CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The application claims the benefit of priority to U.S. Provisional Application No.

62/670,627, filed May 11, 2018, the entirety of which is incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] The need for low cost, high-throughput, methods for nucleic acid sequencing and re sequencing has led to the development "massively parallel sequencing" (MPS) technologies. One commonly used method for sequencing DNA is referred to as "sequencing-by-synthesis" (SBS). See Blackburn et al., Curr, Genomics 16(3):159-174 (2015); Stranneheim and Lundeberg, Biotechnoi. J. 7:1063-73 (2012); and Guo et al., Acc. Chem. Res. 43:551-563 (2010).

[0003] SBS requires the controlled [i.e., one at a time) incorporation of the correct complementary nucleotide opposite the oligonucleotide or DNA template being sequenced. This allows for accurate- sequencing by adding nucleotides in multiple cycles as each nucleotide residue is sequenced one at a time, thus preventing an uncontrolled series of incorporations occurring. In one approach reversible terminator nucleotides (RTs) are used to determine the sequence of the DNA template. In the most commonly used SBS approach, each RT comprises a modified nucleotide that includes (1) a blocking group that ensures that only a single base can be added by a DNA polymerase enzyme to the 3' end of a growing DNA copy strand, and (2) a fluorescent label that can be detected by a camera. In the most common SBS methods, templates and sequencing primers are fixed to a solid support and the support is exposed to each of four DNA nucleotide analogs, each comprising a different fluorophore attached to the nitrogenous base by a cleavable linker, and a 3'-0-azidomethyl group at the 3' -OH position of deoxyribose, and DNA polymerase. Only the correct, complementary base anneals to the target and is subsequently incorporated at the 3' terminus of primer. Nucleotides that have not been incorporated are washed away and the solid support is imaged. TCEP (tris(2-carboxyethyl)phosphine) is introduced to cleave the linker and release the fluorophores and to remove the 3'-0-azidomethyl group, regenerating a 3'-OH. The cycle can then be repeated (Bentley et al., Nature 456, 53-59, 2008). A different fluorescent color label is used for each of the four bases, so that in each cycle of sequencing, the identity of the RT that is incorporated can be identified by its color.

[0004] Despite the widespread use of SBS, improvements are still needed. For example, current SBS methods require expensive reversibly terminated d NTPs (RTs) with a label (e.g., dye) on the base connected by a cleavable linker resulting in a) a chemical scar left on the incorporated bases after label cleavage, b) less efficient incorporation, c) quenching, d) excited dye induced termination of extension, and reducing signal in each sequencing cycle.

BRI EF DESCRI PTION OF TH E DRAWI NGS

[0005] FIG. 1 is an illustration of an exemplary embodiment of a sequencing method of the invention. FIG. 1A shows a polysubstrate (PS). FIG. IB shows a primer extension product (PEP) with a dNTP incorporated at the 3' terminus of the growing strand. FIG. 1C shows a PS with a binding moiety directly bound to a 3' terminus of the growing strand. FIG. ID shows a PS with a binding moiety indirectly bound to a 3' terminus of the growing strand; the PS binding moiety is bound to a primary binder. FIG. IE shows a PS in which the binding moiety is streptavidin (A). The binding moiety is bound to an incorporated d NTP with a biotin affinity tag (B). FIG. I F shows the emission of light by the substrate molecules In the presence of a luminescent enzyme.

[0006] FIG. 2 is a schematic of an exemplary embodiment of a sequencing method of the invention.

[0007] FIG. 3 is a schematic of an exemplary process for the synthesis of a poiysubstrate of the present disclosure (e.g., a streptavidin labeled dextran polymer covalently bound to luciferin).

[0008] FIGS. 4A-4D are schematics showing exemplary 3' blocking groups of a dNTP analog of the present disclosure.

DETAI LED DESCRI PTION OF TH E I NVENTION

1. DEFINITIONS AND TERMS

[0009] The terminology used herein Is for the purpose of describing pa rticular embodiments only, and is not intended to be limiting, because the scope of the present invention will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, i n this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent. I n some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should noi he construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0010] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 0.1 or 1.0, as appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about."

[0011] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of compounds.

[0012] The term“comprising" is intended to mean that the compounds, compositions and methods include the recited elements, but not excluding others. "Consisting essentially of" when used to define compounds, compositions and methods, shall mean excluding other elements that would materially affect the basic and novel characteristics of the claimed invention. "Consisting of" shall mean excluding any element, step, or ingredient not specified in the claim. Embodiments defined by each of these transition terms are within the scope of this invention

[0013] As used herein, "luminescence" refers to emission of light (a detectable signal) by a substance resulting from a chemical reaction. Luminescence results from action of an enzyme or catalyst (e.g., luciferase or horseradish peroxidase) on a substrate molecule [e.g., luciferin or luminol). As used herein luminescence includes chemiluminescence and bioluminescence. Reactions producing luminescence may be called "luminescent reactions." Enzymes involved in these reactions may be called a "luminescent enzyme" or "luminescence producing enzyme".

[0014] As used herein, a "substrate molecule" is a molecule or compound upon which an enzyme acts, including natural and non-naturally occurring proteins and chemical compounds that emit light when acted on by a luminescent enzyme. A substrate molecule includes substrate molecules that are oxidized by an enzyme under suitable conditions to emit light. Examples of substrate molecules include luciferin, colenterzine, luminol and isoiuminol.

[0015] The terms "luminescent enzyme" or "luminescence producing enzyme" refers to an enzyme that catalyzes a reaction that produce light from a substrate molecule attached to a polymer molecule of a polysubstrate. Example of luminescent enzymes include firefly luciferase, Reniiia luciferase, Cypridina luciferase, Aequorin photoprotein, Obelin photoprotein, peroxidase, horseradish peroxidase, and the like. [0016] As used herein, in the context of a nucleotide analog, the terms "unlabeled" and “non- labeled" are used interchangeably and refer to a nucleotide analog lacking a fluorescent moiety (e.g., fluorescent dye).

[0017] As used herein, unless otherwise apparent from confexf, "nonlabled reversible terminator [nucleotide]," "NLRT," "reversible terminator nucleotide," "reversible terminator," "RT," and the like are all used to refer to a sequencing reagent comprising a nucleobase or analog, deoxyribose or analog, and a cleavable blocking group. A noniabied reversible terminator nucleotide may refer to a dNTP [i.e., a substrate for polymerase) or a reversible terminator nucleotide incorporated to into a primer extension product, initially at the 3' terminus and, following additional incorporation cycles, if any, in an "internal" portion of the primer extension product. In some embodiments, a NLRT refers to a RT lacking a fluorescent moiety. NLRTs are described in US Pat. Pub. 2018/0223358 A1 at (but not limited to) 1Hl[0037]-[0046] and [0074]-[0114].

[0018] The terms "reversible blocking group," of a reversible terminator nucleotide may also be referred to as a "removable blocking group," a "cleavable linker," a "blocking moiety," a "blocking group," "reversible terminator blocking group" and the like. A reversible blocking group is a chemical moiety attached to the nucleotide sugar (e.g., deoxyribose), usually at the 3' -O position of the sugar moiety, which prevents addition of a nucleotide by a polymerase at that position. A reversible blocking group can be cleaved by an enzyme (e.g., a phosphatase or esterase), chemical reaction, heat, light, etc., to provide a hydroxyl group at the 3'-position of the nucleoside or nucleotide such that addition of a nucleotide by a polymerase may occur.

[0019] As used herein, a "dNTP analog" includes both naturally occurring deoxyribonucleotide triphosphates and analogs thereof, including analogs with a 3'-0 cleavable blocking group.

[0020] As used herein, in the context of a cleavable blocking group of a nucleotide analog, the designation 3'-0-" is sometimes implied rather than explicit. For example, the terms“azidomethyl", "3'- O-azidomethyl" are interchangeable as will be apparent from context.

[0021] The terms "reversible," "removable," and "cleavable" in reference to a cleavable blocking group have the same meaning.

[0022] The term "reversible blocking group," of a reversible terminator nucleotide may also be referred to as a "removable blocking group," a "cleavable linker," a "blocking moiety," a "blocking group," "reversible terminator blocking group" and the like. A reversible blocking group is a chemical moiety attached to the nucleotide sugar (e.g., deoxyribose), usually at the 3' -O position of the sugar moiety, which prevents addition of a nucleotide by a polymerase at that position. A reversible blocking group can be cleaved by an enzyme (e.g., a phosphatase or esterase), chemical reaction, heat, light, etc., to provide a hydroxyl group at the 3'-position of the nucleoside or nucleotide such that addition of a nucleotide by a polymerase may occur.

[0023] "Antigen" as used herein means a compound that can be specifically bound by an antibody. Some antigens are immunogens (see, janeway, et aL, Immunobiology, 5th Edition, 2001, Garland Publishing). Some antigens are haptens that are recognized by an antibody but which do not elicit an immune response unless conjugated to a protein. Exemplary antigens include NLRTs, reversible- terminator blocking groups, dNTPs, polypeptides, small molecules, lipids, or nucleic acids.

[0024] "Array" or "microarray" means a solid support (or collection of solid supports such as beads) having a surface, preferably but not exclusively a planar or substantially planar surface, which carries a collection of sites comprising nucleic acids (e.g., DNA template molecules or DMA nanobails) such that each site of the collection is spatially defined and not overlapping with other sites of the array; that is, the sites are spatially discrete. The array or microarray can also comprise a non-planar interrogatable structure with a surface such as a bead or a well. The solid support may also comprise oligonucleotides covalently or non-covalently bound to the solid support that hind a nucleic add to the array. Conventional microarray technology is reviewed in, e.g., Scbena, Ed. (2000), Microarrays: A Practical Approach (lRL Press, Oxford). As used herein, "random array" or "random microarray" refers to a microarray where the identity of the nucleic acids is not discernable, at least initially, from their location but may be determined by a particular biochemistry detection technique on the array. See, e.g., U.S. Pat. Nos. 6,396,995; 6,544,732; 6,401,267; and 7,070,927; PCT publications WO 2006/073504 and 2005/082098; and U.S. Pat. Pub. Nos, 2007/0207482 and 2007/0087362.

[0025] "Derivative" or "analog" means a compound or molecule whose core structure is the same as, or closely resembles that of, a parent compound, but which has a chemical or physical modification, such as a different or additional side group, or 2' and or 3' blocking groups.

[0026] With respect to nucleotides and nucleosides this includes derivatives that allow the nucleotide or nucleoside to be linked to another molecule. For example, the base can be a deazapurine. The derivatives should be capable of undergoing Watson-Crick pairing. "Derivative" and "analog" also mean a synthetic nucleotide or nucleoside derivative having modified base moieties and/or modified sugar moieties. Such derivatives and analogs are discussed in, e.g., Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et aL, Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also comprise modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl- phosphonate, phosphoranilidate and phosphoramidate linkages. The analogs should be capable of undergoing Watson-Crick base pairing. For example, deoxyadenosine analogs include didanosine (dd I ) and vidarabine, and adenosine analogs include, BCX4430; deoxycytidine analogs include cytarabine, gemcitabine, emtricitabine (FTC), lamivudine (3TC), and zalcitabine (ddC); guanosine and deoxyguanosine analogs include abacavir, aciclovir, and entecavir; thymidine and deoxythymidine analogs include stavudine (d4T), telbivudine, and zidovudine (azidothymidine, or AZT); and deoxyuridine analogs include idoxuridine and trifluridine. "Derivative", "analog" and "modified" as used herein, may be used interchangeably, and are encompassed by the terms "nucleotide" and "nucleoside" defined herein.

[0027] With respect to chemical compounds and molecules, a “derivative" or "analog" means a compound or molecule whose core structure is the same as, or closely resembles that of, a parent compound or molecule, but which has a chemical or physical modification, such as a different or additional side group or functional group. For example, the term includes a derivative or analog of a substrate molecule (e.g., analogs and derivatives of luciferin) that produce a signal after exposure to a catalyst (e.g., luciferase) under suitable condtions.

[0028] "incorporate" means becoming part of a nucleic acid molecule. In SBS, incorporation of an RT occurs when a polymerase adds an RT to a growing DIMA strand through the formation of a phosphodiester or modified phosphodiester bond between the 3' position of the pentose of one- nucleotide, that is, the 3' terminal nucleotide on the DIMA strand, and the 5' position of the pentose on an adjacent nucleotide, in some embodiments, incorporate includes incorportion of a nucleotide or dNTP analog at the 3' end of a primer extension product.

[0029] As used herein, an "affinity reagent" means a moiety that recognizes (e.g., binds) a base, sugar, cleavable blocking group, or a combination of these components in a dNTP analog incorporated into a nucleic acid molecule in some instances, the incorporated dIMTP analog is the 3' terminal nucleotide of a primer extension product,

[0030] As used herein, an "affinity tag ligand" means a member of a specific binding pair (e.g., biotin) that is attached to a dIMTP analog, for example, by way of a linker. Affinity tag ligands and uses thereof are described, for example, in US Patent Publication 2017/0240961, incorporated herein by reference. In some aspects, after incorporation of the dIMTP analog into a nucleic acid molecule or primer extension product, the incorporated dIMTP analog is bound by exposed to an affinity reagent having a second member of the specific binding pair (e.g., streptavidin), which can facilitate detection of the incorporated dIMTP analog. For example, the binding moiety of a polysubstrate may comprise the affinity tag anti -ligand. Affinity tags are also described in US Pat. Pub. 2018/0223358 Al, e.g.,

[0031] "Label," in ibe context of a labeled affinity reagent, means any atom or molecule that can be used to provide a detectable and/or quantifiable signal. Suitable labels include radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemicaily active molecules, enzymes, cofactors, and enzyme substrates. In some embodiments, the detection label is a molecule containing a charged group (e.g., a molecule containing a cationic group or a molecule containing an anionic group), a fluorescent molecule (e.g., a fluorescent dye), a fluorogenic molecule, or a metal. Optionally, the detection label is a fluorogertic label. A fluorogenic label can be any label that is capable of emitting light when in an unquenched form (e.g , when not quenched by another agent). The fluorescent moiety emits light energy (i.e., fluoresces) at a specific emission wavelength when excited by an appropriate excitation wavelength. When the fluorescent moiety and a quencher moiety are in close proximity, light energy emitted by the fluorescent moiety is absorbed by the quencher moiety. In some embodiments, the fluorogenic dye is a fluorescein, a rhodamine, a phenoxazine, an acridine, a coumarin, or a derivative thereof in some embodiments, the fluorogenic dye is a carboxyfiuorescein. Further examples of suitable fluorogenic dyes include the fluorogenic dyes commercially available under the Alexa Fluor ^* product line (Life Technologies, Carlsbad, CA). Alternatively, non-fluorogenic labels may be used, including without limitation, redoxgenic labels, reduction tags, thio- or thiol-containing molecules, substituted or unsubstituted alkyls, fluorescent proteins, non-fluorescent dyes, and luminescent proteins such as luciferase and horseradish peroxidase.

[0032] "Nucieobase" means a nitrogenous base that can base-pair with a complementary nitrogenous base of a template nucleic acid. Exemplary nucleobases include adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), inosine (I) and derivatives of these. References to thymine herein should be understood to refer equally to uracil unless otherwise clear from context. As used herein, the terms "nucieobase," "nitrogenous base," add“base" are used interchangeably.

[0033] A "naturally occurring nucieobase," as used herein, means adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (Li). In some cases, naturally occurring nucieobase refers to A, C, G and T (the naturally occurring bases found in DNA).

[0034] A "nucleotide" consists of a nucieobase, a sugar, and one or more phosphate groups. They are monomeric units of a nucleic acid sequence in RNA, the sugar is a ribose, and in DNA a deoxyribose, i.e. a sugar lacking a hydroxyl group that is present in ribose. The nitrogenous base is a derivative of purine or pyrimidine. The purines are adenine (A) and guanine (G), and the pyrimidines are cytosine (C) and thymine (T) (or in the context of RNA, uracil (U)). [0035] "Nucleic acid" means a polymer of nucleotide monomers. As used herein, the terms may refer to single- or double-stranded forms. Monomers making up nucleic adds and oligonucleotides are capable of specifically binding to a natural polynucleotide by way of a regular pattern of monomer-to- monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Such monomers and their internucleosidic linkages may be naturally occurring or may be analogs thereof, e.g., naturally occurring or non-naturally occurring analogs. Non-naturally occurring analogs may include, phosphorothioate internucleosidic linkages, bases containing linking groups permitting the attachment of labels, such as haptens, and the like. Whenever a nucleic acid or oligonucleotide is represented by a sequence of letters (upper or lower case), such as "ATGCCTG," it will be understood that the nucleotides are in 5’ to 3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and “T" denotes thymidine, denotes deoxyinosine, "IT denotes uridine, unless otherwise indicated or obvious from context. Unless otherwise noted the terminology and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiiey-Liss, New York, 1999), Usually nucleic acids comprise the natural nucleosides (e.g,, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DMA or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs, e.g,, modified bases, sugars, or internucleosidic linkages.

[0036] "Primer" means an oligonucleotide, either natural or synthetic, which is capable, upon forming a duplex with a polynucleotide template (e.g., a template DNA molecule), of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process are- determined by the sequence of the template polynucleotide. Usually primers are extended by a DNA polymerase in a template dependent manner. Primers usually have a length in the range of from 9 to 40 nucleotides, or in some embodiments, from 14 to 36 nucleotides.

[0037] "Polynucleotide" is used interchangeably with the term "nucleic acid" to mean DNA, RNA, and hybrid and synthetic nucleic acids and may be single-stranded or double-stranded. "Oligonucleotides" are short polynucleotides of between about 6 and about 300 nucleotides in length. "Complementary polynucleotide" refers to a polynucleotide complementary to a target nucleic acid.

[0038] "Solid support" and "support" are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. Microarrays usually comprise at least one planar solid phase support, such as a glass microscope slide. [0039] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymerase" refers to one agent or mixtures of such agents, and reference to "the method" includes reference to equivalent steps and/or methods known to those skilled in the art.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Ail publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, compositions, formulations and methodologies which are described in the publications and which might be used in connection with the presently described invention.

[0041] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0042] In the following description, numerous specific details are set forth to provide a more- thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[0043] Although the present invention is described primarily with reference to specific embodiments, it is also envisioned that other embodiments will become apparent to those skilled in the art upon reading the present disclosure, and it is intended that such embodiments be contained within the present inventive methods.

[0044] The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. i-IV), Using Antibodies: A Laboratory Manual, Ceils: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Flarbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, N.Y., Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, I RL Press, London, Nelson and Cox (2000), Lebninger, Principles of Biochemistry 3rd Ed., W. H. Freeman Pub., New York, N.Y. and Berg et ai. (2002) Biochemistry, 5th Ed., W. H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for ail purposes. 2. OVERVIEW

[0045] This application describes luminescence-based nucleic acid sequencing systems using a polysubstrate (“PS"). Sequencing methods are well known to those of ordinary skill in the art (see Appendix A, [0281]-[0296] . The polysubstrates of the invention may be used in a number of different sequencing methods but find particular application in sequencing by synthesis (SBS) using reversible terminator nucleotide analogs. See, Stranneheim and Lundeberg, Biotechnol J. 7:1063-73 (2.012) and Guo et ai., Acc. Chem. Res. 43:551-563 (2010).

[0046] In one approach, polysubstrates of the invention comprise a polymer molecule, a plurality of substrate molecules attached to the polymer molecule, and a binding moiety attached to the polymer molecule (see, Fig. 1A). In one approach the binding moiety (e.g., antibody) specifically binds a 3' terminal nucleotide of a primer extension product, e.g., as described in US Pat. Pub. 2018/0223358 Al (see, Fig. 1C). Alternatively, the binding moiety can be associated indirectly with a 3' terminal nucleotide of a primer extension product by, for example, specifically binding a primary antibody or other affinity reagent to the 3' terminal nucleotide and binding the binding moiety of the PS to the primary antibody or other affinity reagent (see, Figs. ID and IE).

[0047] Polysubstrates associated with 3' terminal nucleotides of SBS primer extension products produce luminescent signals in the presence of a "luminescent enzyme" (see, Fig. IF). The signal may be used to characterize the 3' terminal nucleotide of a primer extension product (e.g., identify the nucleotide as adenosine, guanosine, cytidine, thymidine, or uridine monophosphate). This information may be used to identify the complementary nucleotide in the primer extension template. Combinations of different PS species may be used in combination for massively parallel sequencing, in one aspect, the PS is used in combination with antibody based SBS methods described in US Pat. Pub. 2018/0223358 Al. [0048] Polysubstrates (PS) and compositions comprising a polysiibstrate(s) are described in this section, in general , a polysubstrate comprises:

(i) a polymer molecule;

(ii) a plurality of substrate molecules attached to the polymer molecule; and

(iii) a binding moiety attached to the polymer molecule.

In some aspects, the substrate molecules are substrates for luminescent enzymes. Thus, in some- embodiments the substrate molecules may be referred to as luminescent enzyme substrates." In some aspects, the binding moiety specifically binds a 3' terminal nucleotide of a primer extension product, such as a primer extension product produced by SBS. In some aspects, the binding moiety binds to a primary binding moiety, and the primary binding moiety specifically binds a 3' terminal nucleotide of a primer extension product. After the PS is bound directly or indirectly to the 3' terminal nucleotide of a primer extension product (see, e.g., Figs. 1C, ID and IE), a luminescent enzyme is contacted with the PS resulting in emission of a detectable signal (see, Fig. IF). By using combinations of polysubstrates with different binding moieties (e.g., binding moieties specific for different nucleobases) and optionally different substrates, luminescent signals may be produced which identify the nucleobase of the 3' terminal nucleotide (e.g., as A, T, G or C).

3.1 Polymer Molecules

[0049] Polymer molecules can be thought of as a scaffold for holding a plurality of substrate molecules (e.g., at least 2, usually at least 5, substrate molecules). Polymer molecules may have a variety of physical structures and chemical compositions. For illustration, polymer molecules (1) may be linear or branched and (2) may be a homopolymer, a heteropolymer or co-polymer. In some embodiments the polymer molecule is, or comprises, a polysaccharide. An exemplary polymer molecule is dextran, a branched polysaccharide, but polymer molecules comprising many other polymers may be used Including polysaccharides, polynucleotides, or polypeptides as well as synthetic polymers such as a polyacrylate, polyhydroxyalkanoate (PHA), polylactic acid (PLA), poly-b-malic acid PMA, polythioester (PTE), and the like. In some embodiments, the polymer molecule is not a polysaccharide.

[0050] Polymer molecules may have a wide variety of lengths or sizes. In some embodiments, the polymer molecule comprises at least five (5) linked monomers. In another embodiment, the polymer comprises about 5 to about 1000 monomers. In some embodiments, the polymer molecule of the polysubstrate comprises a molecular mass of between about 10 KDa and about 10,000 KDa. In some embodiments, the polymer molecule Is dextran with a molecular mass in the range of about 10 KDa to about 2,000 KDa.

3.1.1 Linear polymers

[0051] In some embodiments, suitable polymer molecules for use with the invention are linear polymers. Linear polymers may comprise a single type of chemical bond (e.g., a glycosidic bond) between monomers of the polymer molecule (e.g., dextrin) in some embodiments, a linear polymer is a homopolymer (e.g., cellulose, starch or polyethylene). In some embodiments, a linear polymer is a heteropolymer (e.g., lactose). Suitable linear polymer molecules include cellulose, starch, polyethylene, glucose, mannose, galactose, fructose, lactose, maltose, sucrose, or a combination thereof. In embodiments, the linear polymer includes at least 5 monomers. In another embodiment, the linear polymer includes about 5 to about 1000 monomers.

3.1.2 Branched polymers

[0052] In some embodiments, suitable polymer molecules for use with the invention are branched polymers, such as dextran, glycogen, starch or iamlnarin. in one embodiment, the branched polymer comprises at least two branch points.

3.1.3 Homopolymers

[0053] In some embodiments, the polymer molecule of the polysubstrate is a homopolymer. Suitable homopolymers for use with the invention include glucose, polyethylene or polyacrylate.

3.1.4 Heteropolymers

[0054] In some embodiments, the polymer molecule of the polysubstrate is a heteropolymer. Suitable heteropolymers for use with the invention include sucrose, acrylate or styrene.

3.1.5 Polysaccharide Polymers

[0055] In some embodiments, the polymer molecule is a polysaccharide having a plurality of covalently linked monosaccharides or disaccharides in one embodiment, the polysaccharide polymer includes at least 5 covalently linked sugar monomers in another embodiment, the polysaccharide polymer includes about 5 to about 1000 covalently linked sugar monomers. In some embodiments, the polysaccharide polymer comprises a linear or branched carbohydrate polymer such as, but not limited to, glucose, fructose, mannose, lactose, maltose, sucrose, glyceraldehyde, or a combination thereof.

[0056] In some embodiments, the polysaccharide polymer includes one, or more than one, types of covalent bonds between monomers (e.g. a- or b-glycosidic bonds, N-, C-, S- or O-glycosidic bonds). In some embodiments, the polysaccharide polymer includes a plurality of O-glyosidic bonds. [0057] In some embodiments, the polymer molecule comprises glucose monomers. For example, the polymer molecule can be an glucan (e.g., a- or b-glucan) comprising D-giucose monomers linked by glycosidic bonds. In one embodiment, the glucose polysaccharide is linear. In another embodiment, the glucose polysaccharide is branched. Glucose polysaccharides suitable for use in the invention include D- glucose and/or i-glucose. In one embodiment, the glucose polysaccharide includes at least 5 covalently linked glucose monomers. Glucans suitable for use with the invention include cellulose (b-1,4), chrysoiaminarin (b-1,3), curdlan (b-1,3), iaminarin (b-1,3 and b-1,6), lentinan (b-1,6 and (b-1,3 purified from Lentinus edodes), lichenin (b-1,3 and b-1,4), zymosan (b-1,3), dextran (a- 1,6), glycogen (a-1,4 and a -1,6), pullulan (a -1,4 and a -1,6), amylose (a -1,4), amylopectin (a-1,6), and starch (a -1,4 and a- 1,6)).

3.1.6 Non-Polysaccharide Polymers

[0058] Non-polysaccharide polymers suitable for use in the invention include various polymer structures (e.g., linear or branched) and compositions (e.g., homopolymers or heteropolymers) such as, polymers of proteins, polymers of nucleic acids, and synthetic polymers.

[0059] In some embodiments, the polysubstrate comprises a synthetic non-polysaccharide polymer molecule. Synthetic polymer molecules include homopolymers and heteropolymers including, but not limited to, polyacrylate, polyethylene, polypropylene, polystyrene, polyvinyl chloride, synthetic rubber, phenol formaldehyde resin, neoprene, nylon, polyacrylonitrile, polyester, PVB, silicone, combinations, and derivatives, thereof.

3.2 Substrate Molecules

[0060] The polysubstrate comprises a plurality of substrate molecules attached to the polymer molecule. Substrate molecules are substrates for an enzyme in a reaction that produces a detectable signal, usually a luminescent signal. In a substrate-enzyme combination in which a luminescent signal is produced the enzyme may be referred to as a "luminescent enzyme" and the substrate molecule may be referred to as a "luminescent enzyme substrate." In various embodiments, the action of an enzyme on a substrate molecule results in conversion or modification of the substrate molecule in which the substrate molecule is converted to an intermediate or converted into an excited state. Exemplary reactions are oxidation reactions and reduction reactions. In some embodiments, the substrate- molecule is a protein.

[0061] In some embodiments, the plurality of substrate molecules attached to the polymer molecule is at least 5 substrate molecules. In some embodiments, the plurality of substrate molecules attached to the polymer molecule is at least 5, at least 6, at least 7, at least S, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 500, at least 750, at least 1000, or more substrate molecules

[0062] Various types of substrate molecules are suitable for use in the invention. Luminescent enzyme substrates are of particular interest. Exemplary luminescent enzyme substrates suitable for use in the invention include proteins such as luciferin or coelenterazine and derivatives or analogs of luciferin or coelenterazine, and non-protein molecules such as luminol or isoluminol and derivatives or analogs of luminol or isoluminol. Other suitable substrate molecules include, without limitation, acridinium, 2,2’-Azinobis[3-ethylbenzothiazoline-6-suifonic acid]-diammonium salt (ABTS), o- phenylenediamlne dihydrochloride (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), o-nitrophenyl-b-D- galactopyranoside (ONPG), derivatives or analogs thereof.

3.2.1. Luminol Isoluminol, and PACCs

[0063] In some embodiments, the substrate molecule is luminol. Luminol (e.g., C ₈ H ₇ N ₃ O _2; CAS lMo: 521-31-3) is a compound that exhibits luminescence when combined with an oxidizing agent (e.g., hydrogen peroxide) in the presence of a peroxidase (e.g., horseradish peroxidase (HRP)) or other enzyme or catalyst. This reaction may be referred to as the "luminol reaction." Derivatives of luminol are known in the art and include, but are not limited to, luminol analogs set forth in EP Patent 0515194.

[0064] Luminol is a species within a larger family of compounds known as 2,3-dihydro-l,4- phthalazinediones (PACCs). Members of the PACC family suitable for use with the instant disclosure include compounds set forth in U.S. Patent No: 4,861,778, 5-amlno-6,7,8-trimethoxy- and dimethylamino[ca]benz analogs described in U.S. Patent No. 4,374,925, and Canadian Patent CA1147336.

[0065] Luminol is a substrate of the enzyme, horseradish peroxidase (HRP). Oxidation of luminol by HRP yields several unstable intermediates that emit excess energy as light, which can be detected. The product occurring after light emission can be referred to as 3-aminophtbalate.

[0066] In some embodiments, the substrate molecule is isoluminol. Isoluminol is a derivative of luminol. Isoluminol (e.g., 4-aminophthalhydrazide; CAS No: 3682-14-1) is a compound that exhibits luminescence when combined with an oxidizing agent (e.g., hydrogen peroxide) in the presence of a peroxidase (e.g., horseradish peroxidase) or other enzyme or catalyst. This reaction may be referred to as the "isoluminol reaction". Various derivatives of isoluminol are known including, but not limited to, activated ester derivatives, N-aminobutyl-N-ethyl isoluminol (ABEI), see Palmiolo et al., Tetrahedron Letters, 54:33, 4446-4450 (2013) and derivatives described by Messeri et al., Luminescence, 4:1, 154-158 (1989). Isoluminol analogs suitable for use with the disclosure include, but are not limited to, those set forth in U.S. Patent No. 4,226,993,

3.2.2 Luciferin

[0067] In some embodiments, the substrate molecule is luciferin (e.g., C ₁₁ H ₈ N ₂ O ₃ S ₂ ; CAS lMo: 2591-17- 5). Luciferin is a substrate of the enzyme luciferase. Oxidation of luciferin by luciferase or other enzyme or catalyst yields an excited ketone which emits excess energy as light, which can be detected. The product occurring after light emission can be referred to as oxyluciferin. Luciferins are found in various species of fireflies (e.g., Lampyridae species)), beetles, snails, bacteria, fungi, and marine organisms (e.g., Varguia luciferin and Dinofiagellata luciferin). Notably, different species of luciferin can emit light of different wavelengths. For example light from Photuris pennsyivanica luciferin can be measured at 552 nm (green-yellow) while Pyrophorus plagiophthaiamus luciferin emissions can be measured at 582 nm (orange).

[0068] Derivatives and analogs of luciferin are known in the art. For a review of luciferin synthesis, see, Meroni et al., ARKlVOC 2009 (i) 265-2.88 (2009). Derivatives of luciferin include, but are not limited to, coelenterazine (discussed below), selenoterazine, diphenylterazine, furimazine, and combinations thereof. Additional derivatives of luciferin are known (see, Kiyama et al, Current Topics in Medicinal Chemistry, 16 (24): 2648-2655 (2016)). Analogs of luciferin include, but are not limited to, those described in U.S. Patent No. 8,962,854, PCI Publications WO 2010/106896 and WO2013/027770, EP Patent 1930332, Japanese Patent No. 1,001,380, Takakura et al., Chemistry An Asian Journal, 5(9):2053- 2061 (2010), ioka et al., Chemistry A European Journal, 22(27):9330-9337 (2016), and Ikeda et al., Chemical Communications, 54:1774-1777 (2018).

[0069] Luciferin can be obtained from various commercial sources, for example, as (S)--2-(6-Hydroxy- 2-benzothiazolyl)-2-thiazoline-4-carboxylic acid, 4,5-Dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazole carboxylic add (Catalog Nos: L.9504 and L6152, Sigma).

3.2.3 Coelenterazine

[0070] In some embodiments, the substrate molecule is coelenterazine. Coelenterazine (e.g., C ₂₆ H ₂₁ N ₃ O ₃ ; CAS NO: 55779-48-1) can be found in aquatic organisms including squid, shrimp, ctenophores and radiolarians. Coelenterazine is a substrate for luciferase.

[0071] Derivatives of coelenterazine are known in the art. For example, Vece and Vuocolo substituted the C-3 position to obtain three novel derivatives ( Tetrahedron , 71(46):87Sl-85 (2015)); while Yuan et al., replaced the methylene group at C-8 with an electronic conjugation to produce a shift in luminescent signal by 63 nm relative to native coelenterazine {Chinese Chemical Letters , 27(4):550- 554 (2016)).

[0072] Derivatives of coelenterazine include coelenterazine cp (luminescence intensity 15 times higher than native coelenterazine), coelenterazine F (luminescence intensity 20 times higher and emission spectra about 8 nm longer than native coelenterazine), coelenterazine FCP (luminescence intensity 135 times higher than native coelenterazine), coelenterazine H (luminescence intensity 10 times higher than native coelenterazine), coelenterazine hep (luminescence intensity 190 times higher than native coelenterazine), all available from Biotium, Fremont, CA.

[0073] Other derivatives of coelenterazine include, but are not limited to, bis-deoxy-coelenterazine analogs (see, for example, Nakamura et a I., Tetrahedron Letters, 38:6405 (1997)) and coelenterazine analogs described in U.S. Patent No. 8,809,529, U.S. Patent Publication 2017/0217969, PCT Publication WO2018/022865 and Japanese Patent No. 6,160,716.

3.3 Attachment Of Substrates Molecules To Polymer Molecule

[0074] In some embodiments, a substrate molecule of the polysubstrate is attached to a polymer molecule covalently or noncovalentiy using methods known in the art. In some embodiments, each of the plurality of substrate molecules is attached by a covalent bond at regular intervals across the length of the polymer molecule. In one approach a polymer molecule and/or substrate molecule is functionalized for covalent binding. For example, polyimine compounds such as those disclosed in U.S. Patent No. 8,268,933 can be reacted with synthetic polymers to form functionalized polymer molecules that can attach to substrate molecules in the polysubstrate. In some approaches the substrate molecule and polymer molecule are attached non-covaiently. For example, the substrate molecule can be derivatized with a ligand (one member of a specific binding pair) and the polymer molecule can be derivatized with multiple copies of a corresponding anti-ligand (second member of a specific binding pair) in another approach, polymer molecule monomers can be attached to a substrate and then polymerized to form the polymer molecule-substrate molecule portion of the polysubstrate. A variety of other approaches will be apparent to those of ordinary skill in the art. In some embodiments, about 1% to about 95%, about 5% to about 90%, about 10% to about 80%, about 20% to about 70%, about 30% to about 60%, or about 50%, of the total number (or proportion) of monomers in a polymer molecule are attached to a substrate molecule. In some embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or more, of the total number of monomers in a polymer molecule are attached to a substrate molecule. In some embodiments, the ratio of monomers in the polymer molecule attached to a substrate molecule is 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, or 1:1. 33.1 Homogeneous and Heterogeneous Substrate Molecules

[0075] In some embodiments, the number of substrate molecules attached to a polymer molecule- may be fixed (e.g., constant) or variable. For example, a first PS may contain 10 substrate molecules, while a second PS may contain 100 substrate molecules. In this approach, even if the first and second polysubstrates comprise the same substrate molecule or combination of molecules they can be distinguished based on the intensity of the signal produced. By manipulating the number of substrate molecules present on a PS or within sets of PS, the signal produced by the substrate molecules within the polysubstrate can be modulated. In some cases, increasing the number of substrate molecules attached to the polymer molecule can result in an increase in signal. In some embodiments, decreasing the number of substrate molecules attached to the polymer molecule can result in a decrease in signal.

[0076] In some embodiments, a polysubstrate may contain a single species of substrate molecule (e.g., luminol), i.e., has homogeneous substrate molecules. In another embodiment, a polysubstrate can include two, or more, species of a substrate molecules (e.g., luciferin and luminol; luciferin and coelenterazine; P. pennsylvanica luciferin and P. plagiophthalamus luciferin), i.e., has heterogeneous substrate molecules.

3.4. Binding moiety

[0077] The binding moiety directly or indirectly binds to the incorporated dNTP analog in the primer extension product, so that the association of a (particular) polysubstrate and a (particular) primer extension product, e.g., at a (particular) location on an array, and the resulting signal detected therefrom, identifies the dNTP analog incorporated in the particular primer extension product at the particular location on the array. 3.4.1 Direct Binding (e.g., Using An affinity reagent

[0078] In one approach the Incorporated dNTP analog Is a naturally occurring nucleotide, a reverse terminator nucleotide with a 3' -OH blocking group but without a linker (e.g., deavable linker) by which a fluorophore, dye or affinity tag is linked to the nucieobase or phosphate moiety of the 3' incorporated nucleotide. In one approach the binding moiety is an affinity reagent, e.g., as described in US Pat. Pub. 2018/0223358 Al, especially 1)0115-110167). In some embodiments, the affinity reagent binds to the nucieobase, sugar, deavable blocking group, or combination of these components. As discussed below, affinity reagents may be antibodies, aptamers, affirmers, knottins, fusion proteins and the like, that can specifically bind, or bind to another binding moiety that can specifically recognize and bind a dNTP analog incorporated at the 3' end of a primer extension product. [0079] Alternatively, the dNTP analog may be bound by an affinity reagent (e.g,, mouse anti-dATP) and the binding moiety portion of the polysubstrate (e.g,, goat anti-mouse lgG) may bind the affinity reagent using well known principles used in indirect antibody (or other binding agent) labeling. In some approaches, the binding moiety is an affinity reagent, which can specifically recognize the nucleobase, the sugar (e.g., deoxyribose), the blocking group, or combination thereof in the dNTP analog.

[0080] Affinity reagents and binding moieties bind a target (e.g., dNTP analog) with specificity, in the context of an affinity reagent, "specificity" is the degree to which the affinity reagent discriminates between different molecules (e.g., dNTP analogs) as measured, for example, by relative binding affinities of the affinity reagent for the molecules. With respect to the affinity reagents of the present invention, an affinity reagent should have substantially higher affinity for one dNTP analog (its target) than for other dNTPs (for example, the affinity reagent binds to a C nucleoside analogue but not to A, T or G). Also, the affinity reagent binds to its target nucleoside analog at the 3' end of a polynucleotide when incorporated by a polymerase, but not to a nucleotide base elsewhere on the polynucleotide chain. An affinity reagent is specific for a particular dNTP analog, such as dATP, if in the presence of a plurality of template polynucleotides (e.g., on an array) in which 3'-termini of primer extension products include dATP, dTTP, dCTP, and dGTP; the affinity reagent binds preferentially to dATP under reaction conditions used in SBS sequencing. As used herein, "preferential binding" of an affinity agent to a first structure- compared to a second structure means the affinity agent binds the first structure but does not bind the second structure or binds the second structure less strongly (i.e., with a lower affinity) or less efficiently.

[0081] in some approaches, the binding moiety recognizes and binds an epitope of the newly incorporated dNTP analog. Typically, the dNTP analog is generally one of: dTTP, dCTP, dGTP, dCTP and analogs thereof. Thus, if a binding moiety specifically binds an epitope of dTTP (or analog thereof), the binding moiety does not specifically bind an epitope of dCTP, dGTP, dATP, or analogs thereof.

3.4.2. Anti-dNTP Antibody

[0082] In some approaches, the binding moiety is an antibody (including binding fragments of antibodies, single chain antibodies, bispecific antibodies, and the like), that recognizes the naturally occurring 3' incorporated base or dNTP analog of a primer extension product, e.g., as described in US Pat. Pub. 2018/0223358 Al.

[0083] In the case of affinity reagents that are antibodies, specific binding of the binding moiety can be determined using any assay for antibody binding known in the art, including Western Blot, enzyme- linked immunosorbent assay (ELISA), flow cytometry, or column chromatography. In one approach specific binding is demonstrated using an ELISA type assay. For example, serum antibodies raised against B'-azidomethyl-dC can be serially titrated against a bound substrate of 3'- G-azidomethyl-dC (positive specificity assay) and nucleotide[s) such as 3'-0-azidomethyl-dG or -dA or 3'-OH-dC (negative specificity assay).

[0084] in some approaches, the binding moiety is a primary antibody that directly recognizes the naturally occurring 3' incorporated base or dNTP analog of a primer extension product, as described in US Pat. Pub. 2018/0223358 Al. In some instances, the primary antibody is covalently linked to a polysubstrate, and the PS having the primary antibody can specifically bind the dNTP analog incorporated at the 3' end of the primer extension product. Once bound, the PS is acted on by a luminescent enzyme resulting in the production of a detectable signal, which can be used to identify the dNTP analog incorporated at the 3' end of the primer extension product.

3.4.3 Other Binding Molecules ( e.g Binding Affinity Tags }

[0085] Binding moieties may be any molecule or moiety that binds a defined target molecule with high specifically, in some examples, a binding moiety (e.g., affinity reagent) is an aptamer, knottin, affirmer or fusion protein.

[0086] In some approaches, the binding moiety is an aptamer that recognizes the naturally occurring 3' incorporated base or dNTP analog, as described in US Pat. Pub. 2018/0223358 Al. In some approaches, the binding moiety is an affirmer that recognizes the naturally occurring 3' incorporated base or dNTP analog, as described in US Pat. Pub. 2018/0223358 Al. In some approaches, the binding moiety is a knottin that recognizes the naturally occurring 3' incorporated base or dNTP analog, as described in US Pat. Pub. 2018/0223358 Al. In some approaches, the binding moiety is an antibody fusion affinity reagent that recognizes the naturally occurring 3' incorporated base or dNTP analog, as described in US Pat. Pub. 2018/0223358 Al

3.4.4 Other Ligand-Anti-Ligand Combinations

[0087] in some approaches, a nucleotide analog comprising an affinity tag is used in SBS and the polysubstrate binding moiety binds to the affinity tag. Thus, in some embodiments the binding moiety does not specifically bind to the nucleotide (nucleobase, sugar, or 3ΌH blocking group, as described in US Pat. Pub. 2018/0223358 Al). Any suitable ligand-anti-ligand combination (also called specific binding pairs, SBPs) may be used, where one member of the pair is the binding moiety of the polysubstrate and the second member of the pair is associated with, usually covalently bound, to the 3' incorporated base. Exemplary ligand-anti-iigand/specific binding pairs include biotin/avidin, biotin/streptavidin, antibody/antigen, lectin/carbohydrate, and the like. [0088] In some approaches, an affinity reagent or other binding molecule ("primary binder ) specifically binds to the incorporated 3' terminal nucleotide, and the binding moiety of the polysubstrate specifically binds the primary binder. For example, a 3' incorporated dCTP analog may be bound by a mouse antibody that specifically binds the 3' incorporated dCTP analog, and the polysubstrate binding moiety, e.g., goat anti-mouse IgG, may bind the primary binder. See Table 7 for illustrative examples.

[0089] Detection of 3'-dNTPs using direct and is also described in US Pat. Pub. 2018/0223358 Al.

4. LUMINESCENT ENZYMES

[0090] As described above, the substrate molecules of polysubstrates are acted upon by enzymes, e.g., luminescent enzymes, to produce light [a detectable signal) in a luminescent reaction. The light emitted has a characteristic wavelength (color), intensity (brightness), and duration (kinetics). Detection of light with specific characteristics at a position of a DMA array identifies the polysubstrate(s) bound at that array position, including the specificity of the polysubstrate binding moiety. It will be appreciated that knowledge of the poiysubstrafe binding moiety identifies the incorporated 3'-nucieotide and the complementary nucleotide at the corresponding position of the template strand in one aspect the invention provides a composition comprising:

1. A template DNA;

2. A primer extension product with a 3' terminal nucleotide;

3. A polysubstrate that directly or indirectly binds the 3' terminal nucleotide; and

4. A luminescent enzyme that acts on the substrate molecules of the polysubstrate to produce light (i.e., catalyzes a light producing reaction). Enzymes and, more specifically, enzyme - substrate molecule combinations for producing a luminescent signal are well known and may be adapted to the present invention. Table 1 provides exemplary enzyme-substrate molecule combinations suitable for use with the invention.

[0091] TABLE 1

Substrate molecule class of enzyme species of enzyme

Luciferin, or analog/derivative luciferase Renilla Luciferase

Coelenterazine, or analog/derivative luciferase Renilia Luciferase

Luminol, or analog/derivative peroxidase horseradish peroxidase Isoluminol, or analog/derivative peroxidase horseradish peroxidase [0092] The action of a luminescent enzyme on a substrate molecule results in conversion or modification of the substrate molecule. In some embodiments, the luminescent enzyme causes a substrate molecule to undergo an oxidation or reduction reaction. In some embodiments, the luminescent enzyme converts a substrate molecule into an intermediate compound or excited state.

[0093] Various classes of enzymes are suitable for use with the disclosure, including, but not limited to luminescence producing enzymes. Enzymes suitable for use herein, include, but are not limited to, luciferase, peroxidase, alkaline phosphatase, b-galactosidase, b-giucosidase, b-giucuronidase, b- lactamase, caspase, ceiiulase, cytochrome p450, kinase, lipase, peptidase, phosphatase, phosphoiipidase and protease.

[0094] In some embodiments, the luminescent enzyme is a luciferase. Luciferases can be found in bacteria, insects, fungi, and marine organisms. In some embodiments, luciferase catalyzes the oxidation of a substrate molecule (e.g., luciferin] in a poiysubstrate to produce oxyluciferin and concomitant release of light. Release of light from the poiysubstrate can be detected, for example using a luminometer or any suitable radiant energy-measuring device. Exemplary iuciferase's suitable for use with the invention include Iuciferase's isolated from Firefly, Renilia, Gaussia, and Cypndino (Vargulin).

[0095] Firefly Iuciferase's generally produce light in the yellow and red regions of the emission spectrum. However, various derivatives are known, including Green Renilia luciferase which emits light of a green wavelength. Accordingly, the detectable signal released by a poiysubstrate can be based, in part, on the species of luciferase acting on the substrate molecule.

[0096] Substituted polypeptides or fusion proteins encoding Iuciferase's are known in the art, including, but not limited to, those set forth in U.S. Patent Nos. 5,670,356; 8,003,350; PCI Publication WQ2017/221873 and U.S. Publications US2014/0186918; US2014/0201855; US2015/0152395 and 2018/0057801.

[0097] In some embodiments, the luminescent enzyme is horseradish peroxidase (HRP). HRP substrates include cbromogenic (e.g., TMB, DAB, ABTS) and luminescent substrates (e.g., luminoi and isoluminol). In some embodiments, a luminescent substrate molecule is reacted with HRP in the presence of hydrogen peroxide to produce a signal (e.g., light). HRP can catalyze the oxidation of luminoi to 3-aminophthalate via several intermediates resulting in the emission of light, which can be detected. HRP is commercially available in unmodified or modified forms (see for example, Catalog Nos. 77332 and P1709, Sigma). 5. DISTINGUISHING POLYSUBSTRATES BASED ON DIFFERENCES IN SIGNALS

[0098] Sequencing-by-synthesis methods have been described in which four nucleotides (A, T, G and C) are determined using four colors of signals (four color or four channel sequencing), two colors of signals (two color or two channel sequencing), and one color of signals (one color or one channel sequencing). In variations of these methods dNTPs or dNTP analogs may be combined with templates (e.g., flowed into a flow cell) sequentially or in combination, and four, or fewer than four, such as two or one, images may be required. In some variations detectable labels are disassociated from and/or associated with a template. See WO 2009/097368 and WO 2013/044018, both incorporated herein for ail purposes. All of these methods and others may be carried out using polysubstrates of the invention.

[0099] As described above, light emitted from a poiysubstrate has characteristic properties, including wavelength (color), intensity (brightness), and duration (kinetics). Detection of light with specific characteristics at a position of a DIMA array identifies the polysubstrate(s) bound at that array site, including the specificity of the poiysubstrate binding moiety, and knowledge of the poiysubstrate binding moiety identifies the incorporated 3'-nucleotide and the complementary nucleotide at the corresponding position of the template strand. Thus, polysubstrates specific for different 3' terminal nucleotides (i.e., comprising different binding moieties) can be distinguished based on differences in the substrate molecule composition of the poiysubstrate.

5.1 Wavelength

[0100] As noted above, different substrates, even when acted on by the same luminescent enzyme, produce light of different wavelengths. For example, P. pennsylvanica luciferin generally produces light at about 552. nm, P. plagiophthalamu luciferin produces light at about 582 nm, and coelenterazine produces light at about 460 nm. Thus, three polysubstrates with different (homogenous) substrate- molecules could be distinguished based on wavelength. Analogs of luciferase and luciferase substrates can produce "red-shift" emissions (i.e,, a shift to longer wavelength) of about 620-750 nm (see, Leoning et ai., Nat. Methods. 7(1) :5-6 (2010)). See Tables 3 and 4. The skilled reader will understand that as used herein, a "wavelength" refers to the wavelength maxima, or wavelength of maximum emission intensity, for a compound or reaction, and one of skill in the art will understand that emission is over a wavelength spectrum. The skilled reader will also understand that the wavelength associated with a given compound or reaction may be affected by pH, temperature, or other compounds in a reaction composition. 5.2 intensity

[0101] ln some embodiments, the methods include detecting the intensity of light produced by the polysubstrate. Intensity may vary with the number of substrate molecules associated with the polysubstrate molecule. For example, a polysubstrate with 100 linked luminol molecules with emit light more intensely [or more brightly) than a polysubstrate with 5 linked luminol molecules. Likewise, the intensity of a signal can be controlled or modulated by contacting an array with a mixture of a given polysubstrate (e.g., anti-dATP as the binding moiety, dextran as polymer, and luminol as substrate molecule) and a "cold" analog of the polysubstrate molecule (e.g., anti-dATP as the binding moiety, dextran as polymer, and no substrate molecule). See Table 2.

[0102] TABLE 2

5.3 Duration

[0103] In some embodiments, the methods disclosed herein comprise distinguishing polysubstrates based on the duration of signal from the linked substrate molecules. Substrate molecules when acted on by a luminescent enzyme may result in differing kinetics with respect to the production of light. For example, some substrate molecules may have a rapid rate of signal decay (flash kinetics) or a prolonged rate of signal decay (glow kinetics). For example, Reniila Luciferase Flash Assay Kit, (Catalog No. 16164, ThermoFisher Scientific) and Reniila Luciferase Glow Assay Kit (Catalog No. 16167, ThermoFisher Scientific). See, Brady et al., Anal. Chem., 2001, 73(24):5777-5783. Glow reactions are characterized by a converted substrate formation period on the order of minutes and persistence of light emission at full intensity for up to several hours. Flash reactions are characterized by a substrate formation period and light emission on the order of seconds. A flash luciferase typically provides an intense signal of light that is detected over a short period of time (e.g., seconds) in contrast, a glow luciferase generally provides a less-intense signal of light over a longer duration (e.g., minutes).

[0104] In one approach, duration of signal from substrates molecules of a polysubstrate can be used to distinguish between dNTP analog incorporation. For example, a first polysubstrate may contain a set of flash substrate molecules; while a second polysubstrate may contain a set of glow substrate molecules in this example, both polysubstrates are acted on by the same luminescent enzyme (e.g., luciferase), and yet the first polysubstrate will produce a signal for a shorter duration as compared to the second polysubstrate. As such, the use of duration can be used to distinguish between nucleotides incorporated at the 3' terminal nucleotide of a primer extension product even if the wavelength produced by the substrate molecules is the same.

6. SEQUENCING METHODS

[0105] The instant disclosure provides methods for sequencing one or more DMA template molecules or detecting incorporation of a dNTP analog at the 3' end of a primer extension product.

[0106] In some approaches, methods for detecting incorporation of a dNTP analog at the 3' end of a primer extension product are provided. In some embodiments, the methods include incorporating a dNTP analog at the 3' end of a primer extension product using a DNA polymerase to produce a 3' terminal nucleotide of a primer extension product; associating a polysubstrate with the 3' terminal nucleotide of a primer extension product to form a labeled primer extension product; combining the labeled primer extension product and at least one luminescent enzyme; and detecting a signal produced by the action of the at least one luminescent enzyme on the labeled primer extension product, thereby detecting incorporation of the dNTP analog at the 3' end of the primer extension product.

[0107] In some approaches, the dNTP analog is a reversible terminator nucleotide in some embodiments, the dNTP analog contains a 3'-O reversible blocking group on deoxyribose. In some approaches, the dNTP analog includes an affinity fag ligand (e.g., biotin). In some approaches, the dNTP analog includes a cleavable linker connecting an affinity tag ligand to a nucleobase. In some approaches, the dNTP analog does not contain a fluorophore or fluorescent dye. In some embodiments, the dNTP analog includes a naturally occurring or unmodified nucleoside of DNA or RNA. In some embodiments, the dNTP analog includes an unmodified nucleobase. in some approaches, the dNTP analog includes a dNTP analog that hybridizes to adenosine, guanosine, thymidine, uridine, cytidine, or a deoxyribonucleoside thereof. In some embodiments, the dNTP analog is a mixture of different dNTP analog species. In one approach, a mixture of dNTP analogs includes a dNTP analog for each nucleoside to which the dNTP analog hybridizes (e.g., a dNTP analog for each of A, G, T and C).

[0108] In some approaches, the incorporating includes Incorporating multiple different species of dNTP analogs at the 3' end of a plurality of different primer extension products immobilized on a DNA array using a DNA polymerase to produce a plurality of 3' terminal nucleotides of primer extension products. In some embodiments, the multiple different species of dNTP analogs comprise a dNTP analog that hybridizes to adenosine; a dNTP analog that hybridizes to thymidine and/or uridine; a dNTP analog that hybridizes to cytidine, and a dNTP analog that hybridizes to guanosine. In some approaches, the incorporating includes contacting the DNA array with multiple different species of dNTP analogs simultaneously. In some approaches, the incorporating includes contacting the DNA array with multiple different species of dNTP analogs sequentially.

[0109] In some embodiments, the primer extension products are annealed to a DNA template molecule immobilized on the DNA array. In some embodiments, the DNA template molecule is a DNA nanobali or amplicon from a clonal cluster (e.g., an amplicon produced via bridge amplification or Wildfire amplification).

[0110] In various embodiments, the template polynucleotide is DNA (e.g., cDNA, genomic DNA, or amplification products) or RNA. In various embodiments, the polynucleotide is double stranded or single stranded.

[0111] in some embodiments, the template nucleic acid is immobilized on a solid surface. In some embodiments, the template nucleic acid is immobilized on a substrate (e.g., a bead, flow cell, pad, channel in a microfluidic device and the like). The substrate may comprise silicon, glass, gold, a polymer, PDMF, and the like.

[0112] in some embodiments, the template nucleic acid is immobilized or contained within a droplet (optionally immobilized on a bead or other component within the droplet).

[0113] It will be understood that the method is not limited to a particular form of DNA template molecules, and the DNA template molecules can be any template such as, for example, a DNA concatemer, a dendrimer, a clonal population of templates {e.g., as produced by bridge amplification or Wildfire amplification) or a single polynucleotide molecule. Thus, importantly, the specification should be read as if each reference to a DNA template molecule can alternatively refer to a concatemer template, a clonal population of short linear templates, a single molecule template (e.g., in a zero-mode waveguide), and templates in other forms. [0114] Suitable DMA template molecule, including DNBs, clusters, polonys, and arrays or groups thereof, are further described in U.S. Pat. Nos, WO 2007133831, 8,440,397; 8,445,194; 8,133,719;

8,445,196; 8,445,197; 7,709,197; 12/335,168, 7,901,891; 7,960,104; 7,910,354; 7,910,302; 8,105,771;

7,910,304; 7,906,285; 8,2.78,039; 7,901,890; 7,897,344; 8,2.98,768; 8,415,099; 8,671,811; 7,115,400;

8,236,499, and U.S. Patent Publication Nos. 2015/0353926; 2010/0311602; 2014/0228223; and 2013/0338008, ali of which are hereby incorporated by reference in their entirety for all purposes and particularly for all disclosure related to nucleic acid templates, concatemers and arrays according to the present invention.

[0115] As is well known, high throughput sequencing is automated and reagents may be automatically introduced into a flow ceil containing a template array (or combined with a nucleic acid array in any other manner). Reagents include dNTPs, enzymes such as polymerases, luminescence enzymes, polysubstrates, "cold" analogs of polysubstrates lacking substrate molecules, cofactors, primers, wash buffers, disassociate buffers, reagents required for luminesce reactions, and the like. In each step, including associating or disassociating polysubstrates with primer extension products, it will be understood that temperature, pH, and duration, and the like will be selected to achieve the intended goal.

[0116] ln some approaches, the methods include combining the labeled primer extension product and the at least one luminescent enzyme to form an enzyme-substrate molecule complex. It will be apparent from the context of the disclosure that combining includes any form of combining between the labeled primer extension product and the substrate molecules that facilitates an enzymatic reaction between the luminescent enzyme and the substrate molecules of the polysubstrate.

DETECTING A SIGNAL PRODUCED BY A SUBSTRATE MOLECULE

[0117] In some approaches, the methods include detecting a signal produced by the action of the at least one luminescent enzyme on the labeled primer extension product, thereby detecting incorporation of the dNTP analog at the 3' end of the primer extension product. In some instances, the signal is light. In some embodiments, the signal may vary based on wavelength, duration and/or intensity. The signal may be detected by a device that can detect light energy such as a luminometer, photometer, charge- coupled device (CCD) and complementary metal-oxide semiconductor (CMOS) device, for example, in some embodiments a device described in co-pending U.S. Patent Application Nos. 5/803,077, 62/473,970; 62/560,585; 62/553,614, each of which is incorporated herein by reference. In some embodiments, these devices can be used to detect: (i) the intensity of light released, (ii) the duration (kinetics) of the signal, and/or (iii) the wavelength (color) of light emitted.

[0118] In order to reduce background or interference in detection of the signal from the substrate molecules it is preferred that little or no light is produced by reactions that are not dependent on the presence of the polysubstrate. Under typical luminescence conditions described herein (e.g., for nucleic acid sequencing), luminescence cannot be detected when the luminescent enzyme (e.g., firefly luclferase) is not present in the reaction. 8. DISASSOCIATION

[0119] After imaging, a PS may be disassociated from the primer extension product, usually prior to incorporation of the next dNTP (i.e., prior to generation of a new 3' terminal nucleotide). Polysubstrates may be removed by disrupting the ability of the binding moiety to bind the incorporated 3' terminal nucleotide. Multiple methods for disassociating a polysubstrate from a primer extension product will be apparent to the person of skill depending, in part, on the structure of the binding moiety. For example, many types of binding moieties, including antibody-based binding moieties, can be removed by low pH, high pH, high or low salt, or denaturing agents such as a chaotropic stripping buffer. Other classes of affinity reagents (e.g., aptamers) can be removed by any means known in the art. In addition, affinity reagents can be displaced by introducing an agent that competes with the bound epitope for affinity reagent binding. For example, a polysubstrate for which the binding moiety is streptavidin can be displaced from a dNTP with a biotin affinity tag by adding biotin to the system. Methods for removing affinity reagents are also discussed in US Pat Pub. 2018/0223358 Al.

[0120] In some embodiments binding of the polysubstrate to the 3' terminal nucleotide requires the presence of a 3'-OH blocking group with a cleavable linker and the binding can be disrupted by removing the blocking group. In other embodiments, simultaneous removal of polysubstrates and blocking groups also may occur simultaneously, and may be effected by addition of a solution comprising a blocking group cleaving component (e.g., a phosphine reagent) and an affinity reagent releasing agent (e.g., high salt). Removal of 3ΌH blocking groups is also discussed in US Pat. Pub. 2018/0223358 Al.

[0121] In one approach, the binding moiety is attached to a polymer molecule by a cleavable linker and the substrate molecules may be removed by cleavage of the cleavable linker.

[0122] It will be appreciated that conditions and structures may be selected so that less than all polysubstrates on an array are disassociated under a certain set of conditions. For example, bound polysubstrates specific for dATP ("PS-1") may be diassociated without disassociation of bound polysubstrates specific for dCTP ("PS-2"). This may be used in methods such as "2 image" methods, in which both polysubstrates can comprise the same or similar sets of substrate molecules. For example, a first image may be taken, one polysubstrate removed, and a second image taken. As illustrated in Table 3, two nucleotides can be distinguished using two images and disassociation of one polysubstrate. The same methodology can be used to distinguish 3 or 4 nucleotides using two images.

[0123] TABLE 3

[0124] In another variation, partial dissociation is used. For example, as illustrated in Table 4, it will be appreciated that conditions and structures may be selected so that less than all polysubstrates on an array are disassociated under a certain set of conditions. For example, bound polysubstrates specific for d.ATP ("PS-1") may be diassociated without disassociation of bound polysubstrates specific for dCTP ("PS-2"). This may be used in methods such as "2 image" methods, in which both polysubstrates can comprise the same or similar sets of substrate molecules. As illustrated in Table 4, two nucleotides can be distinguished using two images and disassociation of one polysubstrate. The same methodology can be used to distinguish 3 or 4 nucleotides using two images.

9. REACTION MIXTURES

[0126] Polysubstrates, dNTP analogs, and nucleic acids (e.g , primer extension products, DNA template molecules, primers and oligonucleotides) can be used as components of a reaction mixture. For example, such components can be used in reaction mixtures for nucleic acid sequencing (e.g., sequencing by synthesis or sequencing by ligation). Exemplary reaction mixtures include, but are not limited to, those containing (a) DNA template molecules; (b) primer; (c) a dNTP analog or a mixture of different dNTP analog species; and (d) a polymerase

[0127] The reaction mixture can further optionally contain one or more luminescent enzymes (e.g., luciferase or horseradish peroxidase) in some cases, such a reaction mixture includes (a) DNA template molecules; (b) polymerase; (c) a primer; (d) a mixture of dNTP analogs, wherein the mixture includes a dNTP analog for each of adenosine, guanosine, cytidine and thymidine or uracil; and (e) a luminescent enzyme (e.g., luciferase). in some cases, the dNTP analog Is not labeled with a fiuorophore and contains a 3 '-O reversible blocking group

[0128] In some embodiments, the dNTP analog can contain a 3'-O reversibly blocked nucleoside analog, where the nucleobase is covalently linked to a linker, and the linker is linked to an affinity tag ligand. In some cases, the reaction mixture contains a mixture of nucleoside analogues having different nucleobases, where the nucleobases are covalently linked to a distinguishable affinity tag ligand via a linker. In some cases, the reaction mixture further contains one or more distinguishable affinity reagents. 10. ILLUSTRATIVE EXAMPLES (PROPHETIC)

[0129] Table 5 illustrates a method in which sequence may be determined using 3 colors and one- image:

[0130] TABLE 5

[0131] Table 6 illustrates a method in which sequence may be determined using 3 colors and one- image, in which one base is associated with the absence of a signal.

[0132] TABLE 6

[0133] Table 7 illustrates a method in which sequence may be determined using dNTPs with an affinity tag:

[0134] TABLE 7

[013S] Table 8 illustrates a method in which sequence may be determined using 3 colors and three- images, in which one base is associated with the absence of a signal. This method may be carried out in several steps:

Step 1. Bind polysubstrates to three 3' dNTPs (e.g., A, T and G). Bind no polysubstrate to a fourth dNTP (e.g., C).

Step 2. Flow flash luciferase over array; take a first image detecting iuciferin signal and identifying position with A; wash array.

Step 3. Flow glow luciferase over array; take a second image defecting luminoi signal and identifying position with T; wash array.

Step 4. Flow HRP over array; take a third image detecting coelenterazine signal and identifying position with G; wash array.

Positions with C are identified by the absence of signal.

[0136] TABLE 8

[0137] In an related embodiment, flash Luciferase, glow Luciferase, and FIRP can be flowed together and a single image taken.

[0138] Table 9 illustrates indirect binding of a polysubstrate to a 3' dNTP.

[0139] TABLE 9

[0140] Table 10 Illustrates a method in which sequence may be determined using 2 colors and one image, in which one base is associated with the absence of a signal.

[0141] TABLE 10

[0142] Table 11 Illustrates a method in which sequence may be determined using one color and two images, in which one base is associated with the absence of a signal. The percentage of unlabeled PS (e.g., PS lacking substrate molecules) is modulated resulting in a change in intensity as compared to 100% labeled PS.

[0143] TABLE 11

[0144] Table 12 illustrates a method in which sequence may be determined using one color and two images, in which one base is associated with the absence of a signal. The percentage of unlabeled PS (e.g., PS lacking substrate molecules) is modulated resulting in a change in intensity as compared to 100% labeled PS.

[0145] TABLE 12

[0146] As illustrated In Table 13, two nucleotides can be distinguished using two images and disassociation of one polysubstrate. The same methodology can be used to distinguish 3 or 4 nucleotides using two images.

[0147] Table 11 illustrates a method in which sequence may be determined using one color and two images. The percentage of unlabeled PS (e.g., PS lacking substrate molecules) is modulated resulting in a change in intensity as compared to 100% labeled PS

[0148] TABLE 13

[0149] Table 14 Illustrates a method In which sequence may be determined using one color and two images. The percentage of unlabeled PS [e.g., PS lacking substrate molecules) is modulated resulting in a change in intensity as compared to 100% labeled PS. Additionally, between image 1 and 2, a disulfide bond (S-S) bond in the binding moiety is cleaved, preventing detection of the dNTP analog by the binding moiety, thereby affecting production of signal.

[0150] TABLE 14

[0151] Table 15 illustrates a method in which sequence may be determined using one color and two images. The percentage of unlabeled PS [e.g., PS lacking substrate molecules) is modulated resulting in a change in Intensity as compared to 100% labeled PS.

[0152] TABLE 15

[0153] Table 16 illustrates a method in which sequence may be determined using one or two colors and two images. The percentage of uniabeled PS (e.g., PS lacking substrate molecules) is modulated resulting in a change in intensity as compared to 100% labeled PS. Additionally, two luciferases are used that having distinct kinetic properties. Flash luciferase #1 produces a detectable signal over a short duration in image 1 but is not detected during image 2. in contrast, Glow luciferase #1 does not produce a detectable signal during image 1 but does produce a detectable signal during image 2.

[0154] TABLE 16

[015S] Table 17 illustrates a method in which sequence may be determined using one color and one- image. The percentage of uniabeled PS (e.g., PS lacking substrate molecules] is modulated resulting in a change in intensity as compared to 100% labeled PS.

[0156] TABLE 17

[0157] Table 18 illustrates a method in which sequence may be determined using one color and one image. The percentage of unlabeled PS (e.g., PS lacking substrate molecules) is modulated resulting in a change in intensity as compared to 100% labeled PS.

[0158] TABLE 18

[0159] This application incorporates by reference the entire content of co-pending Application No. 15/862,566, the text of which appeared as US Pat. Pub. 2.018/022.3358 A1 of U.S. Provisional Application No. 62/670,627, and which was published as US 2018/0223358 Al.

[0160] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.