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WO2025230914A1 - Nucléotides avec lieurs auto-immolables déclenchés par des enzymes pour le séquençage par synthèse - Google Patents

Nucléotides avec lieurs auto-immolables déclenchés par des enzymes pour le séquençage par synthèse

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Publication number
WO2025230914A1
WO2025230914A1 PCT/US2025/026687 US2025026687W WO2025230914A1 WO 2025230914 A1 WO2025230914 A1 WO 2025230914A1 US 2025026687 W US2025026687 W US 2025026687W WO 2025230914 A1 WO2025230914 A1 WO 2025230914A1
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WIPO (PCT)
Prior art keywords
nucleotide
group
nucleotides
moiety
solid support
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
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PCT/US2025/026687
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English (en)
Inventor
Stephane EMOND
Antoine FRANCAIS
Cassie ZERBE
Adam Culley
Natasha CRAKE-GHOSAL
Elena CRESSINA
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Illumina Inc
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Illumina Inc
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Publication of WO2025230914A1 publication Critical patent/WO2025230914A1/fr
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Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

  • the present disclosure generally relates to nucleotides, nucleosides, or oligonucleotides comprising enzymatically cleavable self-immolative linker groups and their uses in polynucleotide sequencing methods. Methods of preparing the nucleotides, nucleosides, or oligonucleotides having enzymatically cleavable self-immolative linker groups are also disclosed.
  • Fabricated arrays can also be manufactured by the technique of “spotting” known polynucleotides onto a solid support at predetermined positions (e.g., Stimpson et al., Proc. Natl. Acad. Sci. 92: 6379- 6383, 1995). [0004] One way of determining the nucleotide sequence of a nucleic acid bound to an array is called “sequencing by synthesis” or “SBS”.
  • This technique for determining the sequence of DNA ideally requires the controlled (i.e., one at a time) incorporation of the correct complementary nucleotide opposite the nucleic acid being sequenced.
  • This allows for accurate sequencing by adding nucleotides in multiple cycles as each nucleotide residue is sequenced one at a time, thus preventing an uncontrolled series of incorporations from occurring.
  • the incorporated nucleotide is read using an appropriate label attached thereto before removal of the label moiety and the subsequent next round of sequencing.
  • a structural modification (“blocking group” or “protecting group”) is included in each labeled nucleotide that is added to the growing chain to ensure that only one nucleotide is incorporated.
  • nucleotides which are usually nucleotide triphosphates, generally require a 3 ⁇ -hydroxy protecting group so as to prevent the polymerase used to incorporate it into a polynucleotide chain from continuing to replicate once the base on the nucleotide is added.
  • a 3 ⁇ -hydroxy protecting group so as to prevent the polymerase used to incorporate it into a polynucleotide chain from continuing to replicate once the base on the nucleotide is added.
  • the protecting group should prevent additional nucleotide molecules from being added to the polynucleotide chain whilst simultaneously being easily removable from the sugar moiety without causing damage to the polynucleotide chain.
  • the modified nucleotide needs to be compatible with the polymerase or another appropriate enzyme used to incorporate it into the polynucleotide chain.
  • the ideal protecting group must therefore exhibit long-term stability, be efficiently incorporated by the polymerase enzyme, cause blocking of secondary or further nucleotide incorporation, and have the ability to be removed under mild conditions that do not cause damage to the polynucleotide structure, preferably under aqueous conditions.
  • a nucleotide comprising a nucleobase, a ribose or 2 ⁇ deoxyribose, and a detectable moiety, wherein the detectable moiety is covalently attached to the nucleotide via a self-immolative linker, and wherein the self-immolative linker comprises an optionally substituted benzyl moiety, a leaving group comprising a carbamate moiety or an acetyl moiety, and an enzymatically cleavable moiety.
  • Another aspect of the present disclosure relates to an oligonucleotide or polynucleotide comprising a nucleotide described herein incorporated thereof.
  • Another aspect of the present disclosure relates to a kit comprising a nucleotide described in the present disclosure.
  • Another aspect of the present disclosure relates to a method of preparing a growing polynucleotide complementary to a target single-stranded polynucleotide, comprising incorporating a nucleotide of the present disclosure into a growing complementary polynucleotide.
  • Another aspect of the present disclosure relates to a method of determining the sequences of a plurality of 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 aqueous solution comprising DNA polymerase and one or more of four different types of nucleotides (A, G, C, and T or U; dATP, dGTP, dCTP and dTTP or dUTP) 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 at least one type of nucleotide is a nucleotide of the present disclosure carrying a fluorescent label
  • a further aspect of the present disclosure relates to a method of determining the sequences of a plurality of 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 aqueous solution comprising DNA polymerase and one or more of four different types of nucleotides A, G, C, and T or U 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 at least one type of nucleotide is an unlabeled nucleotide of the present disclosure having a first functional group attached via the self-immolative linker, and wherein each of the one or more
  • FIGs. 1A and 1B are HPLC analysis plots showing self-immolation of nucleotide containing a carbamate arabinofuranoside (c-ABF) linker moiety.
  • FIG.1A shows the HPLC peak of a negative control without enzymatic cleavage
  • FIG. 1B shows the HPLC peak of the nucleotide self-immolation product relative to the starting material.
  • FIG.2 is a line chart showing percent residual starting material as a functional of time of nucleotides comprising various cleavable linker moieties.
  • a-ABF acetal arabinofuranoside
  • Embodiments of the present disclosure relate to nucleosides and nucleotides with an enzymatically cleavable self-immolative linkers for sequencing applications, for example, sequencing-by-synthesis (SBS).
  • SBS sequencing-by-synthesis
  • enzymatic cleavage is likely to be less damaging to DNA and may generate less signal decay during SBS.
  • enzymes for cleaving the self-immolative linker may have improved stability during storage and shipping. For example, such enzymes can be freeze dried. Recombinant SBS cleaving enzymes may be produced at scale from relatively low costs.
  • 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.
  • any “R” group(s) represent substituents that can be attached to the indicated atom. An R group may be substituted or unsubstituted.
  • R 1 and R 2 are defined as selected consisting of hydrogen and alkyl, or R 1 and R 2 together with the atoms to which they are attached form an aryl or carbocyclyl, it is meant that R 1 and R 2 can be selected from hydrogen or alkyl, or alternatively, the substructure has structure: where A is an aryl ring or a the depicted double bond.
  • 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.
  • radical is a di-radical such as “alkylene” or “alkenylene.”
  • 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.
  • “Ca to Cb” in which “a” and “b” are integers refer 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.
  • 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 “C1 to C4 alkyl” group refers to all alkyl groups having from 1 to 4 carbons, that is, CH3-, CH3CH2-, CH3CH2CH2- , (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and (CH3)3C-;
  • a C3 to C4 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 C6, and a range defined by any of the two numbers .
  • C 1 -C 6 alkyl includes C 1 , C 2 , C 3 , C 4 , C 5 and C 6 alkyl, C2-C6 alkyl, C1-C3 alkyl, etc.
  • C2-C6 alkenyl includes C2, C3, C4, C5 and C6 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 C 6 alkynyl, C 2 - 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 C 3 -C 7 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 “C 1 -C 4 alkyl” or similar designations.
  • “C 1 -C 6 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 “C 1 -C 9 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 “C2-C6 alkenyl” or similar designations.
  • C2-C6 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.
  • alkenyl groups include, but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and the like.
  • alkynyl refers to a straight or branched hydrocarbon chain containing one or more triple bonds.
  • the alkynyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term “alkynyl” where no numerical range is designated.
  • the alkynyl group may also be a medium size alkynyl having 2 to 9 carbon atoms.
  • the alkynyl group could also be a lower alkynyl having 2 to 6 carbon atoms.
  • the alkynyl group may be designated as “C 2 -C 6 alkynyl” or similar designations.
  • C 2 -C 6 alkynyl indicates that there are two to six carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl.
  • Typical alkynyl groups include, but are in no way limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.
  • heteroalkyl refers to a straight or branched hydrocarbon chain containing one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the chain backbone.
  • the heteroalkyl group may have 1 to 20 carbon atoms, although the present definition also covers the occurrence of the term “heteroalkyl” where no numerical range is designated.
  • the heteroalkyl group may also be a medium size heteroalkyl having 1 to 9 carbon atoms.
  • the heteroalkyl group could also be a lower heteroalkyl having 1 to 6 carbon atoms.
  • the heteroalkyl group may be designated as “2 to 10 membered heteroalkyl” or similar designations.
  • the heteroalkyl group may contain one or more heteroatoms.
  • “2 to 10 membered heteroalkyl” indicates that the total number of carbon atoms and one or more heteroatoms (excluding hydrogen atoms) in the backbone of the chain is 2 to 10.
  • heteroalkylene refers to an alkylene group, as defined herein, containing one or more heteroatoms in the carbon back bone (i.e., an alkylene group in which one or more carbon atoms is replaced with a heteroatom, for example, nitrogen atom, oxygen atom or sulfur atom).
  • a -CH2- may be replaced with -O-, -S-, or -NH-.
  • Heteroalkylene groups include, but are not limited to ether, thioether, amino-alkylene, and alkylene-amino-alkylene moieties.
  • the heteroalkylene may include one, two, three, four, or five - CH2CH2O- unit(s).
  • 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).
  • 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 “C6-C10 aryl,” “C6 or C10 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).
  • 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.
  • 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, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.
  • a “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, as a substituent, via an alkylene group. Examples include but are not limited to 2-thienylmethyl, 3- thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl.
  • the alkylene group is a lower alkylene group (i.e., a C1-C6 alkylene group).
  • 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, pyrrolidinyl, 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
  • 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.
  • (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. Examples include but are not limited to (tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl, (piperidin-4-yl)propyl, (tetrahydro-2H-thiopyran-4-yl)methyl, and (1,3-thiazinan-4-yl)methyl.
  • (cycloalkyl)alkyl or “(carbocyclyl)alkyl” refers to a cycloalkyl or carbocyclyl group (as defined herein) connected, as a substituent, via an alkylene group. Examples include but are not limited to cyclopropylmethyl, cyclobutylmethyl, cyclopentylethyl, and cyclohexylpropyl.
  • R is selected from the group consisting of hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C 6 -C 10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
  • a “sulfonyl” group refers to an “-SO 2 R” 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 “S-sulfonamido” group refers to a “-SO2NRARB” group in which RA and RB are each independently 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.
  • N-sulfonamido refers to a “-N(RA)SO2RB” group in which RA and Rb are each independently 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.
  • An “amino” group refers to a “-NRARB” 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.
  • a non-limiting example includes free amino (i.e., -NH 2 ).
  • An “aminoalkyl” group refers to an amino group connected via an alkylene group.
  • the term “hydroxy” as used herein refers to a –OH group.
  • cyano refers to a “-CN” group.
  • zido refers to a –N3 group.
  • substituent may be selected from one or more of the indicated substituents.
  • 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.
  • a group is deemed to be “substituted,” it is meant that the group is substituted with one or more 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, and C1-C6 haloalkoxy), 3-10 membered heterocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-
  • a substituent is depicted as a di-radical (i.e., has two points of attachment to the rest of the molecule), it is to be understood that the substituent can be attached in any directional configuration unless otherwise indicated.
  • a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups are 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 or pyrimidine base.
  • Purine bases include adenine (A), deaza adenine (e.g., 7-deaza adenine), guanine (G), deaza guanine (e.g., 7-deaza guanine) and modified derivatives or analogs thereof.
  • 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.
  • 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.
  • 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.
  • purine base is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers.
  • 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, deazapurine, adenine, 7-deaza adenine, guanine, 7-deaza guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid and isoguanine.
  • 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” or “labeled with” 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.
  • self-immolative linker refers to a linker moiety (e.g., a covalent construct) that can degrade spontaneously in response to specific stimuli (e.g., by an enzyme), results in cleavage of two or more chemical bonds. It is to be understood that a self- immolative linker is a type of cleavable linker.
  • “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, phosphoranilidate and phosphoramidate linkages.
  • 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 used herein, the terms “monophosphate,” “diphosphate,” and “triphosphate” sense as understood by those skilled in the art, and include protonated forms.
  • protecting group and “protecting groups” as used herein refer to any atom or group of atoms that is added to a molecule in order to prevent existing groups in the molecule from undergoing unwanted chemical reactions. Sometimes, “protecting group” and “blocking group” can be used interchangeably.
  • phasing refers to a phenomenon in SBS that is caused by incomplete removal of the 3 ⁇ terminators and fluorophores, and failure to complete the incorporation of a portion of DNA strands within clusters by polymerases at a given sequencing cycle. Pre-phasing 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 pre- phasing 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 pre-phasing increases, hampering the identification of the correct base.
  • Pre-phasing can be caused by the presence of a trace amount of unprotected or unblocked 3 ⁇ -OH nucleotides during sequencing by synthesis (SBS). The unprotected 3 ⁇ -OH nucleotides could be generated during the manufacturing processes or possibly during the storage and reagent handling processes.
  • nucleotide analogues which decrease the incidence of pre-phasing is surprising and provides a great advantage in SBS applications over existing nucleotide analogues.
  • the nucleotide analogues provided can result in faster SBS cycle time, lower phasing and pre-phasing values, and longer sequencing read lengths.
  • nucleotides with Enzymatically Cleavable Self-Immolative Linker [0071]
  • One aspect of the present disclosure relates to a nucleotide comprising a nucleobase, a ribose or 2 ⁇ deoxyribose, and a detectable moiety, wherein the detectable moiety is covalently attached to the nucleotide via a self-immolative linker, and wherein the self-immolative linker comprises an optionally substituted benzyl moiety, a leaving group comprising a carbamate moiety or an acetyl moiety, and an enzymatically cleavable moiety.
  • the self-immolative linker has the structure of formula (I): (I), wherein each of L 1 and L 2 is independently an optionally present spacer moiety; X is O or NH; R EC is the enzymatically cleavable moiety; R BZ is H, unsubstituted or substituted C 1 -C 6 alkyl, unsubstituted or substituted C 6 -C 10 aryl, or unsubstituted or substituted 5 to 10 membered heteroaryl; the phenylene moiety is optionally substituted with one or more electron withdrawing groups or one or more electron donating groups; the asterisk indicates the point of connection to the nucleobase; and the detectable moiety is covalently attached to the leaving group, the benzyl moiety (if L 2 is present), each optionally via a spacer L 3 .
  • one or more of the methylene units of the C2-C10 alkylene, the 2 to 20 membered (e.g., 2 to 10 membered, 3 to 8 membered, or 4 to 6 membered) heteroalkylene, the 2 to 20 membered (e.g., 2 to 10 membered, 3 to 8 membered, or 4 to 6 membered) heteroalkenylene, or the 2 to 20 membered (e.g., 2 to 10 membered, 3 to 8 membered, or 4 to 6 membered) heteroalkynylene described herein may be replaced by a ring or ring system selected from unsubstituted or substituted C6-C10 arylene, or unsubstituted or substituted 5 to 10 membered heteroaryl containing 1, 2, 3, 4, 5 or 6 heteroatoms selected from N, O and S.
  • L 2 is not present.
  • the leaving group comprises a carbamate moiety.
  • the detectable moiety is covalently attached to the phenylene moiety, optionally via a spacer L 3 .
  • the self-immolative linker of the nucleotide has the structure of formula (Ia-1) or (Ia-2): (Ia-2), and R 3 is H, an electron donating group, or an electron withdrawing group.
  • the leaving group comprises an acetal moiety.
  • the detectable moiety is covalently attached to the leaving group moiety, optionally via a spacer L 3 .
  • the self-immolative linker of the nucleotide has the structure of formula (Ib-1), (Ib-2) or (Ib-3): or R 2 withdrawing group.
  • the electron donating group is an optionally substituted amino, hydroxy, C 1 -C 6 alkoxy, C 1 -C 6 alkyl or C 2 -C 6 alkenyl. In one embodiment, the electron donating group is methoxy.
  • both of R 1 and R 2 are H.
  • R 1 is H and R 2 is an electron withdrawing group as described herein.
  • R 2 is H and R 1 is an electron donating group as described herein.
  • R 1 is an electron donating group as described herein and R 2 is an electron withdrawing group as described herein.
  • R 3 is H. In some other embodiments, R 3 is an electron donating group as described herein. In some other embodiments, R 3 is an electron withdrawing group as described herein.
  • R BZ is H. In some other embodiments, R BZ is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl). In some other embodiments, R BZ is optionally substituted phenyl. In some embodiments, X is O.
  • X is NH.
  • –X–R EC is –O–glycoside.
  • the –O–glycoside forms an –O–glycosidic bond with the carbon atom of the phenylene to which it is attached.
  • R EC is an arabinofuranosidyl (ABF) group.
  • the self-immolative linker has the structure: .
  • the detectable moiety comprises a functional group that form covalent or noncovalent bonding with a labeling reagent.
  • L 1 is a 2 to 10 membered heteroalkylene spacer, 2 to 10 membered heteroalkenylene spacer, or 2 to 10 membered heteroalkynylene spacer, each containing one to three heteroatoms selected from N, O and S.
  • L 1 comprises In some embodiments, L 3 is a 2 to 10 selected from N, O, and S, wherein one methylene unit is optionally replaced by an optionally substituted phenylene, or an optionally substituted 5 or 6 membered heteroarylene moiety (e.g., a triazole).
  • L 3 . of the nucleotide with linker moiety of formula (I), (Ia- 1), (Ia-2), (Ib-1), (Ib-2) or (Ib-3) the nucleotide comprises a 3 ⁇ blocking group.
  • the 3 ⁇ blocking group is enzymatically cleavable.
  • the 3 ⁇ blocking group and the enzymatic cleavable moiety of the self-immolative linker are removable by a single enzymatic reaction, and the enzymatic reaction results in the self-immolation of the linker and the removal of the detectable moiety of the nucleotide.
  • the 3 ⁇ blocking group is –O–glycoside, forming an –O–glycosidic bond with the 3 ⁇ carbon atom of the nucleotide.
  • the 3 ⁇ blocking group has a structure: (alpha-L-arabinofuranoside), where the squiggle line carbon atom of the ribose or 2 ⁇ deoxyribose.
  • beta- L-arabinofuranoside 3 ⁇ blocking group may also be used.
  • the 3 ⁇ blocking group has a structure (alpha-D-glucoside), (alpha-D-galactoside), beta-D- other embodiments, the removable 3 ⁇ blocking group has a (alpha-D-xyloside) or beta-D- xyloside.
  • the removable 3 ⁇ blocking group has a structure (alpha-N-Acetyl-D-glucosamine), or beta-N-Acetyl-D- the removable 3 ⁇ blocking group has a structure (alpha-D-glucuronide), or beta-D-glucuronide.
  • the self-immolative linker moiety may also include the same moiety(in which X is O in formula (I), (Ia-1), (Ia-2), (Ib-1), (Ib-2) or (Ib-3)) as the 3 ⁇ blocking group described herein such that a single enzymatic reaction results in the self-immolation of the linker and the removal of the detectable moiety of the nucleotide.
  • the detectable moiety is a fluorescent dye.
  • the detectable moiety is a functional group that is capable of attaching to a labeling reagent.
  • the functional group can attach to the labeling reagent via covalent bonding (e.g., a chemical reaction between the functional group of the nucleotide and a reactive group of the labeling reagent to form covalent bonding).
  • the functional group can attach to the labeling reagent via noncovalent interaction (e.g., the functional group is a biotin which can bind to a labeled avidin such as streptavidin.
  • the nucleotide may be a nucleotide triphosphate comprising 2 ⁇ deoxyribose.
  • nucleotide may be a nucleotide triphosphate comprising 2 ⁇ deoxyribose.
  • the oligonucleotide or polynucleotide is at least partially complementary and hybridized to a target polynucleotide immobilized on a surface of a solid support.
  • the solid support comprises an array of a plurality of target polynucleotides immobilized thereon.
  • Enzymes Capable of Cleaving Self-Immolative Linkers may be capable of cleaving self-immolative linkers described herein.
  • glycoside hydrolases or glycosyl hydrolases may be suitable for cleaving linker groups in accordance with the present disclosure.
  • Table 1 lists particular example glycoside hydrolases that can cleave, for example, the glycoside 3 ⁇ blocking group or the enzymatic cleavable moiety of the self-immolative linker.
  • Enzymes in accordance with the present disclosure may be suitable for inclusion in a kit for sequencing by synthesis.
  • Enzymes in accordance with the present disclosure may be suitable in methods of growing a polynucleotide strand.
  • Enzymes in accordance with the present disclosure may be suitable for use in methods of sequencing.
  • Table 1 Exemplary enzymes and corresponding E.C. Number and CAZy family Enzyme Name E.C. Number CAZy Family (as of July 2023) ⁇ -L-arabinofuranosidase E.C.3.2.1.55 GH 2, 3, 10, 43, 51, 54, 62 and 159 1, 1, 7, [0081]
  • the enzymatically triggered self-immolation of nucleotide containing a carbamate/ABF self-immolative linker is illustrated in the scheme A below.
  • the described nucleotide also comprises a detectable fluorescent label.
  • a detectable fluorescent label e.g., a fluorescent dye
  • the label can be conjugated via the self-immolative linker in accordance with the present disclosure by a variety of means including hydrophobic attraction, ionic attraction, and covalent attachment.
  • the detectable label is conjugated to the substrate by covalent attachment. More particularly, the covalent attachment is by means of a self-immolative linker.
  • Various fluorescent dyes may be used in the present disclosure as detectable fluorescent labels, in particularly those dyes that may be excitation by a blue light (e.g., about 450 nm to about 460 nm) or a green light (e.g., about 520 nm to about 540 nm). 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, and bisboron containing heterocycles are disclosed in U.S. Publication Nos.
  • 2018/0094140 2018/0201981, 2020/0277529, 2020/0277670, 2021/0188832, 2022/0033900, 2022/0195517, 2022/0380389, 2023/0313292, and 2023/0416279, each of which is incorporated by reference in its entirety.
  • green dyes including cyanine or polymethine dyes disclosed in International Publication Nos. WO2013/041117, WO2014/135221, WO 2016/189287, WO2017/051201 and WO2018/060482A1, each of which is incorporated by reference in its entirety.
  • Labeled nucleosides and nucleotides are useful for labeling polynucleotides formed by enzymatic synthesis, such as, by way of non-limiting example, in PCR amplification, isothermal amplification, solid phase amplification, polynucleotide sequencing (e.g., solid phase sequencing), nick translation reactions and the like.
  • the dye may be covalently attached to oligonucleotides or nucleotides via the nucleotide base.
  • the labeled nucleotide or oligonucleotide may have the label attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a self-immolative linker moiety.
  • the reference to nucleotides is also intended to be applicable to nucleosides.
  • the present application will also be further described with reference to DNA, although the description will also be applicable to RNA, PNA, and other nucleic acids, unless otherwise indicated.
  • One aspect of the present disclosure relates to a method of preparing a growing polynucleotide complementary to a target single-stranded polynucleotide, comprising incorporating a nucleotide as described herein into a growing complementary polynucleotide.
  • the incorporation of the nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide.
  • the incorporation of the nucleotide is accomplished by a polymerase, a terminal deoxynucleotidyl transferase, or a reverse transcriptase.
  • the method may be used to synthesize oligonucleotide or polynucleotides. In other embodiments, the method is used in the context of SBS.
  • Another aspect of the present disclosure relates to a method of determining the sequences of a plurality of 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 aqueous solution comprising DNA polymerase and one or more of four different types of nucleotides A, G, C, and T or U 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 at least one type of nucle
  • step (d) comprises enzymatically removing the fluorescent label from the incorporated nucleotides.
  • the fluorescent label and the 3 ⁇ blocking group are removed in a single reaction.
  • the method further comprises: (e) washing the solid support after the removal of the 3 ⁇ blocking group and the fluorescent label from the incorporated nucleotides.
  • the method further comprises repeating steps (b) to (e) until the sequences of at least a portion of the target polynucleotides are determined.
  • steps (b) to (e) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles.
  • Another aspect of the present disclosure relates to an alternative sequencing by synthesis method in which at least one labeling reagent is introduced after the incorporation of unlabeled nucleotides.
  • the present disclosure relates to a method of determining the sequences of a plurality of 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 aqueous solution comprising DNA polymerase and one or more of four different types of nucleotides A, G, C, and T or U 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 at least one type of nucleot
  • step (e’) comprises enzymatically removing the one or more fluorescent labels from the incorporated nucleotides.
  • the one or more fluorescent labels and the 3 ⁇ blocking group are removed in a single reaction.
  • the method further comprises: (f’) washing the solid support after the removal of the 3 ⁇ blocking group and the one or more fluorescent labels from the incorporated nucleotides.
  • the method comprises repeating steps (b’) to (e’) until the sequences of at least a portion of the target polynucleotides are determined.
  • steps (b’) to (e’) are repeated at least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 cycles.
  • the fluorescent label is removed by contacting the solid support with an aqueous cleavage solution comprising a glycoside hydrolase or glycosidase that is capable of catalyzing the degradation of the self-immolative linker.
  • the glycoside hydrolase or glycosidase is an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an N-acetyl-glucosaminidase, or a glucuronidase.
  • the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 0.1 ⁇ M.
  • the removal of the fluorescent label is conducted at a temperature of at least about 30°C. In some embodiments, the removal of the fluorescent label is conducted at a pH between about 5 and about 10.
  • each type of nucleotides has a 3 ⁇ -O-glycoside blocking group as described herein.
  • the 3 ⁇ -O-glycoside group is removed by contacting the solid support with an aqueous cleavage solution comprising a glycoside hydrolase or glycosidase that is capable of catalyzing the hydrolysis of the –O–glycosidic bond of the nucleotide.
  • the glycoside hydrolase or glycosidase is an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an n-acetyl-glucosaminidase, or a glucuronidase.
  • the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 ⁇ M or within a range defined by any two of the preceding values.
  • the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 0.1 ⁇ M. In some embodiments, the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 0.5 ⁇ M. In some embodiments, the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 1 ⁇ M. In some embodiments, the concentration of the glycoside hydrolase or glycosidase in the aqueous cleavage solution is at least about 1.5 ⁇ M.
  • step (d) or (e’) comprises enzymatically removing the 3 ⁇ blocking group from the nucleotides incorporated into the extended copy polynucleotides.
  • step (d) or (e’) is conducted at a temperature of 30°C to 100°C.
  • step (d) or (e’) is conducted at a temperature of at least about 35°C.
  • step (d) or (e’) is conducted at a temperature of at least about 37°C.
  • step (d) or (e’) is conducted at a temperature of at least about 60°C. In some embodiments, step (d) or (e’) is conducted at a temperature of at least about 65°C. In some embodiments, step (d) or (e’) is conducted at a temperature of at least about 70°C. In some embodiments, step (d) or (e’) is conducted at a temperature of at least about 80°C.
  • step (d) or (e’) is conducted at a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0, in in a range defined by any two of the preceding values.
  • step (d) or (e’) is conducted at a pH between about 5.5 and about 6.5.
  • step (d) or (e’) is conducted at a pH between about 5.9 and about 6.1.
  • step (d) or (e’) is conducted at a pH of about 6.0.
  • the fourth type of nucleotide is unlabeled.
  • the G nucleotide is unlabeled.
  • G nucleotide has a structure selected from the group consisting of: , wh [0096]
  • the solid support comprises at least 500,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, or 5,000,000 spatially distinguishable sites/cm 2 that comprise multiple copies of target polynucleotides.
  • step (b) or (b’), also referred to as the incorporation step includes contacting a mixture containing one or more nucleotides (e.g., dATP, dCTP, dGTP, and dTTP or dUTP) with a copy polynucleotide/target polynucleotide complex in an incorporation solution comprising a polymerase and one or more buffering agents.
  • nucleotides e.g., dATP, dCTP, dGTP, and dTTP or dUTP
  • the polymerase is a DNA polymerase, such as a mutant of 9°N polymerase (e.g., those disclosed in WO 2005/024010, which is incorporated by reference), for example, Pol 812, Pol 1901, Pol 1558 or Pol 963.
  • the amino acid sequences of Pol 812, Pol 1901, Pol 1558 or Pol 963 DNA polymerases are described, for example, in U.S. Patent Publication Nos. 2020/0131484 A1 and 2020/0181587 A1, both of which are incorporated by reference herein. Additional polymerases that may be used in the method include those disclosed in U.S. Ser. Nos.
  • the one or more buffering agents comprise a primary amine, a secondary amine, a tertiary amine, a natural amino acid, or a non-natural amino acid, or combinations thereof.
  • the buffering agents comprise ethanolamine or glycine, or a combination thereof.
  • the buffer agent comprises or is glycine.
  • the mutant of 9°N polymerase may be engineered for high efficient incorporation of the nucleotide with the 3 ⁇ -O-glycoside blocking group.
  • steps of removing labels and/or blocking groups i.e., steps (d) or (e’)
  • steps of removing labels and/or blocking groups also referred to as the cleaving step
  • steps of removing labels and/or blocking groups also referred to as the cleaving step
  • the cleavage step includes contacting the incorporated nucleotide and the copy polynucleotide strand with a cleavage solution comprising an enzyme described herein.
  • the cleavage solution comprises a catalyst or enzyme capable of cleaving the self-immolative linker group in accordance with the present disclosure.
  • the cleavage solution comprises an enzyme capable of cleaving the self-immolative linker group described herein.
  • the enzyme (e.g., those described in Table 1) is capable of triggering the self-immolative linker to self- immolate, resulting in the removal of the detectable moiety.
  • the cleavage solution comprises a catalyst or enzyme capable of cleaving the blocking group in accordance with the present disclosure.
  • the cleavage solution comprises an enzyme capable of cleaving the blocking group described herein.
  • the cleavable self- immolative linker and the blocking group are removed by the same enzyme.
  • the 3 ⁇ -OH blocking group and the detectable label are removed in a single step of reaction.
  • the self-immolative linker and the 3 ⁇ blocking group are removed in two separate steps.
  • the , the 3 ⁇ blocking group may be removed in a chemical reaction that is separate from the enzymatic reaction resulting in the cleavable of the self-immolative linker.
  • Such 3 ⁇ blocking group may include azidomethyl (- CH 2 N 3 ) or substituted azidomethyl (e.g., -CH(CHF 2 )N 3 or CH(CH 2 F)N 3 ), or allyl, each connecting to the 3’ oxygen atom of the ribose or deoxyribose moiety of the nucleotide.
  • 3 ⁇ blocking groups are disclosed in U.S. Publication No.2020/0216891 A1, which is incorporated by reference in its entirety.
  • Non-limiting examples of the 3 ⁇ blocking group include: (AOM), , , each covalently attached to the 3 ⁇ carbon of the ribose or [0100]
  • the 3 ⁇ blocking group may be removed or deprotected by a chemical reagent to generate a free hydroxy group, for example, in the presence of a water- soluble phosphine reagent.
  • Non-limiting examples include tris(hydroxymethyl)phosphine (THMP), tris(hydroxyethyl)phosphine (THEP) or tris(hydroxylpropyl)phosphine (THP or THPP).
  • 3 ⁇ -acetal blocking groups described herein may be removed or cleaved under various chemical conditions.
  • non-limiting cleaving conditions include a Pd(II) complex, such as Pd chloride dimer, in the presence of a phosphine ligand, for example tris(hydroxymethyl)phosphine (THMP), or tris(hydroxylpropyl)phosphine (THP or THPP).
  • a Pd(II) complex such as Pd chloride dimer
  • a phosphine ligand for example tris(hydroxymethyl)phosphine (THMP), or tris(hydroxylpropyl)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).
  • a 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 [0101]
  • the 3’ blocking group described herein such as allyl or AOM 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 Na2PdCl4, Li2PdCl4, Pd(CH3CN)2Cl2, (PdCl(C3H5))2, [Pd(C3H5)(THP)]Cl, [Pd(C 3 H 5 )(THP) 2 ]Cl, Pd(OAc) 2 , Pd(Ph 3 ) 4 , Pd(dba) 2 , Pd(Acac) 2 , PdCl 2 (COD), Pd(TFA) 2 , Na2PdBr4, K2PdBr4, PdCl2, PdBr2, and Pd(NO3)2.
  • the Pd(0) complex is generated in situ from Na 2 PdCl 4 or K 2 PdCl 4 .
  • the palladium source is allyl palladium(II) chloride dimer [(PdCl(C3H5))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 THP, THMP, PTA, TCEP, bis(p- sulfonatophenyl)phenylphosphine dihydrate potassium salt, or 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 Na2PdCl4 or K2PdCl4 to THP is about 1:3.
  • the molar ratio of Na 2 PdCl 4 or K 2 PdCl 4 to THP is about 1:3.5.
  • the molar ratio of Na2PdCl4 or K2PdCl4 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), N,N,N′,N′- tetraethylethylenediamine (TEEDA), or 2-piperidine ethanol (also known as (2- hydroxyethyl)piperidine, having the structure ), or combinations thereof.
  • the buffer reagent comprises or 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.
  • the polymerase is a DNA polymerase as described herein.
  • the kit comprises a second enzyme for removing the detectable moiety of the nucleotide (i.e., through enzymatically triggered cleavage of the self- immolative linker).
  • the second enzyme can also remove the 3 ⁇ blocking group of the nucleotide.
  • the second enzyme is a glycoside hydrolase or glycosidase.
  • the second enzyme is an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an N-acetyl-glucosaminidase, or a glucuronidase.
  • the second enzyme is an L-arabinofuranosidase, a D-glucosidase, a D- mannosidase, a D-xylosidase, a D-galactosidase, an N-acetyl-D-glucosaminidase, or a D- glucuronidase.
  • the second enzyme is an ⁇ -L-arabinofuranosidase, a ⁇ -L-arabinofuranosidase, an ⁇ -D-glucosidase, a ⁇ -D-glucosidase, an ⁇ -D-mannosidase, a ⁇ -D- mannosidase, an ⁇ -D-xylosidase, a ⁇ -D-xylosidase, an ⁇ -D-galactosidase, a ⁇ -D-galactosidase, an ⁇ -N-acetyl-D-glucosaminidase, a ⁇ -N-acetyl-D-glucosaminidase, an ⁇ -D-glucuronidase, or a ⁇ - D-glucuronidase.
  • the kit further comprises a chemical reagent or a third enzyme for removing the 3 ⁇ blocking group of the nucleotide to generate a free hydroxy group.
  • the kit comprises a third enzyme for removing the blocking group of the nucleotide, wherein the third enzyme is in a separate compartment from the first enzyme.
  • the second enzyme is a glycoside hydrolase or glycosidase that is capable of catalyzing the hydrolysis of a –O–glycosidic bond of the nucleotide, for example a –O–glycosidic bond of the blocking group.
  • the third enzyme is an arabinofuranosidase, a glucosidase, a mannosidase, a xylosidase, a galactosidase, an N-acetyl-glucosaminidase, or a glucuronidase.
  • the third enzyme is an L-arabinofuranosidase, a D- glucosidase, a D-mannosidase, a D-xylosidase, a D-galactosidase, an N-acetyl-D- glucosaminidase, or a D-glucuronidase.
  • the third enzyme is an ⁇ -L- arabinofuranosidase, a ⁇ -L-arabinofuranosidase, an ⁇ -D-glucosidase, a ⁇ -D-glucosidase, an ⁇ -D- mannosidase, a ⁇ -D-mannosidase, an ⁇ -D-xylosidase, a ⁇ -D-xylosidase, an ⁇ -D-galactosidase, a ⁇ -D-galactosidase, an ⁇ -N-acetyl-D-glucosaminidase, a ⁇ -N-acetyl-D-glucosaminidase, an ⁇ -D- glucuronidase, or a ⁇ -D-glucuronidase.
  • the kit may comprise a chemical reagent for cleaving the 3 ⁇ blocking group, including those described herein with respect to the cleavage mix, such as a water soluble phosphine reagent and/or a palladium catalyst.
  • the kit may contain four types of labeled nucleotides (A, C, G and T or U; dATP, dCTP, dGTP and dTTP or dUTP), where one or more of the four types of nucleotides is labeled.
  • each of the four types of nucleotides can be labeled with a compound that is the same or different from the label on the other three nucleotides.
  • a first type of the four types of nucleotides carries a first label
  • a second type of nucleotides carries a second label
  • a third type of nucleotide carries a third label
  • a fourth nucleotide is unlabeled (dark).
  • a first type of nucleotide carries a first label
  • a second type of nucleotide carries a second label
  • a third nucleotide is a mixture of the third type of nucleotide carrying the first label and the third type of nucleotide carrying the second label
  • a fourth nucleotide is unlabeled (dark).
  • one or more of the label nucleotides can have a distinct absorbance maximum and/or emission maximum such that the compound(s) is(are) distinguishable from other compounds.
  • each compound can have a distinct absorbance maximum and/or emission maximum such that each of the compounds is spectrally distinguishable from the other three compounds (or two compounds if the fourth nucleotide is unlabeled). It will be understood that parts of the absorbance spectrum and/or emission spectrum other than the maxima can differ and these differences can be exploited to distinguish the compounds.
  • the kit may be such that two or more of the compounds have a distinct absorbance maximum.
  • the compounds, nucleotides, or kits that are set forth herein may be used to detect, measure, or identify a biological system (including, for example, processes or components thereof).
  • Exemplary techniques that can employ the compounds, nucleotides or kits include sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cellular assay (e.g., cell binding or cell function analysis), or protein assay (e.g., protein binding assay or protein activity assay).
  • the use may be on an automated instrument for carrying out a particular technique, such as an automated sequencing instrument.
  • the sequencing instrument may contain two light sources operating at different wavelengths (e.g., a blue light source at about 450 nm to about 460 nm and a green light source at about 520 nm to about 560 nm).
  • the sequencing instrument may contain a single light source (e.g., a blue light source at about 450 nm to about 460 nm, or a green light source at about 520 nm to about 560 nm).
  • the labeled nucleotide(s) described herein may be supplied in combination with unlabeled or native nucleotides, or any combination thereof.
  • kits comprise a plurality, particularly two, or three, or more particularly four, nucleotides
  • the different nucleotides may be labeled with different dye compounds, or one may be dark, with no dye compounds.
  • the dye compounds are spectrally distinguishable fluorescent dyes.
  • spectrally distinguishable fluorescent dyes refers to fluorescent dyes that emit fluorescent energy at wavelengths that can be distinguished by fluorescent detection equipment (for example, a commercial capillary-based DNA sequencing platform) when two or more such dyes are present in one sample.
  • fluorescent detection equipment for example, a commercial capillary-based DNA sequencing platform
  • the spectrally distinguishable fluorescent dyes can be excited at the same wavelength, such as, for example by the same light source.
  • two of the spectrally distinguishable fluorescent dyes can both be excited at one wavelength and the other two spectrally distinguishable dyes can both be excited at another wavelength.
  • Particular excitation wavelengths for the dyes are between 450–460 nm, 490–500 nm, or 520 nm or above (e.g., 523 nm or 532 nm).
  • one or more types of nucleotides are unlabeled, and wherein the first type of unlabeled nucleotides comprises a first functional moiety, and the kit further comprises a first labeling reagent, wherein the first labeling reagent comprises one or more first detectable labels and a first binding moiety that is capable of specific binding to the first functional moiety of the first type of unlabeled nucleotide.
  • the first functional moiety of the first type of unlabeled nucleotide is bound to the first labeling reagent by either covalent bonding or noncovalent interaction via a self-immolative linker described herein.
  • two or more types of nucleotides are unlabeled.
  • each of the four types of nucleotides is unlabeled, and wherein the second type of unlabeled nucleotides comprises a second functional moiety, and the kit further comprises a second labeling reagent, wherein the second labeling reagent comprises one or more second detectable labels and a second binding moiety that is capable of specific binding to the second functional moiety of the second type of unlabeled nucleotide.
  • the second functional moiety of the second type of unlabeled nucleotide is bound to the second labeling reagent by either covalent bonding or noncovalent interaction via a self-immolative linker described herein.
  • the third type of unlabeled nucleotides comprises a third functional moiety
  • the kit further comprises a third labeling reagent, wherein the third labeling reagent comprises one or more third detectable labels and a third binding moiety that is capable of specific binding to the third functional moiety of the third type of unlabeled nucleotide.
  • the third functional moiety of the third type of unlabeled nucleotide is bound to the third labeling reagent by either covalent bonding or noncovalent interaction via a self-immolative linker described herein.
  • the third type of unlabeled nucleotides comprises a mixture of the third type of unlabeled nucleotides comprising the first functional moiety and the third type of unlabeled nucleotides comprising the second functional moiety, and wherein both the first labeling reagent and the second labeling reagent are capable of specific binding to the third type of unlabeled nucleotides.
  • the fourth type of unlabeled nucleotides is not capable of specific binding with any of the first, second, or third labeling reagent. Post-incorporation labeling kits and methods have been described in U.S. Publication No. 2023/0383342 A1, which is incorporated by reference in its entirety.
  • Non-limiting examples of noncovalent interaction between a functional moiety of the nucleotide and a binding moiety of the labeling reagent include but are not limited to avidin (e.g., streptavidin or neutravidin) and biotin; dinitrophenyl (DNP) moiety and anti-DNP antibody; digoxigenin (DIG) and anti-DIG antibody; ⁇ -N-acetyl glucosamine (O-GlcNAc) and WGA (lectin); alkyl guanine moiety and SNAP-Tag®, alkyl chloride moiety and HaloTag®; 3-nitrotyrosine and anti-nitrotyrosine antibody; nickel or cobalt complex such as Ni-nitrilotriacetic acid (NTA) and His-Tag; zinc complex and oligo- aspartate protein.
  • avidin e.g., streptavidin or neutravidin
  • DNP dinitrophenyl
  • DIG digoxigenin
  • Non-limiting examples of covalent interaction between a functional moiety and a labeling reagent include but are not limited to a biorthogonal reaction 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.
  • one of the functional moiety and the binding moiety comprises or is norbornene, transcyclooctene (TCO), dibenzocyclooctyne (DBCO), or bicyclo[6.1.0]nonyne (BCN), and the other one of the functional moiety and the binding moiety comprises or is azido.
  • one of the functional moiety and the binding moiety comprises or is TCO
  • the other one of the functional moiety and the binding moiety comprises or is an optionally substituted 1,2,4,5-tetrazine moiety.
  • the sequencing methods described herein may also be carried out using unlabeled nucleotides and affinity reagents containing a fluorescent dye described herein.
  • the affinity reagents may bind to an incorporated nucleotide via the enzymatically cleavable linker (e.g., a self-immolative linker) of the incorporated nucleotide.
  • the enzymatically cleavable linker e.g., a self-immolative linker
  • one, two, three or each of the four different types of nucleotides e.g., dATP, dCTP, dGTP and dTTP or dUTP
  • Each of the four types of nucleotides has a 3 ⁇ blocking group to ensure that only a single base can be added by a polymerase to the 3 ⁇ end of the primer polynucleotide. After incorporation of an unlabeled nucleotide, the remaining unincorporated nucleotides are washed away. An affinity reagent is then introduced that specifically recognizes and binds to the incorporated dNTP to provide a labeled extension product comprising the incorporated dNTP. Uses of unlabeled nucleotides and affinity reagents in sequencing by synthesis have been disclosed in WO 2018/129214 and WO 2020/097607.
  • a modified sequencing method of the present disclosure using unlabeled nucleotides may include the following steps: (1) contacting a solid support with sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (2) contacting the solid support with an aqueous solution comprising DNA polymerase and one or more of four different types of unlabeled nucleotides (A, G, C and T or U) under conditions suitable for DNA polymerase-mediated primer extension, wherein each of the nucleotides comprises a 3′ blocking group and at least one type of nucleotide comprising an self-immolative linker in accordance with the present disclosure; (3) contacting the extended copy polynucleotides with a set of affinity reagents under conditions wherein one affinity reagent binds specifically to the incorporated unlabeled nucleotides to provide labeled extended copy polyn
  • the method further comprises removing the affinity reagents from the incorporated nucleotides.
  • removing the affinity reagents from the incorporated nucleotides comprises cleaving the self-immolative linkers.
  • the 3 ⁇ blocking group and the affinity reagent are removed in the same reaction.
  • the method further comprises a step (6) washing the solid support with a third aqueous wash solution.
  • steps (2) through (6) are repeated at least 50, 100, 150, 200, 250 or 300 cycles to determine the target polynucleotide sequences.
  • the set of affinity reagents may comprise a first affinity reagent that binds specifically to the first type of nucleotide, a second affinity reagent that binds specifically to the second type of nucleotide, and a third affinity reagent that binds specifically to the third type of nucleotide.
  • each of the first, second and the third affinity reagents comprises a detectable labeled that is spectrally distinguishable.
  • the affinity reagents may include protein tags, antibodies (including but not limited to binding fragments of antibodies, single chain antibodies, bispecific antibodies, and the like), aptamers, knottins, affimers, or any other known agent that binds an incorporated nucleotide with a suitable specificity and affinity.
  • at least one affinity reagent is an antibody or a protein tag.
  • at least one of the first type, the second type, and the third type of affinity reagents is an antibody or a protein tag comprising one or more detectable labels (e.g., multiple copies of the same detectable label), wherein the detectable label is or comprises a bis-boron dye moiety described herein.
  • Some embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) “Real-time DNA sequencing using detection of pyrophosphate release.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P.
  • PPi inorganic pyrophosphate
  • An image can be obtained after the array is treated with a particular nucleotide type (e.g., A, T, C or G). Images obtained after addition of each nucleotide type will differ with regard to which features in the array are detected. These differences in the image reflect the different sequence content of the features on the array. However, the relative locations of each feature will remain unchanged in the images.
  • the images can be stored, processed and analyzed using the methods set forth herein. For example, images obtained after treatment of the array with each different nucleotide type can be handled in the same way as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods.
  • cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in WO 04/018497 and U.S. Pat. No. 7,057,026, the disclosures of which are incorporated herein by reference.
  • This approach is being commercialized by Solexa (now Illumina, Inc.), and is also described in WO 91/06678 and WO 07/123,744, each of which is incorporated herein by reference.
  • the labels do not substantially inhibit extension under SBS reaction conditions.
  • the detection labels can be removable, for example, by cleavage or degradation. Images can be captured following incorporation of labels into arrayed nucleic acid features. In particular embodiments, each cycle involves simultaneous delivery of four different nucleotide types to the array and each nucleotide type has a spectrally distinct label.
  • each image can then be obtained, each using a detection channel that is selective for one of the four different labels.
  • different nucleotide types can be added sequentially, and an image of the array can be obtained between each addition step.
  • each image will show nucleic acid features that have incorporated nucleotides of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature. However, the relative position of the features will remain unchanged in the images. Images obtained from such reversible terminator-SBS methods can be stored, processed and analyzed as set forth herein. Following the image capture step, labels can be removed, and reversible terminator moieties can be removed for subsequent cycles of nucleotide addition and detection.
  • Some embodiments can utilize detection of four different nucleotides using fewer than four different labels.
  • SBS can be performed utilizing methods and systems described in the incorporated materials of U.S. Pub. No. 2013/0079232.
  • a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in intensity for one member of the pair compared to the other, or based on a change to one member of the pair (e.g.
  • nucleic acid via chemical modification, photochemical modification or physical modification) that causes apparent signal to appear or disappear compared to the signal detected for the other member of the pair.
  • three of four different nucleotide types can be detected under particular conditions while a fourth nucleotide type lacks a label that is detectable under those conditions, or is minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc.).
  • Incorporation of the first three nucleotide types into a nucleic acid can be determined based on presence of their respective signals and incorporation of the fourth nucleotide type into the nucleic acid can be determined based on absence or minimal detection of any signal.
  • one nucleotide type can include label(s) that are detected in two different channels, whereas other nucleotide types are detected in no more than one of the channels.
  • the aforementioned three exemplary configurations are not considered mutually exclusive and can be used in various combinations.
  • An exemplary embodiment that combines all three examples, is a fluorescent-based SBS method that uses a first nucleotide type that is detected in a first channel (e.g. dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type that is detected in a second channel (e.g.
  • sequencing data can be obtained using a single channel.
  • the first nucleotide type is labeled but the label is removed after the first image is generated, and the second nucleotide type is labeled only after a first image is generated.
  • the third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images.
  • Some embodiments can utilize sequencing by ligation techniques. Such techniques utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides.
  • the oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize.
  • images can be obtained following treatment of an array of nucleic acid features with the labeled sequencing reagents. Each image will show nucleic acid features that have incorporated labels of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature, but the relative position of the features will remain unchanged in the images. Images obtained from ligation-based sequencing methods can be stored, processed and analyzed as set forth herein. Exemplary SBS systems and methods which can be utilized with the methods and systems described herein are described in U.S. Pat. Nos.
  • Some embodiments can utilize nanopore sequencing (Deamer, D. W. & Akeson, M. “Nanopores and nucleic acids: prospects for ultrarapid sequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, “Characterization of nucleic acids by nanopore analysis”, Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, “DNA molecules and configurations in a solid-state nanopore microscope” Nat.
  • the target nucleic acid passes through a nanopore.
  • the nanopore can be a synthetic pore or biological membrane protein, such as ⁇ - hemolysin.
  • each base-pair can be identified by measuring fluctuations in the electrical conductance of the pore.
  • Some other embodiments of sequencing methods involve the use the 3 ⁇ blocked nucleotide described herein in nanoball sequencing technique, such as those described in U.S. Patent No. 9,222,132, the disclosure of which is incorporated by reference.
  • RCA rolling circle amplification
  • a large number of discrete DNA nanoballs may be generated.
  • the nanoball mixture is then distributed onto a patterned slide surface containing features that allow a single nanoball to associate with each location.
  • DNA nanoball generation DNA is fragmented and ligated to the first of four adapter sequences.
  • the template is amplified, circularized and cleaved with a type II endonuclease.
  • a second set of adapters is added, followed by amplification, circularization and cleavage.
  • the final product is a circular template with four adapters, each separated by a template sequence.
  • Library molecules undergo a rolling circle amplification step, generating a large mass of concatemers called DNA nanoballs, which are then deposited on a flow cell.
  • Goodwin et al. “Coming of age: ten years of next-generation sequencing technologies,” Nat Rev Genet. 2016;17(6):333-51.
  • Some embodiments can utilize methods involving the real-time monitoring of DNA polymerase activity.
  • Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and ⁇ - phosphate-labeled nucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and 7,211,414, both of which are incorporated herein by reference, or nucleotide incorporations can be detected with zero-mode waveguides as described, for example, in U.S. Pat. No. 7,315,019, which is incorporated herein by reference, and using fluorescent nucleotide analogs and engineered polymerases as described, for example, in U.S. Pat. No.7,405,281 and U.S. Pub. No.
  • FRET fluorescence resonance energy transfer
  • the illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, M. J. et al. “Zero-mode waveguides for single-molecule analysis at high concentrations.” Science 299, 682-686 (2003); Lundquist, P. M. et al. “Parallel confocal detection of single molecules in real time.” Opt. Lett.33, 1026-1028 (2008); Korlach, J. et al.
  • SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in U.S. Pub. Nos.
  • Methods set forth herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used for detecting protons. More specifically, methods set forth herein can be used to produce clonal populations of amplicons that are used to detect protons. [0124]
  • the above SBS methods can be advantageously carried out in multiplex formats such that multiple different target nucleic acids are manipulated simultaneously.
  • different target nucleic acids can be treated in a common reaction vessel or on a surface of a particular substrate.
  • the target nucleic acids can be in an array format.
  • the target nucleic acids can be typically bound to a surface in a spatially distinguishable manner.
  • the target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle or binding to a polymerase or other molecule that is attached to the surface.
  • the array can include a single copy of a target nucleic acid at each site (also referred to as a feature) or multiple copies having the same sequence can be present at each site or feature.
  • Multiple copies can be produced by amplification methods such as, bridge amplification or emulsion PCR as described in further detail below.
  • the methods set forth herein can use arrays having features at any of a variety of densities including, for example, at least about 10 features/cm 2 , 100 features/cm 2 , 500 features/cm 2 , 1,000 features/cm 2 , 5,000 features/cm 2 , 10,000 features/cm 2 , 50,000 features/cm 2 , 100,000 features/cm 2 , 1,000,000 features/cm 2 , 5,000,000 features/cm 2 , or higher.
  • An advantage of the methods set forth herein is that they provide for rapid and efficient detection of a plurality of target nucleic acid in parallel.
  • an integrated system of the present disclosure can include fluidic components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, the system comprising components such as pumps, valves, reservoirs, fluidic lines and the like.
  • a flow cell can be configured and/or used in an integrated system for detection of target nucleic acids. Exemplary flow cells are described, for example, in U.S. Pub. No.2010/0111768 and US Ser. No.13/273,666, each of which is incorporated herein by reference.
  • one or more of the fluidic components of an integrated system can be used for an amplification method and for a detection method.
  • one or more of the fluidic components of an integrated system can be used for an amplification method set forth herein and for the delivery of sequencing reagents in a sequencing method such as those exemplified above.
  • an integrated system can include separate fluidic systems to carry out amplification methods and to carry out detection methods. Examples of integrated sequencing systems that are capable of creating amplified nucleic acids and also determining the sequence of the nucleic acids include, without limitation, the MiSeq TM platform (Illumina, Inc., San Diego, CA) and devices described in US Ser.
  • Arrays in which polynucleotides have been directly attached to silica-based supports are those for example disclosed in WO 00/06770 (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 WO 2005/047301 (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 WO 00/31148, WO 01/01143, WO 02/12566, WO 03/014392, U.S. Pat. No. 6,465,178 and WO 00/53812, 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 WO 2005/065814, which is incorporated herein by reference.
  • 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). Attachment to beads or microparticles can be useful for sequencing applications. Bead libraries can be prepared where each bead contains different DNA sequences.
  • Templates 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.
  • the method of the disclosure is applicable to all types of high-density arrays, including single-molecule arrays, clustered arrays, and bead arrays.
  • Labeled nucleotides 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.
  • labeled nucleotides 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 WO 00/18957 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 the nucleotides labeled with dye compounds of the disclosure.
  • the labeled nucleotides 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.
  • cAbf-TFA O O O Br NHTFA NHTFA
  • the protected cABf linker (cABf-TFA) was synthesized in 6 steps from 4- hydroxy 2-bromo benzaldehyde, as described in Scheme 1. Briefly, 4-hydroxy 2-bromo benzaldehyde was glycosylated using tetraacetyl L-arabinofuranose and boron trifluoride diethyl etherate, to obtain the intermediate 1. This intermediate was functionalized with a 3-(N- trifluoroacetamido)propynyl group by Sonogashira cross-coupling with N- propargyltrifluoroacetamide to obtain compound 2.
  • intermediate 3 was treated with carbonyl diimidazole (CDI), followed by glycine methyl ester to introduce the carbamate group on the benzylic hydroxyl group (compounds 4).
  • CDI carbonyl diimidazole
  • glycine methyl ester to introduce the carbamate group on the benzylic hydroxyl group (compounds 4).
  • Full deprotection of intermediate 4 was accomplished by treating with aqueous LiOH.
  • cAbf-TFA was obtained by coupling compound 5 with 6-(trifluoroacetamido)hexanoic acid with TSTU as the coupling agent.
  • the final product cABf-TFA was characterized by 1 H NMR and MS.
  • reaction contained 50 ⁇ M c-ABF ffT and 5 ⁇ M enzyme in 100 mM sodium citrate pH 5.5.
  • the reaction was incubated for 0 or 1 minute at 60°C and 20 ⁇ l of sample was combined with 20 ⁇ l of 500 mM self-immolation buffer (CAPS pH 11) and incubated for 5 minutes at 60°C prior to analysis by HPLC (YMC Triart C18, 250 X 4.5 mL, S-5 ⁇ m, 12 nm, 0.1M TEAB/100% Acetonitrile) to monitor the production of the self-immolation product and depletion of intact c- Abf ffT.
  • HPLC YMC Triart C18, 250 X 4.5 mL, S-5 ⁇ m, 12 nm, 0.1M TEAB/100% Acetonitrile
  • FIGs. 1A and 1B are HPLC analysis of the resulting test samples.
  • FIG. 1B illustrates the resulting self-immolation product relative to the starting material after the c-ABF ffT was incubated in the presence of ABFase GH51 for 1 minute.
  • FIG. 1A illustrates the HPLC peak of the starting material c-ABF ffT as a negative control. The HPLC analysis showed that the c-ABF ffT was subjected to enzymatically triggered self-immolation.
  • each linker test substrate (AOL, LN3 and c-ABF) at 0.1 mM were made in a solution of 50 mM buffer (glycine pH 9.9 or sodium citrate pH 5.5), 50 mM NaCl, 6 mM MgSO4 and 1 mM EDTA (in glycine buffer solutions only). These were incubated in a heating block at 60°C in the dark for 16 days.
  • Example 4 Enzymatic Cleavage of Acetal-ABF Linker
  • a linker including an acetal leaving group with the ABF group (referred to herein as acetal-ABF or a-ABF) was investigated.
  • [ 0146] intended to model an acetal leaving similar conditions as in Example 1 followed by HPLC analysis were performed to monitor the reaction of interest of the model substrate.
  • FIGs.3A–3C plot HPLC analysis of the resulting test samples.

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Abstract

Des modes de réalisation de la présente divulgation concernent des molécules nucléotidiques ayant un groupe lieur auto-immolable clivable par voie enzymatique. L'invention concerne également des procédés et des kits pour des applications de séquençage utilisant de tels nucléotides.
PCT/US2025/026687 2024-04-29 2025-04-28 Nucléotides avec lieurs auto-immolables déclenchés par des enzymes pour le séquençage par synthèse Pending WO2025230914A1 (fr)

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Citations (90)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006678A1 (fr) 1989-10-26 1991-05-16 Sri International Sequençage d'adn
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
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
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
WO2001057248A2 (fr) 2000-02-01 2001-08-09 Solexa Ltd. Groupes de polynucleotides et leur utilisation dans le sequencage
US6274320B1 (en) 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
US6287768B1 (en) 1998-01-07 2001-09-11 Clontech Laboratories, Inc. Polymeric arrays and methods for their use in binding assays
US6288220B1 (en) 1998-03-05 2001-09-11 Hitachi, Ltd. DNA probe array
US6287776B1 (en) 1998-02-02 2001-09-11 Signature Bioscience, Inc. Method for detecting and classifying nucleic acid hybridization
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
US6306597B1 (en) 1995-04-17 2001-10-23 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
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
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
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
US6969488B2 (en) 1998-05-22 2005-11-29 Solexa, Inc. System and apparatus for sequential processing of analytes
US7001792B2 (en) 2000-04-24 2006-02-21 Eagle Research & Development, Llc Ultra-fast nucleic acid sequencing device and a method for making and using the same
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
US7211414B2 (en) 2000-12-01 2007-05-01 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
WO2007123744A2 (fr) 2006-03-31 2007-11-01 Solexa, Inc. Systèmes et procédés pour analyse de séquençage par synthèse
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US7329492B2 (en) 2000-07-07 2008-02-12 Visigen Biotechnologies, Inc. Methods for real-time single molecule sequence determination
US20080108082A1 (en) 2006-10-23 2008-05-08 Pacific Biosciences Of California, Inc. Polymerase enzymes and reagents for enhanced nucleic acid sequencing
WO2008069973A2 (fr) * 2006-12-01 2008-06-12 The Trustees Of Columbia University In The City Of New York Séquençage en quatre couleurs de l'adn par synthèse utilisant des terminateurs nucléotidiques réversibles, fluorescents et clivables
US7405281B2 (en) 2005-09-29 2008-07-29 Pacific Biosciences Of California, Inc. Fluorescent nucleotide analogs and uses therefor
US20090026082A1 (en) 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090127589A1 (en) 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100227327A1 (en) * 2008-08-08 2010-09-09 Xiaoliang Sunney Xie Methods and compositions for continuous single-molecule nucleic acid sequencing by synthesis with fluorogenic nucleotides
US20100282617A1 (en) 2006-12-14 2010-11-11 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays
WO2013041117A1 (fr) 2011-09-23 2013-03-28 Illumina Cambrigde Ltd. Colorants pour le marquage de ligands moléculaires
US20130079232A1 (en) 2011-09-23 2013-03-28 Illumina, Inc. Methods and compositions for nucleic acid sequencing
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
US9222132B2 (en) 2008-01-28 2015-12-29 Complete Genomics, Inc. Methods and compositions for efficient base calling in sequencing reactions
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
WO2018129214A1 (fr) 2017-01-04 2018-07-12 Complete Genomics, Inc. Séquençage par étapes par des terminateurs réversibles non marqués ou des nucléotides naturels
US20180201981A1 (en) 2016-12-22 2018-07-19 Illumina Cambridge Limited Coumarin compounds and their uses as fluorescent labels
US20200131484A1 (en) 2018-10-31 2020-04-30 Illumina, Inc. Polymerases, compositions, and methods of use
WO2020097607A1 (fr) 2018-11-09 2020-05-14 Mgi Tech Co., Ltd. Séquençage massivement parallèle à l'aide de nucléotides non marqués
US20200181587A1 (en) 2018-12-05 2020-06-11 Illumina, Inc. Polymerases, compositions, and methods of use
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
US20210188832A1 (en) 2017-10-16 2021-06-24 Illumina Cambridge Limited Secondary amine-substituted coumarin compounds and their uses as fluorescent labels
WO2021259881A1 (fr) * 2020-06-22 2021-12-30 Illumina Cambridge Limited Nucléosides et nucléotides avec groupe de blocage acétal 3'
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
US20230313292A1 (en) 2022-03-29 2023-10-05 Illumina Cambridge Limited Chromenoquinoline dyes and uses in sequencing
US20230332197A1 (en) 2022-03-31 2023-10-19 Illumina Singapore Pte. Ltd. Nucleosides and nucleotides with 3' vinyl blocking group
US20230383342A1 (en) 2022-05-31 2023-11-30 Illumina Cambridge Limited Compositions and methods for nucleic acid sequencing
US20230416279A1 (en) 2022-06-28 2023-12-28 Illumina Cambridge Limited Fluorescent dyes containing fused tetracyclic bis-boron heterocycle and uses in sequencing

Patent Citations (92)

* 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)
WO1991006678A1 (fr) 1989-10-26 1991-05-16 Sri International Sequençage d'adn
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
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
US6306597B1 (en) 1995-04-17 2001-10-23 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
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
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
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
US6969488B2 (en) 1998-05-22 2005-11-29 Solexa, Inc. System and apparatus for sequential processing of analytes
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
US6274320B1 (en) 1999-09-16 2001-08-14 Curagen Corporation Method of sequencing a nucleic acid
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
US7001792B2 (en) 2000-04-24 2006-02-21 Eagle Research & Development, Llc Ultra-fast nucleic acid sequencing device and a method for making and using the same
US7329492B2 (en) 2000-07-07 2008-02-12 Visigen Biotechnologies, Inc. Methods for real-time single molecule sequence determination
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
US7211414B2 (en) 2000-12-01 2007-05-01 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
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
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
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
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US7405281B2 (en) 2005-09-29 2008-07-29 Pacific Biosciences Of California, Inc. Fluorescent nucleotide analogs and uses therefor
US20100111768A1 (en) 2006-03-31 2010-05-06 Solexa, Inc. Systems and devices for sequence by synthesis analysis
WO2007123744A2 (fr) 2006-03-31 2007-11-01 Solexa, Inc. Systèmes et procédés pour analyse de séquençage par synthèse
US20080108082A1 (en) 2006-10-23 2008-05-08 Pacific Biosciences Of California, Inc. Polymerase enzymes and reagents for enhanced nucleic acid sequencing
WO2008069973A2 (fr) * 2006-12-01 2008-06-12 The Trustees Of Columbia University In The City Of New York Séquençage en quatre couleurs de l'adn par synthèse utilisant des terminateurs nucléotidiques réversibles, fluorescents et clivables
US20090026082A1 (en) 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090127589A1 (en) 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20100282617A1 (en) 2006-12-14 2010-11-11 Ion Torrent Systems Incorporated Methods and apparatus for detecting molecular interactions using fet arrays
US9222132B2 (en) 2008-01-28 2015-12-29 Complete Genomics, Inc. Methods and compositions for efficient base calling in sequencing reactions
US20100227327A1 (en) * 2008-08-08 2010-09-09 Xiaoliang Sunney Xie Methods and compositions for continuous single-molecule nucleic acid sequencing by synthesis with fluorogenic nucleotides
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20130079232A1 (en) 2011-09-23 2013-03-28 Illumina, Inc. Methods and compositions for nucleic acid sequencing
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
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
WO2018060482A1 (fr) 2016-09-30 2018-04-05 Illumina Cambridge Limited Nouveaux colorants fluorescents et leurs utilisations en tant que biomarqueurs
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
WO2018129214A1 (fr) 2017-01-04 2018-07-12 Complete Genomics, Inc. Séquençage par étapes par des terminateurs réversibles non marqués ou des nucléotides naturels
US20210188832A1 (en) 2017-10-16 2021-06-24 Illumina Cambridge Limited Secondary amine-substituted coumarin compounds and their uses as fluorescent labels
US20200131484A1 (en) 2018-10-31 2020-04-30 Illumina, Inc. Polymerases, compositions, and methods of use
WO2020097607A1 (fr) 2018-11-09 2020-05-14 Mgi Tech Co., Ltd. Séquençage massivement parallèle à l'aide de nucléotides non marqués
US20200181587A1 (en) 2018-12-05 2020-06-11 Illumina, Inc. Polymerases, compositions, and methods of use
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
WO2021259881A1 (fr) * 2020-06-22 2021-12-30 Illumina Cambridge Limited Nucléosides et nucléotides avec groupe de blocage acétal 3'
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
US20230313292A1 (en) 2022-03-29 2023-10-05 Illumina Cambridge Limited Chromenoquinoline dyes and uses in sequencing
US20230332197A1 (en) 2022-03-31 2023-10-19 Illumina Singapore Pte. Ltd. Nucleosides and nucleotides with 3' vinyl blocking group
US20230383342A1 (en) 2022-05-31 2023-11-30 Illumina Cambridge Limited Compositions and methods for nucleic acid sequencing
US20230416279A1 (en) 2022-06-28 2023-12-28 Illumina Cambridge Limited Fluorescent dyes containing fused tetracyclic bis-boron heterocycle and uses in sequencing

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
COCKROFT, S. LCHU, J.AMORIN, M.GHADIRI, M. R.: "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution", J. AM. CHEM. SOC., vol. 130, 2008, pages 818 - 820, XP055097434, DOI: 10.1021/ja077082c
DEAMER, D. W.AKESON, M.: "Nanopores and nucleic acids: prospects for ultrarapid sequencing", TRENDS BIOTECHNOL., vol. 18, 2000, pages 147 - 151, XP004194002, DOI: 10.1016/S0167-7799(00)01426-8
DEAMER, D.D. BRANTON: "Characterization of nucleic acids by nanopore analysis", ACC. CHEM. RES., vol. 35, 2002, pages 817 - 825, XP002226144, DOI: 10.1021/ar000138m
FODOR ET AL., TRENDS BIOTECH., vol. 12, 1994, pages 19 - 26
GOODWIN ET AL.: "Coming of age: ten years of next-generation sequencing technologies", NAT REV GENET., vol. 17, no. 6, 2016, pages 333 - 51, XP093225787, DOI: 10.1038/nrg.2016.49
HEALY, K.: "Nanopore-based single-molecule DNA analysis.", NANOMED., vol. 2, 2007, pages 459 - 481, XP009111262, DOI: 10.2217/17435889.2.4.459
KORLACH, J. ET AL.: "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures", PROC. NATL. ACAD. SCI. USA, vol. 105, 2008, pages 1176 - 1181
LEVENE, M. J. ET AL.: "Zero-mode waveguides for single-molecule analysis at high concentrations", SCIENCE, vol. 299, 2003, pages 682 - 686, XP002341055, DOI: 10.1126/science.1079700
LI, J.M. GERSHOWD. STEINE. BRANDINJ. A. GOLOVCHENKO: "DNA molecules and configurations in a solid-state nanopore microscope", NAT. MATER., vol. 2, 2003, pages 611 - 615, XP009039572, DOI: 10.1038/nmat965
LUNDQUIST, P. M. ET AL.: "Parallel confocal detection of single molecules in real time", OPT. LETT., vol. 33, 2008, pages 1026 - 1028, XP001522593, DOI: 10.1364/OL.33.001026
METZKER ET AL., NUCLEIC ACIDS RESEARCH, vol. 22, no. 20, 1994, pages 4259 - 4267
NATURE, vol. 437, 2005, pages 376 - 380
RONAGHI, M., KARAMOHAMED, S., PETTERSSON, B., UHLEN, M. AND NYREN, P.: "Real-time DNA sequencing using detection of pyrophosphate release", ANALYTICAL BIOCHEMISTRY, vol. 242, no. 1, 1996, pages 84 - 9, XP002388725, DOI: 10.1006/abio.1996.0432
RONAGHI, M.: "Pyrosequencing sheds light on DNA sequencing", GENOME RES., vol. 11, no. 1, 2001, pages 3 - 11, XP000980886, DOI: 10.1101/gr.11.1.3
RONAGHI, M.UHLEN, M.NYREN, P: "A sequencing method based on real-time pyrophosphate", SCIENCE, vol. 281, no. 5375, 1998, pages 363, XP002135869, DOI: 10.1126/science.281.5375.363
SCHEIT: "Nucleotide Analogs", 1980, JOHN WILEY & SON
SCIENCE, vol. 309, no. 5741, 2005, pages 1728 - 1732
SONI, G. V.MELLER, ''A.: "Progress toward ultrafast DNA sequencing using solid-state nanopores.", CLIN. CHEM., vol. 53, 2007, pages 1996 - 2001, XP055076185, DOI: 10.1373/clinchem.2007.091231
STIMPSON ET AL., PROC. NATL. ACAD. SCI., vol. 92, 1995, pages 6379 - 6383
UHLMAN ET AL., CHEMICAL REVIEWS, vol. 90, 1990, pages 543 - 584

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