WO2025230760A1 - Reversibly terminated nucleotides with modified phosphate chains, and methods of making and using the same - Google Patents

Abstract

In some examples, a method includes contacting a duplex between a first polynucleotide and a second polynucleotide with a plurality of nucleotides. Each nucleotide of the plurality may include a sugar; a nucleobase coupled to the sugar; an alpha phosphate group coupled to the sugar; and a label coupled to the alpha phosphate group. The method may include using a polymerase to couple a first nucleotide of the plurality to the duplex. The method may include detecting the label of the first nucleotide coupled to the duplex. The method may include decoupling the label from the alpha phosphate group of the first nucleotide.

Description

REVERSIBLY TERMINATED NUCLEOTIDES WITH MODIFIED PHOSPHATE

CHAINS, AND METHODS OF MAKING AND USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Patent Application No. 63/640.367. filed April 30, 2024 and entitled “Reversibly Terminated Nucleotides with Modified Phosphate Chains, and Methods of Making and Using the Same,” the entire contents of which are incorporated by reference herein.

FIELD

[0002] This application relates to nucleotides.

BACKGROUND

[0003] Modified nucleotides are key drivers of various sequencing technologies. For example, some sequencing technologies track the incorporation of fluorophore-labelled nucleotides. The sequence of DNA is determined by reading the emission from nucleotide-specific fluorophores during each incorporation cycle.

[0004] It may be desirable to modify nucleotides to include different moieties. Traditional methods of installing modifications may use intermediates that are costly and involve multi- step syntheses.

SUMMARY

[0005] Reversibly terminated nucleotides with modified phosphate chains, and methods of making and using the same, are provided herein.

[0006] Some examples herein provide a method. The method may include contacting a duplex between a first polynucleotide and a second polynucleotide with a plurality of nucleotides. Each nucleotide of the plurality' may include a sugar; a nucleobase coupled to the sugar; an alpha phosphate group coupled to the sugar; and a label coupled to the alpha phosphate group. The method may include using a polymerase to couple a first nucleotide of the plurality’ to the duplex. The method may include detecting the label of the first nucleotide coupled to the duplex. The method may include decoupling the label from the alpha phosphate group of the first nucleotide.

[0007] In some examples, decoupling the label from the alpha phosphate group leaves behind a PO4" or PO4H group to which the polymerase adds another nucleotide of the plurality.

[0008] In some examples, the label is coupled to the alpha phosphate group via a linker. In some examples, the linker has a length between about 1 nm and about 20 nm. In some examples, the linker is coupled to the alpha phosphate group via a cleavable group. In some examples, the cleavable group is the same as a reversible terminator coupled to a 3' position of the sugar.

[0009] In some examples, the sugar comprises ribose or deoxyribose.

[0010] In some examples, the method further includes, after decoupling the label from the alpha phosphate group of the first nucleotide, using a polymerase to couple a second nucleotide of the plurality to the first nucleotide.

[0011] In some examples, each nucleotide of the plurality further comprising a reversible terminator coupled to the sugar, the method comprising decoupling the reversible terminator from the sugar of the first nucleotide. In some examples, the label is decoupled from the alpha phosphate group of the first nucleotide using a first reagent, and wherein the reversible terminator is decoupled from the sugar of the first nucleotide using a second reagent. In some examples, the first reagent is the same as the second reagent. In some examples, the first reagent is different than the second reagent.

[0012] Some examples herein provide a composition. The composition may include a duplex between a first polynucleotide and a second polynucleotide. The composition may include a plurality of nucleotides in contact with the duplex. Each nucleotide of the plurality may include a sugar; a nucleobase coupled to the sugar; an alpha phosphate group coupled to the sugar; and a label coupled to the alpha phosphate group. The composition may include a polymerase to couple a first nucleotide of the plurality to the duplex.

[0013] In some examples, the label is decouplable from the alpha phosphate group of the first nucleotide using a reagent. In some examples, decoupling the label from the alpha phosphate group leaves behind a POi" or PO4H group to which the polymerase adds another nucleotide of the plurality.

[0014] In some examples, the label is coupled to the alpha phosphate group via a linker. In some examples, the linker has a length between about 1 nm and about 20 nm. In some examples, the linker is coupled to the alpha phosphate group via a cleavable group. In some examples, the cleavable group is the same as a reversible terminator coupled to a 3' position of the sugar.

[0015] In some examples, the sugar comprises ribose or deoxyribose.

[0016] In some examples, the polymerase is further to couple a second nucleotide of the plurality to the first nucleotide.

[0017] In some examples, each nucleotide of the plurality further comprising a reversible terminator coupled to the sugar, the reversible terminator being decouplable from the sugar of the first nucleotide. In some examples, the label is decouplable from the alpha phosphate group of the first nucleotide using a first reagent, and wherein the reversible terminator is decouplable from the sugar of the first nucleotide using a second reagent. In some examples, the first reagent is the same as the second reagent. In some examples, the first reagent is different than the second reagent.

[0018] It is to be understood that any respective features/examples of each of the aspects of the disclosure as described herein can be implemented together in any appropriate combination, and that any features/examples from any one or more of these aspects can be implemented together with any of the features of the other aspect(s) as described herein in any appropriate combination to achieve the benefits as described herein.

BRIEF DESCRIPTION OF DRAWINGS

[0019] FIG. 1 A schematically illustrates a previously known reversibly terminated nucleotide.

[0020] FIG. I B schematically illustrates an example structure formed during sequencing-by- synthesis (SBS) using the reversibly terminated nucleotide of FIG. 1A.

[0021] FIG. 2A schematically illustrates an example reversibly terminated nucleotide with modified phosphate chain provided herein. [0022] FIG. 2B schematically illustrates an example structure formed during SBS using the reversibly terminated nucleotide of FIG. 2A.

[0023] FIGS. 3A-3F schematically illustrate example reversibly terminated nucleotides with different example modified phosphate chains provided herein.

[0024] FIG. 4 schematically illustrates an example structure formed during SBS using another example reversibly terminated nucleotide.

[0025] FIGS. 5A-5E schematically illustrate additional example reversibly terminated nucleotide with different example modified phosphate chains provided herein.

[0026] FIG. 6 illustrates a flow of operations in an example method of using a reversibly terminated nucleotide such as described with reference to FIGS. 2A-2B, 3A-3F, 4, or 5A-5E.

[0027] FIGS. 7A-7F illustrate example methods of making reversibly terminated nucleotides such as described with reference to FIGS. 2A-2B and 3A-3F.

[0028] FIGS. 8A-8D illustrate example methods of making reversibly terminated nucleotides such as described with reference to FIGS. 4 and 5A-5E.

DETAILED DESCRIPTION

[0029] Disclosed herein are reversibly terminated nucleotides with modified phosphate chains, and methods of making and using the same. It may be desirable to incorporate modifications into the phosphate chains of nucleotides. For example, modifying the phosphate chains of nucleoside triphosphates may allow the tracking of the enzymatic synthesis of DNA.

Illustratively, phosphate chains including labels, such as dyes (e.g., fluorophores) may be used to track the synthesis.

[0030] As noted above, some previous methods for synthesizing modified nucleotides use advanced intermediates that are costly and involve multi-step syntheses. Other previous methods involve the functionalization of triphosphate substrates. This can lead to phosphate chain degradation, which in turn may lead to poor overall yields. In comparison, the presently disclosed nucleotides with a modified polyphosphate chain may be made without the use advanced intermediates. Instead, commercially available nucleosides or nucleotide monophosphates can be used as a starting point. Further, modifications that previously used multi-step syntheses can be prepared separately and installed en masse on the nucleotide using the methods provided herein. Still further, the number of steps after formation of the triphosphate are reduced herein, which may improve overall yields over the yields of previous methods. Additionally, the present nucleotides may be configured so that the dye coupled to the triphosphate chain may be removed without leaving behind any residual moiety coupled to the growing phosphodiester chain of the polynucleotide. As such, the present nucleotides are expected to decrease error rates in SBS.

[0031] After explaining selected terms used in the present application, nonlimiting examples of the present reversibly terminated nucleotides with modified phosphate chains will be provided, as will nonlimiting examples of their uses and their methods of manufacture.

Terms

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term ■‘including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have,” “has,” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, 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.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but can include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but can also include additional features or components.

[0033] The terms “substantially”, “approximately”, and “about” used throughout this Specification are used to describe and account for small fluctuations, such as due to variations in processing. For example, they can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%. [0034] As used herein, terms such as “covalently coupled” or “covalently bonded” refer to the forming of a chemical bond that is characterized by the sharing of pairs of electrons between atoms. For example, a covalently coupled molecule refers to a molecule that forms chemical bonds with a substrate, as compared to coupling to the surface via other means, for example, a non-covalent bond such as electrostatic interaction.

[0035] The term “halogen” or “halo.” as used herein, means fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being examples.

[0036] As used herein, “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 4 carbon atoms. The alkyl group may be designated as “Ci-4 alkyl” or similar designations. By way of example only, “Ci-4 alkyl” or “Ci-4alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

[0037] As used herein, “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 4 carbon atoms. The alkenyl group may be designated as “C2-4 alkenyl” or similar designations. By way of example only, “C2-4 alkenyl” indicates that there are two to four carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-l-yl, propen-2-yl, propen-3-yl, buten-l-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-l-yl, 2-methyl- propen-l-yl, 1-ethyl-ethen-l-yl, 2-methyl-propen-3-yL buta-1, 3-dienyl, buta-l,2,-dienyl, and buta-l,2-dien-4-yl. Typical alkenyl groups include, but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and the like.

[0038] Groups that include an alkenyl group include optionally substituted alkenyl, cycloalkenyl, and heterocycloalkenyl groups.

[0039] As used herein, “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 “alky nyl” 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 4 carbon atoms. The alkynyl group may be designated as “C2-4 alkynyl” or similar designations. By way of example only, “C2-4 alkynyl” or “C2-4alkynyl” indicates that there are two to four carbon atoms in the alky nyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-l-yl, propyn-2-yl, butyn-l-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.

[0040] Groups that include an alkynyl group include optionally substituted alkynyl, cycloalkynyl. and heterocycloalkynyl groups.

[0041] As used herein, “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 examples, the aryl group has 6 to 10 carbon atoms. The aryl group may be designated as “Ce-io aryl,” “Ce or C10 aryl,” or similar designations. Examples of aryl groups include, but are not limited to. phenyl, naphthyl, azulenyl, and anthracenyl.

[0042] As used herein, “heterocycle” refers to a cyclic compound which includes atoms of carbon along with another atom (heteroatom), for example nitrogen, oxygen or sulfur. Heterocycles may be aromatic (heteroaryl) or aliphatic. An aliphatic heterocycle may be completely saturated or may contain one or more or two or more double bonds, for example the heterocycle may be a heterocycloalkyl. The heterocycle may include a single heterocyclic ring or multiple heterocyclic rings that are fused.

[0043] As used herein, “heteroaryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the ring backbone. When the heteroaryl is a ring system, every’ ring in the system is aromatic. The heteroaryl group may have 5-18 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heteroaryl” where no numerical range is designated. In some examples, 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. Examples of 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, isoquinlinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl. isoindolyl, and benzothienyl.

[0044] As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0045] As used herein, “cycloalkenyl” or “cycloalkene” means a carbocyclyl ring or ring system having at least one double bond, wherein no ring in the ring system is aromatic. An example is cyclohexenyl or cyclohexene. Another example is norbomene or norbomenyl.

[0046] As used herein, “heterocycloalkenyl” or “heterocycloalkene” means a carbocyclyl ring or ring system with at least one heteroatom in ring backbone, having at least one double bond, wherein no ring in the ring system is aromatic. In some examples, heterocycloalkenyl or heterocycloalkene ring or ring system is 3 -membered. 4-membered. 5-membered, 6- membered, 7-membered, 8-membered, 9-membered, or 10-membered.

[0047] As used herein, “cycloalkynyl” or “cycloalkyne” means a carbocyclyl ring or ring system having at least one triple bond, wherein no ring in the ring system is aromatic. An example is cyclooctyne. Another example is bicyclononyne. Another example is dibenzocyclooctyne (DBCO). [0048] As used herein, “heterocycloalkynyl” or “heterocycloalkyne” means a carbocyclyl ring or ring system with at least one heteroatom in ring backbone, having at least one triple bond, wherein no ring in the ring system is aromatic. In some examples, heterocycloalkynyl or heterocycloalkyne ring or ring system is 3-membered, 4-membered, 5-membered, 6- membered, 7-membered, 8-membered, 9-membered, or 10-membered.

[0049] As used herein, “heterocycloalkyl” means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocycloalkyls may be joined together in a fused, bridged or spiro-connected fashion. Heterocycloalkyls may have any degree of saturation provided that at least one heterocyclic ring in the ring system is not aromatic. The heterocycloalkyl 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 “heterocycloalkyl” where no numerical range is designated. The heterocycloalkyl group may also be a medium size heterocycloalkyl having 3 to 10 ring members. The heterocycloalkyl group could also be a heterocycloalkyl having 3 to 6 ring members. The heterocycloalkyl group may be designated as “3-6 membered heterocycloalkyl” or similar designations. In some six membered monocyclic heterocycloalkyls, the heteroatom(s) are selected from one up to three of O, N or S, and in some five membered monocyclic heterocycloalkyls. the heteroatom(s) are selected from one or two heteroatoms selected from O, N, or S. Examples of heterocycloalkyl rings include, but are not limited to, azepinyl, acridinyL carbazolyL cinnolinyl, dioxolanyL imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3- oxathianyl, 1.4-oxathiinyl, 1.4-oxathianyl, 277-1.2-oxazinyl. trioxanyl, hexahydro- 1,3, 5- triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3 -dithiolyl, 1,3-dithiolanyL isoxazolinyL isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazoli dinonyl, thiazolinyl, thiazolidinyl, 1,3- oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl. tetrahydro- 1.4-thiazinyl. thiamorpholinyl. dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

[0050] As used herein, the term “nucleoside” is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some examples also includes a nucleobase. A nucleoside that lacks a nucleobase can be referred to as “abasic.” Nucleosides include deoxyribonucleosides, modified deoxyribonucleosides, ribonucleosides, modified ribonucleosides, peptide nucleosides, modified peptide nucleosides, modified phosphate sugar backbone nucleosides, and mixtures thereof. Examples of nucleosides include adenosine, thymidine, cytidine, guanosine, uridine, deoxyadenosine, deoxythymidine, deoxycytidine, deoxyguanosine, and deoxyuridine.

[0051] As used herein, the term "‘nucleoside” also is intended to encompass any nucleoside analogue which is a type of nucleoside that includes a modified nucleobase and/or sugar compared to naturally occurring nucleosides. Example modified nucleobases include inosine, xanthine, hypoxanthine, 5 -methylcytosine, 5-hydroxymethyl cytosine, 2-aminoadenine, 6- methyl adenine, 6-methyl guanine, 4-thiouracil, 8-hydroxyl adenine or guanine, 7- methylguanine. 7-methyladenine, 8-azaguanine. 8-azaadenine, or the like. Example modified nucleobases also include isocytosine, isoguanine, 2-aminopurine 2-propyl guanine, 2-propyl adenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-thiouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine 5-halo substituted uracil or cytosine, 7-deazaguanine, 7-deazaadenine, 3- deazaguanine, 3-deazaadenine or the like.

[0052] As used herein, the term “nucleotide” is intended to mean a molecule that includes a sugar and at least one of a phosphate group, a phosphoramidate, and a phosphorothioate. In some examples a nucleotide also includes a nucleobase. A nucleotide that lacks a nucleobase can be referred to as “abasic.” Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides adenosine triphosphate (ATP), thymidine triphosphate (TTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxy cytidine triphosphate (dCTP). deoxyguanosine triphosphate (dGTP). and deoxyuridine triphosphate (dUTP).

[0053] As used herein, the term “nucleotide” also is intended to encompass any nucleotide analogue which is a type of nucleotide that includes a modified nucleobase. sugar and/or phosphate moiety compared to naturally occurring nucleotides. Example modified nucleobases include inosine, xanthine, hypoxanthine, 5-methylcytosine, 5 -hydroxymethyl cytosine, 2- aminoadenine, 6-methyl adenine, 6-methyl guanine, 4-thiouracil, 8-hydroxyl adenine or guanine, 7-methylguanine, 7-methyladenine. 8-azaguanine, 8-azaadenine. or the like. Example modified nucleobases also include isocytosine, isoguanine, 2-aminopurine 2-propyl guanine, 2- propyl adenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-thiouracil, 5-halocytosine, 5- propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine. 8-thioalkyl adenine or guanine 5-halo substituted uracil or cytosine. 7-deazaguanine, 7-deazaadenine, 3- deazaguanine, 3-deazaadenine or the like. As is known in the art, certain nucleotide analogues cannot become incorporated into a polynucleotide, for example, nucleotide analogues such as adenosine '-phosphosulfate. Nucleotides can include any suitable number of phosphates, e.g.. three, four, five, six, or more than six phosphates.

[0054] As used herein, the term “polynucleotide” refers to a molecule that includes a sequence of nucleotides that are bonded to one another. A polynucleotide is one nonlimiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), and analogues thereof. A polynucleotide can be a single stranded sequence of nucleotides, such as RNA or single stranded DNA, a double stranded sequence of nucleotides, such as double stranded DNA, DNA that is folded to form a hairpin that is partially single stranded and partially double stranded, double-stranded amalgamations in which there are molecules that are non-covalently coupled to one another (e.g., via reversible hydrogen binding), and/or can include a mixture of a single stranded and double stranded sequences of nucleotides. Double stranded DNA (dsDNA) includes genomic DNA. and PCR and amplification products. Single stranded DNA (ssDNA) can be converted to dsDNA and vice-versa. Polynucleotides can include non-naturally occurring DNA, such as enantiomeric DNA. The precise sequence of nucleotides in a polynucleotide can be known or unknow n. The following are examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, expressed sequence tag (EST) or serial analysis of gene expression (SAGE) tag), genomic DNA. genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer or amplified copy of any of the foregoing. [0055] As used herein, a “polymerase” is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind a primer and a single stranded target polynucleotide, and can sequentially add nucleotides to the growing primer to form a “complementary copy” polynucleotide having a sequence that is complementary to that of the target polynucleotide. DNA polymerases may bind to the target polynucleotide and then move down the target polynucleotide sequentially adding nucleotides to the free hydroxyl group at the 3' end of a growing polynucleotide strand. DNA polymerases may synthesize complementary DNA molecules from DNA templates. RNA polymerases may synthesize RNA molecules from DNA templates (transcription). Other RNA polymerases, such as reverse transcriptases, may synthesize cDNA molecules from RNA templates. Still other RNA polymerases may synthesize RNA molecules from RNA templates, such as RdRP. Polymerases may use a short RNA or DNA strand (primer), to begin strand growth. Some polymerases may displace the strand upstream of the site where they are adding bases to a chain. Such polymerases may be said to be strand displacing, meaning they have an activity that removes a complementary strand from a template strand being read by the polymerase.

[0056] Example DNA polymerases include Bst DNA polymerase, 9° Nm DNA polymerase, Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I (Large), (Klenow) fragment, Klenow fragment (3'-5' exo-), T4 DNA polymerase, T7 DNA polymerase, Deep VentR™ (exo-) DNA polymerase. Deep VentR™ DNA polymerase, DyNAzyme™ EXT DNA, DyNAzyme™ II Hot Start DNA Polymerase, Phusion™ High-Fidelity DNA Polymerase, Therminator™ DNA Polymerase, Therminator™ II DNA Polymerase, VentR® DNA Polymerase, VentR® (exo-) DNA Polymerase, RepliPHI™ Phi29 DNA Polymerase, rBst DNA Polymerase. rBst DNA Polymerase (Large), Fragment (IsoTherm™ DNA Polymerase), MasterAmp™ AmpliTherm™, DNA Polymerase, Taq DNA polymerase, Tth DNA polymerase, TH DNA polymerase, Tgo DNA polymerase, SP6 DNA polymerase, Tbr DNA polymerase, DNA polymerase Beta, ThermoPhi DNA polymerase, and Isopol™ SD+ polymerase. In specific, nonlimiting examples, the polymerase is selected from a group consisting of Bst, Bsu, and Phi29. Some polymerases have an activity that degrades the strand behind them (3' exonuclease activity)- Some useful polymerases have been modified, either by mutation or otherwise, to reduce or eliminate 3' and/or 5' exonuclease activity. [0057] Example RNA polymerases include RdRps (RNA dependent, RNA polymerases) that catalyze the synthesis of the RNA strand complementary to a given RNA template. Example RdRps include polioviral 3Dpol, vesicular stomatitis virus L, and hepatitis C virus NS5B protein. Example RNA Reverse Transcriptases. A non-limiting example list to include are reverse transcriptases derived from Avian Myelomatosis Virus (AMV), Murine Moloney Leukemia Virus (MMLV) and/or the Human Immunodeficiency Virus (HIV), telomerase reverse transcriptases such as (hTERT), SuperScript™ III, SuperScript™ IV Reverse Transcriptase, ProtoScript® II Reverse Transcriptase.

[0058] The terms “polynucleotide” and “oligonucleotide” are used interchangeably herein. The different terms are not intended to denote any particular difference in size, sequence, or other property unless specifically indicated otherwise. For clarity of description the terms can be used to distinguish one species of polynucleotide from another when describing a particular method or composition that includes several polynucleotide species.

[0059] As used herein, the term “polymer” refers to a molecule including many repeated subunits or recurring units. Non-limiting examples of polymer structures include linear, branched, or hyper-branched polymers. Polymers as described herein can be linear, branched, hyper-branched or dendritic. Different classes of polymer backbones include, but are not limited to, polyacrylamides, polyacrylates, polyurethanes, polysiloxanes, silicones, polyacroleins, polyphosphazenes, polyisocyanates, poly-ols, polysaccharides, polypeptides, and combinations thereof. A polymer can include one or more moieties that can react with one or more other moieties to form a covalent bond.

[0060] All ranges may include the upper and lower values, and all ranges and ratio limits disclosed herein may be combined. It is to be understood that unless specifically stated otherwise, references to “a,” “an,” and/or “the” may include one or more than one and that reference to an item in the singular may also include the item in the plural. Unless otherwise indicated, the terms “first,” “second.” etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to, e.g., a “second” item does not require or preclude the existence of, e.g., a “first” or lower-numbered item, and/or, e.g.. a “third” or higher-numbered item. Further, reference to, e.g., a “first” item and a “second” item does not mean that there are no intervening items, and such intervening items may be present. Reversibly terminated nucleotides with modified phosphate chains, and methods of using the same

[0061] Some commercially available SBS platforms use an approach in which nucleotides are modified at two positions. First, a reversible terminator is coupled to the 3'-OH of the nucleotide’s sugar, which inhibits a polymerase from adding another nucleotide to an extended primer until the reversible terminator is removed. Second, a dye is coupled to the nucleotide’s nitrogenous base, which allows the base which was added to be optically identified. For example, FIG. 1A schematically illustrates a previously know n reversibly terminated nucleotide 100. FIG. IB schematically illustrates an example structure formed during SBS using the reversibly terminated nucleotide of FIG. 1A. As may be seen in FIG. 1A. nucleotide 100 includes sugar 110, nucleobase 112. and triphosphate group 114. Nucleobase 1 12 may be bound to the 1 ' carbon of sugar 1 10. Triphosphate group 1 14 may be coupled to the 5' carbon of sugar 110. The 3'-O- of sugar 110 may be coupled to a reversible terminator 124 via bond 125, e.g., an azidomethyl moiety in the example shown in FIG. 1 A. Nucleobase 112 is coupled to dye 126 via an elongated group including first portion 120 including dye 126, and second portion 122 which is coupled to first portion 120 at bond 121 and to nucleobase 122 via bond 127.

[0062] During SBS, a polymerase (not shown) adds a particular nucleotide 100 to the 3' end of an extended primer, based on the sequence of a template strand. More specifically, a duplex between first polynucleotide 140 and second polynucleotide 150 is contacted with a plurality of nucleotides 100 having different bases 112 than one another. Based on the sequence of template strand 140, the polymerase couples the alpha phosphate of triphosphate group 114 of a nucleotide 100 to the 3'-OH of a previously added nucleotide of second polynucleotide 150, and releases pyrophosphate. The dye 126 then is optically detected, and used to identity the nucleobase 112 which was added. The dye 126 then is removed using a first chemical reagent that decouples first portion 120 from second portion 122 at bond 121 to form modified second portion 122’ which remains coupled to the nucleobase as shown in FIG. IB via bond 127. Additionally, the reversible terminator 124 is removed using the same chemical reagent as the removal of dye 126, or a second chemical reagent that decouples reversible terminator 124 from sugar 110 at bond 125 to generate a 3 '-OH group. Another nucleotide 100 may be added in similar fashion to the 3'-OH group, the dye 126 of which is then detected and removed, the reversible terminator similarly removed, and the process then repeated. [0063] As shown in FIG. IB, as the respective dyes are removed from different nucleobases being added to the 3' end of the extended primer, modified second portion 122’ remains coupled to the nucleobase 112. The residual modified second portion 122' may be referred to as “scarring.” Without wishing to be bound by any theory, it is believed that the buildup of modified second portions 122’ (that is, the buildup of scarring) on the extended primer may increase the physical demand on the polymerase as it works to incorporate into the extended primer additional nucleotides, which themselves bring additional scarring. As such, and without wishing to be bound by any theory, it is believed that the buildup of modified second portions 122’ (that is, the buildup of scarring) may cause an increase in the error rate with increased length of sequencing. Additionally, the structural and synthetic complexity associated with modifying the nucleobase as shown in FIG. 1 A, coupled with a demand for relatively high purity and manufacturing scalability of the nucleotides, increases the cost of SBS.

[0064] As provided herein, these and other problems are solved using the presently provided nucleotides, which are modified at two locations. First, similarly as shown in FIG. 1A. a reversible terminator is coupled to the present nucleotide’s 3' carbon of the sugar, which inhibits a polymerase from adding another nucleotide to an extended primer until the reversible terminator is removed. Second, rather than coupling the label (e.g., dye) to the nitrogenous base as in FIG. 1 A, the label may be coupled to the present nucleotide’s alpha phosphate group via a linker. The label is detected, which allows the nucleobase which was added to be identified, e.g., optically identified in a similar manner as described with reference to FIGS. 1 A-1B. As provided herein, a reagent may be used to remove the label without leaving any scarring on the nucleobase. and indeed without leaving any scarring on any other portion of the nucleotide. More specifically, rather than leaving behind a residual modified second portion 122’ as described with reference to FIGS. 1 A-1B, the dye is removed from the present alpha phosphate group by completely removing the linker so as to leave behind a standard POT group to which the polymerase readily may add another such nucleotide. Because the nucleobase need not be modified, and because no residual group remains attached to the nucleotide that otherwise may cause polymerase errors such as described with reference to FIGS. 1 A-1B, the error rate of the SBS is expected to decrease. As such, the length of sequencing reads with satisfactory⁷ error rates are therefore expected to increase. [0065] In some examples, the present nucleotides may provide still further benefits over nucleotides such as described with reference to FIGS. 1A-1B. For example, in the present nucleotides, the linker used to couple the label (e.g., dye) to the alpha phosphate group may have any suitable length without concern for scarring (and thus for concern about such scarring causing subsequent nucleotide addition issues), because the linker is completely removed. As such, the linker may be made sufficiently long to reduce or inhibit the nucleobase from quenching the label (e.g.. dye). This feature may be used, for example, to couple labels to G nucleotides which otherwise are known to quench fluorophores and thus may otherwise be left ■‘dark.” As another example, in some examples of the present nucleotides, the same reagent may be used to remove both the reversible terminator at the 3' carbon of the sugar, and the linker and dye coupled to the alpha phosphate group. This feature may be used to decrease the cost of SBS, because two different reagents need not be used to separately remove the dye and the reversible terminator in a manner such as described with reference to FIGS. 1 A-1B. Furthermore, this feature may be used to decrease the time needed to complete an SBS cycle, because two different fluidic cycles need not be used to separately remove the dye and the reversible terminator in a manner such as described with reference to FIGS. 1 A-1B.

[0066] As will be discussed below, example nucleotides provided herein may include a sugar, a nucleobase coupled to the sugar, an alpha phosphate group coupled to the sugar, and a label coupled to the alpha phosphate group. A polymerase may be used to couple the nucleotide to a duplex between a first polynucleotide and a second polynucleotide in a manner similar to that described with reference to FIGS. 1 A-1B. The label of the nucleotide coupled to the duplex may be detected. The label then may be decoupled from the alpha phosphate group of the first nucleotide. In some examples, decoupling the label from the alpha phosphate group leaves behind a POT or PO4H group to which the polymerase adds another such nucleotide.

[0067] Various examples of the present nucleotides, and examples of their use, now will be described with reference to FIGS. 2A-2B, 3A-3F, 4, 5A-5E, and 6. Various example methods of making the present nucleotides will be described further below with reference to FIGS. 7A- 7F and 8A-8D.

[0068] FIG. 2A schematically illustrates an example reversibly terminated nucleotide with modified phosphate chain provided herein. FIG. 2B schematically illustrates an example structure formed during SBS using the reversibly terminated nucleotide of FIG. 2A. [0069] As may be seen in FIG. 2A, nucleotide 200 includes sugar 210, nucleobase 212, and alpha phosphate group 215. In the nonlimiting example shown in FIG. 2 A, alpha phosphate group 215 is part of a triphosphate group 214. Nucleobase 212 may be bound to the 1' carbon of sugar 210, and may include any suitable naturally occurring nucleobase, or may include a modified nucleobase, some nonlimiting examples of which are described elsewhere herein. The 3' carbon of sugar 210 may be coupled to -OR group 224 via a respective bond (bond not specifically illustrated). In examples in which nucleotide 200 is reversibly terminated, the R group may be or include a reversible terminator such as described elsewhere herein (e.g.. in Table 1), or otherw ise known in the art. However, while many examples described herein refer to the present nucleotides as being “reversibly terminated,” it will be appreciated that the nucleotides need not necessarily be reversibly terminated. For example, the R group may be H. Sugar 210 may include any suitable number of carbons, e.g., may be or include a ribose or deoxyribose, or may be configured as described elsewhere herein.

[0070] Reversible terminator 224 may be removable using any suitable reagent(s) and condition(s). Non-limiting examples of reversible terminators 224 that may be used in nucleotide 200, and suitable reagents for removing such reversible terminators, are listed in Table 1:

Table 1

[0071] In contrast to nucleobase 1 12 described with reference to FIGS. 1 A-1 B, nucleobase 212 may lack any label (e.g., dye). Instead, in nucleotide 200, label (e.g., dye 226, such as a fluorescent dye) may be coupled to alpha phosphate group 215, and the alpha phosphate group 215 may be coupled to the 5' carbon of sugar 210. More specifically, as illustrated in FIG. 2A. label (e.g., dye) 226 may be coupled to alpha phosphate 215 via cleavable group Y 221. Cleavable group Y may include any suitable heteroatom (e.g., O or N) which is directly coupled to the phosphorous atom of alpha phosphate group 215. Cleavable group Y may be coupled to label (e.g., dye) 226 via any suitable via linker 225. In various examples, linker 225 may include a saturated or unsaturated alkyl chain, a polymer, or both a saturated or unsaturated alkyl chain and a polymer. Nonlimiting examples of polymers suitable for use in linker 225 include a synthetic peptide or a synthetic polymer. Nonlimiting examples of a synthetic polymer include an ethylene glycol polymer, which may be referred to as poly(ethylene glycol). In various examples in which linker 225 includes a polymer, the polymer may include about 1 to about 50 repeating units, illustratively about 1 to about 10 repeating units, or about 11 to about 20 repeating units, or about 21 to about 50 repeating units. In various examples in which linker 225 includes an alkyl chain of n methylene units, n may be any suitable number, e.g., may be about 1 to about 50, illustratively about 1 to about 10, or about 11 to about 20, or about 21 to about 50. Linker 225 coupling label (e.g., dye) 226 to cleavable group Y 221 may have any suitable length, e.g., a length of about 1 to about 20 nm. Indeed, as noted further above, because the linker is completely removed after label (e.g., dye) 226 is detected, the linker may be made sufficiently long to reduce or inhibit the nucleobase from quenching the label (e.g., dye).

[0072] During SBS, a polymerase (not shown) adds a particular nucleotide 200 to the 3' end of an extended primer, based on the sequence of a template strand. More specifically, in a manner similar to that described with reference to FIGS. 1 A-l B, a duplex between a first polynucleotide (template strand, not shown in FIG. 2A) and second polynucleotide (extended primer) 150 is contacted with a plurality of nucleotides 200 having different bases 212 than one another. As illustrated in FIG. 2B, based on the sequence of the template strand (not specifically illustrated), the polymerase couples the alpha phosphate 215 of triphosphate group 214 of a nucleotide 200 to the 3'-OH of a previously added nucleotide of second polynucleotide 150, and releases pyrophosphate. The dye 226 then is optically detected ("Image" operation shown in FIG. 2B), and used to identify the nucleobase 212 which was added. The dye 226 then is removed using a first chemical reagent that decouples linker 225 from alpha phosphate 215 and leaves a standard POT or PCLH group coupled to the sugar as shown in FIG. 2B. Additionally, the reversible terminator 224 is removed using a second chemical reagent that decouples reversible terminator 224 from sugar at bond 225 to generate a 3 '-OH group.

[0073] In some examples, the cleavable group Y 221 is the same as a reversible terminator group R 224 coupled to a 3'-O- of the sugar. As such, in these examples, the first reagent is the same as the second reagent, meaning that only a single reagent need be used to remove the dye and the reversible terminator, and that such removals occur approximately simultaneously. In other examples, the cleavable group Y 221 is different than the reversible terminator group R 224 coupled to a 3' carbon of the sugar, but a single reagent still may be used to remove the dye and the reversible terminator, and such removals may occur approximately simultaneously. As noted above, this feature may decrease the cost of SBS, because two different reagents need not be used to separately remove the label (e.g., dye) and the reversible terminator, and also may decrease the time needed to complete an SBS cycle, because two different fluidic cycles need not be used to separately remove the label (e.g., dye) and the reversible terminator. However, in still other examples, the cleavable group Y 221 is different than the reversible terminator group R 224 coupled to a 3' carbon of the sugar; different first and second reagents are used to respectively remove the dye and the reversible terminator; and such removals may occur at different times than one another (e.g., where the first and second reagents are applied in sequence), or may occur approximately simultaneously (e.g.. where the first and second reagents are applied at the same time). Another nucleotide 200 may be added in similar fashion to the 3'-OH group, the dye 226 of which is then detected and removed, the reversible terminator similarly removed, and the process then repeated.

[0074] In contrast to FIG. IB, in which modified second portion 122’ remains coupled to the nucleobase 112, label (e.g., dye) is removed without leaving any scarring on the nucleobase, and indeed without leaving any scarring on any other portion of the nucleotide. Because no residual group remains attached to the nucleotide (whether on the nucleobase or otherwise) that otherwise may cause polymerase errors such as described with reference to FIGS. 1 A-1B, the error rate of the SBS is expected to decrease. As such, the length of sequencing reads with satisfactory error rates are therefore expected to increase.

[0075] Some non-limiting examples of cleavable group Y 221 and linker 225 now will be described with reference to FIGS. 3A-3F. Still further variations will be described with reference to FIGS. 4 and 5A-5E.

[0076] FIGS. 3A-3F schematically illustrate example reversibly terminated nucleotides with different example modified phosphate chains provided herein. In the nonlimiting example illustrated in FIG. 3A, cleavable group Y is azidomethyl. In the nonlimiting example illustrated in FIG. 3B, cleavable group Y is AOM. In the nonlimiting example illustrated in FIG. 3C, cleavable group Y is methylvinyl. In the nonlimiting example illustrated in FIG. 3D. cleavable group Y is vinyl. In the nonlimiting example illustrated in FIG. 3E, cleavable group Y is phosphoroamidate. Nonlimiting examples of reagent(s) that respectively may be used to remove such cleavable groups Y are provided in Table 1. Nonlimiting examples of reversible terminator groups R 224 are illustrated in FIG. 3F or are listed in Table 1. FIGS. 3A-3E illustrate a nonlimiting example in which linkers 225 include an alkyl chain (e.g., n-butyl group), as well as moiety 310 coupling the dye 226 to the alkyl chain. In the nonlimiting example illustrated in FIGS. 3A-3E, moiety 310 is or includes an amide bond. However, it will be appreciated that interactions between any suitable hapten and hapten-binding partner may be used to couple the dye 226 to any suitable linker 225. Nonlimiting examples of haptens and hapten-binding partners are provided in Table 2.

Table 2

[0077] In examples in which cleavable group Y is of the same type as reversible terminator R, it will be appreciated that the same reagent(s) may be used to remove such groups approximately simultaneously with one another. Illustratively, in nonlimiting examples in which both Y and R are AZM, THP, TCEP, or other suitable phosphine may be used to remove dye 226 and reversible terminator R 224 at approximately the same time as one another, without the need for separate reagents or separate fluidic operations. In nonlimiting examples in which both Y and R are AOM, Pd(O) may be used to remove dye 226 and reversible terminator R 224 at approximately the same time as one another, without the need for separate reagents or separate fluidic operations. In nonlimiting examples in which both Y and R are methylvinyl, Pd(0) may be used to remove dye 226 and reversible terminator R 224 at approximately the same time as one another, without the need for separate reagents or separate fluidic operations. In nonlimiting examples in which both Y and R are vinyl, tetrazine may be used to remove dye 226 and reversible terminator R 224 at approximately the same time as one another, without the need for separate reagents or separate fluidic operations. In nonlimiting examples in which both Y and R are amine, buffered nitrous acid may be used to remove dye 226 and reversible terminator R 224 at approximately the same time as one another, without the need for separate reagents or separate fluidic operations. It will be appreciated that in other examples, Y and R may be of different types than one another, and may be removed using different reagents than one another, or in some cases may be removed using the same reagent as one another. For example, Pd(O) may be used as a reagent in examples where one of Y and R is AOM and the other of Y and R is methylvinyl. Or, for example, buffered nitrous acid may be used as a reagent in examples where one of Y and R is vinyl and the other of Y and R is amine.

[0078] While FIGS. 3A-3D illustrate nonlimiting examples in which the cleavable group Y 221 is directly bonded to an oxygen of the alpha phosphate group 215, other variations are contemplated. For example, FIG. 3E illustrates an example in which the cleavable group Y includes a secondary amine group that is directly bonded to the phosphorous atom of the alpha phosphate group. Still further variations now will be descnbed with reference to FIGS. 4 and 5A-5E. Regardless of the particular variation which is used, the present cleavable groups Y 221 may be removed using a reagent that leaves behind a natural alpha phosphate group (POE or PO4H).

[0079] FIG. 4 schematically illustrates an example structure formed during SBS using another example reversibly terminated nucleotide. In the nonlimiting example show n in FIG. 4, label 226 (e.g., dye) is coupled to the alpha phosphate group via an imino phosphate moiety. More specifically, in this example cleavable group Y may include an imino group (=N-) which is coupled to (i) the phosphorous atom of the alpha phosphate group, and (ii) moiety X, which in various examples may be or include -SO2-, -C(=O), -CH=CH-, -CH2-, -phenyl-, -guanidine-, or a heterocyclic analogue (e.g., a heterocycle with a ring structure formed by three, four, five, six, or more than six atoms). In some examples, regardless of the particular moiety X which is used, the same group of cleavage reagents may be used, although the kinetics and efficiency of the cleavage may differ. In some examples, varying degrees of heat may be used to tune the cleavage kinetics to be similar, regardless of the particular reagent and particular moiety X which are used. The moiety X may be coupled to label 226 via a linker (not specifically illustrated) which may be configured in a manner such as described elsewhere herein. Dye 226 may be removable using any suitable reagent(s) and condition(s). Non-limiting examples of reagents that may be used to remove dye 226 by replacing the imino-X group with an oxygen to form a phosphate group (POT or PO4H) include acidic buffers, acids, metal salts, and/or heat. In some examples, the acidic buffer is selected from the group consisting of TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid), CAPSO (3-(Cyclohexylamino)-2- hydroxy-1 -propanesulfonic acid), AMPSO (N-(l,l-Dimethyl-2-hydroxyethyl)-3-amino-2- hydroxypropanesulfonic acid), CHES (2-(Cyclohexylamino)ethanesulfonic acid), bis-tris- propane (1.3-Bis[tris(hydroxymethyl)methylamino]propane), ethanolamine, HEPES (4-(2- hy droxy ethyl)- 1 -piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), and borate. In some examples, the acid is selected from the group consisting of HC1 and H2SO4. In some examples, the metal is selected from the group consisting of silver salt (e.g., AgNOs). palladium salt, copper salt, cadmium salt, or gold salt.

[0080] FIGS. 5A-5E schematically illustrate additional example reversibly terminated nucleotides with different example modified phosphate chains provided herein. In the nonlimiting example illustrated in FIG. 5A, moiety X is -(C=O)-. In the nonlimiting example illustrated in FIG. 5B, moiety X is -CH=CH-. In the nonlimiting example illustrated in FIG. 5C, moiety X is -CH2-. In the nonlimiting example illustrated in FIG. 5D, moiety' X is -SO2- phenyl-. In the nonlimiting example illustrated in FIG. 5E, moiety X is -phenyl- which is optionally fully or partially fluorinated. Nonlimiting examples of reversible terminator groups R 224 are listed in Table 1. FIGS. 5A-5E illustrate a nonlimiting example in which linkers 225 include an alkyl chain (e.g., w-butyl group), as well as moiety 310 coupling the dye 226 to the alkyl chain. Interactions between any suitable hapten and hapten-binding partner may be used to couple the dye 226 to any suitable linker (not specifically illustrated) which is coupled to moiety X. Nonlimiting examples of haptens and hapten-binding partners are provided in Table 2.

[0081] FIG. 6 illustrates a flow of operations in an example method of using a reversibly terminated nucleotide such as described with reference to FIGS. 2A-2B, 3A-3F, 4, or 5A-5E. [0082] Method 600 illustrated in FIG. 6 includes contacting a duplex between a first polynucleotide and a second polynucleotide with a plurality of nucleotides (operation 610). Each nucleotide of the plurality’ may include a sugar. For example, in a manner such as described with reference to FIGS. 2A-2B, 3A-3F, 4, and 5A-5F, nucleotide 200 may include sugar 210. Nonlimiting examples of the sugar include ribose and deoxyribose. Other nonlimiting examples are provided elsewhere herein. Each nucleotide of the plurality' also may include a nucleobase coupled to the sugar. For example, in a manner such as described with reference to FIGS. 2A-2B, 3A-3F, 4. and 5A-5F, nucleotide 200 may include nucleobase 212. In some examples, the nucleobase is a naturally occurring nucleobase. In other examples, the nucleobase is a non-naturally occurring nucleobase. In either of such cases, the nucleobase may lack any label, in contrast to nucleobase 112 described with reference to FIGS. 1A-1B. Each nucleotide of the plurality’ also may include an alpha phosphate group coupled to the sugar, and a label coupled to the alpha phosphate group. For example, in a manner such as described with reference to FIGS. 2A-2B, 3A-3F, 4, and 5A-5F, nucleotide 200 may include alpha phosphate group 215 to which label (e g., dye) 226 is coupled, for example via linker 225 which is disposed between alpha phosphate group 215 and label 226.

[0083] Method 600 also may include using a polymerase to couple a first nucleotide of the plurality' to the duplex (operation 620). For example, in a manner such as described with reference to FIGS. 2A-2B, and 4, the polymerase may couple a first nucleotide 200 of the plurality to an extended primer 150, based on the sequence of a template strand, in a manner similar to that described with reference to FIGS. 1 A-1B but using nucleotide 200 instead of nucleotide 100. In some examples, the nucleotide may include a reversible terminator that inhibits the polymerase from coupling a second nucleotide of the plurality to the first nucleotide, unless and until the reversible terminator of the first nucleotide is removed.

[0084] Method 600 may include detecting the label of the first nucleotide coupled to the duplex (operation 630). For example, the label may include a dye that is optically detected using suitable circuitry. Optionally, the dye may’ fluoresce in response to optical excitation, and the circuitry detects the fluorescence. The detected label may be used to identify the particular nucleobase of the nucleotide. For example, different labels respectively may be coupled to nucleotides with different nucleobases than one another. Method 600 also may include decoupling the label from the alpha phosphate group of the first nucleotide (operation 640). For example, in a manner such as described with reference to FIGS. 2A-2B, 3A-3F, 4, and 5A-5F, the cleavable group Y may be cleaved so as to remove label 226 from nucleotide 200. In some examples, decoupling the label from the alpha phosphate group leaves behind a PO4" or PO4H group to which the polymerase may add another nucleotide of the plurality.

[0085] Further example configurations of nucleotide 200 now will be described.

[0086] In various examples, sugar 210 may include a five-carbon sugar. Illustratively, sugar 210 may include a natural sugar, such as ribose or deoxyribose. In other examples, sugar 210 may include a six carbon sugar, e.g., pyranose. Optionally, sugar 210 is a non-naturally occurring sugar, such as threose (as in threose nucleic acid, TNA). In various examples, sugar 210 may include an acyclic sugar moiety, e.g.. may include glycerol (as in glycol nucleic acid, GNA). In various examples, sugar 210 may include a fluorine atom or a methoxy group bound to the 2' carbon of the sugar. Note that sugar 210 may be, but not necessarily be, cyclic. From the examples herein, it may be understood that sugar 210 illustratively may be a three-carbon sugar, a four-carbon sugar, a five-carbon sugar, or a six carbon sugar, and may be cyclic or acyclic.

[0087] In various examples, nucleobase 212 may include a naturally occurring nucleobase such as adenine, cytosine, guanine, thymine, or uracil. It will be appreciated, however, that other naturally occurring nucleobases may be used. For example, nucleobase 212 may include a methylated nucleobase such as 5-methylcytosine, 5-hydroxymethylcytosine, 5- formylcytosine, 5-carboxylcytosine, 4-methylcytosine, 6-methyladenine, 8-oxoguanine, or 8- oxoadenine. In some examples, nucleobase 212 may include inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl guanine, 4-thiouracil. 8-hydroxyl adenine, 8-hydroxyl guanine, 7- methylguanine, 7-methyladenme, 8-azaguanine, 8-azaadenme, or the like. In other examples, nucleobase 112 may include a non-naturally occurring nucleobase, such as 5-(l,6- heptadiynyl)uracil, 5-(2-carboxyvinyl)uracil, 5-(l,6-heptadiynyl)cytosine, 8-(l,6- diaminohexany l)adenine. 2-( 1 ,6-diaminohexany l)guanine. 5-(7-( 1 ,2,3-triazole)hept- 1 - ynyl)uracil. 5-(N-(6-aminohexyl)acrylamide)uracil, 5-(methylacrylamido)uracil, 5-(N- allylmethylamino)uracil, 5-(N-allylacetamidyl)uracil, 5-(7-(l ,2,3-triazole)hept-l - ynyl)cytosine, 5-(methylacetamido)cytosine, 8-(l ,6-diaminohexanyl)adenine, or 2-(l,6- diaminohexanyl)guanine. In some examples, nucleobase 112 may include isocytosine, isoguanine, 2-aminopurine, 2-propyl guanine, 2-propyl adenine. 2-thiouracil, 2-thiothymine, 2- thiocytosine, 5-thiouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine, 8-thiol guanine, 8-thioalkyl adenine, 8-thioalkyl guanine, 5-halo substituted uracil, 5-halo substituted cytosine, 7-deazaguanine, 7-deazaadenine. 3- deazaguanine, 3-deazaadenine, or the like.

[0088] In various examples, nucleotide 200 may include a substituent (not specifically illustrated) coupled between a beta phosphate and a gamma phosphate of triphosphate group 214. Without wishing to be bound by any theory, it is believed that such a substituent may affect the stability of nucleotide 200. Illustratively, and without wishing to be bound by any theory, it is believed that the presence of a modification on the alpha phosphate may alter the electron density across the phosphate groups, potentially resulting in instability, and that a substituent coupled between the beta phosphate and the gamma phosphate of triphosphate group 214 may potentially rebalance the electron density to regain sufficient stability of the triphosphate against degradation (loss of phosphate groups). In nonlimiting examples, such a substituent may be or include an oxygen atom, a selenium atom, a sulfur atom, an amine, a methylene, a fluoromethylene, a difluoromethylene, a dichloromethylene, or a dibromomethylene.

Methods of making reversibly terminated nucleotides with modified phosphate chains

[0089] It will be appreciated that nucleotides such as described with reference to FIGS. 2A-2B, 3A-3F, 4, and 5A-5E may be prepared in any suitable manner, some nonlimiting examples of which will now be provided.

[0090] The general synthesis workflow for introducing the alpha phosphate group to the nucleotide via phosphoramidite chemistry is shown below :

[0091] In this synthetic scheme, the alcohol precursor is reacted with a phosphorus(III) chloride reagent and undergoes SN2 reaction to replace the chloride substituent. The phosphorus(III) intermediate is activated with 4,5-dicyanoimidazole (DCI), or 5-(ethylthio)- IH-tetrazole (ETT) or 5-benzylthio-lH-tetrazole (BTT), allowing the 5 -OH nucleoside to form a phosphorus(III) nucleoside. The phosphorus(III) nucleoside is further oxidized to the air-stable phosphorus(V) nucleoside using tert-butyl hydroperoxide (TBHP). Subsequent treatment with di ethylamine removes the cyanoethyl protecting group on the phosphorus group to form the desired nucleoside monophosphate with the alpha phosphorus substituent.

[0092] FIGS. 7A-7F illustrate example methods of making reversibly terminated nucleotides such as described with reference to FIGS. 2A-2B and 3A-3F. FIG. 7A shows an example method in which the alcohol precursor with a tert-butyloxy carbonyl protected amine group is reacted with a phosphorus(III) chloride reagent and undergoes SN2 reaction to replace the chloride substituent. The phosphorus(III) intermediate is activated with 4.5-dicyanoimidazole (DCI), or 5-(ethylthio)-lH-tetrazole (ETT) or 5-benzylthio-lH-tetrazole (BTT), allowing the 5 ’-OH nucleoside to form a phosphorus(III) nucleoside. The phosphorus(III) nucleoside is further oxidized to the air-stable phosphorus(V) nucleoside using te/7-butyl hydroperoxide (TBHP). Subsequent treatment with diethylamine removes the cyanoethyl protecting group on the phosphorus group to form the desired nucleoside monophosphate with the alpha phosphorus substituent. Acidification removes the /e/7-butylo\ carbonyl protecting group, and the free amine is subjected to a diazotransfer reaction to form an azido group. Deprotection of the 3‘-O-ter/-butyldiphenylsilyl nucleoside monophosphate with tetra-n-butylammonium fluoride (TBAF) form the 3 ’-OH, which is treated with a mixture of acetic acid and acetic anhydride in dimethylsulfoxide (DMSO) to form the 3’-O-methylthiomethyl (MTM) protecting group. Chlorination of the 3’-0-MTM group with sulfuryl chloride followed by sodium azide converts the MTM group to an azidomethyl (AZM) group. Subsequent activation of the monophosphate group and quenching with pyrophosphate forms the 3 ’-AZM nucleotide triphosphate. The terminal NHTFA group is deprotected with ammonium hydroxide and coupled to an activated A-hydroxysuccinimidyl ester dye conjugate to yield the desired dye labelled nucleotide triphosphate.

[0093] FIG. 7B show s an example method in which an aliphatic alcohol precursor is oxidized to the corresponding aldehyde, following by a nucleophilic addition with vinyl magnesium bromide to afford the allyl alcohol intermediate. The allyl alcohol is reacted with a phosphorus(III) chloride reagent to form the phosphorus(III) intermediate which is activated with 4,5-dicyanoimidazole (DCI), or 5-(ethylthio)-lH-tetrazole (ETT) or 5-benzylthio-lH- tetrazole (BTT), allowing the 5 ’-OH nucleoside to form a phosphorus(III) nucleoside. The phosphorus(III) nucleoside is further oxidized to the air-stable phosphorus(V) nucleoside using /e/7-butyl hydroperoxide (TBHP). Subsequent treatment with diethylamine removes the cyanoethyl protecting group on the phosphorus group to form the nucleoside monophosphate with the alpha phosphorus substituent. Subsequent activation of the monophosphate group and quenching with pyrophosphate forms the 3’-A0M nucleotide triphosphate. The terminal NHTFA group on the alpha phosphate arm is deprotected with ammonium hydroxide and coupled to an activated A-hydroxysuccinimidyl ester dye conjugate to yield the desired dye labelled nucleotide triphosphate.

[0094] FIG. 7C shows an example method in which a 3’-alkoxime monophosphate nucleoside is activated with A. A -di cyclohexylcarbodiimide (DCC) and cross coupled with an aliphatic amine linker in the presence of tri ethylamine and tert-butanol in water to form the phosphoramidate intermediate. Subsequent activation of the monophosphate group and quenching with pyrophosphate forms the 3’-alkoxime nucleotide triphosphate. The alkoxime group is removed with aqueous hydroxylamine solution (HONH2) to form the oxyamine group. The terminal NHTFA group on the alpha phosphate arm is deprotected with ammonium hydroxide and coupled to an activated A-hydroxysuccinimidyl (NHS) ester dye conjugate to yield the desired dye labelled nucleotide triphosphate.

[0095] FIG. 7D shows an example method in which a nucleotide triphosphate with a modified alpha phosphate P-0 handle may be prepared. The 5’-OH nucleoside is first activated with dicyanoimidazole (DCI), or 5-(ethylthio)-lH-tetrazole (ETT) and coupled to a reactive phosphorus(III) reagent. Oxidation of the phosphorus(III) to the air-stable phosphorus(V) is achieved by treating with iodine solution, and further reaction with 1.8- Diazabicyclo[5.4.0]undec-7-ene (DBU) removes the cyanoethyl protecting group on the phosphorus center to form the modified nucleotide monophosphate. Activation of the phosphate group with trifluoroacetic anhydride and treating with 1 -methylimidazole (NMI) forms an imidazolinium intermediate. A subsequent SN2 reaction with pyrophosphate (PPi) and quenching of the reaction with triethylammonium acetate (TEAA) affords the desired nucleotide triphosphate.

[0096] FIG. 7E shows an example method in which a nucleotide triphosphate with a modified alpha phosphate P-N handle may be prepared. The nucleotide monophosphate is activated with M A -dicyclohexylcarbodiimide (DCC) and coupled to an aliphatic amine linker in the presence of triethylamine and tert-butanol in water to form the phosphorami date monophosphate intermediate. The phosphorami date group is activated with 1,1 ’ -carbonyldiimidazole (CDI) in pyridine and quenched with pyrophosphate (PPi) to form the desired nucleotide triphosphate.

[0097] FIG. 7F shows an example method in which a nucleotide triphosphate with a modified alpha phosphate P-C handle may be prepared. The phosphate linker is activated with N,N’- diisopropyl carbodiimide (DIC) in the presence of diisopropylethylamine (DIPEA) and 4- dimethylaminopyridine (DMAP), and coupled to the 5 ’-OH nucleoside to form the nucleoside intermediate. Further treatment with 1,1 ’-carbonyldiimidazole (CDI) in pyridine and quenching with pyrophosphate (PPi) affords the desired nucleotide triphosphate.

[0098] FIGS. 8A-8D illustrate example methods of making reversibly terminated nucleotides such as described with reference to FIGS. 4 and 5A-5E. FIG. 8 A shows an example method in which a nucleotide triphosphate with an alpha phosphorus-imino bond may be prepared. The 5 ’-OH nucleoside is treated with a phosphorus(III) reagent to form the phosphorus(III) nucleoside, and subsequent Staudinger reaction with an azido linker provides the air-stable phosphorus-imino nucleotide monophosphate. The monophosphate is coupled to an activated pyrophosphate intermediate to form the desired phosphorus-imino nucleotide triphosphate. In another method, the 5 ’-OH nucleoside can be treated with a cyclic phosphite reagent to form a reactive cyclic phosphite nucleoside. A Staudinger reaction with an azido linker and subsequent hydrolysis will form the desired phosphorus-imino nucleotide triphosphate. In another method, the 5 ’-OH nucleoside can be treated with a cyclic phosphite ester reagent to form a reactive cyclic phosphite ester nucleoside. A Staudinger reaction with an azido linker and subsequent hydrolysis will form the desired phosphorus-imino nucleotide triphosphate.

[0099] FIG. 8B shows an example method in which a 3’-O-vinyl nucleotide triphosphate with a phosphorus-imino modification may be prepared. The 5 ’-OH nucleotide is protected with te/7-butylchlorodiphenylsilane (TBDPSC1) and the 3 ’-acetyl group is deprotected with ammonium hydroxide (NH4OH). The 3 ’-OH intermediate can either be treated with 1,2- dichloroethane followed by sodium te/7-butoxide, or treated with potassium vinyltrifluoroborate followed by te/7-butylammonium fluoride, to form the 3'-O-vinyl nucleoside. The nucleoside is reacted with a phosphorus(III) reagent and subjected to Staudinger reaction with an azido linker. The fluorenylmethoxy (Fm) protecting groups are removed by treatment with piperidine. Sequential reaction with activated dipyridinium pyrophosphate, hydrolysis, and dye conjugation will form the dye-labelled 3’-O-vinyl nucleotide triphosphate.

[0100] FIG. 8C shows an example method in which a 3’-allyloxymethyl (AOM) nucleotide triphosphate with a phosphorus-imino modification may be prepared. The 5 ’-OH nucleoside is reacted with a phosphorus(III) reagent and subjected to Staudinger reaction with an azido linker. The fluorenylmethoxy (Fm) protecting groups are removed by treatment with piperidine. Sequential reaction with activated pyridinium pyrophosphate, hydrolysis, and dye conjugation will form the dye-labelled 3’-A0M nucleotide triphosphate.

[0101] FIG. 8D shows an example method in which a 3 ’-aminooxy nucleotide triphosphate with a phosphorus-imino modification may be prepared. The 5 ’-OH nucleoside is reacted with a phosphorus(III) reagent and subjected to Staudinger reaction with an azido linker. The fluorenylmethoxy (Fm) protecting groups are removed by treatment with piperidine. The nucleotide monophosphate is sequentially treated with activated dipyridinium pyrophosphate, hydrolysis, and dye conjugation. The 3’-oxime protecting group is removed with aqueous hydroxylamine solution (HONH2) to form the dye-labelled 3 ’-aminooxy nucleotide triphosphate.

Additional comments

[0102] It is to be understood that any respective features/examples of each of the aspects of the disclosure as described herein can be implemented together in any appropriate combination, and that any features/examples from any one or more of these aspects can be implemented together with any of the features of the other aspect(s) as described herein in any appropriate combination to achieve the benefits as described herein.

[0103] While various illustrative examples are described above, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the invention. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method, comprising: contacting a duplex between a first polynucleotide and a second polynucleotide with a plurality of nucleotides, each nucleotide of the plurality comprising: a sugar; a nucleobase coupled to the sugar; an alpha phosphate group coupled to the sugar; and a label coupled to the alpha phosphate group; using a polymerase to couple a first nucleotide of the plurality' to the duplex; detecting the label of the first nucleotide coupled to the duplex; and decoupling the label from the alpha phosphate group of the first nucleotide.

2. The method of claim 1, wherein decoupling the label from the alpha phosphate group leaves behind a POT or PO4H group to which the polymerase adds another nucleotide of the plurality.

3. The method of claim 1 or claim 2, wherein the label is coupled to the alpha phosphate group via a linker.

4. The method of claim 3. wherein the linker has a length between about 1 nm and about 20 nm.

5. The method of claim 3 or claim 4, wherein the linker is coupled to the alpha phosphate group via a cleavable group.

6. The method of claim 5, wherein the cleavable group is the same as a reversible terminator coupled to a 3' position of the sugar.

7. The method of any one of claims 1 to 6, wherein the sugar comprises nbose or deoxyribose.

8. The method of any one of claims 1 to 7, further comprising, after decoupling the label from the alpha phosphate group of the first nucleotide, using a polymerase to couple a second nucleotide of the plurality to the first nucleotide.

9. The method of any one of claims 1 to 8, each nucleotide of the plurality further comprising a reversible terminator coupled to the sugar, the method comprising decoupling the reversible terminator from the sugar of the first nucleotide.

10. The method of claim 9, wherein the label is decoupled from the alpha phosphate group of the first nucleotide using a first reagent, and wherein the reversible terminator is decoupled from the sugar of the first nucleotide using a second reagent.

11. The method of claim 10, wherein the first reagent is the same as the second reagent.

12. The method of claim 10, wherein the first reagent is different than the second reagent.

13. A composition, comprising: a duplex between a first polynucleotide and a second polynucleotide; a plurality of nucleotides in contact with the duplex, each nucleotide of the plurality comprising: a sugar; a nucleobase coupled to the sugar; an alpha phosphate group coupled to the sugar; and a label coupled to the alpha phosphate group; and a polymerase to couple a first nucleotide of the plurality to the duplex.

14. The composition of claim 13, wherein the label is decouplable from the alpha phosphate group of the first nucleotide using a reagent.

15. The composition of claim 14. wherein decoupling the label from the alpha phosphate group leaves behind a POT or PO4H group to which the polymerase adds another nucleotide of the plurality.

16. The composition of any one of claims 13 to 15, wherein the label is coupled to the alpha phosphate group via a linker.

17. The composition of claim 16, wherein the linker has a length between about 1 nm and about 20 nm.

18. The composition of claim 16 or claim 17, wherein the linker is coupled to the alpha phosphate group via a cleavable group.

19. The composition of claim 18, wherein the cleavable group is the same as a reversible terminator coupled to a 3' position of the sugar.

20. The composition of any one of claims 13 to 19, wherein the sugar comprises ribose or deoxyribose.

21. The composition of any one of claims 13 to 20, wherein the polymerase is further to couple a second nucleotide of the plurality to the first nucleotide.

22. The composition of any one of claims 13 to 21, each nucleotide of the plurality further comprising a reversible terminator coupled to the sugar, the reversible terminator being decouplable from the sugar of the first nucleotide.

23. The composition of claim 22, wherein the label is decouplable from the alpha phosphate group of the first nucleotide using a first reagent, and wherein the reversible terminator is decouplable from the sugar of the first nucleotide using a second reagent.

24. The composition of claim 23, wherein the first reagent is the same as the second reagent.

25. The composition of claim 23. wherein the first reagent is different than the second reagent.