Compositions including functional groups coupled to substrates, and methods of making the same

Abstract

In one example, an unsaturated cyclic diketone is coupled to a substrate and reacts with an indole or indazole comprising a first functional group to form a first adduct coupling the first functional group to the substrate. In another example, an unsaturated cyclic diketone is coupled to a substrate and reacts with a diene comprising a functional group to form an adduct coupling the functional group to the substrate. In another example, an indole or indazole is coupled to a substrate and reacts with an unsaturated cyclic diketone comprising an oligonucleotide to form an adduct coupling the oligonucleotide to the substrate. In yet another example, a diene is coupled to a substrate and reacts with an unsaturated cyclic diketone comprising an oligonucleotide to form an adduct coupling the oligonucleotide to the substrate.

Description

[Technical Field]

[0001] This application relates to coupling a functional group to a substrate. [Previous Technology]

[0002] Polymer-coated substrates are used in many technical applications. For example, implantable medical devices may be coated with bio-inert polymers. In another example, polymer-coated substrates are used for the preparation and / or analysis of biomolecules. Molecular analysis, such as certain nucleic acid sequencing methods, relies on the attachment of nucleic acid strands to the polymer-coated surface of the substrate. The sequence of the attached nucleic acid strands can then be determined by a variety of different methods known in this art.

[0003] In some sequencing methods, such as sequencing-by-synthesis (SBS), a polymer is coated onto the surface of a substrate (such as a flow channel), and then an oligonucleotide primer (e.g., single-stranded DNA or ssDNA) is grafted onto the polymer.

[0004] Polymer surfaces (and their preparation) are generally compatible with a wide range of sequencing and detection methods, including different chemical conditions, temperatures, optical detection methods, capture fraction density, and other parameters, and are generally stable under various storage and shipping conditions. Some polymer materials used in these molecular biology methods employ side-attached azido groups, which react with olefin or alkyne groups on the surface of the substrate and / or the oligonucleotide to be grafted in copper-mediated cycloaddition reactions. However, residual copper may have cytotoxic effects in biologically relevant environments. In DNA sequencing applications, copper can damage DNA in some cases, thereby reducing sequencing yield and data quality. Furthermore, copper-catalyzed reactions are typically copper-intensive and therefore expensive, and may not be efficient or fast enough to ensure adequate polymer attachment and localization on the substrate surface. Therefore, there is a need for surface polymer coatings with improved properties (such as increased reaction efficiency) that result in reduced residual copper. [Summary of the Invention]

[0005] The examples provided herein relate to compositions containing functional groups coupled to a substrate and methods for manufacturing the same. Methods for using such compositions are also disclosed.

[0006] Some examples herein provide methods for coupling a functional group to a substrate. The method may include providing an unsaturated cyclic diketone coupled to the substrate, and reacting the unsaturated cyclic diketone with an indole or indazole comprising a first functional group to form a first adduct coupling the first functional group to the substrate.

[0007] In some examples, the unsaturated cyclic diketone is: wherein L includes a linker connected to the substrate and each X is independently CH or N. In some examples, the unsaturated cyclic diketone is a triazoline diketone:

[0008] In some instances, the unsaturated cyclic diketone is maleic diimide. In some instances, the unsaturated cyclic diketone is 4-cyclopenten-1,3-diketone.

[0009] In some instances, indole or indazole is: wherein F1 includes a first functional group; R is H, an electron-withdrawing group, or an electron-donating group; and Z is CH or N. In some instances, indole is 1H-indole: . In some instances, indole is 1H-indazole: .

[0010] In some instances, the first adduct is: wherein L includes a connector attached to the substrate and each X is independently CH or N. In some instances, the first adduct is:

[0011] In some instances, the method further includes heating the first adduct to regenerate the cyclic unsaturated diketone coupled to the substrate.

[0012] In some examples, the method further includes reacting the first adduct with a diene comprising a second functional group to form a second adduct coupling the second functional group to a substrate. In some examples, the diene comprises a 1,3-diene. In some examples, the 1,3-diene is: wherein F2 comprises a second functional group. In some examples, the second adduct is: wherein L comprises a linker connected to the substrate.

[0013] In some instances, the second functional group is selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels. In some instances, the second functional group is an oligonucleotide.

[0014] In some instances, the first functional group is selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels. In some instances, the first functional group is an oligonucleotide.

[0015] In some instances, the substrate comprises a polymer disposed on a solid support. In some instances, the polymer is functionalized to include a polyhedral oligomeric silsesquioxane (POSS).

[0016] In some instances, providing an unsaturated cyclic diketone coupled to a substrate includes: providing a 4-substituted ureaazole coupled to a substrate, and oxidizing the 4-substituted ureaazole to form a triazolinone.

[0017] Some examples herein provide a composition comprising a substrate and an adduct coupled to the substrate: wherein L comprises a linker connected to the substrate; F1 comprises a first functional group; each X is independently CH or N; R is H, an electron-withdrawing group or an electron-donating group; and Z is CH or N.

[0018] In some instances, the adduct is:

[0019] In some instances, the first functional group is selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels. In some instances, the first functional group is an oligonucleotide.

[0020] In some instances, the substrate comprises a polymer disposed on a solid support. In some instances, the polymer is functionalized to comprise a polyhedral oligomeric silsesquioxane (POSS).

[0021] Some examples herein provide methods for coupling functional groups to a substrate. The method may include providing an unsaturated cyclic diketone coupled to the substrate, and reacting the unsaturated cyclic diketone with a diene including the functional group to form an adduct that couples the functional group to the substrate.

[0022] In some examples, the unsaturated cyclic diketone is: where L is a linker attached to the substrate and each X is independently CH or N. In some examples, the unsaturated cyclic diketone is a triazoline diketone; in some examples, the unsaturated cyclic diketone is maleic anhydride; in some examples, the unsaturated cyclic diketone is 4-cyclopenten-1,3-diketone.

[0023] In some examples, the diene includes 1,3-diene. In some examples, the 1,3-diene is: wherein F2 includes a functional group. In some examples, the adduct is: wherein L includes a linker attached to a substrate.

[0024] In some instances, the functional group is selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels. In some instances, the functional group is an oligonucleotide.

[0025] In some instances, the substrate comprises a polymer disposed on a solid support. In some instances, the polymer is functionalized to include polyhedral oligomeric silsesquioxanes (POSS).

[0026] In some instances, providing an unsaturated cyclic diketone coupled to a substrate includes: providing a 4-substituted ureaazole coupled to a substrate, and oxidizing the 4-substituted ureaazole to form a triazolinone.

[0027] Some examples herein provide a composition comprising a substrate and an adduct coupled to the substrate: wherein L comprises a linker connected to the substrate, F2 comprises a functional group, and each X is independently CH or N.

[0028] In some instances, the adduct is:

[0029] In some instances, the adduct is:

[0030] In some instances, the adduct is:

[0031] In some instances, the functional group is selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels. In some instances, the functional group is an oligonucleotide.

[0032] In some examples, the substrate comprises a polymer disposed on a solid support. In some examples, the polymer is functionalized to include polyhedral oligomeric silsesquioxanes (POSS).

[0033] Some examples herein provide a method for coupling a functional group to a substrate. The method may include providing an indole or indazole coupled to the substrate, and reacting the indole or indazole with a first unsaturated cyclic diketone comprising an oligonucleotide to form a first adduct that couples the oligonucleotide to the substrate.

[0034] In some instances, the first unsaturated cyclic diketone is: , where F3 comprises the oligonucleotide and each X is independently CH or N. In some instances, the first unsaturated cyclic diketone is a triazoline diketone: .

[0035] In some instances, the indole or indazole is: , where Z is CH or N, L includes a linker attached to the substrate, and R is H, an electron-withdrawing group, or an electron-donating group. In some instances, the indole is 1H-indole: where L is a linker attached to the substrate. In some instances, the first adduct is: where F3 includes the oligonucleotide, and each X is independently CH or N.

[0036] In some examples, the method further includes heating the first adduct to regenerate the indole or indazole coupled to the substrate. In some examples, the method further includes reacting the indole or indazole with a second unsaturated cyclic diketone after regenerating the indole or indazole coupled to the substrate to form a second adduct. In some examples, the second unsaturated cyclic diketone includes a functional group. In some examples, the functional group is selected from the group consisting of: a second oligonucleotide, a hydrophilic molecule, a hydrophilic macromolecule, a catalyst, and a label.

[0037] In some instances, the substrate comprises a polymer disposed on a solid support. In some instances, the polymer is functionalized to comprise a polyhedral oligomeric silsesquioxane (POSS).

[0038] In some instances, the method further includes providing a 4-substituted ureaazole comprising an oligonucleotide and oxidizing the 4-substituted ureaazole to form an unsaturated cyclic diketone triazolinone comprising an oligonucleotide.

[0039] Some examples herein provide a composition comprising a substrate and an adduct coupled to the substrate: wherein L comprises a linker connected to the substrate, F3 comprises an oligonucleotide, each X is independently CH or N, and Z is CH or N.

[0040] In some instances, the adduct is:

[0041] In some instances, the substrate comprises a polymer disposed on a solid support. In some instances, the polymer is functionalized to comprise a polyhedral oligomeric silsesquioxane (POSS).

[0042] Some examples herein provide a method for coupling a functional group to a substrate. The method may include providing a diene coupled to the substrate and reacting the diene with an unsaturated cyclic diketone comprising an oligonucleotide to form a first adduct that couples the oligonucleotide to the substrate.

[0043] In some instances, the unsaturated cyclic diketone is: , where F3 comprises an oligonucleotide and each X is independently CH or N.

[0044] In some instances, the unsaturated cyclic diketone is a triazoline diketone. In some instances, the unsaturated cyclic diketone is a maleic diamide.

[0045] In some instances, the unsaturated cyclic diketone is 4-cyclopenten-1,3-diketone:

[0046] In some instances, the diene comprises a 1,3-diene. In some instances, the 1,3-diene is: , wherein L comprises a linker attached to a substrate. In some instances, the adduct is: wherein L comprises a linker attached to a substrate, each X is independently CH or N, and F3 comprises the oligonucleotide.

[0047] In some instances, the adduct is:

[0048] In some instances, the substrate comprises a polymer disposed on a solid support. In some instances, the polymer is functionalized to include polyhedral oligomeric silsesquioxanes (POSS).

[0049] In some instances, the method further includes providing a 4-substituted ureaazole comprising an oligonucleotide and oxidizing the 4-substituted ureaazole to form an unsaturated cyclic diketone triazolinone comprising an oligonucleotide.

[0050] Some examples herein provide a composition comprising a substrate and an adduct coupled to the substrate: wherein L comprises a linker connected to the substrate, each X is independently CH or N, and F3 is an oligonucleotide.

[0051] In some instances, the adduct is:

[0052] In some instances, the substrate comprises a polymer disposed on a solid support. In some instances, the polymer is functionalized to comprise a polyhedral oligomeric silsesquioxane (POSS).

[0053] It should be understood that any individual feature / example of each of the embodiments of the invention described herein may be implemented together in any suitable combination, and any feature / example of any one or more of such embodiments may be implemented together with any feature of any of the other embodiments described herein in any suitable combination to achieve the benefits described herein.

Implementation Method

[0059] Cross-Reference to Related Applications This application claims the right of U.S. Provisional Patent Application No. 63 / 133,955, filed January 5, 2021, entitled “Compositions Including Functional Groups Coupled to Substrates, and Methods of Making the Same”, the entire contents of which are incorporated herein by reference.

[0060] The examples provided herein relate to compositions containing functional groups coupled to a substrate and methods for manufacturing the same. Methods for using such compositions are also disclosed.

[0061] For example, some previously known methods for coupling oligonucleotide primers to a substrate may include using relatively irritating reagents, such as cyanuric chloride, hydrazine, or diethyl ether, to prepare the substrate. Alternatively, some previously known coupling chemistry methods utilize a hydrazone formation reaction between a hydrazine-functionalized glass substrate and an oligonucleotide modified with a 5'-aldehyde. Therefore, this chemical reaction can be carried out at a relatively low pH of about 5, which may be problematic for oligonucleotide stability and may increase the likelihood of initiating non-specific binding between the positively charged oligonucleotide and the glass substrate. Furthermore, the hydrazone bond can be formed reversibly, thus increasing the likelihood that the oligonucleotide can be gradually decoupled from the glass substrate. In some other previously known methods, the oligonucleotide primer is coupled to a polymeric substrate (such as PAZAM) using relatively irritating "click chemistry" reagents (such as Cu(I)) and a relatively high pH of about 7 to 11 (which may also be problematic for oligonucleotide stability).

[0062] In contrast, this article provides a method for preparing a functionalized surface that can accept one or more functional groups, such as but not limited to oligonucleotides, via one or more alternative "click chemistry" reactions under approximately neutral or slightly alkaline pH conditions. The term "click chemistry" refers to a reaction that meets one or more criteria, such as having a relatively high yield, being applicable over a relatively wide range, producing relatively easily removable byproducts, being relatively easy to perform, being carried out using relatively easily removable solvents and reagents, and / or being carried out using relatively mild solvents and reagents. Alternatively or additionally, the "click chemistry" reaction may be thermodynamically advantageous and may specifically produce a single product.

[0063] In some instances, the functional group can be coupled to the substrate via a reaction between an unsaturated cyclic diketone and a diene, indazole, or indole. In some specific examples provided herein, the unsaturated cyclic diketone used in the embodiments of the invention can be a heterocyclic compound comprising an azo moiety linked to two carbonyl functional groups, for example having a structure such that it may be called a triazoline dione (TAD), wherein L comprises a linker linked to the substrate, and the functional group can be coupled to the linker using a diene or indole to couple the functional group to the substrate. The structure of the TAD molecule can stabilize the azo functional group via electronic conjugation. However, the electron-withdrawing carbonyl group and the symmetry of the electronic system may produce orbital-controlled electrophilic reactivity, resembling in some states a carbene or singlet oxygen. Therefore, TAD molecules readily participate in ultrafast Diels-Alder reactions and olefinic reactions, providing selective and predictable covalent linkage reactions with relatively high yields at relatively low temperatures (e.g., at or below room temperature, for example, at or below about 20°C) under isomolar conditions, requiring a catalyst. TAD molecules exhibit highly kinetic-biased behavior towards electron-rich π systems, resulting in relatively good selectivity for indoles and / or substituted dienes. Furthermore, the adducts of TAD with indoles or dienes form robust heterocyclic skeletons compatible with a wide range of applications (such as, but not limited to, those presented herein). As an additional feature, TAD molecules can be visually colored, while their adducts with indoles or dienes may be colorless, providing an analytically available method for assessing reaction efficiency. Other non-limiting examples of unsaturated cyclic diketones that can be used in the compositions and methods of the present invention are provided elsewhere herein.

[0064] In some instances, surface-coupled TAD or other unsaturated cyclic diketones react with indoles having structures such as 1H-indoles, wherein F1 comprises a functional group. In non-limiting instances, such indoles can react reversibly with unsaturated cyclic diketones such as TAD in a "reversible click" reaction, for example, to form adducts, which can be called Michael-addition adducts of TAD and 1H-indoles, via which the functional group F1 is coupled to the substrate. Other non-limiting examples of indoles and indazoles that can react with other unsaturated cyclic diketones are provided elsewhere herein.

[0065] In some instances, surface-coupled TAD reacts with a 1,3-diene having a structure such as trans, trans-1,3-hexadiene, wherein F2 comprises a functional group. In some instances, this diene can react substantially irreversibly with an unsaturated cyclic diketone such as TAD in an "ultra-click" reaction, for example, to form an adduct, which can be called a Diels-Alder cycloaddition product, and via which the functional group F2 is coupled to the substrate. Alternatively, the diene can react substantially irreversibly with an adduct of a "reversible click" reaction between TAD or other unsaturated cyclic diketone and indole or indazole in an "trans-click" reaction, for example, to form an adduct via which the functional group F2 is coupled to the substrate, and in this reaction the indole is substituted, thereby causing the functional group F1 to dissociate from the substrate.

[0066] It should be noted that reactions such as those described herein can be used to couple any suitable number and type of functional groups to a substrate at different times. For example, a "reversible click" reaction can be used to couple a first functional group (F1) to a substrate via an unsaturated cyclic diketone (e.g., TAD) and indole or indazole, and a "trans-click" reaction can subsequently be used to cause the first functional group to dissociate from the substrate via indole or indazole and to couple a second functional group (F2) to the surface via diene. Or, for example, because the "reversible click" reaction is reversible, an unsaturated cyclic diketone (e.g., TAD) coupled to the surface can be regenerated by heating the adduct of the "reversible click" reaction to a suitable temperature to induce the dissociation of indole or indazole. Therefore, an unsaturated cyclic diketone (e.g., TAD) coupled to the surface can then be used to couple another functional group to the substrate by reacting with another indole or indazole in another "reversible click" reaction (which itself can be reversible), or by reacting with a diene in an "ultra-click" reaction to couple yet another functional group to the substrate.

[0067] While some examples may include the use of TAD or other unsaturated cyclic diketones coupled to the substrate via linkers, it should be understood that in other examples, the unsaturated cyclic diketones may be functionalized and coupled to the substrate via reactions such as "reversible click," "ultra-click," or "trans-click." Illustratively, TAD has a structure such that F3 comprises a functional group and is coupled to the substrate via a diene, indole, or indazole coupled to the substrate. In some examples, indole reacting with solution-based TAD may have a structure where L comprises a linker attached to the substrate. Indole may react with TAD in a "reversible click" reaction to form an adduct: which is another Michael addition adduct of TAD and 1H-indole, via which the functional group F3 is coupled to the substrate.

[0068] In some instances, the diene reacting with solution-based TAD may be a 1,3-diene having the following structure: wherein L includes a linker attached to a substrate. The diene may react with TAD or other unsaturated cyclic diketones in a substantially irreversible "ultra-click" reaction, for example, to form an adduct: which is another Diels-Alder cycloaddition product, and the functional group F3 is coupled to the substrate via it.

[0069] Similarly, as described above, because the "reversible click" reaction is reversible, the surface-coupled indole or indazole can be regenerated by heating the adduct of the "reversible click" reaction to a suitable temperature to dissociate TAD or other unsaturated cyclic diketones having functional groups attached thereto. Therefore, the indole or indazole can then be used to react with another unsaturated cyclic diketone (e.g., TAD) in another "reversible click" reaction (which itself can be reversible) to couple another functional group to the substrate.

[0070] First, some terms used herein will be briefly explained. Then, some example compositions containing functional groups coupled to a substrate, and examples of their manufacture and use, will be described. Terminology

[0071] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term "including" and other forms such as "include / includes / included" is not restrictive. The use of the term "having" and other forms such as "have / has / had" is not restrictive. As used in this specification, whether in transitional phrases or body of a claim, the term "comprise(s) / comprising" shall be understood to have an open meaning. That is, the term shall be interpreted synonymously with the phrases "at least having" or "at least including". For example, when used in the context of a method, the term "comprising" means that the method includes at least the described steps, but may include other steps. When used in the context of a compound, composition, or apparatus, the term "comprising" means that the compound, composition, or apparatus includes at least the described features or components, but may also include other features or components.

[0072] The terms “substantially,” “about,” and “approximately” as used throughout this specification are used to describe and account for small fluctuations such as those attributable to variations in the process. For example, they may 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%.

[0073] As used herein, the term "array" refers to different groups of molecules attached to one or more substrates such that different molecules can be distinguished from each other based on their relative positions. An array may include different molecules located at different addressable sites on the substrates. Alternatively or additionally, an array may include individual substrates each carrying one or more different molecules, wherein different molecules can be identified based on the position of the substrate on the surface to which it is attached or based on the position of the substrate in a liquid.

[0074] As used herein, the terms "covalent attachment" or "covalent bond" refer to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently attached molecule refers to a molecule that forms a chemical bond with a substrate by attaching to a surface through other means, such as non-covalent bonding, such as electrostatic interactions.

[0075] As used herein, "a" and "b" being integers, "Ca to Cb" or "Ca-b" refers to the number of carbon atoms in a specified group. That is, the group may contain "a" to "b" (including the endpoints) carbon atoms. Thus, for example, "C1 to C4 alkyl" or "C1-4 alkyl (C1-4alkyl / C1-4alkyl)" refers to all alkyl groups having 1 to 4 carbon atoms, namely CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and (CH3)3C-.

[0076] As used herein, the term "halogen" or "halogen group" means fluorine, chlorine, bromine or iodine, with fluorine and chlorine being examples.

[0077] As used herein, "alkyl" refers to a fully saturated (i.e., free of double or triple bonds) straight or branched hydrocarbon chain. An alkyl group may have 1 to 20 carbon atoms (wherever it appears herein, numerical ranges such as "1 to 20" refer to integers within a given range; for example, "1 to 20 carbon atoms" means that an alkyl group may consist of 1, 2, 3, etc., up to and including 20 carbon atoms, but the definition of this invention also covers the presence of the term "alkyl" where no numerical range is specified). An alkyl group may also be a medium-sized alkyl group having 1 to 9 carbon atoms. An alkyl group may also be a low-carbon alkyl group having 1 to 4 carbon atoms. An alkyl group may be referred to as "C1-4 alkyl" or similar names. For example only, "C1-4 alkyl (C1-4alkyl / C1-4alkyl)" indicates that the alkyl chain contains one to four carbon atoms, meaning the alkyl chain is selected from the following groups: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, secondary butyl, and tertiary butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and similar groups.

[0078] As used herein, "alkenyl" refers to a straight-chain or branched hydrocarbon chain containing one or more double bonds. An alkenyl group can have 2 to 20 carbon atoms, but the definition of this invention also covers the presence of the term "alkenyl" where no numerical range is specified. An alkenyl group can also be a medium-sized alkenyl group having 2 to 9 carbon atoms. An alkenyl group can also be a low-carbon alkenyl group having 2 to 4 carbon atoms. An alkenyl group can be represented as "C2-4 alkenyl" or a similar name. For example, "C2-4 alkenyl" indicates that the alkenyl chain contains two to four carbon atoms, meaning the alkenyl chain is selected from the following groups: vinyl, 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-vinyl-1-yl, 2-methyl-propen-3-yl, but-1,3-dienyl, but-1,2-dienyl, and but-1,2-dien-4-yl. Typical alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and hexenyl and similar groups.

[0079] The group including alkenyl groups includes, as appropriate, substituted alkenyl, cycloalkenyl and heterocyclic alkenyl groups.

[0080] As used herein, "alkynyl" refers to a straight-chain or branched hydrocarbon chain containing one or more linkages. An alkynyl group can have 2 to 20 carbon atoms, but the definition of this invention also covers the presence of the term "alkynyl" where no numerical range is specified. An alkynyl group can also be a medium-sized alkynyl group having 2 to 9 carbon atoms. An alkynyl group can also be a low-carbon alkynyl group having 2 to 4 carbon atoms. An alkynyl group can be represented as "C2-4 alkynyl" or a similar name. By way of example only, "C2-4 alkynyl (C2-4alkynyl / C2-4alkynyl)" indicates that the alkynyl chain contains two to four carbon atoms, 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 not limited to, ethynyl, propynyl, butynyl, pentyynyl, and hexynyl and similar groups.

[0081] The group including alkynyl includes, where appropriate, substituted alkynyl, cycloalkynyl and heterocyclic alkynyl.

[0082] As used herein, "aryl" refers to an aromatic ring or ring system containing only carbon in its ring backbone (i.e., two or more fused rings sharing two adjacent carbon atoms). When an aryl is a ring system, each ring in the system is an aromatic ring. Aryl groups can have 6 to 18 carbon atoms, but the definition of this invention also covers the presence of the term "aryl" where no numerical range is specified. In some instances, aryl groups have 6 to 10 carbon atoms. Aryl groups can be represented as "C6-10 aryl," "C6 or C10 aryl," or similar names. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulel, and anthracene.

[0083] As used herein, "heterocycle" means a cyclic compound comprising a carbon atom and another atom (heteroatom) (e.g., nitrogen, oxygen, or sulfur). Heterocycles can be aromatic (heteroaryl) or aliphatic. Aliphatic heterocycles can be fully saturated or may contain one or more or two or more double bonds; for example, a heterocycle can be a heterocyclic alkyl group. Heterocycles can include a single heterocycle or multiple fused heterocycles.

[0084] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) containing one or more heteroatoms (i.e., elements other than carbon, including but not limited to nitrogen, oxygen, and sulfur) in the ring backbone. When a heteroaryl is a ring system, each ring in the system is an aromatic ring. A heteroaryl may have 5 to 18 ring members (i.e., the number of atoms constituting the ring backbone, including carbon atoms and heteroatoms), but the definition of this invention also covers the presence of the term "heteroaryl" where no numerical range is specified. In some instances, a heteroaryl has 5 to 10 ring members or 5 to 7 ring members. A heteroaryl may be referred to as "5 to 7-membered heteroaryl," "5 to 10-membered heteroaryl," or similar names. Examples of heteroaryl rings include, but are not limited to, furanyl, thiophene, terazolyl, pyrroleyl, acezolyl, thiazolyl, imidazolyl, pyrazolyl, isoacezolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridyl, terazolyl, pyrimidinyl, pyridyl, triazolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoacezolyl, benzothiazolyl, indolyl, isoindolyl, and benzothiaphenyl.

[0085] As used herein, "cycloalkyl" means a fully saturated carbocyclic ring or cyclic system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0086] As used herein, "cycloalkenyl" or "cycloalkene" means a carbocyclic ring or ring system having at least one double bond, wherein no ring in the ring system is an aromatic ring. An example is cyclohexenyl or cyclohexene. Another example is norcamphene or norenyl.

[0087] As used herein, "heterocyclic alkenyl" or "heterocyclic alkene" means a carbocyclic ring or ring system having at least one heteroatom in the ring backbone, having at least one double bond, wherein no ring in the ring system is an aromatic ring. In some instances, the heterocyclic alkenyl or heterocyclic alkene ring or ring system is 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, or 10-membered.

[0088] As used herein, "cycloynyl" or "cycloynyl" means a carbocyclic ring or ring system having at least one cyclic bond, wherein no ring in the ring system is an aromatic ring. An example is cyclooctyne. Another example is bicyclic nonyne. Another example is dibenzocyclooctyne (DBCO).

[0089] As used herein, "heterocyclic ynyl" or "heterocyclic ynyl" means a carbocyclic ring or ring system having at least one heteroatom in the ring backbone, having at least one parabolic bond, wherein no ring in the ring system is an aromatic ring. In some instances, the heterocyclic ynyl or heterocyclic ynyl ring or ring system is 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, or 10-membered.

[0090] As used herein, "heterocyclic alkyl" means a non-aromatic cyclic ring or cyclic system containing at least one heteroatom in the ring backbone. Heterocyclic alkyl groups can be joined together by fusion, bridging, or spirolinking. Heterocyclic alkyl groups can have any degree of saturation, limited by the condition that at least one heterocycle in the cyclic system is not an aromatic ring. Heterocyclic alkyl groups can have 3 to 20 ring members (i.e., the number of atoms constituting the ring backbone, including carbon atoms and heteroatoms), but the definition of this invention also covers the presence of the term "heterocyclic alkyl" where no numerical range is specified. Heterocyclic alkyl groups can also be of medium size with 3 to 10 ring members. Heterocyclic alkyl groups can also be heterocyclic alkyl groups with 3 to 6 ring members. Heterocyclic alkyl groups can be referred to as "3 to 6-membered heterocyclic alkyl groups" or similar names. In some six-membered monocyclic heterocyclic alkyl groups, the heteroatom is selected from one to three of O, N, or S, and in some five-membered monocyclic heterocyclic alkyl groups, the heteroatom is selected from one or two heteroatoms selected from O, N, or S. Examples of heterocyclic alkyl rings include, but are not limited to, azirrolyl, acridine, carbazoyl, oxolinyl, dioxopentyl, dihydroimidazoyl, imidazodinyl, α-linylyl, epoxyethyl, oxacycloheptanyl, thioheptanyl, piperidinyl, piperidine, dioxypiperidine, pyrrolidone, pyrrolididinedione, 4-piperidone, pyrazolinyl, pyrazolidine, 1,3-dioxacyclohexenyl, 1,3-dioxacyclohexylalkyl, 1,4-dioxacyclohexenyl, 1,4-dioxacyclohexylalkyl, 1,3-oxothiohexylalkyl, 1,4-oxothiohexyl, 1,4-oxothiohexyl, 1,4-oxothiohexyl, 1,4-oxothiohexyl, 1,4-oxothiohexyl, and 1,4-oxothiohexylyl. Alkyl, 2H-1,2-alkyl, trialkyl, hexahydro-1,3,5-trialkyl, 1,3-m-dioxacyclopentenyl, 1,3-dioxacyclopentenyl, 1,3-dithiocyclopentenyl, 1,3-dithioalkyl, isoalkylazolinyl, isoalkylazolinyl, alkylazolinyl, alkylazolinyl, alkylazolinone, thiazolinyl, thiazolinyl, 1,3-oxothiocyclopentenyl, indololinyl, isoindololinyl, tetrahydrofuranyl, tetrahydropiperanyl, tetrahydrothiopiperanyl, tetrahydro-1,4-thiazolinyl, thiazolinyl, dihydrobenzofuranyl, benzimidazolinyl, and tetrahydroquinoline.

[0091] As used herein, a substituted group is derived from an unsubstituted parent group, wherein one or more hydrogen atoms have been replaced by another atom or group. Unless otherwise specified, when a group is considered "substituted," it means that the group is substituted by one or more substituents independently selected from: C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocycloyl (substituted, as appropriate, with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7-carbocyclo-C1-C6-alkyl (substituted, as appropriate, with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy). Substitution), 5 to 10-membered heterocyclic groups (substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy as appropriate), 5 to 10-membered heterocyclic -C1-C6-alkyl (substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy as appropriate), aryl (substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy as appropriate), aryl(C1-C6)alkyl (substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy as appropriate), aryl(C1-C6)alkyl (substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy as appropriate) 6-alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy substituted), 5 to 10 heteroaryl (substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy as appropriate), 5 to 10 heteroaryl (C1-C6) alkyl (substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy as appropriate), halogen, cyano, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy (C1-C6) alkyl (i.e. ether) Aryloxy, thiohydro (mercapto), halogen (C1-C6) alkyl (e.g., -CF3), halogen (C1-C6) alkoxy (e.g., -OCF3), C1-C6 alkylthio, arylthio, amino, amino (C1-C6) alkyl, nitro, O-aminomethoxy, N-aminomethoxy, O-thiaminomethoxy, N-thiaminomethoxy, C-aminomethoxy, N-aminomethoxy, S-sulfomethoxy, N-sulfomethoxy, C-carboxyl, O-carboxyl, acetyl, cyano, isocyano, thiocyanate, isothiocyanate, sulfinyl, sulfonyl, and side oxygen (=O). Whenever a descriptive group is "substituted as appropriate," the group may be substituted with the above-mentioned substituents.

[0092] When the compounds disclosed herein have at least one stereoisomeric center, they may exist individually as mirror-image or non-mirror-image isomers or as mixtures of such isomers (including racemates). The isolation of individual isomers or the selective synthesis of individual isomers is achieved by applying various methods well known to those skilled in the art. When the compounds disclosed herein are to be understood as existing in tautomeric form, all tautomeric forms are included within the scope of the described structure. Unless otherwise indicated, all such isomers and mixtures thereof are included within the scope of the compounds disclosed herein. Furthermore, the compounds disclosed herein may exist in one or more crystalline or amorphous forms. Unless otherwise indicated, all such forms are included within the scope of the compounds disclosed herein, including any polymorphic forms. Additionally, some of the compounds disclosed herein may form solvates with water (i.e., hydrates) or common organic solvents. Unless otherwise indicated, such solvates are included within the scope of the compounds disclosed herein.

[0093] As used herein, the term "nucleotide" is intended to mean a molecule that includes a sugar and at least one phosphate ester group, and in some instances, a nucleobase. A nucleotide lacking a nucleobase may be called "base-free". Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate ester sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytosine monophosphate (CMP), cytosine diphosphate (CDP), cytosine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), and deoxyadenosine monophosphate. (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0094] As used herein, the term "nucleotide" is also intended to encompass any nucleotide analogue, relative to naturally occurring nucleotides, including those with modified nucleobases, sugars, and / or phosphate esters. Examples of modified nucleobases include inosine, xathanine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymidine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azoline, and so on. Azouracil, 6-azocytosine, 6-azothymidine, 5-uracil, 4-thiouracil, 8-halogenated adenine or guanine, 8-aminoadenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyadenine or guanine, 5-halogenated uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, or analogs thereof. As is known in the art, certain nucleotide analogs cannot be incorporated into polynucleotides, such as nucleotide analogs like adenosine 5'-phosphate sulfate. Nucleotides may include any suitable number of phosphate esters, such as three, four, five, six, or more than six phosphate esters.

[0095] As used herein, the term "polynucleotide" refers to a molecule comprising a sequence of nucleotides bonded together and is used interchangeably with the term "oligonucleotide." The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. Unless otherwise specifically indicated, the different terms are not intended to indicate any particular difference in size, sequence, or other characteristics. For clarity of description, these terms may be used to distinguish one polynucleotide species from another when describing a particular method or composition comprising several polynucleotide species. A polynucleotide is a non-limiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), and the like. A polynucleotide may be a single-stranded nucleotide sequence such as RNA or single-stranded DNA, a double-stranded nucleotide sequence such as double-stranded DNA, or may include a mixture of single-stranded and double-stranded nucleotide sequences. 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 mirror-image isomorphic DNA. The precise nucleotide sequence in a polynucleotide can be known or unknown. Examples of polynucleotides include: genes or gene fragments (e.g., probes, primers, expressed sequence tags (ESTs) or serial analysis of gene expression (SAGE) tags), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plastids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the foregoing.

[0096] As used herein, "polymerase" is intended to mean an enzyme that assembles the active site of a polynucleotide by polymerizing nucleotides into polynucleotides. A polymerase can bind to an initiated single-stranded target polynucleotide and sequentially add nucleotides to a growth primer to form a "complementary replica" polynucleotide having a sequence complementary to the sequence of the target polynucleotide. Another polymerase or the same polymerase can then form a replica of the target nucleotide by forming a replica of the complementary replica polynucleotide. Either of these replicas may be referred to herein as an "amplifier". A DNA polymerase can bind to the target polynucleotide and then move the target polynucleotide down, sequentially adding nucleotides to the free hydroxyl group at the 3' end of the growth polynucleotide strand (growth amplifier). DNA polymerases can synthesize complementary DNA molecules from a DNA template, and RNA polymerases can synthesize RNA molecules from a DNA template (transcription). Polymerases can use short RNA or DNA strands (primers) to initiate strand growth. Some polymerases can cause upstream strand shifting, in which they add bases to the strand. Such polymerases may be referred to as strand-shifting polymerases, meaning they possess the activity of removing the complementary strand from the template strand by the polymerase read. Examples of polymerases with strand-shifting activity include, but are not limited to, Bacillus stearothermophilus (Bst) polymerase, exo-Klenow polymerase, or large fragments of sequencing-level T7 exo-polymerase. Some polymerases degrade the preceding strand, effectively replacing it with the following growing strand (5' end nuclease activity). Some polymerases have the activity of degrading the following strand (3' end nuclease activity). Some applicable polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' end nuclease activity.

[0097] As used herein, the term "introducer" refers to a polynucleotide to which nucleotides can be added via a free 3' OH group. The introducer length can be any suitable number of bases and can include any suitable combination of natural and non-natural nucleotides. The target polynucleotide may include an "adaptor" that hybridizes to the introducer (having a sequence complementary to the introducer) and can be amplified by adding nucleotides to the free 3' OH group of the introducer to produce a complementary duplicate polynucleotide. The introducer may be coupled to a substrate.

[0098] In some instances, the primers used on the substrate surface are P5 and P7 primers, available from Illumina, Inc. In some instances, the P5 and P7 primer sequences may have the following sequences: Paired read groups:

[0099] P5: 5'-AATGATACGGCGACCACCGAGAUCTACAC-3' (SEQ ID NO: 1)

[0100] P7: 5'-CAAGCAGAAGACGGCATACGAG*AT-3' (SEQ ID NO:2) Single read segment group:

[0101] P5: 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 3)

[0102] P7: 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO:4) where G* is G or 8-side-oxyguanine.

[0103] In some instances, the attached oligonucleotide (such as a primer or a P5 or P7 primer) includes a linker or spacer at the 5' end. Such linkers or spacers may be included to allow chemical or enzymatic cleavage, or to impart other desired properties, such as enabling covalent attachment to a polymer or solid support, or to act as spacers to position the cleavage site at an optimal distance from the solid support. In some cases, 10 spacer nucleotides may be positioned between the P5 or P7 primer and the attachment site to the polymer or solid support. In some instances, polyT spacers are used, but other nucleotides and combinations thereof may also be used. In one instance, the spacer is a 6T to 10T spacer. In some instances, the linker includes a cleavable nucleotide comprising a chemically cleavable functional group, such as vicinal diol or allyl T.

[0104] As used herein, the term "amplifier" when referring to polynucleotides is intended to mean a product that replicates the polynucleotide, wherein the product has a nucleotide sequence that is substantially identical or substantially complementary to at least a portion of the nucleotide sequence of the polynucleotide. "Amplification" refers to the process of producing an amplifier of the polynucleotide. The first amplifier of the target polynucleotide may be a complementary copy. Additional amplifiers are copies of the target polynucleotide or generated from the first amplifier after its generation. Subsequent amplifiers may have a sequence that is substantially complementary to or substantially identical to the target polynucleotide. It should be understood that a few mutations in the polynucleotide may occur during the generation of the amplifier (e.g., due to amplification artifacts).

[0105] As used herein, the term "silane" refers to an organic or inorganic compound containing one or more silicon atoms. Non-limiting examples of inorganic silane compounds are SiH4 or halogenated SiH4, wherein hydrogen is substituted by one or more halogen atoms. Non-limiting examples of organic silane compounds are X-RC-Si(ORD)3, wherein X is a non-hydrolyzable organic group, such as amino, vinyl, epoxy, methpropylene, sulfur, alkyl, alkenyl, or alkynyl; RC is a spacer, for example -(CH2)n-, wherein n is 0 to 1000; each RD is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclic, optionally substituted aryl, optionally substituted 5 to 10-membered heteroaryl, and optionally substituted 5 to 10-membered heterocyclic, as defined herein. In some instances, the silane can be crosslinked such that the oxygen atom of the -ORD group of X-RC-Si(ORD)3 attaches to the silicon atom of the adjacent organosilicon compound X-RC-Si(ORD)3. Furthermore, the silane compound can be attached to the substrate surface by covalently bonding the X-RC-Si(ORD)3 portion to oxygen atoms on the surface. Therefore, in some instances, the described silane includes the following structure:

[0106] As used herein, the term "silane" may include mixtures of different silane compounds. In some instances, X is norenyl. In some instances, X is bicyclic nonynyl. In some instances, X is an alkene- or alkyne-containing group. In some instances, X is an alkene or alkyne. In some instances, the RC linker is a C2-6 alkyl group.

[0107] As used herein, the term "substrate" refers to a material including a solid support. A substrate may include a polymer defining or disposed on a solid support. Examples of substrate materials may include glass, silicon dioxide, plastic, quartz, metal, metal oxide, organosilicone (e.g., polyhedral organic silsesquioxane (POSS)), polyacrylate, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of a POSS may be the POSS described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. Illustratively, POSS-containing monomers can be rapidly polymerized to a gel point to provide a POSS resin (a polymer functionalized to include POSS) on which soft materials can be functionalized. In some instances, the substrate used in this application includes silicon dioxide-based substrates, such as glass, fused silica, or other silicon dioxide-containing materials. In some instances, the substrate may include silicon, silicon nitride, or hydrogenated silica. In some instances, the substrate used in this application includes plastic materials or components, such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). Examples of plastic materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some instances, the substrate is or includes silicon dioxide-based materials or plastic materials or combinations thereof. In certain instances, the substrate has at least one surface comprising glass or a silicon-based polymer. In some instances, the substrate may include a metal. In some of these instances, the metal is gold. In some instances, the substrate has at least one surface comprising a metal oxide. In one instance, the surface comprises tantalum oxide or tin oxide. Acrylamide, ketones, or acrylates may also be used as substrate materials or components. Other substrate materials may include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramics, polyimide, quartz, resins, polymers, and copolymers. In some instances, the substrate and / or substrate surface may be or include quartz. In some other instances, the substrate and / or substrate surface may be or include semiconductors, such as GaAs or ITO. The foregoing list is intended to illustrate and not limit the scope of this application. The substrate may comprise a single material or a plurality of different materials. The substrate may be a composite or a laminate. In some instances, the substrate comprises an organosilicone material. The substrate may be flat, round, spherical, rod-shaped, or any other suitable shape. The substrate may be rigid or flexible. In some instances, the substrate is a bead or a channel.

[0108] In some instances, the substrate includes a patterned surface. A "patterned surface" refers to the arrangement of different regions in or on the exposed layer of the substrate. For example, one or more regions may be features containing one or more trapping leads. These features may be separated by gaps in which no trapping leads are present. In some instances, the pattern may be a column and row arrangement of xy-format features. In some instances, the pattern may be a repeating feature and / or gap region arrangement. In some instances, the pattern may be a randomly arranged feature and / or gap region. In some instances, the substrate includes an array of holes (recesses) on its surface. These holes may be provided by substantially vertical sidewalls. In some instances, the substrate includes an array of pillars (protrusions) on its surface. The holes and pillars may be manufactured using a variety of techniques commonly known in the art, including but not limited to photolithography, stamping, molding, nanoimprint lithography, and micro-etching. As those skilled in the art will understand, the techniques used will depend on the composition and shape of the array substrate. To illustrate, a column with a diameter between about 50 nm and about 500 nm can be called a nanocolumn and can have a height similar to its diameter.

[0109] Features in the patterned surface of the substrate may include a series of features on glass, silicone, plastic, or other suitable materials (e.g., pores such as micropores or nanopores, or pillars such as nanopillars), with patterned, covalently linked gels, such as poly(N-(5-azidoacetylammonopentyl)acrylamide-co-acrylamide) (PAZAM). This process produces gel pads for sequencing, which can be stable during sequencing operations with multiple cycles. The covalent link between the polymer and the pores can help maintain the gel in the structured features throughout the entire lifespan of the structured substrate during multiple uses. However, in many instances, the gel need not be covalently linked to the pores. For example, under certain conditions, silane-free acrylamide (SFA) that is not covalently attached to any part of the structured substrate can be used as the gel material.

[0110] In a specific example, the structured substrate can be manufactured by patterning a suitable material with pores (e.g., micropores or nanopores), coating the patterned material with a gel material (e.g., PAZAM, SFA, or chemically modified variants thereof, such as an azide form of SFA (azido-SFA)), and polishing the surface of the gel-coated material, for example by chemical or mechanical polishing, thereby retaining the gel in the pores but removing or inactivating substantially all of the gel from the interstitial regions on the surface of the structured substrate between the pores. Primers can be attached to the gel material. A solution comprising multiple target polynucleotides (e.g., fragmented human genomes or portions thereof) can then be contacted with the polished substrate, such that individual target polynucleotides will be seeded in individual pores by interacting with primers attached to the gel material; however, due to the absence or inactivation of the gel material, the target polynucleotides will not occupy the interstitial regions. The amplification of the target polynucleotides may be limited to the pores because the absence or inactivation of the gel in the interstitial regions may inhibit the outward migration of growth clusters. The process is adjustable and utilizes conventional micron or nanometer manufacturing methods, making it easy to manufacture.

[0111] Patterned substrates may include, for example, holes provided in a wafer or wafer. The etched patterns and geometries of these holes can take many different shapes and sizes, and such features can be physically or functionally separable from each other. Particularly suitable substrates with such structural features include patterned substrates with selectable sizes of solid particles (such as microspheres). An example of a patterned substrate with these features is an etched substrate used in conjunction with BEAD ARRAY technology (Illumina, Inc., San Diego, Calif.). Nano-imprint lithography (NIL) can be used to provide the holes.

[0112] In some instances, the substrate described herein forms at least a portion of the flow channel or is located in or coupled to the flow channel. The flow channel may include flow chambers divided into a plurality of channels or a plurality of segments. Examples of flow channels and substrates that can be used in the methods and compositions described herein for manufacturing flow channels include, but are not limited to, those purchased from Illumina, Inc. (San Diego, CA).

[0113] As used herein, the term "structure" refers to a compound bonded to a substrate, such as a copolymer. Copolymers may be covalently bonded to a substrate, for example, via an azide group.

[0114] As used herein, the term "polymer" refers to a molecule comprising a plurality of repeating subunits or repeating units. Non-limiting examples of polymer structures include linear, branched, or hyperbranched polymers. Non-limiting examples of linear polymers include block copolymers or random / statistical copolymers. Non-limiting examples of branched polymers include star polymers, star-shaped or star-shaped block polymers (including both hydrophobic and hydrophilic segments), H-shaped polymers (including both hydrophobic and hydrophilic segments), dumbbell-shaped polymers, comb-shaped polymers, brush-shaped polymers, dendritic polymers, ladder-shaped polymers, and dendritic polymers. Polymers may be crosslinked or lightly crosslinked. Polymers described herein may be linear, branched, hyperbranched, or dendritic. Polymers described herein may also be in the form of polymer nanoparticles. Other examples of polymer architectures include, but are not limited to, cyclic block polymers and coil-cycle-coil polymers. Polymers having more than one type of repeating unit may be configured as block copolymers, random copolymers, or alternating copolymers or mixtures thereof. The final copolymer structure can be in various architectures, including, for example, random copolymers, block copolymers, comb polymers, or star polymer architectures. Different categories of polymer backbones include, but are not limited to, polyacrylamide, polyacrylates, polyurethanes, polysiloxanes, silicones, polyacrylaldehyde, polyphosphonates, polyisocyanates, polyols, polysaccharides, polypeptides, and combinations thereof. In some instances, the polymer comprises a polyacrylamide backbone. In some other instances, the polymer comprises a polyacrylate backbone. In still other instances, the polymer comprises a polyurethane backbone. In yet another instance, the polymer comprises a polyphosphonate backbone. In still yet another instance, the polymer comprises a dendritic polymer backbone.

[0115] As used herein, the term "luciferoid" is intended to mean a molecule that responds to light of a second wavelength different from the first wavelength and emits light of the first wavelength. The light emitted by a luciferoid can be called "fluorescence" and can be detected by suitable optical circuitry.

[0116] As used herein, "detecting" fluorescence means receiving light from a phosphorescent cluster, generating an electrical signal based on the received light, and using the electrical signal to determine the light received from the phosphorescent cluster. Any suitable optical detection circuit can be used to detect fluorescence, which may include an optical detector that generates an electrical signal based on the light received from the phosphorescent cluster and electronic circuitry that uses the electrical signal to determine the light received from the phosphorescent cluster. As an example, the optical detector may include an active-pixel sensor (APS), which includes an amplified array of photodetectors configured to generate an electrical signal based on the light received by the photodetector. The APS may be based on complementary metal-oxide-semiconductor (CMOS) technology known in this art. CMOS-based detectors may include field-effect transistors (FETs), such as metal-oxide-semiconductor field-effect transistors (MOSFETs). In specific instances, a CMOS imager (CMOS-SPAD) with a single-photon burst diode can be used, for example, to perform fluorescence lifetime imaging (FLIM). In other instances, the optical detector may include photodiodes such as burst photodiodes, charge-coupled devices (CCDs), cryogenic photon detectors, reverse-biased light-emitting diodes (LEDs), photoresistors, photocrystals, photovoltaic cells, photomultiplier tubes (PMTs), quantum dot photoconductors, or photodiodes, or the like. The optical detection circuitry may further include any suitable combination of hardware and software operatively communicating with the optical detector to receive electrical signals from it and to be configured to detect fluorescence based on these signals, such as light from fluorophores detected by the optical detector. For example, the electronic circuitry may include memory and a processor coupled to the memory. The memory may store instructions for causing the processor to receive signals from the optical sensor and use such signals to detect fluorescent clusters. For example, such instructions may cause the processor to use signals from the optical sensor to determine whether fluorescence is emitted within the field of view of the optical sensor and use such determination to determine the presence of a fluorescent cluster.

[0117] As used herein, the term "adduct" is intended to mean the product of a chemical reaction between two or more molecules, wherein the product contains all the atoms of the reacting molecules.

[0118] As used herein, the term "linker" is intended to mean one or more molecules through which one element is attached to another element. For example, a linker enables a molecule to attach to a substrate. Linkers can be covalent or non-covalent. Non-limiting examples of covalent linkers include alkyl chains, polyethers, amides, esters, aryl groups, polyaryl groups, and the like. Non-limiting examples of non-covalent linkers include host-guest wedges, cyclodextrin / norcamphene, adamantane-β-CD containment wedges, DNA hybridization interactions, and similar mechanisms.

[0119] As used herein, the term "functional group" is intended to mean one or more molecules that can interact with one or more other molecules. As used herein, an element referred to as "functionalization" means that the element includes a functional group. For example, a functional group can be covalently bonded to one or more other molecules, such as reacting reversibly or substantially irreversibly with one or more other molecules to form a product. Or, for example, a functional group can non-covalently associate with one or more other molecules. Non-limiting examples of functional groups include oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels. Illustratively, a functional group including an oligonucleotide (such as an initiator) can hybridize with another oligonucleotide (such as a polynucleotide to be amplified or sequenced). Alternatively, a functional group including a label can include a luciferase or a Förster resonance energy transfer (FRET) complex, and the detection of fluorescence from the luciferase or FRET complex can be used to characterize the molecule to which the label is attached. Compositions containing functional groups coupled to a substrate and methods for manufacturing the same

[0120] As mentioned above and described in more detail below, the compositions and methods of the present invention provide a convenient way to couple any suitable functional group to a substrate, and in some instances, common reactive components are used to sequentially couple different functional groups to the substrate. In examples such as those described with reference to Figures 1A to 1E and Figures 2A to 2C, an unsaturated cyclic diketone is coupled to the substrate via a linker, and a functionalized indole, functionalized indazole, or functionalized diene is reacted with it to form a reactive adduct, through which the functional group is coupled to the substrate. In other examples, such as those described with reference to Figures 3A to 3B, an indole or indazole is coupled to the substrate via a linker, and a functionalized unsaturated cyclic diketone is reacted with it to form a reactive adduct, through which the functional group is coupled to the substrate. In other examples, such as those described with reference to Figures 4A and 4B, the diene is coupled to the substrate via a linker, and a functionalized unsaturated cyclic diketone reacts with it to form a reactive adduct, through which the functional group is coupled to the substrate.

[0121] Figures 1A to 1E illustrate example compositions and operations used in processes for coupling functional groups to a substrate. Although Figures 1A to 1E illustrate specific examples of molecules that can be used in the compositions and operations of the present invention, it should be understood that other molecules may be suitable for use.

[0122] Referring now to FIG. 1A, composition 100 includes a plurality of unsaturated cyclic diketones 110 coupled to substrate 101 via individual linkers (L). Illustratively, the unsaturated cyclic diketones may have the following structure: , wherein L includes a linker connected to substrate 101 and each X is independently CH or N. In some examples, as illustrated in FIG. 1A, the unsaturated cyclic diketone 110 may include a TAD molecule having the following structure: . In other examples, the unsaturated cyclic diketone 110 may include a maleic anhydride molecule having the following structure: . In still other examples, the unsaturated cyclic diketone 110 may include a 4-cyclopenten-1,3-dione molecule having the following structure: .

[0123] Non-limiting examples of connectors (L) are provided elsewhere herein. Substrate 101 may include a polymer disposed on a solid support, or may include a solid support on which no polymer is disposed. In some instances, the solid support may include any substrate material such as described elsewhere herein. The polymer (if included) may include any suitable polymer such as described elsewhere herein, illustratively, a polymer functionalized to include POSS. The substrate may be silane-treated to facilitate the capture of polymers and / or unsaturated cyclic diketones. L may be coupled to substrate 101 in any suitable manner (e.g., coupled to a solid support or coupled to a polymer disposed on a solid support). For example, a polymer brush may be grown using monomers having the relevant functional groups.

[0124] An unsaturated cyclic diketone can react with an indole or indazole molecule including a first functional group (F1) to form a first adduct that couples the first functional group to a substrate. For example, as illustrated in FIG1A, a TAD molecule 110 can react with an indole molecule 120 including a first functional group (F1) to form a first adduct that couples the first functional group to a substrate. The unsaturated cyclic diketone can contact the indole or indazole molecule 120 dissolved in any suitable solvent (e.g., a polar protic solvent, such as water or alcohol; or a polar aprotic solvent, such as acetonitrile, ester, or ether) at any suitable reaction temperature, for example at room temperature, wherein the solvent is compatible with the diketone molecule and the indole or indazole molecule. In the non-limiting example illustrated in FIG1A, the indole molecule 120 may have the following structure: it may be called 1H-indole, and wherein F1 includes the first functional group. However, unsaturated cyclic diketones can react with any suitable indole or indazole, for example, molecules having the following structure: where R is H, an electron-withdrawing group, or an electron-donating group, and Z is CH or N. Illustratively, indazoles can have the following structure: they can be called 1H-indazoles.

[0125] The first functional group may be or include any suitable one or more molecules, such as those described elsewhere herein. In non-limiting examples, the first functional group (F1) may be selected from the group consisting of: oligonucleotides, hydrophilic molecules (such as PEG), hydrophilic macromolecules, catalysts, and labels. Illustratively, the first functional group (F1) may be or include oligonucleotides.

[0126] The reaction of an unsaturated cyclic diketone with indole or indazole can provide a composition comprising a plurality of first adduct molecules 130 having the following structure: which may be referred to as a Michael addition adduct, wherein X, Z, R, L, and F1 are defined elsewhere herein. Illustratively, in a particular example shown in Figure 1B, the reaction of TAD molecule 110 with indole molecule 120 can provide a composition 100' comprising a plurality of first adduct molecules 130 having the following structure: F1 can be readily coupled to other adducts of the substrate, for example, using a Michael addition reaction between an unsaturated cyclic diketone and indole or indazole, based on the teachings provided herein.

[0127] The reaction between the unsaturated cyclic diketone molecule 110 and the indole or indazole molecule 120 can be reversible and may be referred to as a "reversible click" reaction. Therefore, in some instances, the adduct molecule 130 can be heated to a suitable temperature to regenerate the unsaturated cyclic diketone molecule 110 coupled to the substrate and cause the indole or indazole molecule 120 (and the first functional group F1 coupled thereto) to dissociate in a manner such that, as shown in Figure 1C, the TAD molecule remains coupled to the substrate 101 while the indole molecule dissociates. For example, the substrate 101 can be heated to a temperature of at least 50°C, or a temperature of about 50°C to about 100°C, or a temperature of about 60°C to about 90°C, or a temperature of about 60°C to about 80°C to regenerate the unsaturated cyclic diketone molecule 110 coupled to the substrate and cause the indole or indazole molecule 120 to dissociate. As an alternative, after regenerating the unsaturated cyclic diketone molecule 110, the diketone molecule can react with another functionalized indole or indazole in a manner as described with reference to Figures 1A to 1C. Such reactions are similarly reversible, and thus the unsaturated cyclic diketone molecule 110 can be regenerated for use in other reactions.

[0128] It should be noted that the reactivity of adduct molecule 130 may be significantly less than that of unsaturated cyclic diketone 110, for example, significantly less than that of TAD. Therefore, indole or indazole molecule 120 can be considered as “protecting” the unsaturated cyclic diketone (e.g., TAD molecule). Thus, in some instances, unsaturated cyclic diketone 110 can be considered as “deprotected” by heating to reverse the “reversible click” reaction, or by reacting adduct molecule 130 with diene in a “trans click” reaction, as further described below. In this regard, it should be noted that indole or indazole molecule 120 used as a protecting agent may or may not include functional groups. Additionally, in some instances, only a subset of indole or indazole molecules are dissociated from adduct molecule 130 (whether by “trans click” or “reversible click”), and the remaining indole molecules coupled to the substrate can continue to protect the unsaturated cyclic diketone from participating in inappropriate chemical or biochemical reactions. In some instances, the indole or indazole molecule 120 includes functional groups such as hydrophilic molecules (such as PEG) that can inhibit substrate fouling and, in instances where the substrate is used for polynucleotide sequencing, can help improve sequencing quality for longer sequencing operations.

[0129] An unsaturated cyclic diketone can react with a diene including a second functional group (F2) to form a second adduct that couples the second functional group to a substrate. The diene may include a 1,3-diene, which may include substitutions on any carbon atom of the diene, and may include any suitable heteroatom substitution process conceivable to improve reactivity. In some instances, as illustrated in Figure 1D, TAD molecule 110 may react with a 1,3-diene 140 including a second functional group (F2) to form a second adduct that couples the second functional group to a substrate. Before TAD molecule 110 is regenerated in a manner as described with reference to Figures 1A to 1C, TAD molecule 110 may have, but does not necessarily have, been pre-reacted with indole 120 to form adduct 130. In the composition 102 illustrated in Figure 1D, TAD molecule 110 can contact 1,3-diene molecule 140 dissolved in any suitable solvent (e.g., a polar protic solvent, such as water or alcohol, or a polar aprotic solvent, such as acetonitrile, ester or ether), which is compatible with both the TAD molecule and the diene molecule.

[0130] In the non-limiting example illustrated in Figure 1D, the 1,3-diene molecule 140 may have the following structure: (trans,trans-1,3-hexadiene). However, it should be understood that many other dienes may be suitably used. For example, diene molecule 140 may actually be a 2,4-diene, for example having the following structure: (trans-trans-2,4-hexadiene). In other examples, the diene may include Danishefsky's diene, for example having the following structure: (1-methoxy-3-trimethylsilyloxy-but-1,3-diene). In other examples, the diene may include derivatives of Daniszewski diene, such as Brassard diene, for example having the structure: (1,3-alkoxy-1-trimethylsilaneoxy-1,3-butadiene), wherein at least one of the R groups may include F2 and the other of the R groups may include, illustratively, a methyl or ethyl group; or such as Rawal diene, for example having the structure: (1-dialkylamino-3-trimethylsilaneoxy-1,3-butadiene), wherein at least one of the R groups may include F2 and the other of the R groups may include, illustratively, a methyl or ethyl group. Chan diene may be used similarly.

[0131] In some instances, the second functional group (F2) of the diene may be selected from the group consisting of oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels. Illustratively, the second functional group is an oligonucleotide. In some instances where the unsaturated cyclic diketone 110 is regenerated from the adduct 130 before reacting with the diene molecule 140, the second functional group (F2) of the diene molecule 140 may differ from the first functional group (F1) of the indole molecule 120. Illustratively, the first functional group may include an oligonucleotide and the second functional group may include elements other than oligonucleotides, such as hydrophilic molecules, hydrophilic macromolecules, catalysts, or labels. Alternatively, the first functional group may include a first oligonucleotide and the second functional group may include a second oligonucleotide different from the first oligonucleotide. Alternatively, the second functional group (F2) of the diene molecule 140 may be the same as the first functional group (F1) of the indole molecule 120.

[0132] In the non-limiting example illustrated in Figure 1E, the [4+2] cyclization reaction of TAD molecule 110 can provide a composition 102' comprising a plurality of second adduct molecules 150, which may have the following structure: wherein L is a linker attached to the substrate and F2 is a second functional group. However, based on the teachings of the present invention, other adducts of reactions between other unsaturated cyclic diketones and / or other dienes are conceivable. Illustratively, the reaction of an unsaturated cyclic diketone with trans, trans-1,3-hexadiene can produce an adduct: wherein X is CH or N. In the example where the unsaturated cyclic diketone is maleic anhydride, the adduct may have the following structure: In the example where the unsaturated cyclic diketone is 4-cyclopenten-1,3-diketone, the adduct may have the following structure:

[0133] The reaction between an unsaturated cyclic diketone 110 (e.g., TAD) and a diene molecule 140 (e.g., 1,3-diene) to form a second adduct molecule 150, a non-limiting example of which is illustrated in FIG. 1E, can be substantially irreversible. When such a reaction is carried out via the process illustrated in FIG. 1D (e.g., by contacting the unsaturated cyclic diketone 110 with the diene molecule 140), such a reaction can be referred to as an "ultra-click" reaction. However, the second adduct molecule 150 illustrated in FIG. 1E can alternatively be obtained via a different reaction process. More specifically, the diene molecule 140 can actually react with the first adduct molecule 130, for example by contacting the composition 100' with a suitable solvent comprising the diene molecule 140 in a manner as illustrated in FIG. 1F. In composition 101'' illustrated in Figure 1F, diene molecule 140 may replace indole or indazole molecule 120 from adduct molecule 130, causing the first functional group F1 to dissociate from substrate 101 and the second functional group F2 to couple to the substrate, thereby forming the second adduct molecule 150 of composition 102' illustrated in Figure 1E. Therefore, indole molecule 120 can be considered as "protecting" unsaturated cyclic diketone 110 (such as TAD) before adduct molecule 130 reacts with diene molecule 140.

[0134] It should be understood that, as described with reference to Figures 1A to 1F, the unsaturated cyclic diketone 110 coupled to the substrate 101 can be prepared using any suitable combination of operations. For example, the TAD molecule 110 can be prepared via an oxidation mechanism via a 4-substituted ureaazole (wherein the 4-substituent of the ureaazole is coupled to the substrate, for example, via linker L). For example, Figures 2A to 2C schematically illustrate example compositions and operations for coupling functional groups to a substrate. As illustrated in Figure 2A, the substrate 101 can be functionalized to include isocyanate 111 coupled thereto via linker L, for example using commercially available isocyanates from SiSiB® Silicones-PCC Group (Nanjing, China). Isocyanate 111 can be contacted with and condensed with a compound 112 having the following structure: it can be called N-aminoethoxycarbamate or hydrazine carbamate. In some instances, compound 112 can be obtained by reacting hydrazine (N2H4) with diethyl carbonate.

[0135] As illustrated in Figure 2B, the reaction product 113 of isocyanate 111 and compound 112 may have the following structure: where L is a linker attached to the substrate. The reaction product 113 may then undergo a base-mediated cyclization reaction to form a 4-substituted ureaazole 114, coupled to the substrate in a manner as illustrated in Figure 2C, and having the following structure: where L is a linker attached to the substrate. The 4-substituted ureaazole 114 may be oxidized to form a TAD molecule 110 coupled to the substrate 101 via linker L, as described with reference to Figures 1A and 1D. A wide variety of oxidation conditions may be used to convert the 4-substituted ureaazole 114 into the TAD molecule 110, such as oxidation using in-situ generated N2O4, superacid conditions, hypervalent iodide species, potassium peroxymonosulfate, hypochlorite, 1,4-diazabicyclo[2.2.2]octanebromide (DABCO-Br) or chlorate.

[0136] It should be noted that the reactivity of 4-substituted ureazole 114 can be significantly less than that of TAD molecule 110. Therefore, 4-substituted ureazole 114 can be considered as providing a "protecting" group and can be oxidized to form TAD molecule 110 only before the intended reaction of the TAD molecule with a diene or indole is described, as in reference to Figures 1A to 1F. Schemes for coupling other types of unsaturated cyclic diketones, such as those described elsewhere herein, can be suitably used.

[0137] As further mentioned above, although in some instances the unsaturated cyclic diketone is coupled to the substrate and reacts with the functionalized diene, indazole or indole in solution, in other instances the diene, indazole or indole may be coupled to the substrate and react with the functionalized unsaturated cyclic diketone in solution.

[0138] For example, Figures 3A and 3B schematically illustrate an example composition and operation for coupling a functional group to a substrate in another process. Referring now to Figure 3A, composition 300 includes a plurality of indole or indazole molecules 320 coupled to substrate 301 via individual linkers (L). Indole or indazole molecules 320 may have the following structure: where Z is CH or N; L includes a linker connected to the substrate; and R is H, an electron-withdrawing group, or an electron-donating group. In a non-limiting example illustrated in Figure 3A, indole molecule 320 may have the following structure: where L is a linker connected to substrate 301. Referring to Figures 1A to 1F, other example structures of indole and indazole are provided. Non-limiting examples of L and substrate are provided elsewhere herein. Substrate 301 may include a polymer (e.g., a polymer functionalized to include POSS) disposed on a solid support, or may include a solid support on which no polymer is disposed.

[0139] Indole or indazole molecules can react with unsaturated cyclic diketones including a functional group (F3) to form adducts that couple the functional group to a substrate. For example, indole molecule 320 can react with TAD molecule 310 including a functional group (F3) in a manner as illustrated in Figure 3A to form an adduct that couples the functional group to a substrate. For example, indole or indazole molecule 320 can contact unsaturated cyclic diketone 310 dissolved in any suitable solvent (e.g., a polar protic solvent, such as water or alcohol, or a polar aprotic solvent, such as acetonitrile, ester, or ether), which is compatible with both the unsaturated cyclic diketone molecule and the indole or indazole molecule.

[0140] Unsaturated cyclic diketones may have the following structure: wherein X is CH or N, and wherein F3 includes a functional group. In the non-limiting example illustrated in Figure 3A, the unsaturated cyclic diketone may include a TAD molecule 310 having the following structure: . Alternatively, the unsaturated cyclic diketone may include maleic anhydride or 4-cyclopenten-1,3-dione, which is functionalized to include F3 at the 4 position. The functional group may be or include any suitable one or more molecules, such as those described elsewhere herein. In a non-limiting example, the functional group (F3) may be selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels. Illustratively, the functional group (F3) may be or include oligonucleotides.

[0141] The reaction of an unsaturated cyclic diketone in solution with an indole or indazole molecule coupled to a substrate can provide a composition comprising a plurality of adduct molecules having the following structure: wherein Z is CH or N; L includes a linker attached to the substrate; R is H, an electron-withdrawing group, or an electron-donating group; F3 includes a functional group, and each X is independently CH or N. In the non-limiting example illustrated in Figure 3B, the reaction of TAD molecule 310 with indole molecule 320 can provide a composition 300' comprising a plurality of adduct molecules 330 having the following structure: wherein L is a linker attached to the substrate and F3 is a functional group.

[0142] As noted elsewhere herein, the reaction between an unsaturated cyclic diketone and an indole or indazole molecule can be reversible and may be referred to as a "reversible click" reaction. Therefore, in some instances, the adduct of such a reaction can be heated to a suitable temperature to regenerate the indole or indazole molecule coupled to the substrate and cause the unsaturated cyclic diketone (and the functional group F3 coupled thereto) to dissociate in a manner similar to that described with reference to Figure 1C. For example, substrate 301 can be heated to a temperature of at least 50°C, or about 50°C to about 100°C, or about 60°C to about 90°C, or about 60°C to about 80°C, or about 80°C to about 100°C, or about 90°C to about 100°C to regenerate the indole or indazole molecule 320 coupled to the substrate and cause the dissociation of the unsaturated cyclic diketone (e.g., TAD) molecule 310. As an alternative, after regenerating the indole or indazole molecule 320, the indole or indazole molecule can react with another functionalized unsaturated cyclic diketone molecule in a manner as described with reference to Figure 3A. Such a reaction can be similarly reversible, and thus the indole or indazole molecule 320 can be regenerated for other reactions, such as for reacting with a second unsaturated cyclic diketone molecule to form a second adduct. The second unsaturated cyclic diketone molecule may include functional groups, such as those selected from the group consisting of: a second oligonucleotide, a hydrophilic molecule, a hydrophilic macromolecule, a catalyst, and a label. The second oligonucleotide may have the same sequence or a different sequence as the oligonucleotide coupled to the earlier coupled unsaturated cyclic diketone. In examples of unsaturated cyclic diketones including TAD, TAD molecules can be prepared in a manner similar to that described with reference to Figures 2A to 2C, for example by providing a 4-substituted ureaazole, wherein the 4-substituent is a functional group (such as an oligonucleotide), and oxidizing the 4-substituted ureaazole to form a TAD including the functional group F3 (such as an oligonucleotide).

[0143] Figures 4A and 4B schematically illustrate example compositions and operations for coupling functional groups to a substrate in another process. More specifically, a diene can be coupled to the substrate, and an unsaturated cyclic diketone including a functional group can react with the diene to couple the functional group to the surface. Examples of diene molecules provided with reference to Figures 1A to 1F can be functionalized to include a linker L that couples the diene to the surface, rather than including the functional group itself. Referring now to Figure 4A, composition 400 includes a plurality of diene molecules 440 coupled to substrate 401 via individual linkers (L). In the illustrated example, diene molecule 440 may have the following structure: where L is a linker attached to substrate 401, but any other suitable 1,3-diene, 2,4-diene, Daniszewski diene, Brassal diene, or Laval diene may be used instead. Non-limiting examples of L and substrate are provided elsewhere herein. The substrate 401 may include a polymer disposed on a solid support (e.g., a polymer functionalized to include POSS), or may include a solid support on which no polymer is disposed.

[0144] Diene molecule 440 is reacted with an unsaturated cyclic diketone (e.g., TAD) molecule 410, including a functional group (F3), in a manner as illustrated in FIG. 4A, to form an adduct that couples the functional group to the substrate. For example, the diene molecule 440 coupled to the substrate may be contacted with an unsaturated cyclic diketone molecule 410 dissolved in any suitable solvent (e.g., a polar protic solvent, such as water or an alcohol, or a polar aprotic solvent, such as acetonitrile, an ester, or an ether), the solvent being compatible with both the unsaturated cyclic diketone molecule and the diene molecule. The unsaturated cyclic diketone molecule may have the following structure: where X is CH or N, and F3 includes a functional group. In the non-limiting example illustrated in FIG. 4A, the unsaturated cyclic diketone may include a TAD molecule 410 having the following structure: where F3 includes a functional group. In other examples, the unsaturated cyclic diketone may include maleic anhydride: or 4-cyclopenten-1,3-dione:. The functional group may be or include any suitable one or more molecules, as described elsewhere herein. In non-limiting examples, the functional group (F3) may be selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels. Illustratively, the functional group (F3) may be or include oligonucleotides.

[0145] The reaction of an unsaturated cyclic diketone with a diene molecule can provide a composition comprising a plurality of adduct molecules having the following structure: wherein each X is independently CH or N; L comprises a linker connected to a substrate; and F3 comprises a functional group. In the non-limiting example illustrated in FIG4B, the reaction of TAD molecule 410 with diene molecule 440 can provide a composition 400' comprising a plurality of adduct molecules 430 having the following structure: Other adducts between other dienes and other unsaturated cyclic diketones can be readily envisioned based on the teachings herein. The reaction between an unsaturated cyclic diketone and a diene molecule can be substantially irreversible and can be referred to as an "overspeed click" reaction. Methods using compositions containing functional groups coupled to a substrate

[0146] As noted elsewhere herein, an oligonucleotide is a non-limiting example of a functional group that can be coupled to a substrate in a manner such as that described with reference to Figures 1A-1F, 2A-2C, 3A-3B, or 4A-4B. Oligonucleotides coupled to a substrate in a manner such as that described herein can be used in a variety of amplification techniques. Examples of techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), or random prime amplification (RPA), or combinations thereof. In some instances, one or more primers used for amplification can be coupled to the substrate. Bridge amplification or dynamic exclusion amplification (ExAmp) can be achieved using two or more types of attachment primers, wherein the amplicon can form a bridge-like structure between two attachment primers that side-attach a replicated template sequence. Amplification can also be performed using an amplification primer attached to the substrate and a second primer in solution (e.g., emulsion PCR).

[0147] Alternatively or concurrently, oligonucleotides coupled to a substrate in a manner as described herein can be used to determine the sequence of a target polynucleotide. For example, the target polynucleotide can be coupled (e.g., hybridized) to one of a plurality of primers covalently bonded to the substrate in a manner as described herein. The target polynucleotide can be amplified using the plurality of primers to form a cluster of substrate-binding amplicons. This cluster of substrate-binding amplicons can be contacted with a labeled nucleotide (e.g., a fluorescently labeled nucleotide) and a polymerase, such that a detectable signal (e.g., fluorescence) is generated simultaneously with the incorporation of the nucleotide by the polymerase, and this signal can be used to identify the nucleotide and thereby determine the nucleotide sequence of the target polynucleotide. Working Example

[0148] Other examples are further detailed in the following examples, which are not intended to limit the scope of the patent application in any way. Example 1. Block copolymer coupled to TAD comprising oligonucleotides.

[0149] In one example, a first block copolymer (BCP1) comprising a diene is prepared using the following reaction procedure, and the diene is subsequently reacted with a TAD comprising an oligonucleotide (P5 or P7 primer) to form a second block copolymer (BCP2):

[0150] The NH2 group of BCP2 reacts with the substrate to covalently couple BCP2 to a glass or silicon dioxide support. The reaction proceeds on the attached isocyanate, activated carboxylic acid, or Michael acceptor. A clear indicator of the TAD-diene reaction is present because the TAD molecules are dark (purple-red) and the solution gradually becomes colorless as they react (are consumed). The concentration of TAD molecules, including oligonucleotides, is monitored using UV or colorimetric methods and can be performed instantly.

[0151] From this reaction process, it can be understood that an unsaturated cyclic diketone (such as TAD) including the desired functional group (such as an oligonucleotide (e.g., an initiator)) can react with a diene coupled to a substrate (such as a polymer disposed on a solid support), thereby coupling the functional group to the substrate. Example 2. Patterned pores coupled to a TAD including an oligonucleotide.

[0152] Figure 5 schematically illustrates an example composition and operation for coupling functional groups to a substrate in another process. The composition (a) illustrated in Figure 5 includes a glass solid support on which a polymer resin is disposed, which is patterned using nanoimprint lithography (NIL) to form pores. The method of patterning using NIL is described in WO2018 / 119053 and WO2018 / 118932, the entire contents of which are incorporated herein by reference. The method of preparing the substrate is described in WO2014 / 133905, the entire contents of which are incorporated herein by reference. In an example of the invention, the pores comprise a polymer coupled to a 4-substituted ureaazole ("ureaazole polymer"), as described with reference to Figures 2A to 2C. Composition (a) is oxidized using DABCO-Br to form composition (b), wherein the pores comprise a polymer coupled to a TAD molecule ("TAD polymer"), as described with reference to Figure 1A. Composition (b) is reacted with a non-functionalized indole molecule ("indole protectant") to form composition (c), wherein the pores comprise a polymer coupled to an adduct for the TAD-indole reaction, as described with reference to Figure 1B, but with functional group F1 omitted. Composition (c) is reacted with a diene of Example 3, functionalized to include an oligonucleotide initiator (P5 / P7) ("P5 / P7-diene grafting / displacement"), in a manner as described with reference to Figure 1F to form composition (d), wherein the pores comprise a polymer coupled to a TAD-diene adduct, as described with reference to Figure 1E. In some examples, a portion of the nanopores will be functionalized in this method, leaving some remaining TAD portion available for the reaction.

[0153] The indole "reversible click" reaction forming composition (c) is mild and can be carried out at room temperature. A large excess of indole molecules can be used to provide complete or substantially complete protection of the polymer-coupled TAD molecules, thereby preventing or inhibiting subsequent reactions of these molecules during downstream chemical or biochemical operations. Furthermore, the "trans-click" reaction of the indole-TAD adduct of composition (c) with the functionalized diene to form composition (d) can be a specific and substantially irreversible interchange reaction, which is also mild, non-etching, and therefore compatible with many different types of substrates, and can be carried out within minutes at room temperature. The oligonucleotide-functionalized diene replaces at least some of the indole protecting agent molecules previously coupled to the substrate.

[0154] From this reaction process, it can be understood that a diene including a desired functional group (such as an oligonucleotide (e.g., an initiator)) can react with an indole-protected TAD coupled to a substrate (such as a polymer disposed on a solid support), thereby coupling the functional group to the substrate. Example 3. Block copolymer coupled to a diene including an oligonucleotide.

[0155] In another example, a first block copolymer (BCP1) comprising 4-substituted ureazole was prepared using the following reaction procedure, followed by oxidation of ureazole with DABCO-Br (obtained by reacting DABCO with Br2 in a suitable solvent such as DCM or CHCl3) to form a third block copolymer (BCP3) comprising TAD, and TAD was reacted with a diene comprising an oligonucleotide (P5 or P7 primer) to form a fourth block copolymer (BCP4):

[0156] The NH2 groups of BCP4 react with the substrate to covalently couple BCP4 to a solid support as described in Example 1. Additional reactions are carried out according to the process described below to produce a polymer containing TAD / unsaturated cyclic diketones. The first process shown below uses reversible addition-fragmentation chain-transfer (RAFT) polymerization. These polymers are then reacted with dienes to form oligomeric functionalized materials that are surface-attached using a similar chemical method (i.e., via covalent attachment of the remaining TAD units, not all of which are exhausted during the polymer coating step, thus leaving a large remainder for reaction with the diene oligomer). The subsequent process shown below details a method for producing the monomer Az-TAD used in the first reaction process below.

[0157] From these reaction processes, it can be understood that dienes including desired functional groups (such as oligonucleotides (e.g., initiators)) can react with ureazole-protected unsaturated cyclic diketones (such as TAD) coupled to a substrate (such as a polymer disposed on a solid support), thereby coupling the functional groups to the substrate. Other notes

[0158] It should be understood that any individual feature / example of each of the embodiments of the invention described herein may be implemented together in any suitable combination, and any feature / example of any one or more of such embodiments may be implemented together with any feature of any of the other embodiments described herein in any suitable combination to achieve the benefits described herein.

[0159] Although various illustrative examples have been described above, it will be apparent to those 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. [Simplified Explanation of the Diagram]

[0054] Figures 1A to 1F schematically illustrate example compositions and operations used in a process for coupling functional groups to a substrate.

[0055] Figures 2A to 2C schematically illustrate example compositions and operations for another process of coupling functional groups to a substrate.

[0056] Figures 3A and 3B schematically illustrate example compositions and operations for another process of coupling functional groups to a substrate.

[0057] Figures 4A and 4B schematically illustrate example compositions and operations used in another process for coupling functional groups to a substrate.

[0058] Figure 5 schematically illustrates an example composition and operation for coupling a functional group to a substrate in another process. [Sequence List]

[0161] <![CDATA[ <110> Illumina, Inc. (USA) and Illumina Cambridge Limited (UK) <![CDATA[ <120> Compositions containing functional groups coupled to a substrate and methods for manufacturing the same thereof <![CDATA[ <140> TW111100168]]> <![CDATA[ <141> 2022-01-04 <![CDATA[ <150> US 63 / 133,955 <![CDATA[ <151> 2021-01-05 <![CDATA[ <160> 4 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 29]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polynucleotides <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (23)..(23)]]> <![CDATA[ <223> n is U]]> <![CDATA[ <400> 1]]> aatgatacgg cgaccaccga ganctacac 29 <![CDATA[ <210> 2]]> <![CDATA[ <211> 24]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polynucleotides <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (twenty two)..(22)]]> <![CDATA[ <223> G* represents G or 8-side-oxyguanine. <![CDATA[ <400> 2]]> caagcagaag acggcatacg anat 24 <![CDATA[ <210> 3]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polynucleotides <![CDATA[ <400> 3]]> aatgatacgg cgaccaccga 20 <![CDATA[ <210> 4]]> <![CDATA[ <211> 21]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Synthetic polynucleotides <![CDATA[ <400> 4]]> caagcagaag acggcatacg a 21.

Claims

1. A method for coupling a functional group to a substrate, the method comprising: providing an unsaturated cyclic diketone coupled to the substrate; and reacting the unsaturated cyclic diketone with an indole or indazole including a first functional group to form a first adduct coupling the first functional group to the substrate.

2. The method of claim 1, wherein the unsaturated cyclic diketone is: wherein L comprises a linker connected to the substrate and each X is independently CH or N.

3. The method of claim 2, wherein the unsaturated cyclic diketone is a triazoline diketone: .

4. The method of claim 2, wherein the unsaturated cyclic diketone is a maleic diacetylimine: .

5. The method of claim 2, wherein the unsaturated cyclic dione is 4-cyclopenten-1,3-dione: .

6. The method of any one of claims 1 to 5, wherein the indole or indazole is: wherein F1 contains the first functional group; R is H, an electron-withdrawing group or an electron-donating group; and Z is CH or N.

7. The method of claim 6, wherein the indole is 1H-indole: .

8. The method of claim 6, wherein the indole is 1H-indazole: .

9. The method of claim 6, wherein the first adduct is: wherein L comprises a connector connected to the substrate and each X is independently CH or N.

10. The method of claim 9, wherein the first adduct is: .

11. The method of any one of claims 1 to 5, further comprising heating the first adduct to regenerate the cyclic unsaturated diketone coupled to the substrate.

12. The method of any one of claims 1 to 5, further comprising reacting the first adduct with a diene including a second functional group to form a second adduct that couples the second functional group to the substrate.

13. The method of claim 12, wherein the diene comprises 1,3-diene.

14. The method of claim 13, wherein the 1,3-diene is: wherein F2 contains the second functional group.

15. The method of claim 14, wherein the second adduct is: wherein L comprises a connector connected to the substrate.

16. The method of claim 12, wherein the second functional group is selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels.

17. The method of claim 16, wherein the second functional group is an oligonucleotide.

18. The method of any one of claims 1 to 5, wherein the first functional group is selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels.

19. The method of claim 18, wherein the first functional group is an oligonucleotide.

20. The method of any one of claims 1 to 5, wherein the substrate comprises a polymer disposed on a solid support.

21. The method of claim 20, wherein the polymer is functionalized to include polyhedral oligomeric silsesquioxane (POSS).

22. The method of any one of claims 1 to 5, wherein providing the unsaturated cyclic diketone coupled to the substrate comprises: providing a 4-substituted ureaazole coupled to the substrate; and oxidizing the 4-substituted ureaazole to form a triazolinone.

23. A composition comprising: a substrate; and an adduct coupled to the substrate: wherein L comprises a linker connected to the substrate; F1 comprises a first functional group; each X is independently CH or N; R is H, an electron-withdrawing group or an electron-donating group; and Z is CH or N.

24. The composition of claim 23, wherein the adduct is: .

25. The composition of claim 23 or claim 24, wherein the first functional group is selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels.

26. The composition of claim 25, wherein the first functional group is an oligonucleotide.

27. The composition of claim 23 or 24, wherein the substrate comprises a polymer disposed on a solid support.

28. The composition of claim 27, wherein the polymer is functionalized to include polyhedral oligomeric silsesquioxane (POSS).

29. A method for coupling a functional group to a substrate, the method comprising: providing an unsaturated cyclic diketone coupled to the substrate; and reacting the unsaturated cyclic diketone with a diene including the functional group to form an adduct that couples the functional group to the substrate; wherein the substrate comprises a polymer disposed on a solid support; and wherein the polymer is functionalized to include polyhedral oligomeric silsesquioxane (POSS).

30. The method of claim 29, wherein the unsaturated cyclic diketone is: wherein L is a linker connected to the substrate and each X is independently CH or N.

31. The method of claim 30, wherein the unsaturated cyclic diketone is a triazoline diketone: .

32. The method of claim 30, wherein the unsaturated cyclic diketone is a maleic diacetylimine: .

33. The method of claim 30, wherein the unsaturated cyclic dione is 4-cyclopenten-1,3-dione: .

34. The method of any one of claims 29 to 33, wherein the diene comprises 1,3-diene.

35. The method of claim 34, wherein the 1,3-diene is: wherein F2 contains the functional group.

36. The method of claim 35, wherein the adduct is: wherein L comprises a connector connected to the substrate.

37. The method of any one of claims 29 to 33, wherein the functional group is selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels.

38. The method of claim 37, wherein the functional group is an oligonucleotide.

39. The method of any one of claims 29 to 33, wherein providing the unsaturated cyclic diketone coupled to the substrate comprises: providing a 4-substituted ureaazole coupled to the substrate; and oxidizing the 4-substituted ureaazole to form a triazolinone.

40. A composition comprising: a substrate; and an adduct coupled to the substrate: wherein L comprises a linker connected to the substrate, F2 comprises a functional group, and each X is independently CH or N; wherein the substrate comprises a polymer disposed on a solid support; and wherein the polymer is functionalized to include polyhedral oligomeric silsesquioxane (POSS).

41. The composition of claim 40, wherein the adduct is: .

42. The composition of claim 40, wherein the adduct is: .

43. The composition of claim 40, wherein the adduct is: .

44. The composition of any one of claims 40 to 43, wherein the functional group system is selected from the group consisting of: oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels.

45. The composition of claim 44, wherein the functional group is an oligonucleotide.

46. ​​A method for coupling a functional group to a substrate, the method comprising: providing an indole or indazole coupled to the substrate; and reacting the indole or indazole with a first unsaturated cyclic diketone comprising an oligonucleotide to form a first adduct that couples the oligonucleotide to the substrate.

47. The method of claim 46, wherein the first unsaturated cyclic diketone is: , wherein F3 comprises the oligonucleotide and each X is independently CH or N.

48. The method of claim 47, wherein the first unsaturated cyclic diketone is a triazolinone: .

49. The method of any one of claims 46 to 48, wherein the indole or indazole is: , wherein Z is CH or N, L contains a linker connected to the substrate, and R is H, an electron-withdrawing group, or an electron-donating group.

50. The method of claim 49, wherein the indole is 1H-indole: where L is a linker connected to the substrate.

51. The method of claim 49, wherein the first adduct is: wherein F3 contains the oligonucleotide, and each X is independently CH or N.

52. The method of any one of claims 46 to 48, further comprising heating the first adduct to regenerate the indole or indazole coupled to the substrate.

53. The method of claim 52, further comprising, after regenerating the indole or indazole coupled to the substrate, reacting the indole or indazole with a second unsaturated cyclic diketone to form a second adduct.

54. The method of claim 53, wherein the second unsaturated cyclic diketone includes a functional group.

55. The method of claim 54, wherein the functional group is selected from the group consisting of: second oligonucleotides, hydrophilic molecules, hydrophilic macromolecules, catalysts, and labels.

56. The method of any one of claims 46 to 48, wherein the substrate comprises a polymer disposed on a solid support.

57. The method of claim 56, wherein the polymer is functionalized to include polyhedral oligomeric silsesquioxane (POSS).

58. The method of any one of claims 46 to 48, further comprising: providing a 4-substituted ureazole comprising the oligonucleotide; and oxidizing the 4-substituted ureazole to form an unsaturated cyclic diketone triazolinone comprising the oligonucleotide.

59. A composition comprising: a substrate; and an adduct coupled to the substrate: , wherein L comprises a linker connected to the substrate, F3 comprises an oligonucleotide, each X is independently CH or N, and Z is CH or N.

60. The composition of claim 59, wherein the adduct is: .

61. The composition of claim 59 or claim 60, wherein the substrate comprises a polymer disposed on a solid support.

62. The composition of claim 61, wherein the polymer is functionalized to include polyhedral oligomeric silsesquioxane (POSS).

63. A method for coupling a functional group to a substrate, the method comprising: providing a diene coupled to the substrate; and reacting the diene with an unsaturated cyclic diketone comprising an oligonucleotide to form a first adduct that couples the oligonucleotide to the substrate.

64. The method of claim 63, wherein the unsaturated cyclic diketone is: , wherein F3 contains the oligonucleotide and each X is independently CH or N.

65. The method of claim 64, wherein the unsaturated cyclic diketone is a triazolinone: .

66. The method of claim 64, wherein the unsaturated cyclic diketone is a maleic diacetylimine: .

67. The method of claim 64, wherein the unsaturated cyclic dione is 4-cyclopenten-1,3-dione: .

68. The method of any one of claims 63 to 67, wherein the diene comprises 1,3-diene.

69. The method of claim 66, wherein the 1,3-diene is: , wherein L comprises a connector attached to the substrate.

70. The method of claim 69, wherein the adduct is: wherein L comprises a linker connected to the substrate, each X is independently CH or N, and F3 comprises the oligonucleotide.

71. The method of claim 70, wherein the adduct is: .

72. The method of any one of claims 63 to 67 and 69 to 71, wherein the substrate comprises a polymer disposed on a solid support.

73. The method of claim 72, wherein the polymer is functionalized to include polyhedral oligomeric silsesquioxane (POSS).

74. The method of any one of claims 63 to 67 and 69 to 71, further comprising: providing a 4-substituted ureazole comprising the oligonucleotide; and oxidizing the 4-substituted ureazole to form an unsaturated cyclic diketone triazolinone comprising the oligonucleotide.

75. A composition comprising: a substrate; and an adduct coupled to the substrate: wherein L comprises a linker connected to the substrate, each X is independently CH or N, and F3 is an oligonucleotide.

76. The composition of claim 75, wherein the adduct is: .

77. The composition of claim 75 or 76, wherein the substrate comprises a polymer disposed on a solid support.

78. The method of claim 77, wherein the polymer is functionalized to include polyhedral oligomeric silsesquioxane (POSS).

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