group is a lower alkylene group (i.e., a C1-C6 alkylene group).
  • aryloxy refers to RO- in which R is an aryl, as defined above, such as but not limited to phenyl.
  • heteroaryl refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the ring backbone. When the heteroaryl is a ring system, every ring in the system is aromatic.
  • the heteroaryl group may have 5-18 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heteroaryl” where no numerical range is designated.
  • the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members.
  • the heteroaryl group may be designated as “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similar designations.
  • heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzoimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.
  • carbocyclyl means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system backbone. When the carbocyclyl is a ring system, two or more rings may be joined together in a fused, bridged or spiro-connected fashion. Carbocyclyls may have any degree of saturation provided that at least one ring in a ring system is not aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls.
  • the carbocyclyl group may have 3 to 20 carbon atoms, although the present definition also covers the occurrence of the term “carbocyclyl” where no numerical range is designated.
  • the carbocyclyl group may also be a medium size carbocyclyl having 3 to 10 carbon atoms.
  • the carbocyclyl group could also be a carbocyclyl having 3 to 6 carbon atoms.
  • the carbocyclyl group may be designated as “C3-C6 carbocyclyl” or similar designations.
  • carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro- indene, bicycle[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.
  • cycloalkyl means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • heterocyclyl means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined together in a fused, bridged or spiro-connected fashion. Heterocyclyls may have any degree of saturation provided that at least one ring in the ring system is not aromatic. The heteroatom(s) may be present in either a non-aromatic or aromatic ring in the ring system.
  • the heterocyclyl group may have 3 to 20 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heterocyclyl” where no numerical range is designated.
  • the heterocyclyl group may also be a medium size heterocyclyl having 3 to 10 ring members.
  • the heterocyclyl group could also be a heterocyclyl having 3 to 6 ring members.
  • the heterocyclyl group may be designated as “3-6 membered heterocyclyl” or similar designations.
  • the heteroatom(s) are selected from one up to three of O, N or S, and in preferred five membered monocyclic heterocyclyls, the heteroatom(s) are selected from one or two heteroatoms selected from O, N, or S.
  • heterocyclyl rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2- oxazinyl, trioxanyl, hexa
  • (aryl)alkyl refer to an aryl group, as defined above, connected, as a substituent, via an alkylene group, as described above.
  • the alkylene and aryl group of an aralkyl may be substituted or unsubstituted. Examples include but are not limited to benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.
  • the alkylene is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene unit(s).
  • heteroarylalkyl refers to a heteroaryl group, as defined above, connected, as a substituent, via an alkylene group, as defined above.
  • the alkylene and heteroaryl group of heteroaralkyl may be substituted or unsubstituted. Examples include but are not limited to 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, and their benzo-fused analogs.
  • the alkylene is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene unit(s).
  • (heterocyclyl)alkyl refer to a heterocyclic or a heterocyclyl group, as defined above, connected, as a substituent, via an alkylene group, as defined above.
  • the alkylene and heterocyclyl groups of a (heterocyclyl)alkyl may be substituted or unsubstituted.
  • alkylene is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene unit(s).
  • (carbocyclyl)alkyl refer to a carbocyclyl group (as defined herein) connected, as a substituent, via an alkylene group.
  • alkylene is an unsubstituted straight chain containing 1, 2, 3, 4, 5, or 6 methylene unit(s).
  • alkoxyalkyl or “(alkoxy)alkyl” refers to an alkoxy group connected via an alkylene group, such as C2-C8 alkoxyalkyl, or (C1-C6 alkoxy)C1-C6 alkyl, for example, –(CH 2 ) 1-3 -OCH 3.
  • -O-alkoxyalkyl or “-O-(alkoxy)alkyl” refers to an alkoxy group connected via an –O-(alkylene) group, such as –O-(C 1 -C 6 alkoxy)C 1 -C 6 alkyl, for example, –O-(CH2)1-3-OCH3.
  • haloalkyl refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono-haloalkyl, di-haloalkyl, and tri- haloalkyl).
  • haloalkyl refers to an alkoxy group in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono-haloalkoxy, di-haloalkoxy and tri- haloalkoxy).
  • Such groups include but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy and 1-chloro-2-fluoromethoxy, 2-fluoroisobutoxy.
  • a haloalkoxy may be substituted or unsubstituted.
  • An “amino” group refers to a –NH2 group.
  • the term “mono-substituted amino group” as used herein refers to an amino (–NH 2 ) group where one of the hydrogen atom is replaced by a substituent.
  • di-substituted amino group refers to an amino (–NH2) group where each of the two hydrogen atoms is replaced by a substituent.
  • RA and RB are independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, aralkyl, or heterocyclyl(alkyl), as defined herein.
  • R is selected from the group consisting of hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
  • a “sulfonyl” group refers to an “-SO2R” group in which R is selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
  • a “sulfonate” group refers to a “-SO3 ⁇ ” group.
  • a “sulfate” group refers to “-SO4 ⁇ ” group.
  • a “S-sulfonamido” group refers to a “-SO2NRARB” group in which RA and RB are each independently selected from hydrogen, C 1- C 6 alkyl, C 2- C 6 alkenyl, C 2- C 6 alkynyl, C 3- C 7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
  • N-sulfonamido refers to a “-N(RA)SO2RB” group in which RA and R b are each independently selected from hydrogen, C 1- C 6 alkyl, C 2- C 6 alkenyl, C 2- C 6 alkynyl, C 3- C7 carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
  • An O-carbamyl may be substituted or unsubstituted.
  • An N-carbamyl may be substituted or unsubstituted.
  • An O-thiocarbamyl may be substituted or unsubstituted.
  • An N-thiocarbamyl may be substituted or unsubstituted.
  • alkylamino or “(alkyl)amino” refers to an amino group wherein one or both hydrogen is replaced by an alkyl group.
  • An “(alkoxy)alkyl” group refers to an alkoxy group connected via an alkylene group, such as a “(C1-C6 alkoxy) C1-C6 alkyl” and the like.
  • hydroxy refers to a –OH group.
  • cyano refers to a “-CN” group.
  • zido refers to a –N 3 group.
  • isonitrile refers to a “–N+ ⁇ C ⁇ ” group.
  • a substituted group is derived from the unsubstituted parent group in which there has been an exchange of one or more hydrogen atoms for another atom or group.
  • substituents independently selected from C 1 -C 6 alkyl, C 1 -C 6 alkenyl, C 1 -C 6 alkynyl, C 1 -C 6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1- C 6 haloalkyl, and C 1 -C 6 haloalkoxy), C 3 -C 7 carbocyclyl-C 1 -C 6 -alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl
  • a “nucleotide” includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. They are monomeric units of a nucleic acid sequence.
  • the sugar is a ribose, and in DNA a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present in ribose.
  • the nitrogen containing heterocyclic base can be purine, deazapurine, or pyrimidine base.
  • Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof, such as 7-deaza adenine or 7-deaza guanine.
  • Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof.
  • the C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine.
  • nucleotide conjugate generally refers to a nucleotide labeled with a fluorescent moiety, optionally through a cleavage linker as described herein. In some embodiment, when a nucleotide conjugate is described as an unlabeled nucleotide, such nucleotide does not include a fluorescent moiety. In some further embodiments, an unlabeled nucleotide conjugate also does not have a cleavable linker. [0070] As used herein, a “nucleoside” is structurally similar to a nucleotide but is missing the phosphate moieties.
  • nucleoside analogue An example of a nucleoside analogue would be one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule.
  • the term “nucleoside” is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, a ribonucleoside comprising a ribose moiety and a deoxyribonucleoside comprising a deoxyribose moiety.
  • a modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced with a carbon and/or a carbon has been replaced with a sulfur or an oxygen atom.
  • a “nucleoside” is a monomer that can have a substituted base and/or sugar moiety. Additionally, a nucleoside can be incorporated into larger DNA and/or RNA polymers and oligomers.
  • the term “purine base” is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers.
  • the term “pyrimidine base” is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers.
  • a non-limiting list of optionally substituted purine-bases includes purine, adenine, guanine, deazapurine, 7-deaza adenine, 7-deaza guanine.
  • pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (e.g., 5-methylcytosine).
  • oligonucleotide or polynucleotide when described as “comprising” a nucleoside or nucleotide described herein, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide.
  • nucleoside or nucleotide when a nucleoside or nucleotide is described as part of an oligonucleotide or polynucleotide, such as “incorporated into” an oligonucleotide or polynucleotide, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide.
  • the covalent bond is formed between a 3 ⁇ hydroxy group of the oligonucleotide or polynucleotide with the 5 ⁇ phosphate group of a nucleotide described herein as a phosphodiester bond between the 3 ⁇ carbon atom of the oligonucleotide or polynucleotide and the 5 ⁇ carbon atom of the nucleotide.
  • the term “cleavable linker” is not meant to imply that the whole linker is required to be removed.
  • the cleavage site can be located at a position on the linker that ensures that part of the linker remains attached to the detectable label and/or nucleoside or nucleotide moiety after cleavage.
  • “derivative” or “analog” means a synthetic nucleotide or nucleoside derivative having modified base moieties and/or modified sugar moieties. Such derivatives and analogs are discussed in, e.g., Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990.
  • Nucleotide analogs can also comprise modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphonate, and phosphoramidate linkages. “Derivative,” “analog” and “modified” as used herein, may be used interchangeably, and are encompassed by the terms “nucleotide” and “nucleoside” defined herein. [0075] As used herein, the term “phosphate” is used in its ordinary sense as understood OH O P O by those skilled in the art, and includes its protonated forms (for example, O- and OH O P O OH ).
  • a compound such as a nucleotide conjugate described herein may exist in ionized form, e.g., containing a -CO2 ⁇ , -SO3 ⁇ or -O ⁇ . If a compound contains a positively or negatively charged substituent group, it may also contain a negatively or positively charged counterion such that the compound as a whole is neutral. In other aspects, the compound may exist in a salt form, where the counterion is provided by a conjugate acid or base.
  • the term “orthogonal” in the context of chemical reaction it refers to the situation when there are two pairs of substances and each substance can interact with their respective partner, but does not interact with either substance of the other pair.
  • the first and the second functional groups it refers to that the first functional groups will selectively react with detectable labels (e.g., fluorescent dyes), while the second functional groups will have little or no reactivity towards the same chemical entities that are reactive to the first functional groups.
  • the term “phasing” refers to a phenomenon in SBS that is caused by incomplete removal of the 3 ⁇ terminators and fluorophores, and/or failure to complete the incorporation of a portion of DNA strands within clusters by polymerases at a given sequencing cycle. Prephasing is caused by the incorporation of nucleotides without effective 3 ⁇ terminators, wherein the incorporation event goes 1 cycle ahead due to a termination failure. Phasing and prephasing cause the measured signal intensities for a specific cycle to consist of the signal from the current cycle as well as noise from the preceding and following cycles. As the number of cycles increases, the fraction of sequences per cluster affected by phasing and prephasing increases, hampering the identification of the correct base.
  • Prephasing can be caused by the presence of a trace amount of unprotected or unblocked 3 ⁇ -OH nucleotides during sequencing by synthesis (SBS).
  • SBS sequencing by synthesis
  • the unprotected 3 ⁇ -OH nucleotides could be generated during the manufacturing processes or possibly during the storage and reagent handling processes.
  • Certain embodiments of the present disclosure relate to a water soluble polymer scaffold, comprising: a plurality of first functional groups that are capable of reacting specifically with a plurality of detectable labels to form covalent bonding; a plurality of unsubstituted or substituted polyacrylamide spacer units; one or more second functional groups that is capable of reacting specifically with a biomolecule to form covalent bonding; and optionally one or more reactive oxygen species (ROS) quencher moieties.
  • ROS reactive oxygen species
  • the water soluble polymer scaffold described herein may be prepared by Reversible addition fragmentation chain transfer (RAFT) polymerization, which is a reversible deactivation radical polymerization (RDRP), or a living/controlled polymerization.
  • RAFT Reversible addition fragmentation chain transfer
  • RDRP reversible deactivation radical polymerization
  • FIG. 1A A general composition of the polymers synthesized by RAFT polymerization is illustrated in FIG. 1A. As shown in FIG.
  • the first functional groups sites for dye attachment
  • second functional groups sites for PIL
  • monomer spacers i.e., unsubstituted or substituted polyacrylamide spacer units
  • optional ROS quencher moieties may be distributed randomly along the water soluble polymer scaffold.
  • the polymer scaffold may further comprise chemically orthogonal end capping groups. The monomer units with various functionalities were reacted together at the same time to form a linear polymer, which means that the dye spacing is statistical.
  • the water soluble polymer scaffold may be a block copolymer, where the first functional groups, monomer spacers, and optional ROS quencher moieties are distributed along a first block for dye attachments, and a second block includes the second functional groups as handles for PIL.
  • the ROS quencher moieties can quench reactive- oxygen species. Such quenching may help protect the detectable labels from damage due to presence of reactive-oxygen species.
  • the plurality of first functional groups, the plurality of unsubstituted or substituted polyacrylamide spacer units, the one or more second functional groups, and the optionally present ROS quencher moieties are evenly or randomly distributed along the polymer scaffold.
  • the plurality of first functional groups, the plurality of unsubstituted or substituted polyacrylamide spacer units, and the optionally present ROS quencher moieties are evenly or randomly distributed along the polymer scaffold, and the one or more second functional groups are present at a terminal portion of the polymer scaffold.
  • the detectable label is or comprises a fluorescent dye.
  • the first functional groups are capable of reacting with the plurality of detectable labels via an orthogonal or a biorthogonal reaction.
  • the orthogonal or biorthogonal reaction is selected from the group consisting of a [3+2] dipolar cycloaddition, a Diels-Alder cycloaddition, a [4+1] cycloaddition, a phosphine ligation, or condensation with 2-acylphenyl boronic acid.
  • the plurality of detectable labels each comprises -NH-NH 2 , unsubstituted or substituted alkynyl, unsubstituted or substituted C5-C16 cycloalkynyl, unsubstituted or substituted 5 to 16 membered heterocycloalkynyl, unsubstituted or substituted C 5 -C 16 cycloalkenyl, unsubstituted or substituted 5 to 16 membered heterocycloalkenyl, substituted vinyl, isonitrile, substituted boronic acid moiety, substituted yet further embodiments, the embodiments, the first functional groups of the polymer scaffold are present in one or more repeating units of formula (I): ), wherein R 1 is H or C1-C4 alkyl.
  • each first hereo tly attached to an acrylamide repeating unit of the polymer scaffold.
  • the plurality of detectable labels each comprises alkynyl, unsubstituted or substituted dibenzocyclooctyne , unsubstituted or substituted bicyclo[6.1.0]nonyne (BCN) moiety, norbornene moiety.
  • the plurality of first functional groups comprise -O- NH2, unsubstituted or substituted alkynyl, unsubstituted or substituted C5-C16 cycloalkynyl, unsubstituted or substituted 5 to 16 membered heterocycloalkynyl, unsubstituted or substituted C5-C16 cycloalkenyl, unsubstituted or substituted 5 to 16 membered heterocycloalkenyl, substituted vinyl, isonitrile, substituted boronic acid moiety, substituted .
  • the unsubstituted or substituted polyacrylamide spacer unit has the structure of formula (II): ), wherein R 2 is H or C1-C4 alkyl; and each R 3a and R 3b is independently H, unsubs uted C 1 -C 6 alkyl.
  • each R 2 , R 3a and R 3b is H.
  • R 2 and R 3a are H, and R 3 is C1-C6 alkyl substituted with one or more substituents selected from a phosphate, -C(O)OH, -C(O)O ⁇ , -SO3H or -SO3 ⁇ .
  • R 3b embodiments, R 2 is H, and each R 3a and R 3b is unsubstituted or R 3a and R 3b are methyl).
  • the one or more second functional groups are capable of reacting with the biomolecule via an orthogonal or biorthogonal reaction.
  • the orthogonal or biorthogonal reaction is selected from the group consisting of a [3+2] dipolar cycloaddition, a Diels-Alder cycloaddition, a [4+1] cycloaddition, a phosphine ligation, or condensation with 2-acylphenyl boronic acid.
  • the substituted tetrazine is a phenyl tetrazine, pyrimidyl tetrazine, methyl tetrazine, pyridyl tetrazine, t-butyl tetrazine, which are further described below.
  • the second functional groups are substituted tetrazine.
  • one of the one or more second functional groups and the biomolecule comprises or is selected from alkynyl, unsubstituted or substituted DBCO moiety, unsubstituted or substituted BCN moiety, unsubstituted or substituted norbornene moiety, unsubstituted or substituted transcyclooctene (TCO) moiety, primary isonitrile (e.g. ), tertiary isonitrile (e.g. ), vinyl boronic acid ( ), 2-acylphenyl boronic acid ( ), phosphine amine (e.g. ), or phosphine thiol (e.g.
  • the other of the one or more second functional groups and the biomolecule comprises or is selected from azido ( , wherein the phenyl ring may be optionally substituted), pyrimidyl tetrazine wherein the pyrimidyl ring is o ptionally substituted), methyl ), pyridyl tetrazine or where the pyridyl ring is optionally substituted), t-butyl ) , triazine, cyclopropenone ( ), cyclopropenium ), DTO ( I n or is selected from norbornene, TCO, DBCO, BCN, or optionally substituted triphenylphosphine, and the other one of the one or more second functional groups and the biomolecule comprises or is azido.
  • the one or more second functional groups comprises or is norbornene, TCO, DBCO, or BCN, and the biomolecule comprises or is azido.
  • the first functional groups comprise or is azido
  • the first reactive moiety comprises or is selected from norbornene, TCO, DBCO, or BCN moiety.
  • one of the one or more second functional groups and the biomolecule comprises or is isonitrile, and the other of the one or more second functional groups and the biomolecule comprises or is a substituted tetrazine.
  • one of the one or more second functional groups and the biomolecule comprises or is TCO, and the other one of the one or more second functional groups and the biomolecule comprises or is a substituted tetrazine described herein.
  • the one or more second functional groups and the biomolecule comprises or is an amino hydrazine moiety, and the other the one or more second functional groups and the biomolecule comprises or is a 2-acylphenyl boronic acid moiety.
  • the one or more second functional groups of the polymer scaffold are present in one or more repeating units of formula (III): wherein alkyl; alkylene or 2 to 12 membered heteroalkylene containing one to four heteroatoms selected from O, N and S; and R 5 is a substituted 1,2,4,5-tetrazine moiety.
  • L is ethylene, and R 5 is .
  • the one or more biomolecule comprises tetrazine.
  • the polymer scaffold comprises one or more thiol containing ROS quencher moieties.
  • Certain embodiments of the present disclosure relate to a labeling reagent comprising a plurality of detectable labels covalently attached to the water soluble polymer scaffold in accordance with the present disclosure, through the plurality of first functional groups of the water soluble polymer scaffold.
  • the labeling reagent is covalently attached to a biomolecule through the one or more second functional groups of the water soluble polymer scaffold.
  • the biomolecule is a nucleotide, oligonucleotide or polynucleotide.
  • the nucleotide, oligonucleotide or polynucleotide is immobilized on a solid support.
  • the solid support comprises an array of immobilized oligonucleotides or polynucleotides.
  • Certain embodiments of the present disclosure relate to a method of determining the sequences of a plurality of different target polynucleotides, comprising: (a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b) contacting the solid support with an incorporation mixture comprising DNA polymerase and one more of four different types of nucleotides under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides; wherein each of the four types of nucleotides comprises a 3′ blocking group; and the incorporation mixture comprises a first type of unlabeled nucleotide having a first reactive moiety covalently attached to the first type of unlabeled nucleotide; (
  • the first reactive moiety of the first type of unlabeled nucleotides comprises unsubstituted or substituted TCO moiety and wherein the one or more second functional groups of the first labeling reagent comprises unsubstituted or substituted 1,2,4,5-tetrazine moiety.
  • the first reactive moiety is covalently attached to the nucleobase of the first type of unlabeled nucleotide via a cleavable linker.
  • the incorporation mixture comprises a second type of labeled nucleotide, and a third type of labeled nucleotide.
  • the incorporation mixture comprises a second type of unlabeled nucleotide having a second reactive moiety covalently attached to the second type of unlabeled nucleotide, and a third type of labeled nucleotide.
  • the incorporation mixture comprises a second type of unlabeled nucleotide having a second reactive moiety covalently attached to the second type of unlabeled nucleotide, and a mixture of a third type of unlabeled nucleotide having a first reactive moiety covalently attached to the third type of unlabeled nucleotide and a third type of unlabeled nucleotide having a second reactive moiety covalently attached to the third type of unlabeled nucleotide.
  • step (c) further comprises contacting the extended copy polynucleotides with a second labeling reagent comprising one or more second detectable labels and the second labeling reagent is configured to reacts specifically with the second reactive moiety to form covalent bonding.
  • the incorporation mixture comprises a second type of unlabeled nucleotide having a second reactive moiety covalently attached to the second type of unlabeled nucleotide, and a third type of unlabeled nucleotide having a third reactive moiety covalently attached to the third type of unlabeled nucleotide.
  • step (c) further comprises contacting the extended copy polynucleotides with a second labeling reagent comprising one or more second detectable labels and the second labeling reagent is configured to react specifically with the second reactive moiety of the second type of unlabeled nucleotides to form covalent bonding, and a third labeling reagent comprising one or more third detectable labels and the third labeling reagent is configured to react specifically with the third reactive moiety of the third type of unlabeled nucleotides to form covalent bonding.
  • the incorporation mixture comprises a fourth type of unlabeled nucleotide, wherein the fourth type of unlabeled nucleotide is not capable of reacting with any of the labeling reagents.
  • step (e) also removes the detectable labels of the incorporated nucleotides.
  • the detectable labels and the 3 ⁇ blocking groups of the incorporated nucleotides are removed in a single chemical reaction.
  • the method comprises (f) washing the solid support with an aqueous wash solution.
  • steps (b) to (f) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 cycles to determine the target polynucleotide sequences.
  • the four types of nucleotides comprise dATP, dCTP, dGTP and dTTP or dUTP, or non-natural nucleotide analogs thereof.
  • kits [0096] Certain embodiments of the present disclosure relate to a kit for sequencing application, comprising: an incorporation mixture composition comprising one or more of four different types of nucleotides each comprising a 3′ blocking group, wherein a first type of unlabeled nucleotide having a first reactive moiety covalently attached to the first type of unlabeled nucleotide; and a first labeling reagent in accordance with the present disclosure, comprising a plurality of first detectable labels, wherein the one or more second functional groups of the first labeling reagent is capable of reacting specifically with the first reactive moiety of the first type of unlabeled nucleotides to form covalent bonding.
  • one of the second functional group and the first reactive moiety comprises unsubstituted or substituted transcyclooctene (TCO), and the other one of the second functional group and the first reactive moiety comprises an unsubstituted or substituted tetrazine moiety.
  • the first reactive moiety of the first type of unlabeled nucleotides comprises TCOand the one or more second functional groups of the first labeling reagent comprises a substituted 1,2,4,5-tetrazine moiety.
  • the kit comprises a second type of labeled nucleotide, and a third type of labeled nucleotide.
  • the kit comprises a second type of unlabeled nucleotide having a second reactive moiety covalently attached to the second type of unlabeled nucleotide, and a third type of labeled nucleotide.
  • the kit comprises a second type of unlabeled nucleotide having a second reactive moiety covalently attached to the second type of unlabeled nucleotide, and a mixture of a third type of unlabeled nucleotide having a first reactive moiety covalently attached to the third type of unlabeled nucleotide and a third type of unlabeled nucleotide having a second reactive moiety covalently attached to the third type of unlabeled nucleotide.
  • the kit further comprises a second labeling reagent comprising one or more second detectable labels and the second labeling reagent is capable of reacting specifically with the second reactive moiety to form covalent bonding.
  • the kit comprises a second type of unlabeled nucleotide having a second reactive moiety covalently attached to the second type of unlabeled nucleotide, and a third type of unlabeled nucleotide having a third reactive moiety covalently attached to the third type of unlabeled nucleotide.
  • the kit further comprises a second labeling reagent comprising one or more second detectable labels and the second labeling reagent is capable of reacting specifically with the second reactive moiety of the second type of unlabeled nucleotide to form covalent bonding, and a third labeling reagent comprising one or more third detectable labels and the third labeling reagent is capable of reacting specifically with the third reactive moiety of the third type of unlabeled nucleotide to form covalent bonding.
  • the kit comprises a fourth type of unlabeled nucleotide, wherein the fourth type of unlabeled nucleotide is not capable of reacting with any labeling reagents.
  • the incorporation mixture composition further comprises a DNA polymerase.
  • the four different types of nucleotides are distinguishable using a single light source.
  • the four different types of nucleotides are distinguishable using two light sources operating at two different wavelengths.
  • one light source has a wavelength of about 450 nm to about 460 nm, and the other light source has a wavelength of about 520 nm to about 540 nm.
  • Fluorescent Dyes Various fluorescent dyes may be used in the present disclosure as detectable labels for the post incorporation labeling reagents described herein, in particularly those dyes that may be excitation by a blue light or a green light. These dyes may also be referred to as “blue dyes” and “green dyes” respectively. Examples of various type of blue dyes, including but not limited to coumarin dyes, chromenoquinoline dyes, naphthalimide dyes, and bisboron containing heterocycles are disclosed in U.S.
  • Non- limiting examples of the blue dyes include: , . [0102] Examples of green dyes including cyanine or polymethine dyes disclosed in International Publication Nos. WO2013/041117, WO2014/135221, WO2016/189287, WO2017/051201 and WO2018/060482A1, each of which is incorporated by reference in its entirety.
  • Non-limiting examples of the green dyes include: , , and salts, mesomeric forms, and optionally substituted [0103]
  • the fluorescent dyes described herein may be further modified to introduce one or more substituents (such as -SO3H, -OH, -C(O)OH, -C(O)OR, where R is unsubstituted or substituted C 1 -C 6 alkyl) to improve the hydrophilicity of the dyes while maintaining the signal intensity of the dye.
  • coumarin dye A may be further modified to improve the hydrophilicity of the compound a salt thereof (where -SO3H is in i onized 3 ⁇ Blocking Groups [0104]
  • the nucleotide described herein may also have a 3 ⁇ blocking group covalently attached to the deoxyribose sugar of the nucleotide.
  • 3 ⁇ blocking group are disclosed in WO2002/029003, WO2004/018497 and WO2014/139596.
  • the blocking group may be azidomethyl (-CH2N3) or substituted azidomethyl (e.g., -CH(CHF2)N3 or CH(CH2F)N3), or allyl, each connecting to the 3 ⁇ -oxygen atom of the deoxyribose moiety.
  • the 3 ⁇ blocking group is azidomethyl, forming 3 ⁇ -OCH2N3 with the 3 ⁇ carbon of the ribose or deoxyribose.
  • the 3 ⁇ hydroxy protecting group such as azidomethyl may be removed or deprotected by using a water-soluble phosphine reagent to generate a free 3 ⁇ - OH.
  • Non-limiting examples include tris(hydroxymethyl)phosphine (THMP), tris(hydroxyethyl)phosphine (THEP) or tris(hydroxypropyl)phosphine (THP or THPP).
  • 3 ⁇ -acetal blocking groups described herein may be removed or cleaved under various chemical conditions.
  • non-limiting cleaving condition includes a Pd(II) complex, such as Pd(OAc)2 or allylPd(II) chloride dimer, in the presence of a phosphine ligand, for example tris(hydroxymethyl)phosphine (THMP), or tris(hydroxypropyl)phosphine (THP or THPP).
  • a Pd(II) complex such as Pd(OAc)2 or allylPd(II) chloride dimer
  • a phosphine ligand for example tris(hydroxymethyl)phosphine (THMP), or tris(hydroxypropyl)phosphine (THP or THPP).
  • blocking groups containing an alkynyl group may also be removed by a Pd(II) complex (e.g., Pd(OAc)2 or allyl Pd(II) chloride dimer) in the presence of a phosphine ligand (e.g., THP or THMP).
  • Pd(II) complex e.g., Pd(OAc)2 or allyl Pd(II) chloride dimer
  • a phosphine ligand e.g., THP or THMP
  • Palladium Cleavage Reagents [0107]
  • the 3 ⁇ blocking group such as allyl or AOM as described herein may be cleaved by a palladium catalyst.
  • the Pd catalyst is water soluble.
  • a Pd(0) complex e.g., Tris(3,3′,3′′- phosphinidynetris(benzenesulfonato)palladium(0) nonasodium salt nonahydrate.
  • the Pd(0) complex may be generated in situ from reduction of a Pd(II) complex by reagents such as alkenes, alcohols, amines, phosphines, or metal hydrides.
  • Suitable palladium sources include Na 2 PdCl 4 , Li 2 PdCl 4 , Pd(CH 3 CN) 2 Cl 2, (PdCl(C 3 H 5 )) 2 , [Pd(C 3 H 5 )(THP)]Cl, [Pd(C3H5)(THP)2]Cl, Pd(OAc)2, Pd(Ph3)4, Pd(dba)2, Pd(Acac)2, PdCl2(COD), Pd(TFA)2, Na 2 PdBr 4 , K 2 PdBr 4 , PdCl 2 , PdBr 2 , and Pd(NO 3 ) 2 .
  • the Pd(0) complex is generated in situ from Na2PdCl4 or K2PdCl4.
  • the palladium source is allyl palladium(II) chloride dimer [(PdCl(C 3 H 5 )) 2 ].
  • the Pd(0) complex is generated in an aqueous solution by mixing a Pd(II) complex with a phosphine.
  • Suitable phosphines include water soluble phosphines, such as tris(hydroxypropyl)phosphine (THP), tris(hydroxymethyl)phosphine (THMP), 1,3,5-triaza-7-phosphaadamantane (PTA), bis(p- sulfonatophenyl)phenylphosphine dihydrate potassium salt, tris(carboxyethyl)phosphine (TCEP), and triphenylphosphine-3,3 ⁇ ,3 ⁇ ⁇ -trisulfonic acid trisodium salt.
  • the palladium catalyst is prepared by mixing [(Allyl)PdCl]2 with THP in situ.
  • the molar ratio of [(Allyl)PdCl]2 and the THP may be about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10.
  • the molar ratio of [(Allyl)PdCl]2 to THP is 1:10.
  • the palladium catalyst is prepared by mixing a water soluble Pd reagent such as Na2PdCl4 or K2PdCl4 with THP in situ.
  • the molar ratio of Na2PdCl4 or K2PdCl4 and THP may be about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10.
  • the molar ratio of Na 2 PdCl 4 or K 2 PdCl 4 to THP is about 1:3.
  • the molar ratio of Na2PdCl4 or K2PdCl4 to THP is about 1:3.5.
  • the molar ratio of Na 2 PdCl 4 or K 2 PdCl 4 to THP is about 1:2.5.
  • one or more reducing agents may be added, such as ascorbic acid or a salt thereof (e.g., sodium ascorbate).
  • the cleavage mixture may contain additional buffer reagents, such as a primary amine, a secondary amine, a tertiary amine, a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof.
  • the buffer reagent comprises ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylethanolamine (DMEA), 2- diethylethanolamine (DEEA), N,N,N′,N′-tetramethylethylenediamine(TEMED), or N,N,N′,N′- tetraethylethylenediamine (TEEDA), or 2-piperidine ethanol (also known as (2- hydroxyethyl)piperidine, having the ), or combinations thereof.
  • the buffer reagent comprises embodiment, the buffer reagent comprises or is (2-hydroxyethyl)piperidine.
  • the buffer reagent contains one or more inorganic salts such as a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof.
  • the inorganic salt is a sodium salt.
  • Palladium (Pd) Scavengers [0109] Pd has the capacity to stick on DNA, mostly in its inactive Pd(II) form, which may interfere with the binding between DNA and polymerase, causing increased phasing.
  • a post- cleavage wash composition that includes a Pd scavenger compound may be used following the deblocking step. For example, PCT Publication No.
  • Pd scavengers such as 3,3’-dithiodipropionic acid (DDPA) and lipoic acid (LA) may be included in the scan composition and/or the post-cleavage wash composition.
  • DDPA 3,3’-dithiodipropionic acid
  • LA lipoic acid
  • the use of these scavengers in the post- cleave washing solution has the purpose of scavenging Pd(0), converting Pd(0) to the inactive Pd(II) form, thereby improving the prephasing value and sequencing metrics, reducing signal degrade, and extend sequencing read length.
  • Pd scavengers include both Pd(0) and Pd(II) scavengers, which are described in details in U.S. Publication No.
  • the first/second/third reactive moiety of the nucleotide described herein is covalently attached to the nucleobase of the nucleotide via a cleavable linker.
  • cleavable linker is not meant to imply that the whole linker is required to be removed.
  • the cleavage site can be located at a position on the linker that ensures that part of the linker remains attached to the dye and/or substrate moiety after cleavage.
  • Cleavable linkers may be, by way of non-limiting example, electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, cleavable under reductive conditions (for example disulfide or azide containing linkers), oxidative conditions, cleavable via use of safety-catch linkers and cleavable by elimination mechanisms.
  • the use of a cleavable linker to attach the dye compound to a substrate moiety ensures that the label can, if required, be removed after detection, avoiding any interfering signal in downstream steps.
  • WO2004/018493 examples of which include linkers that may be cleaved using water-soluble phosphines or water-soluble transition metal catalysts formed from a transition metal and at least partially water-soluble ligands. In aqueous solution the latter form at least partially water-soluble transition metal complexes.
  • Such cleavable linkers can be used to connect bases of nucleotides to labels such as the dyes set forth herein.
  • Particular linkers include those disclosed in PCT Publication No.
  • WO2004/018493 such as those that include moieties of the formulae: (wherein Q is a C1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C1-C10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).
  • the linkers connect the bases of nucleotides to labels such as, for example, the dye compounds described herein. [0113] Additional examples of linkers include those disclosed in U.S. Publication No.
  • linker moieties illustrated herein may comprise the whole or partial linker structure between the nucleotides/nucleosides and the labels.
  • Additional examples of linkers are disclosed in U.S. Publication No. 2020/0216891 A1, which is incorporated by reference in its entirety: O O R , or herein B is a nucleobase; n is 1, nyl, or –O-C2-C6 alkynyl; and R comprises the first, the second, or the third reactive moiety described herein, which may contain additional linker and/or spacer structure.
  • the first, the second, or the third reactive moiety described herein is covalently bound to the labeling reagent described herein by reacting with the one or more second functional groups of the water soluble polymer scaffold.
  • the cleavable linker comprises (“AOL” linker moiety) where Z is –O-allyl.
  • the nucleotide may contain multiple cleavable linkers repeating units (e.g., k is 1, 2, 3, 45, 6, 7, 8, 9 or 10).
  • the first, second or third reactive moiety may be attached to any position on the nucleotide base, for example, through a linker.
  • Watson-Crick base pairing can still be carried out for the resulting analog.
  • Particular nucleobase labeling sites include the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base.
  • a linker group may be used to covalently attach a dye to the nucleotide.
  • the unlabeled nucleotide may be enzymatically incorporable and enzymatically extendable. Accordingly, a linker moiety may be of sufficient length to connect the nucleotide to the compound such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by a nucleic acid replication enzyme.
  • the linker can also comprise a spacer unit, such as one or more PEG unit(s) (- OCH2CH2-)n, where n is an integer of 1-20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • the spacer distances for example, the nucleotide base from a cleavage site or label.
  • a unlabeled nucleotides described herein may have the formula: [0118] where R is the first, moiety described herein; B is a nucleobase, such as, for example uracil, adenine, 7-deaza adenine, guanine, 7- deaza guanine, and the like; L is a linker; -OR' is monophosphate, diphosphate, triphosphate, thiophosphate, a phosphate ester analog, –O– attached to a reactive phosphorous containing group, or –O– protected by a blocking group; R'' is H or OH; and R''' is H, a 3' hydroxy blocking group described herein, or -OR''' forms a phosphoramidite.
  • R is the first, moiety described herein
  • B is a nucleobase, such as, for example uracil, adenine, 7-deaza adenine, guanine, 7- deaza
  • R' is an acid-cleavable hydroxy protecting group which allows subsequent monomer coupling under the .
  • the block can be due to steric hindrance or can be due to a combination of size, charge and structure, whether or not the dye is attached to the 3 ⁇ position of the sugar.
  • the use of a blocking group allows polymerization to be controlled, such as by stopping extension when an unlabeled nucleotide is incorporated.
  • the linker and blocking group are both present and are separate moieties.
  • the linker and blocking group are both cleavable under the same or substantially similar conditions.
  • deprotection and deblocking processes may be more efficient because only a single treatment will be required to remove both the dye compound and the blocking group.
  • a linker and blocking group need not be cleavable under similar conditions, instead being individually cleavable under distinct conditions.
  • Non-limiting exemplary unlabeled nucleotides as described herein include: ribose or deoxyribose moiety as described above, or a ribose or deoxyribose moiety with the 5 ⁇ position substituted with mono-, di- or tri- phosphates; R represents the first, second or third reactive moiety described herein.
  • non-limiting exemplary unlabeled nucleotide containing a reactive moiety covalently attached via a cleavable linker are shown below: , ,
  • PG stands , 2, 3, 4, 5, 6, 7, 8, 9, or 10; and m is 0, 1, 2, 3, 4, or 5.
  • –O–PG is AOM.
  • –O–PG is –O–azidomethyl.
  • m is 5.
  • m is 0.
  • m is 2.
  • p is 1, 2, 3, 4 or 5. refers to the connection point of the first/second/third reactive moiety with the cleavable linker as a result of a reaction between an amino group of the linker moiety and the carboxyl group of the first/second/third reactive moiety.
  • the nucleotide may be attached to the reactive moiety via more than one of the same cleavable linkers (such as LN3-LN3, sPA-sPA, AOL-AOL).
  • the reactive moiety may be attached to the nucleotidevia two or more different cleavable linkers (such sPA-LN3, sPA-sPA-LN3, sPA-LN3-LN3, etc.).
  • the linker may further include additional PEG spacers as described herein, for example, between R and –(CH2) m -.
  • the nucleotide is a nucleotide triphosphate.
  • the nucleotide has a 2 ⁇ deoxyribose.
  • a synthetic step is carried out and may optionally comprise incubating a template or target polynucleotide strand with a reaction mixture comprising fluorescently labeled nucleotides of the disclosure.
  • a polymerase can also be provided under conditions which permit formation of a phosphodiester linkage between a free 3' hydroxy group on a polynucleotide strand annealed to the template or target polynucleotide strand and a 5' phosphate group on the labeled nucleotide.
  • a synthetic step can include formation of a polynucleotide strand as directed by complementary base pairing of nucleotides to a template/target strand.
  • the detection step may be carried out while the polynucleotide strand into which the labeled nucleotides are incorporated is annealed to a template/target strand, or after a denaturation step in which the two strands are separated. Further steps, for example chemical or enzymatic reaction steps or purification steps, may be included between the synthetic step and the detection step.
  • polynucleotide strand incorporating the labeled nucleotide(s) may be isolated or purified and then processed further or used in a subsequent analysis.
  • polynucleotide strand incorporating the labeled nucleotide(s) as described herein in a synthetic step may be subsequently used as labeled probes or primers.
  • the product of the synthetic step set forth herein may be subject to further reaction steps and, if desired, the product of these subsequent steps purified or isolated.
  • Suitable conditions for the synthetic step will be well known to those familiar with standard molecular biology techniques.
  • a synthetic step may be analogous to a standard primer extension reaction using nucleotide precursors, including the labeled nucleotides as described herein, to form an extended polynucleotide strand (primer polynucleotide strand) complementary to the template/target strand in the presence of a suitable polymerase enzyme.
  • the synthetic step may itself form part of an amplification reaction producing a labeled double stranded amplification product comprised of annealed complementary strands derived from copying of the primer and template polynucleotide strands.
  • Other exemplary synthetic steps include nick translation, strand displacement polymerization, random primed DNA labeling, etc.
  • a particularly useful polymerase enzyme for a synthetic step is one that is capable of catalyzing the incorporation of the labeled nucleotides as set forth herein.
  • a variety of naturally occurring or mutant/modified polymerases can be used.
  • a thermostable polymerase can be used for a synthetic reaction that is carried out using thermocycling conditions, whereas a thermostable polymerase may not be desired for isothermal primer extension reactions.
  • Suitable thermostable polymerases which are capable of incorporating the labeled nucleotides according to the disclosure include those described in WO 2005/024010 or WO06120433, each of which is incorporated herein by reference.
  • polymerase enzymes need not necessarily be thermostable polymerases, therefore the choice of polymerase will depend on a number of factors such as reaction temperature, pH, strand-displacing activity and the like.
  • the disclosure encompasses methods of nucleic acid sequencing, re-sequencing, whole genome sequencing, single nucleotide polymorphism scoring, any other application involving the detection of the modified nucleotide or nucleoside labeled with dyes set forth herein when incorporated into a polynucleotide.
  • SBS generally involves sequential addition of one or more nucleotides or oligonucleotides to a growing polynucleotide chain in the 5' to 3' direction using a polymerase or ligase in order to form an extended polynucleotide chain complementary to the template/target nucleic acid to be sequenced.
  • the identity of the base present in one or more of the added nucleotide(s) can be determined in a detection or “imaging” step.
  • the identity of the added base may be determined after each nucleotide incorporation step.
  • the sequence of the template may then be inferred using conventional Watson-Crick base-pairing rules.
  • the sequence of a template/target polynucleotide is determined by detecting the incorporation of one or more nucleotides into a nascent strand complementary to the template polynucleotide to be sequenced through the detection of fluorescent label(s) attached to the incorporated nucleotide(s).
  • Sequencing of the template polynucleotide can be primed with a suitable primer (or prepared as a hairpin construct which will contain the primer as part of the hairpin), and the nascent chain is extended in a stepwise manner by addition of nucleotides to the 3' end of the primer in a polymerase-catalyzed reaction.
  • each of the different nucleotide triphosphates may be labeled with a unique fluorophore and also comprises a blocking group at the 3' position to prevent uncontrolled polymerization.
  • one of the four nucleotides may be unlabeled (dark).
  • the polymerase enzyme incorporates a nucleotide into the nascent chain complementary to the template/target polynucleotide, and the blocking group prevents further incorporation of nucleotides. Any unincorporated nucleotides can be washed away and the fluorescent signal from each incorporated nucleotide can be “read” optically by suitable means, such as a charge-coupled device using light source excitation and suitable emission filters. The 3'- blocking group and fluorescent dye compounds can then be removed (deprotected) (simultaneously or sequentially) to expose the nascent chain for further nucleotide incorporation.
  • the method utilizes the incorporation of fluorescently labeled, 3' blocked nucleotides A, G, C, and T into a growing strand complementary to the immobilized polynucleotide, in the presence of DNA polymerase.
  • the polymerase incorporates a base complementary to the target polynucleotide but is prevented from further addition by the 3' blocking group.
  • the label of the incorporated nucleotide can then be determined, and the blocking group removed by chemical cleavage to allow further polymerization to occur.
  • the nucleic acid template to be sequenced in a SBS reaction may be any polynucleotide that it is desired to sequence.
  • the nucleic acid template for a sequencing reaction will typically comprise a double stranded region having a free 3' hydroxy group that serves as a primer or initiation point for the addition of further nucleotides in the sequencing reaction.
  • the region of the template to be sequenced will overhang this free 3' hydroxy group on the complementary strand.
  • the overhanging region of the template to be sequenced may be single stranded but can be double- stranded, provided that a “nick” is present on the strand complementary to the template strand to be sequenced to provide a free 3' OH group for initiation of the sequencing reaction. In such embodiments, sequencing may proceed by strand displacement.
  • a primer bearing the free 3' hydroxy group may be added as a separate component (e.g., a short oligonucleotide) that hybridizes to a single-stranded region of the template to be sequenced.
  • the primer and the template strand to be sequenced may each form part of a partially self-complementary nucleic acid strand capable of forming an intra-molecular duplex, such as for example a hairpin loop structure.
  • Hairpin polynucleotides and methods by which they may be attached to solid supports are disclosed in PCT Publication Nos. WO0157248 and WO2005/047301, each of which is incorporated herein by reference.
  • Nucleotides can be added successively to a growing primer, resulting in synthesis of a polynucleotide chain in the 5' to 3' direction.
  • the nature of the base which has been added may be determined, particularly but not necessarily after each nucleotide addition, thus providing sequence information for the nucleic acid template.
  • a nucleotide is incorporated into a nucleic acid strand (or polynucleotide) by joining of the nucleotide to the free 3' hydroxy group of the nucleic acid strand via formation of a phosphodiester linkage with the 5' phosphate group of the nucleotide.
  • the nucleic acid template to be sequenced may be DNA or RNA, or even a hybrid molecule comprised of deoxynucleotides and ribonucleotides.
  • the nucleic acid template may comprise naturally occurring and/or non-naturally occurring nucleotides and natural or non- natural backbone linkages, provided that these do not prevent copying of the template in the sequencing reaction.
  • the nucleic acid template to be sequenced may be attached to a solid support via any suitable linkage method known in the art, for example via covalent attachment.
  • template polynucleotides may be attached directly to a solid support (e.g., a silica-based support).
  • the surface of the solid support may be modified in some way so as to allow either direct covalent attachment of template polynucleotides, or to immobilize the template polynucleotides through a hydrogel or polyelectrolyte multilayer, which may itself be non-covalently attached to the solid support.
  • Arrays in which polynucleotides have been directly attached to a support for example, silica-based supports such as those disclosed in WO00/06770 (incorporated herein by reference), wherein polynucleotides are immobilized on a glass support by reaction between a pendant epoxide group on the glass with an internal amino group on the polynucleotide.
  • polynucleotides can be attached to a solid support by reaction of a sulfur-based nucleophile with the solid support, for example, as described in W02005/047301 (incorporated herein by reference).
  • a still further example of solid-supported template polynucleotides is where the template polynucleotides are attached to hydrogel supported upon silica-based or other solid supports, for example, as described in WO00/31148, WO01/01143, WO02/12566, WO03/014392, U.S. Pat. No. 6,465,178 and WO00/53812, each of which is incorporated herein by reference.
  • a particular surface to which template polynucleotides may be immobilized is a polyacrylamide hydrogel.
  • Polyacrylamide hydrogels are described in the references cited above and in WO2005/065814, which is incorporated herein by reference. Specific hydrogels that may be used include those described in WO2005/065814 and U.S. Pub. No. 2014/0079923.
  • the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl) acrylamide-co- acrylamide)).
  • DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Pat. No. 6,172,218 (which is incorporated herein by reference).
  • Bead libraries can be prepared where each bead contains different DNA sequences. Exemplary libraries and methods for their creation are described in Nature, 437, 376-380 (2005); Science, 309, 5741, 1728- 1732 (2005), each of which is incorporated herein by reference. Sequencing of arrays of such beads using nucleotides set forth herein is within the scope of the disclosure. [0137] Template(s) that are to be sequenced may form part of an “array” on a solid support, in which case the array may take any convenient form. Thus, the method of the disclosure is applicable to all types of high-density arrays, including single-molecule arrays, clustered arrays, and bead arrays.
  • Nucleotides labeled with dye compounds of the present disclosure may be used for sequencing templates on essentially any type of array, including but not limited to those formed by immobilization of nucleic acid molecules on a solid support.
  • nucleotides labeled with dye compounds of the disclosure are particularly advantageous in the context of sequencing of clustered arrays.
  • clustered arrays distinct regions on the array (often referred to as sites, or features) comprise multiple polynucleotide template molecules.
  • the multiple polynucleotide molecules are not individually resolvable by optical means and are instead detected as an ensemble.
  • each site on the array may comprise multiple copies of one individual polynucleotide molecule (e.g., the site is homogenous for a particular single- or double-stranded nucleic acid species) or even multiple copies of a small number of different polynucleotide molecules (e.g., multiple copies of two different nucleic acid species).
  • Clustered arrays of nucleic acid molecules may be produced using techniques generally known in the art.
  • WO 98/44151 and WO00/18957 describe methods of amplification of nucleic acids wherein both the template and amplification products remain immobilized on a solid support in order to form arrays comprised of clusters or “colonies” of immobilized nucleic acid molecules.
  • the nucleic acid molecules present on the clustered arrays prepared according to these methods are suitable templates for sequencing using nucleotides labeled with dye compounds of the disclosure.
  • Nucleotides labeled with dye compounds of the present disclosure are also useful in sequencing of templates on single molecule arrays.
  • single molecule array refers to a population of polynucleotide molecules, distributed (or arrayed) over a solid support, wherein the spacing of any individual polynucleotide from all others of the population is such that it is possible to individually resolve the individual polynucleotide molecules.
  • the target nucleic acid molecules immobilized onto the surface of the solid support can thus be capable of being resolved by optical means in some embodiments. This means that one or more distinct signals, each representing one polynucleotide, will occur within the resolvable area of the particular imaging device used.
  • Single molecule detection may be achieved wherein the spacing between adjacent polynucleotide molecules on an array is at least 100 nm, more particularly at least 250 nm, still more particularly at least 300 nm, even more particularly at least 350 nm.
  • each molecule is individually resolvable and detectable as a single molecule fluorescent point, and fluorescence from said single molecule fluorescent point also exhibits single step photobleaching.
  • the terms “individually resolved” and “individual resolution” are used herein to specify that, when visualized, it is possible to distinguish one molecule on the array from its neighboring molecules. Separation between individual molecules on the array will be determined, in part, by the particular technique used to resolve the individual molecules.
  • nucleotides labeled with dye compounds of the disclosure may be used in automated fluorescent sequencing protocols, particularly fluorescent dye-terminator cycle sequencing based on the chain termination sequencing method of Sanger and co-workers.
  • Dye-labelled dried scaffolds were resuspended in 3 mL DPBS.
  • DMTMM (10x equiv. COOH sites, in 1 mL DPBS) was added and the mixture shaken for 10 mins at room temperature .
  • Tz-NH2 (10x equiv. COOH sites, in 1 mL DPBS) was added and the reaction carried out with shaking for 5 hrs. Tangential flow filtration (TFF) was used to purify the final product poly(acrylamide)-based scaffold A.
  • DP50 degree of polymerization of 50
  • Poly(acrylamide)-based scaffolds were synthesized having 2 dyes, 5 dyes, or 10 dyes per scaffold.
  • a solution brightness assay was developed to quantify the effects of brightness multiplication afforded by utilizing multiple fluorophores on a single nucleotide label.
  • a UV-vis absorption spectrum was used to quantify the concentration of equivalent individual fluorophore units in a sample.
  • a fluorescence spectrum was used to quantify the brightness afforded by the sample.
  • FIG. 2A plots fluorescence intensity maxima of each poly(acrylamide)-based scaffold as a function of UV absorbance maxima in universal scan mixture in the solution brightness assay described herein. Tracking across to the y axis from the data point demonstrates the experimental brightness per scaffolded dye.
  • FIG.2B plots brightness per dye moiety and brightness per scaffold relative to brightness of a single dye molecule (DBCO-NR550C4).
  • DBCO-NR550C4 single dye molecule
  • the 5-dye DP50 scaffold was about 4 times brighter than a single DBCO-NR550C4 molecule.
  • brightness per dye moiety was 0.61 of DBCO-NR550C4 brightness and fluorescence quenching was 39%.
  • the 10-dye scaffold was about 6 times brighter than a single DBCO-NR550C4 molecule.
  • Example 3 Sequencing by Synthesis using a Two-Dye Statistical Copolymer [0153] A water soluble polymer scaffold A1 was prepared (also referred to as “the statistical copolymer”) according to the description in Example 1, where the monomer units are randomly distributed.
  • the water soluble polymer scaffold A1 was a statistical copolymer having the structure: , including two , polyacrylamide spacer units, and second functional groups (e.g., the tetrazine handles) were distributed along the structure of the statistical copolymer A1.
  • the statistical copolymer was subjected to SBS alongside an unlabeled ffC including a TCO reactive moiety capable of binding to the tetrazine group of the statistical copolymer.
  • SBS runs were also conducted with standard ffN set (in which only G nucleotide is dark, also referred to as “Standard 1”) and a second ffN set having an unlabeled ffC having a reactive moiety capable of binding to a labeling reagent having a tetrazine moiety in a post-incorporation labeling SBS workflow (also referred to as “Standard 2”).
  • the sequencer used in this experiment is NextSeq 2000.
  • FIG.3A is a scatter plot of sequencing by synthesis (SBS) runs using standard SBS reagents with a set of nucleotides (dark G, ffC-DB-AOL-NR550S0 (a known green dye), ffT-DB-AOL-coumarin dye A, ffA-DB-AOL-BL-coumarin dye A, and ffA-AOL-AOL- NR550S0), and each of the nucleotides in the incorporation mix has a 3 ⁇ AOM blocking group.
  • the scatter plot was generated at cycle 26.
  • FIG.3B is a SBS scatter plot using a post incorporation labeling method with a set of nucleotides (dark G, unlabeled ffC with a TCO reactive moiety (ffC- AOL-AOL-TCO), ffT-DB-AOL-coumarin dye A, ffA-DB-AOL-BL-coumarin dye A, and ffA- AOL-AOL-NR550S0), and a labeling reagent with a tetrazine moiety (Tz-NR550C4) in 5 ⁇ M concentration.
  • Each of the nucleotides in the incorporation mix has a 3 ⁇ AOM blocking group.
  • the scatter plot was generated at cycle 31.
  • 3C is a SBS scatter plots using a post incorporation labeling method with the 2-dye labeled polymer scaffold A1 as a PIL labeling reagent and a set of nucleotides (dark G, unlabeled ffC with a TCO reactive moiety (ffC-AOL- AOL-TCO), ffT-DB-AOL-coumarin dye A, ffA-DB-AOL-BL-coumarin dye A, and ffA-AOL- AOL-NR550S0), and each of the nucleotides in the incorporation mix has a 3 ⁇ AOM blocking group.
  • the scatter plot was generated at cycle 36.
  • Example 4 Sequencing by Synthesis using a 5-Dye Diblock Copolymer
  • Another water soluble block copolymer scaffold was prepared (also referred to in the context of Example 4 as “the diblock copolymer”).
  • the water soluble polymer scaffold has two separate blocks with the structure:
  • nd including five spacer units were distributed randomly within the first block of the diblock copolymer.
  • the tetrazine moieties that are used as PIL handles i.e., second functional groups
  • the diblock copolymer was subjected to SBS alongside an unlabeled ffC including a TCO reactive moiety capable of binding to the tetrazine group of the diblock copolymer.
  • FIGs.4A–4C are the scatter plots resulting from the three SBS runs discussed above.
  • FIG. 4A–4C are the scatter plots resulting from the three SBS runs discussed above.
  • FIG. 4A was generated using Standard 1 at cycle 26.
  • FIG. 4B was generated using Standard 2 at cycle 30.
  • FIG. 4C was generated using the diblock copolymer as labeling reagent ⁇ ⁇ ⁇ at cycle 53.
  • the results show that signal corresponding to ffC can be differentiated using the diblock copolymer.
  • tetrazine units in a block Another benefit afforded by utilizing tetrazine units in a block is that there is likely to be significantly less chance of the multiple tetrazine sites cross-linking between different clusters. This is because of the decreased steric accessibility of other tetrazine units that are close by to one that has completed a successful PIL event. Tetrazine units in a block also mean that there is no linker space between units, providing a length restriction to binding multiple templates. This contrasts with the likely behavior of a polymer with statistically distributed tetrazine units, where the increased space between tetrazine units (facilitated by spacer monomers), twinned with increased steric accessibility, indicates that it could likely ligate multiple templates.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Des modes de réalisation de la présente divulgation concernent des procédés et des kits de séquençage par synthèse utilisant des réactions chimiques orthogonales ou biorthogonales entre des nucléotides non marqués et des réactifs de marquage post-incorporation marqués à plusieurs colorants contenant des échafaudages polymères solubles dans l'eau.
PCT/US2025/024973 2024-04-19 2025-04-16 Échafaudages polymères solubles dans l'eau pour marquage de colorant Pending WO2025221895A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463636485P 2024-04-19 2024-04-19
US63/636,485 2024-04-19

Publications (1)

Publication Number Publication Date
WO2025221895A1 true WO2025221895A1 (fr) 2025-10-23

Family

ID=95656397

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/024973 Pending WO2025221895A1 (fr) 2024-04-19 2025-04-16 Échafaudages polymères solubles dans l'eau pour marquage de colorant

Country Status (1)

Country Link
WO (1) WO2025221895A1 (fr)

Citations (71)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993017126A1 (fr) 1992-02-19 1993-09-02 The Public Health Research Institute Of The City Of New York, Inc. Nouvelles configurations d'oligonucleotides et utilisation de ces configurations pour le tri, l'isolement, le sequençage et la manipulation des acides nucleiques
WO1995011995A1 (fr) 1993-10-26 1995-05-04 Affymax Technologies N.V. Reseaux de sondes d'acide nucleique sur des microplaquettes biologiques
US5429807A (en) 1993-10-28 1995-07-04 Beckman Instruments, Inc. Method and apparatus for creating biopolymer arrays on a solid support surface
US5436327A (en) 1988-09-21 1995-07-25 Isis Innovation Limited Support-bound oligonucleotides
WO1995035505A1 (fr) 1994-06-17 1995-12-28 The Board Of Trustees Of The Leland Stanford Junior University Procede et appareil pour fabriquer des microensembles d'echantillons biologiques
US5561071A (en) 1989-07-24 1996-10-01 Hollenberg; Cornelis P. DNA and DNA technology for the construction of networks to be used in chip construction and chip production (DNA-chips)
EP0742287A2 (fr) 1995-05-10 1996-11-13 McGall, Glenn H. Sondes d'acide nucleique modifiés
US5583211A (en) 1992-10-29 1996-12-10 Beckman Instruments, Inc. Surface activated organic polymers useful for location - specific attachment of nucleic acids, peptides, proteins and oligosaccharides
US5658734A (en) 1995-10-17 1997-08-19 International Business Machines Corporation Process for synthesizing chemical compounds
US5661040A (en) * 1993-07-13 1997-08-26 Abbott Laboratories Fluorescent polymer labeled conjugates and intermediates
EP0799897A1 (fr) 1996-04-04 1997-10-08 Affymetrix, Inc. (a California Corporation) Méthodes et compositions pour sélectionner tag acides nucléiques et épreuves correspondantes
WO1998044151A1 (fr) 1997-04-01 1998-10-08 Glaxo Group Limited Methode d'amplification d'acide nucleique
US5837858A (en) 1993-10-22 1998-11-17 The Board Of Trustees Of The Leland Stanford Junior University Method for polymer synthesis using arrays
US5874219A (en) 1995-06-07 1999-02-23 Affymetrix, Inc. Methods for concurrently processing multiple biological chip assays
US5919523A (en) 1995-04-27 1999-07-06 Affymetrix, Inc. Derivatization of solid supports and methods for oligomer synthesis
WO2000006770A1 (fr) 1998-07-30 2000-02-10 Solexa Ltd. Biomolecules en rangees et leur utilisation dans une procedure de sequençage
WO2000018957A1 (fr) 1998-09-30 2000-04-06 Applied Research Systems Ars Holding N.V. Procedes d'amplification et de sequençage d'acide nucleique
WO2000031148A2 (fr) 1998-11-25 2000-06-02 Motorola, Inc. Hydrogels de polyacrylamide obtenus a partir de prepolymeres reactifs
WO2000053812A2 (fr) 1999-03-12 2000-09-14 President And Fellows Of Harvard College Amplification par replique de reseaux d'acides nucleiques
US6136269A (en) 1991-11-22 2000-10-24 Affymetrix, Inc. Combinatorial kit for polymer synthesis
WO2000063437A2 (fr) 1999-04-20 2000-10-26 Illumina, Inc. Detection de reactions d'acide nucleique sur microsupports de billes en reseau
WO2001001143A2 (fr) 1999-06-25 2001-01-04 Motorola Inc. Procedes et compositions permettant de fixer des biomolecules a des supports solides, a des hydrogels et a des matrices d'hydrogels
US6172218B1 (en) 1994-10-13 2001-01-09 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
WO2001057248A2 (fr) 2000-02-01 2001-08-09 Solexa Ltd. Groupes de polynucleotides et leur utilisation dans le sequencage
US6287776B1 (en) 1998-02-02 2001-09-11 Signature Bioscience, Inc. Method for detecting and classifying nucleic acid hybridization
US6288220B1 (en) 1998-03-05 2001-09-11 Hitachi, Ltd. DNA probe array
US6287768B1 (en) 1998-01-07 2001-09-11 Clontech Laboratories, Inc. Polymeric arrays and methods for their use in binding assays
US6291193B1 (en) 1998-06-16 2001-09-18 Millennium Pharmaceuticals, Inc. MTbx protein and nucleic acid molecules and uses therefor
US6297006B1 (en) 1997-01-16 2001-10-02 Hyseq, Inc. Methods for sequencing repetitive sequences and for determining the order of sequence subfragments
US6346413B1 (en) 1989-06-07 2002-02-12 Affymetrix, Inc. Polymer arrays
WO2002012566A2 (fr) 2000-08-09 2002-02-14 Motorola, Inc. Utilisation et evaluation d'une photocycloaddition [2+2] dans une immobilisation d'oligonucleotides sur une matrice hydrogel tridimensionnelle
US6355431B1 (en) 1999-04-20 2002-03-12 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays
WO2002029003A2 (fr) 2000-10-06 2002-04-11 The Trustees Of Columbia University In The City Of New York Methode d'analyse massivement parallele destinee a decoder l'adn et l'arn
US6416949B1 (en) 1991-09-18 2002-07-09 Affymax, Inc. Method of synthesizing diverse collections of oligomers
US20020094586A1 (en) * 2001-01-05 2002-07-18 Sensors For Medicine And Science, Inc. Detection of analytes
US20020102578A1 (en) 2000-02-10 2002-08-01 Todd Dickinson Alternative substrates and formats for bead-based array of arrays TM
US6465178B2 (en) 1997-09-30 2002-10-15 Surmodics, Inc. Target molecule attachment to surfaces
US6482591B2 (en) 1994-10-24 2002-11-19 Affymetrix, Inc. Conformationally-restricted peptide probe libraries
US6514751B2 (en) 1998-10-02 2003-02-04 Incyte Genomics, Inc. Linear microarrays
WO2003014392A2 (fr) 2001-08-09 2003-02-20 Amersham Biosciences Ab Utilisation et evaluation d'une photoaddition [2+2] dans l immobilisation d'oligonucleotides sur une matrice d'hydrogel tridimensionnelle
US6524793B1 (en) 1995-10-11 2003-02-25 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method
US6610482B1 (en) 1989-06-07 2003-08-26 Affymetrix, Inc. Support bound probes and methods of analysis using the same
WO2004018493A1 (fr) 2002-08-23 2004-03-04 Solexa Limited Nucleotides marques
WO2004018497A2 (fr) 2002-08-23 2004-03-04 Solexa Limited Nucleotides modifies
WO2005024010A1 (fr) 2003-09-11 2005-03-17 Solexa Limited Polymerases modifies pour l'incorporation amelioree d'analogues nucleotidiques
WO2005047301A1 (fr) 2003-11-07 2005-05-26 Solexa Limited Ameliorations apportees a des reseaux polynuceotidiques
WO2005065814A1 (fr) 2004-01-07 2005-07-21 Solexa Limited Arrangements moleculaires modifies
WO2006120433A1 (fr) 2005-05-10 2006-11-16 Solexa Limited Polymerases ameliorees
WO2013041117A1 (fr) 2011-09-23 2013-03-28 Illumina Cambrigde Ltd. Colorants pour le marquage de ligands moléculaires
US20140079923A1 (en) 2012-06-08 2014-03-20 Wayne N. George Polymer coatings
WO2014135221A1 (fr) 2013-03-08 2014-09-12 Illumina Cambridge Ltd Composés de polyméthine et leur utilisation comme marqueurs fluorescents
WO2014139596A1 (fr) 2013-03-15 2014-09-18 Illumina Cambridge Limited Nucléosides ou nucléotides modifiés
US20160040225A1 (en) 2014-08-08 2016-02-11 Illumina Cambridge Limited Modified nucleotide linkers
US20160122816A1 (en) * 2014-10-31 2016-05-05 Illumina Cambridge Limited Novel polymers and dna copolymer coatings
WO2016189287A1 (fr) 2015-05-22 2016-12-01 Illumina Cambridge Ltd Composés de polyméthine à longs déplacements de stokes et leur utilisation en tant que marqueurs fluorescents
WO2017051201A1 (fr) 2015-09-25 2017-03-30 Illumina Cambridge Limited Composés polyméthines et leur utilisation en tant que marqueurs fluorescents
US20180094140A1 (en) 2016-09-30 2018-04-05 Illumina Cambridge Limited Fluorescent dyes and their uses as biomarkers
US20180201981A1 (en) 2016-12-22 2018-07-19 Illumina Cambridge Limited Coumarin compounds and their uses as fluorescent labels
WO2020126593A1 (fr) 2018-12-17 2020-06-25 Illumina Cambridge Limited Compositions destinées à être utilisées dans le séquençage de polynucléotides
US20200216891A1 (en) 2018-12-26 2020-07-09 Illumina Cambridge Limited Nucleosides and nucleotides with 3'-hydroxy blocking groups
US20200277529A1 (en) 2019-03-01 2020-09-03 Illumina Cambridge Limited Exocyclic amine substituted coumarin compounds and uses as fluorescent labels
US20200277670A1 (en) 2019-03-01 2020-09-03 Illumina Cambridge Limited Tertiary amine substituted coumarin compounds and uses as fluorescent labels
WO2021069000A1 (fr) * 2019-10-09 2021-04-15 I.T.A. Intertact S.R.O. Polymère fluorescent, sonde fluorescente et kit de conjugaison pour analyse fonctionnelle avancée de cellules en hématologie, immunologie et microbiologie, leur procédé de préparation et leur utilisation
US20210187470A1 (en) * 2019-12-23 2021-06-24 llumina, Inc. Nanoparticle with single site for template polynucleotide attachment
US20210188832A1 (en) 2017-10-16 2021-06-24 Illumina Cambridge Limited Secondary amine-substituted coumarin compounds and their uses as fluorescent labels
US20220033900A1 (en) 2020-07-28 2022-02-03 Illumina Cambridge Limited Substituted coumarin dyes and uses as fluorescent labels
US20220195517A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Long stokes shift chromenoquinoline dyes and uses in sequencing applications
US20220380389A1 (en) 2021-05-05 2022-12-01 Illumina Cambridge Limited Fluorescent dyes containing bis-boron fused heterocycles and uses in sequencing
US20220396832A1 (en) 2021-05-20 2022-12-15 Illumina Cambridge Limited Compositions and methods for sequencing by synthesis
US20230313292A1 (en) 2022-03-29 2023-10-05 Illumina Cambridge Limited Chromenoquinoline dyes and uses in sequencing
US20230383342A1 (en) 2022-05-31 2023-11-30 Illumina Cambridge Limited Compositions and methods for nucleic acid sequencing

Patent Citations (72)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5436327A (en) 1988-09-21 1995-07-25 Isis Innovation Limited Support-bound oligonucleotides
US6346413B1 (en) 1989-06-07 2002-02-12 Affymetrix, Inc. Polymer arrays
US6610482B1 (en) 1989-06-07 2003-08-26 Affymetrix, Inc. Support bound probes and methods of analysis using the same
US5561071A (en) 1989-07-24 1996-10-01 Hollenberg; Cornelis P. DNA and DNA technology for the construction of networks to be used in chip construction and chip production (DNA-chips)
US6416949B1 (en) 1991-09-18 2002-07-09 Affymax, Inc. Method of synthesizing diverse collections of oligomers
US6136269A (en) 1991-11-22 2000-10-24 Affymetrix, Inc. Combinatorial kit for polymer synthesis
WO1993017126A1 (fr) 1992-02-19 1993-09-02 The Public Health Research Institute Of The City Of New York, Inc. Nouvelles configurations d'oligonucleotides et utilisation de ces configurations pour le tri, l'isolement, le sequençage et la manipulation des acides nucleiques
US5583211A (en) 1992-10-29 1996-12-10 Beckman Instruments, Inc. Surface activated organic polymers useful for location - specific attachment of nucleic acids, peptides, proteins and oligosaccharides
US5661040A (en) * 1993-07-13 1997-08-26 Abbott Laboratories Fluorescent polymer labeled conjugates and intermediates
US5837858A (en) 1993-10-22 1998-11-17 The Board Of Trustees Of The Leland Stanford Junior University Method for polymer synthesis using arrays
WO1995011995A1 (fr) 1993-10-26 1995-05-04 Affymax Technologies N.V. Reseaux de sondes d'acide nucleique sur des microplaquettes biologiques
US5429807A (en) 1993-10-28 1995-07-04 Beckman Instruments, Inc. Method and apparatus for creating biopolymer arrays on a solid support surface
WO1995035505A1 (fr) 1994-06-17 1995-12-28 The Board Of Trustees Of The Leland Stanford Junior University Procede et appareil pour fabriquer des microensembles d'echantillons biologiques
US6172218B1 (en) 1994-10-13 2001-01-09 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US6482591B2 (en) 1994-10-24 2002-11-19 Affymetrix, Inc. Conformationally-restricted peptide probe libraries
US5919523A (en) 1995-04-27 1999-07-06 Affymetrix, Inc. Derivatization of solid supports and methods for oligomer synthesis
EP0742287A2 (fr) 1995-05-10 1996-11-13 McGall, Glenn H. Sondes d'acide nucleique modifiés
US5874219A (en) 1995-06-07 1999-02-23 Affymetrix, Inc. Methods for concurrently processing multiple biological chip assays
US6524793B1 (en) 1995-10-11 2003-02-25 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method
US5658734A (en) 1995-10-17 1997-08-19 International Business Machines Corporation Process for synthesizing chemical compounds
EP0799897A1 (fr) 1996-04-04 1997-10-08 Affymetrix, Inc. (a California Corporation) Méthodes et compositions pour sélectionner tag acides nucléiques et épreuves correspondantes
US6297006B1 (en) 1997-01-16 2001-10-02 Hyseq, Inc. Methods for sequencing repetitive sequences and for determining the order of sequence subfragments
WO1998044151A1 (fr) 1997-04-01 1998-10-08 Glaxo Group Limited Methode d'amplification d'acide nucleique
US6465178B2 (en) 1997-09-30 2002-10-15 Surmodics, Inc. Target molecule attachment to surfaces
US6287768B1 (en) 1998-01-07 2001-09-11 Clontech Laboratories, Inc. Polymeric arrays and methods for their use in binding assays
US6287776B1 (en) 1998-02-02 2001-09-11 Signature Bioscience, Inc. Method for detecting and classifying nucleic acid hybridization
US6288220B1 (en) 1998-03-05 2001-09-11 Hitachi, Ltd. DNA probe array
US6291193B1 (en) 1998-06-16 2001-09-18 Millennium Pharmaceuticals, Inc. MTbx protein and nucleic acid molecules and uses therefor
WO2000006770A1 (fr) 1998-07-30 2000-02-10 Solexa Ltd. Biomolecules en rangees et leur utilisation dans une procedure de sequençage
WO2000018957A1 (fr) 1998-09-30 2000-04-06 Applied Research Systems Ars Holding N.V. Procedes d'amplification et de sequençage d'acide nucleique
US6514751B2 (en) 1998-10-02 2003-02-04 Incyte Genomics, Inc. Linear microarrays
WO2000031148A2 (fr) 1998-11-25 2000-06-02 Motorola, Inc. Hydrogels de polyacrylamide obtenus a partir de prepolymeres reactifs
WO2000053812A2 (fr) 1999-03-12 2000-09-14 President And Fellows Of Harvard College Amplification par replique de reseaux d'acides nucleiques
US6355431B1 (en) 1999-04-20 2002-03-12 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays
WO2000063437A2 (fr) 1999-04-20 2000-10-26 Illumina, Inc. Detection de reactions d'acide nucleique sur microsupports de billes en reseau
WO2001001143A2 (fr) 1999-06-25 2001-01-04 Motorola Inc. Procedes et compositions permettant de fixer des biomolecules a des supports solides, a des hydrogels et a des matrices d'hydrogels
WO2001057248A2 (fr) 2000-02-01 2001-08-09 Solexa Ltd. Groupes de polynucleotides et leur utilisation dans le sequencage
US20020102578A1 (en) 2000-02-10 2002-08-01 Todd Dickinson Alternative substrates and formats for bead-based array of arrays TM
WO2002012566A2 (fr) 2000-08-09 2002-02-14 Motorola, Inc. Utilisation et evaluation d'une photocycloaddition [2+2] dans une immobilisation d'oligonucleotides sur une matrice hydrogel tridimensionnelle
WO2002029003A2 (fr) 2000-10-06 2002-04-11 The Trustees Of Columbia University In The City Of New York Methode d'analyse massivement parallele destinee a decoder l'adn et l'arn
US20020094586A1 (en) * 2001-01-05 2002-07-18 Sensors For Medicine And Science, Inc. Detection of analytes
WO2003014392A2 (fr) 2001-08-09 2003-02-20 Amersham Biosciences Ab Utilisation et evaluation d'une photoaddition [2+2] dans l immobilisation d'oligonucleotides sur une matrice d'hydrogel tridimensionnelle
WO2004018493A1 (fr) 2002-08-23 2004-03-04 Solexa Limited Nucleotides marques
WO2004018497A2 (fr) 2002-08-23 2004-03-04 Solexa Limited Nucleotides modifies
WO2005024010A1 (fr) 2003-09-11 2005-03-17 Solexa Limited Polymerases modifies pour l'incorporation amelioree d'analogues nucleotidiques
WO2005047301A1 (fr) 2003-11-07 2005-05-26 Solexa Limited Ameliorations apportees a des reseaux polynuceotidiques
WO2005065814A1 (fr) 2004-01-07 2005-07-21 Solexa Limited Arrangements moleculaires modifies
WO2006120433A1 (fr) 2005-05-10 2006-11-16 Solexa Limited Polymerases ameliorees
WO2013041117A1 (fr) 2011-09-23 2013-03-28 Illumina Cambrigde Ltd. Colorants pour le marquage de ligands moléculaires
US20140079923A1 (en) 2012-06-08 2014-03-20 Wayne N. George Polymer coatings
WO2014135221A1 (fr) 2013-03-08 2014-09-12 Illumina Cambridge Ltd Composés de polyméthine et leur utilisation comme marqueurs fluorescents
WO2014139596A1 (fr) 2013-03-15 2014-09-18 Illumina Cambridge Limited Nucléosides ou nucléotides modifiés
US20160040225A1 (en) 2014-08-08 2016-02-11 Illumina Cambridge Limited Modified nucleotide linkers
US20160122816A1 (en) * 2014-10-31 2016-05-05 Illumina Cambridge Limited Novel polymers and dna copolymer coatings
WO2016189287A1 (fr) 2015-05-22 2016-12-01 Illumina Cambridge Ltd Composés de polyméthine à longs déplacements de stokes et leur utilisation en tant que marqueurs fluorescents
WO2017051201A1 (fr) 2015-09-25 2017-03-30 Illumina Cambridge Limited Composés polyméthines et leur utilisation en tant que marqueurs fluorescents
US20180094140A1 (en) 2016-09-30 2018-04-05 Illumina Cambridge Limited Fluorescent dyes and their uses as biomarkers
WO2018060482A1 (fr) 2016-09-30 2018-04-05 Illumina Cambridge Limited Nouveaux colorants fluorescents et leurs utilisations en tant que biomarqueurs
US20180201981A1 (en) 2016-12-22 2018-07-19 Illumina Cambridge Limited Coumarin compounds and their uses as fluorescent labels
US20210188832A1 (en) 2017-10-16 2021-06-24 Illumina Cambridge Limited Secondary amine-substituted coumarin compounds and their uses as fluorescent labels
WO2020126593A1 (fr) 2018-12-17 2020-06-25 Illumina Cambridge Limited Compositions destinées à être utilisées dans le séquençage de polynucléotides
US20200216891A1 (en) 2018-12-26 2020-07-09 Illumina Cambridge Limited Nucleosides and nucleotides with 3'-hydroxy blocking groups
US20200277529A1 (en) 2019-03-01 2020-09-03 Illumina Cambridge Limited Exocyclic amine substituted coumarin compounds and uses as fluorescent labels
US20200277670A1 (en) 2019-03-01 2020-09-03 Illumina Cambridge Limited Tertiary amine substituted coumarin compounds and uses as fluorescent labels
WO2021069000A1 (fr) * 2019-10-09 2021-04-15 I.T.A. Intertact S.R.O. Polymère fluorescent, sonde fluorescente et kit de conjugaison pour analyse fonctionnelle avancée de cellules en hématologie, immunologie et microbiologie, leur procédé de préparation et leur utilisation
US20210187470A1 (en) * 2019-12-23 2021-06-24 llumina, Inc. Nanoparticle with single site for template polynucleotide attachment
US20220033900A1 (en) 2020-07-28 2022-02-03 Illumina Cambridge Limited Substituted coumarin dyes and uses as fluorescent labels
US20220195517A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Long stokes shift chromenoquinoline dyes and uses in sequencing applications
US20220380389A1 (en) 2021-05-05 2022-12-01 Illumina Cambridge Limited Fluorescent dyes containing bis-boron fused heterocycles and uses in sequencing
US20220396832A1 (en) 2021-05-20 2022-12-15 Illumina Cambridge Limited Compositions and methods for sequencing by synthesis
US20230313292A1 (en) 2022-03-29 2023-10-05 Illumina Cambridge Limited Chromenoquinoline dyes and uses in sequencing
US20230383342A1 (en) 2022-05-31 2023-11-30 Illumina Cambridge Limited Compositions and methods for nucleic acid sequencing

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
NATURE, vol. 437, 2005, pages 376 - 380
SCHEIT: "Nucleotide Analogs", 1980, JOHN WILEY & SON
SCIENCE, vol. 309, no. 5741, 2005, pages 1728 - 1732
UHLMAN ET AL.: "Chemical Reviews", vol. 90, 1990, pages: 543 - 584

Similar Documents

Publication Publication Date Title
US20230383342A1 (en) Compositions and methods for nucleic acid sequencing
US12043637B2 (en) Fluorescent dyes containing bis-boron fused heterocycles and uses in sequencing
KR102776582B1 (ko) 치환된 쿠마린 염료 및 형광 표지로서의 용도
JP7307686B2 (ja) 第二級アミン置換クマリン化合物、および蛍光標識としてのそれらの使用
KR20230031837A (ko) 3' 아세탈 블로킹기를 갖는 뉴클레오시드 및 뉴클레오티드
KR20230121556A (ko) 긴 스토크스 이동 크로메노퀴놀린 염료 및 서열분석 응용에서의 용도
CN112638925A (zh) 具有3’-羟基封闭基团的核苷和核苷酸以及它们在多核苷酸测序方法中的用途
US12031178B2 (en) Cyclooctatetraene containing dyes and compositions
KR20230121555A (ko) 알킬피리디늄 쿠마린 염료 및 서열분석 응용에서의 용도
KR20230123871A (ko) 핵산 서열분석을 위한 방법 및 조성물
CN119213083A (zh) 用于减少测序期间的光损伤的组合物和方法
CN112639128A (zh) 利用光可切换标记进行核酸测序的方法和组合物
WO2025221895A1 (fr) Échafaudages polymères solubles dans l'eau pour marquage de colorant
WO2025136890A1 (fr) Nanoparticules d'hydrogel servant d'échafaudage de marquage dans séquençage
US20230304086A1 (en) Labeled avidin and methods for sequencing
US20250075269A1 (en) Compositions and methods for nucleic acid sequencing
WO2025106715A1 (fr) Polymères conjugués ou points de polymères en tant que marqueurs fluorescents
WO2024206407A2 (fr) Colorants de naphtalimide et utilisations dans séquençage d'acides nucléiques
WO2025144711A1 (fr) Colorants polyméthiniques tricycliques pour le séquençage d'acides nucléiques
WO2024123866A1 (fr) Nucléosides et nucléotides à groupes bloquants 3´ et lieurs clivables
WO2025188906A1 (fr) Nucléotides d'adénosine modifiés