Nanoparticles with polynucleotide binding sites and methods for making same

Abstract

The present disclosure relates to nanoparticles comprising a first layer comprising a first polymer and a first plurality of accessory oligonucleotides, a second layer comprising a second polymer and a single template site for binding to a template polynucleotide, and a third layer comprising a third polymer and a second plurality of accessory oligonucleotides. Also described herein are methods for producing the above-described nanoparticles, including "dip-coating," e.g., sequentially immersing a surface having wettable nanodomains in different polymer solutions. Also described herein are methods for producing nanoparticles by forming nanoparticles in nanowells and subsequently releasing the nanoparticles from the nanowells. Also described herein are methods for connecting nanoparticles to a substrate and amplifying a template polynucleotide using a polymerase.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 375,965, filed September 16, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure generally relates to multiphase nanoparticles having three polymer layers, each layer containing a single template site for binding to multiple accessory oligonucleotides or template polynucleotides. Methods for making such nanoparticles and attaching such nanoparticles to a substrate are also described herein. [Background technology]

[0003] Many sequencing platforms use "sequencing by synthesis" (SBS) technology and fluorescence-based methods for detection. In some instances, a template polynucleotide is attached to the surface of a substrate in a process known as seeding. Multiple copies of the template polynucleotide may be synthesized in proximity to the initially "seeded" template in a process called clustering. Nascent copies of the clustered polynucleotides are then synthesized under conditions that emit a signal identifying each nucleotide as it attaches to the nascent strand. Clustering of multiple copies of the seeded template polynucleotide in proximity to the initially seeded location results in amplification of the signal generated during polymerization that can be visualized, improving detection.

[0004] Seeding and clustering work well when template polynucleotides from libraries with distinct sequences are seeded or attached to surface locations sufficiently distant from one another so that clustering results in spatially separated clusters of copied polynucleotides, each resulting from the seeding of a single template polynucleotide; this condition is commonly referred to as monoclonality. When two different template polynucleotides are seeded very close to one another on the surface of a substrate, clustering may result in spatially adjacent or mixed populations of copied polynucleotides (a condition commonly referred to as polyclonality), which may prevent the imaging system used in the SBS process from distinguishing them as separate clusters. Obtaining unambiguous sequence information from polyclonal clusters, if present, may be more difficult, time-consuming, expensive, less efficient, and require more complex data analysis. Furthermore, in simultaneous paired-end read (SPEAR) sequencing, ensuring sufficient spatial separation of Read 1 and Read 2 primers may be important to avoid polyclonality.

[0005] The present disclosure is directed to overcoming these and other deficiencies in the art. Summary of the Invention

[0006] The present disclosure offers advantages, benefits, and other alternatives over known compositions and methods by providing nanoparticles and methods for making same that ensure monoclonality during clustering prior to sequencing.

[0007] In some embodiments, the nanoparticles comprise a first layer comprising a first polymer and a first plurality of accessory moieties, a second layer comprising a second polymer comprising a single template moiety for binding to a template polynucleotide, and a third layer comprising a third polymer and a second plurality of accessory moieties, the second layer being between the first and third layers.

[0008] In one embodiment, the first polymer is a hydrophilic polymer. In another embodiment, the hydrophilic polymer is selected from natural polyacrylamide, polyethyleneimine, polypeptide, polysaccharide, polyvinyl alcohol, polyacrylic acid, polyallylamine, polystyrene sulfonate, or polyoxazoline. In yet another embodiment, the third polymer is a lipophilic polymer. In yet another embodiment, the lipophilic polymer is selected from isopropylacrylamide, acrylic, epoxy, polyethylene, polystyrene, polyvinyl, polymethylsulfonate, polyurethane, and fluorinated polymer. In further embodiments, the first polymer and the third polymer are each independently selected from poly(vinylidene fluoride), polystyrene, epoxy polymer, (meth)acrylate polymer, polydimethylsiloxane, SiO2-containing polymer, poly(lactic acid-co-glycolic acid) polymer, perfluorinated polymer, azapa-co-acrylamide polymer, and the like. polymer, PAZNAM), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) polymer, poly(o-nitrobenzyl-masked acrylamide-co-acrylamide) copolymer, poly(benzopyrone-masked acrylamide-co-acrylamide) copolymer, poly(aminotriazole-acrylamide-co-acrylamide) copolymer, poly(thiotriazole-acrylamide-co-acrylamide) copolymer, poly(alkenyltriazole-acrylamide-co-acrylamide) copolymer, and thiol / ene crosslinkable monomer mixtures.

[0009] In yet further embodiments, the first polymer is polyacrylamide and the third polymer is isopropylacrylamide. In still further embodiments, the second polymer is a copolymer of the first polymer and the third polymer. In some embodiments, the second polymer is a hydrophilic or lipophilic polymer. In other embodiments, the second polymer is a methacrylate.

[0010] In yet another embodiment, the nanoparticles further comprise one or both of a first plurality of accessory oligonucleotides linked to the first plurality of accessory moieties and a second plurality of accessory oligonucleotides linked to the second plurality of accessory moieties. In yet another embodiment, one or both of the first plurality of accessory oligonucleotides and the second plurality of accessory oligonucleotides comprise one or both of a forward primer and a reverse primer, wherein the sequence of the forward primer and the sequence of the reverse primer enable amplification of a template polynucleotide by a polymerase. In a further embodiment, the forward primer of the first plurality of accessory oligonucleotides and the reverse primer of the second plurality of accessory oligonucleotides, or the reverse primer of the first plurality of accessory oligonucleotides and the forward primer of the second plurality of accessory oligonucleotides, are cleavable, and the other primer of the first and second plurality of accessory oligonucleotides is non-cleavable.

[0011] In a further embodiment, the single template site comprises a type of connection site for a template anchor oligonucleotide or a template anchor oligonucleotide.

[0012] In one aspect, a method comprises attaching the nanoparticles of any one of claims 1-14 to a substrate and amplifying a template polynucleotide using a polymerase. In one embodiment, the substrate comprises a nanowell. In another embodiment, the substrate is silanized TiO2 or fluoro-silane TiO2. In yet another embodiment, the attaching comprises hybridizing a forward primer or a reverse primer to the oligonucleotide attached to the substrate.

[0013] In one embodiment, a method of making nanoparticles includes coating wettable nano-domains on a substrate surface with a soluble polymer, the wettable nano-domains being separated by non-wettable gaps; coating the soluble polymer with a first polymer, the first polymer comprising a first plurality of accessory moieties; coating the first polymer with a second polymer, the second polymer comprising a single template moiety; coating the second polymer with a third polymer, the third polymer comprising a second plurality of accessory moieties; and solubilizing the soluble polymer to release the nanoparticles.

[0014] In some embodiments, (i) the first plurality of accessory sites comprises a first plurality of accessory oligonucleotides, and (ii) the second plurality of accessory sites comprises a second plurality of accessory oligonucleotides. In other embodiments, (i) the first plurality of accessory sites comprises a first type of connection site for the first plurality of accessory oligonucleotides, but not for the second plurality of accessory oligonucleotides, and (ii) the second plurality of accessory sites comprises a second type of connection site for the second plurality of accessory oligonucleotides, but not for the first plurality of accessory oligonucleotides.

[0015] In yet another embodiment, the method further comprises, after solubilizing, one or more of: attaching a first plurality of accessory oligonucleotides to the first plurality of accessory sites and attaching a second plurality of accessory oligonucleotides to the second plurality of accessory sites. In yet another embodiment, the single template site comprises a type of attachment site or template anchor oligonucleotide for a template anchor oligonucleotide, and the method further comprises attaching the template anchor oligonucleotide to the single template site.

[0016] In further embodiments, the soluble polymer is a sulfonate, a sugar, or a phenol. In yet further embodiments, the soluble polymer is poly(sodium 4-styrenesulfonate) or poly-4-vinylphenol.

[0017] In certain embodiments, the method includes forming a nanoparticle within the nanowell, the forming including polymerizing a first polymer, where the first polymer comprises a first plurality of accessory moieties; polymerizing a second polymer on the first polymer, where the second polymer comprises a single template moiety; and polymerizing a third polymer on the second polymer, where the third polymer comprises a second plurality of accessory moieties.

[0018] In one embodiment, one or both of (i) the first plurality of accessory sites comprises a first plurality of accessory oligonucleotides, and (ii) the second plurality of accessory sites comprises a second plurality of accessory oligonucleotides.

[0019] In another embodiment, one or both of (i) the first plurality of accessory sites include a first type of connection site for the first plurality of accessory oligonucleotides but not for the second plurality of accessory oligonucleotides, and (ii) the second plurality of accessory sites include a second type of connection site for the second plurality of accessory oligonucleotides but not for the first plurality of accessory oligonucleotides.

[0020] In yet another embodiment, the single template site comprises a connection site type or template anchor oligonucleotide for a template anchor oligonucleotide, and the method further comprises connecting the template anchor oligonucleotide to the single template site.

[0021] In yet another embodiment, the method further comprises releasing the nanoparticles from the nanowells, wherein releasing comprises mechanically releasing the nanoparticles.

[0022] In some embodiments, the method further comprises polymerizing a soluble polymer before polymerizing the first polymer. In other embodiments, the method further comprises releasing the nanoparticles from the nanowells, wherein releasing comprises solubilizing the soluble polymer. In yet other embodiments, the soluble polymer is a sulfonate, a sugar, or a phenol. In yet other embodiments, the soluble polymer is poly(sodium 4-styrenesulfonate) or poly-4-vinylphenol.

[0023] In some embodiments, the first polymer is a hydrophilic polymer. In other embodiments, the hydrophilic polymer is selected from natural polyacrylamide, polyethyleneimine, polypeptides, polysaccharides, polyvinyl alcohol, polyacrylic acid, polyallylamine, polystyrene sulfonate, or polyoxazoline. In yet other embodiments, the second polymer is a copolymer of the first polymer and a third polymer. In yet other embodiments, the second polymer is a hydrophilic polymer or a lipophilic polymer. In further embodiments, the second polymer comprises a methacrylate. In yet further embodiments, the third polymer is a lipophilic polymer. In still further embodiments, the lipophilic polymer is selected from isopropylacrylamide, acrylic, epoxy, polyethylene, polystyrene, polyvinyl, polymethylsulfonate, polyurethane, and fluorinated polymer.

[0024] In certain embodiments, the first polymer and the third polymer are each independently selected from poly(vinylidene fluoride), polystyrene, an epoxy polymer, a (meth)acrylate polymer, a polydimethylsiloxane, a SiO2-containing polymer, a poly(lactic-co-glycolic acid) polymer, a perfluorinated polymer, an azapa-co-acrylamide polymer (PAZNAM), a poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) polymer, a poly(o-nitrobenzyl-masked acrylamide-co-acrylamide) copolymer, a poly(benzopyrone-masked acrylamide-co-acrylamide) copolymer, a poly(aminotriazole-acrylamide-co-acrylamide) copolymer, a poly(thiotriazole-acrylamide-co-acrylamide) copolymer, a poly(alkenyltriazole-acrylamide-co-acrylamide) copolymer, and a thiol / ene crosslinkable monomer mixture.

[0025] In another embodiment, the method further comprises connecting the single template polynucleotide to a single template site on a second polymer. In yet another embodiment, the first plurality of accessory oligonucleotides and the second plurality of accessory oligonucleotides comprise a forward primer and a reverse primer, respectively, or a reverse primer and a forward primer, respectively, wherein the sequence of the forward primer and the sequence of the reverse primer allow for amplification of the template polynucleotide by a polymerase.

[0026] In certain embodiments, a method of forming nanoparticles includes coating wettable nanodomains of a substrate with a soluble polymer, wherein the wettable nanodomains are separated by non-wettable gaps; coating the soluble polymer with a first polymer comprising a first plurality of accessory moieties; coating the first polymer with a second polymer comprising a single template moiety for binding to a template polynucleotide; coating the second polymer with a third polymer comprising a second plurality of accessory moieties; connecting the first plurality of accessory oligonucleotides to the first plurality of accessory moieties; connecting the second plurality of accessory oligonucleotides to the second plurality of accessory moieties; and solubilizing the soluble polymer.

[0027] In some embodiments, conjugating a first plurality of accessory oligonucleotides to a first plurality of accessory moieties occurs before coating the soluble polymer with the first polymer, after coating the soluble polymer with the first polymer and before solubilizing, or after solubilizing. In other embodiments, conjugating a second plurality of accessory oligonucleotides to a second plurality of accessory moieties occurs before coating the second polymer with the third polymer, after coating the second polymer with the third polymer and before solubilizing, or after solubilizing.

[0028] In yet another embodiment, the soluble polymer is a sulfonate, a sugar, or a phenol. In yet another embodiment, the soluble polymer is poly(sodium 4-styrenesulfonate) or poly-4-vinylphenol.

[0029] In some embodiments, the first polymer is a hydrophilic polymer. In other embodiments, the hydrophilic polymer is selected from natural polyacrylamide, polyethyleneimine, polypeptides, polysaccharides, polyvinyl alcohol, polyacrylic acid, polyallylamine, polystyrene sulfonate, or polyoxazoline. In yet other embodiments, the second polymer is a copolymer of the first polymer and a third polymer. In yet other embodiments, the second polymer is a hydrophilic polymer or a lipophilic polymer. In further embodiments, the second polymer comprises a methacrylate. In yet further embodiments, the third polymer is a lipophilic polymer. In still further embodiments, the lipophilic polymer is selected from isopropylacrylamide, acrylic, epoxy, polyethylene, polystyrene, polyvinyl, polymethylsulfonate, polyurethane, and fluorinated polymer.

[0030] In certain embodiments, the first polymer and the third polymer are each independently selected from poly(vinylidene fluoride), polystyrene, an epoxy polymer, a (meth)acrylate polymer, a polydimethylsiloxane, a SiO2-containing polymer, a poly(lactic-co-glycolic acid) polymer, a perfluorinated polymer, an azapa-co-acrylamide polymer (PAZNAM), a poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) polymer, a poly(o-nitrobenzyl-masked acrylamide-co-acrylamide) copolymer, a poly(benzopyrone-masked acrylamide-co-acrylamide) copolymer, a poly(aminotriazole-acrylamide-co-acrylamide) copolymer, a poly(thiotriazole-acrylamide-co-acrylamide) copolymer, a poly(alkenyltriazole-acrylamide-co-acrylamide) copolymer, and a thiol / ene crosslinkable monomer mixture.

[0031] In another embodiment, the method further comprises connecting the single template polynucleotide to a single template site on a second polymer. In another embodiment, the first plurality of accessory oligonucleotides and the second plurality of accessory oligonucleotides comprise a forward primer and a reverse primer, respectively, or a reverse primer and a forward primer, respectively, wherein the sequence of the forward primer and the sequence of the reverse primer allow for amplification of the template polynucleotide by a polymerase. [Brief explanation of the drawings]

[0032] [Figure 1A] 1 shows a scheme illustrating a method for making nanoparticles for use in SBS systems. [Figure 1B] 1 shows a scheme illustrating a method for making nanoparticles for use in SBS systems. [Figure 2] 1 shows a flow diagram of a method for attaching nanoparticles disclosed herein to a substrate to amplify a template polynucleotide. [Figure 3] 1 shows a flow diagram of a method for producing nanoparticles as disclosed herein on the surface of a substrate. [Figure 4] 1 shows a flow diagram of a method for making the nanoparticles disclosed herein in nanowells. [Figure 5] FIG. 1 shows a flow diagram of a method for making nanoparticles as disclosed herein in nanowells that include a soluble layer. [Figure 6] 1 shows the reorganization of nanoparticles upon exposure to heat. [Figure 7] 1 is a scheme illustrating the overall workflow for fabricating nanoparticles as disclosed herein using lithographic templates.

[0033] It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein. DETAILED DESCRIPTION OF THE INVENTION

[0034] Certain aspects of the present disclosure relate to nanoparticles comprising: a first layer comprising a first polymer and a first plurality of accessory oligonucleotides; a second layer comprising a second polymer comprising a single template site for binding to a template polynucleotide; and a third layer comprising a third polymer and a second plurality of accessory oligonucleotides, wherein the second layer is between the first and third layers.

[0035] In certain embodiments, the first polymer and the third polymer are each independently selected from poly(vinylidene fluoride), polystyrene, an epoxy polymer, a (meth)acrylate polymer, a polydimethylsiloxane, a SiO2-containing polymer, a poly(lactic-co-glycolic acid) polymer, a perfluorinated polymer, an azapa-co-acrylamide polymer (PAZNAM), a poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) polymer, a poly(o-nitrobenzyl-masked acrylamide-co-acrylamide) copolymer, a poly(benzopyrone-masked acrylamide-co-acrylamide) copolymer, a poly(aminotriazole-acrylamide-co-acrylamide) copolymer, a poly(thiotriazole-acrylamide-co-acrylamide) copolymer, a poly(alkenyltriazole-acrylamide-co-acrylamide) copolymer, and a thiol / ene crosslinkable monomer mixture.

[0036] In another embodiment, the first polymer can be a hydrophilic polymer. Non-limiting examples of hydrophilic polymers include polyethyleneimine, polypeptides, polysaccharides, polyvinyl alcohol, polyacrylic acid, polyallylamine, polystyrene sulfonate, polyoxazoline, etc. In yet another embodiment, the first polymer is natural polyacrylamide.

[0037] In another embodiment, the third polymer can be a lipophilic polymer. Non-limiting examples of lipophilic polymers include acrylic, epoxy, polyethylene, polystyrene, polyvinyl, polymethylsulfonate, polyurethane, fluorinated polymers, etc. In a non-limiting example, the first polymer and the third polymer can be polyacrylamide and isopropylacrylamide, respectively.

[0038] In another embodiment, a first plurality of accessory oligonucleotides may be covalently attached to a first polymer and / or a second plurality of accessory oligonucleotides may be covalently attached to a second polymer. In yet another embodiment, the covalent bond may be selected from an amine-NHS ester bond site, an amine-imido ester bond site, an amine-pentofluorophenyl ester bond site, an amine-hydroxymethylphosphine bond site, a carboxylcarbodiimide bond site, a thiol-maleimide bond site, a thiol-haloacetyl bond site, a thiolpyridyl disulfide bond site, a thiol-thiosulfonate bond site, a thiol-vinyl sulfone bond site, an aldehyde-hydrazide bond site, an aldehyde-alkoxyamine bond site, a hydroxyisocyanate bond site, an azide-alkyne bond site, an azide-phosphine bond site, a transcyclooctene-tetrazine bond site, a norbornene-tetrazine bond site, an azide-cyclooctyne bond site, an azide-norbornene bond site, an oxime bond site, and any combination of two or more of the foregoing.

[0039] In further embodiments, a first plurality of accessory oligonucleotides may be non-covalently attached to a first polymer and / or a second plurality of accessory oligonucleotides may be non-covalently attached to a second polymer, hi still further embodiments, the non-covalent attachment comprises a hybrid attachment comprising a non-covalently attached peptide selected from one or both of a coiled-coil attachment and an avidin-biotin attachment.

[0040] In one embodiment, the first plurality of accessory oligonucleotides and the second plurality of accessory oligonucleotides each comprise a forward primer and a reverse primer, or each comprise a reverse primer and a forward primer, wherein the sequence of the forward primer and the sequence of the reverse primer allow amplification of the template polynucleotide by a polymerase.

[0041] In some embodiments, the second polymer can be a hydrophilic or lipophilic polymer. Non-limiting examples of hydrophilic polymers include polyethyleneimine, polypeptides, polysaccharides, polyvinyl alcohol, polyacrylic acid, polyallylamine, polystyrene sulfonate, polyoxazoline, etc. Non-limiting examples of lipophilic polymers include acrylic, epoxy, polyethylene, polystyrene, polyvinyl, polymethylsulfonate, polyurethane, fluorinated polymers, etc. In another embodiment, the second polymer can be a methacrylate. In yet another embodiment, the second polymer can include a methacrylate having a small molecular weight (Mw). In yet another embodiment, the methacrylate may have a Mw of about 0 kDa to about 60 kDa, including any and all subranges, such as about 1 to about 50 kDa, about 1 to about 40 kDa, about 1 to about 30 kDa, about 1 to about 20 kDa, about 1 to about 10 kDa, about 10 to about 50 kDa, about 20 to about 50 kDa, about 30 to about 50 kDa, about 40 to about 50 kDa, about 50 to about 60 kDa, etc. Molecular weight (Mw) refers to the "weight average" and may be determined by size exclusion chromatography.

[0042] In yet another embodiment, the single template polynucleotide binding site may be of a different chemistry or structure / moiety than the way the first plurality of accessory oligonucleotides or the second plurality of oligonucleotides may be attached to the first polymer or the third polymer, respectively. In yet another embodiment, the chemistry or structure of how the first plurality of accessory oligonucleotides or the second plurality of accessory oligonucleotides may be attached to the first polymer or the third polymer, respectively, may be different from and incompatible with the chemistry or structure of how a single template polynucleotide binds to a single template polynucleotide binding site on a second polymer. In another embodiment, the chemistry or structure of how the first plurality of accessory oligonucleotides are attached to the first polymer may be different from and incompatible with the chemistry or structure of how the second plurality of accessory oligonucleotides are attached to the third polymer. In yet another embodiment, the chemistry or structure of a single template polynucleotide binding site may be different from the way the first plurality of accessory oligonucleotides or the second plurality of oligonucleotides are attached to the first polymer and the third polymer, respectively, and the chemistry or structure of the way the first plurality of accessory oligonucleotides are attached to the first polymer may be different from and incompatible with the chemistry or structure of the way the second plurality of accessory oligonucleotides are attached to the third polymer. In other words, the three different chemistries or structures may be selected from among each other, such that each layer has only one chemistry or structure suitable for attaching to one type of template or oligonucleotide, and there are no cross-connections between layers. Examples of chemistries or structures that can be used to form nanoparticles with orthogonal bonds on three sides are described in more detail below.

[0043] The template polynucleotide can be a polynucleotide obtained from a sample (e.g., a polydeoxyribonucleic acid isolated from a sample) or a cDNA molecule copied from an mRNA molecule obtained from a sample. The SBS process can be performed, for example, to determine the nucleotide sequence of the template polynucleotide or to identify one or more polymorphisms or variations in the genetic sequence of the template polynucleotide compared to a reference sequence. A library can be prepared from one or more samples, and the library contains a plurality of template polynucleotides obtained from one or more samples. The template polynucleotide can be obtained by obtaining a polynucleotide sequence that is a portion of a sequence present in a sample or copied from a sample. If sequence information for a plurality of template polynucleotides in the library is collected and analyzed for the sample from which the library was obtained, then by sequencing the plurality of template polynucleotides in the SBS process, sequence, genotype, or other sequence-related information can be determined for the template polynucleotides.

[0044] Template polynucleotide can be any given length suitable for obtaining sequencing information in SBS process.For example, template polynucleotide can be about 50 nucleotides long, about 75 nucleotides long, about 100 nucleotides long, about 125 nucleotides long, about 150 nucleotides long, about 175 nucleotides long, about 200 nucleotides long, about 225 nucleotides long, about 250 nucleotides long, about 275 nucleotides long, about 300 nucleotides long, about 325 nucleotides long, about 350 nucleotides long, about 375 nucleotides long, about 400 nucleotides long, about 425 nucleotides long, about 450 nucleotides long, about 475 nucleotides long, about 500 nucleotides long, about 525 nucleotides long, about 550 nucleotides long, about 575 nucleotides long, about 600 nucleotides long, about 625 nucleotides long, about 650 nucleotides long, about 675 nucleotides long, about 700 nucleotides long, about 725 nucleotides long, about 750 nucleotides long, about 775 nucleotides long, about 800 nucleotides long. , about 825 nucleotides in length, about 850 nucleotides in length, about 875 nucleotides in length, about 900 nucleotides in length, about 925 nucleotides in length, about 950 nucleotides in length, about 975 nucleotides in length, about 1,000 nucleotides in length, about 1,025 nucleotides in length, about 1,050 nucleotides in length, about 1,075 nucleotides in length, about 1,100 nucleotides in length, about 1,125 nucleotides in length, about 1,150 nucleotides in length, about 1,175 nucleotides in length The fragment may be about 1,200 nucleotides in length, about 1,225 nucleotides in length, about 1,250 nucleotides in length, about 1,275 nucleotides in length, about 1,300 nucleotides in length, about 1,325 nucleotides in length, about 1,350 nucleotides in length, about 1,375 nucleotides in length, about 1,400 nucleotides in length, about 1,425 nucleotides in length, about 1,450 nucleotides in length, about 1,475 nucleotides in length, about 1,500 nucleotides in length, or longer.

[0045] The template polynucleotide may be processed as part of the process of obtaining the template polynucleotide from a sample. Part of the processing may include adding a polynucleotide sequence, such as to the 5-prime, 3-prime, or both ends of the template, to aid in the subsequent SBS process. As further disclosed herein, the template polynucleotide may be further modified by adding features that facilitate or allow bond formation with sites on nanoparticles. For example, the single template site of the second polymer may be an oligonucleotide attached to the second polymer, where the oligo is complementary to a portion of the template polynucleotide. In some embodiments, the template polynucleotide may have a sequence added during processing, including a sequence that can hybridize to an oligonucleotide attached to the second polymer.

[0046] The attachment of a single template polynucleotide or accessory oligonucleotide to its respective polymer may be achieved by including a moiety or structure on the template polynucleotide or accessory oligonucleotide that is complementary to a moiety or structure on the respective polymer, meaning that they are configured to bind to each other, either covalently or non-covalently, to form a connection therebetween. Cross-reactivity between the moiety or structure attached to the template polynucleotide and the moiety or structure of the first or second polymer must be avoided to prevent attachment of more than one template polynucleotide to the nanoparticle. Furthermore, cross-reactivity between the moiety or structure attached to the accessory oligonucleotide and the moiety or structure of the second polymer must be avoided to prevent occupation of a single template polynucleotide binding site by an accessory oligonucleotide. In other words, the attachment of each of the first plurality of oligonucleotides, the second plurality of oligonucleotides, and the single template polynucleotide to the first polymer, the third polymer, and the second polymer, respectively, may each utilize mutually exclusive moieties, chemistries, or structures.

[0047] For example, the nanoparticles may comprise a first polymer, which may comprise a hydrophilic natural polyacrylamide functionalized with multiple azide side chains. The nanoparticles may further comprise a second polymer, which may comprise a low Mw methacrylate and may be functionalized / pre-grafted with a template site primer, represented by Px, which is a primer used for orthogonal hybridization to hybridize to a portion of the template polynucleotide. The nanoparticles may further comprise a third polymer, which may comprise a lipophilic polymer such as isopropylacrylamide and may be functionalized with multiple amine side chains. The multiple azide side chains of the first polymer may be used to connect a first plurality of accessory oligonucleotides, each of which is functionalized with an alkyne group to facilitate azide-alkyne cycloaddition. The azide-alkyne cycloaddition may be Cu(I)-catalyzed (CuAAC) or Cu-free click (DBCO). The amine side chains of the third polymer may be used to connect a second plurality of accessory oligonucleotides, each oligonucleotide functionalized with an N-hydroxysuccinimide ester to facilitate NHC coupling. The accessory oligonucleotides may include primer P5 or primer P7, as described in more detail below. In some embodiments, each accessory oligonucleotide in the first plurality of accessory oligonucleotides is lead 1 primer P7, and each accessory oligonucleotide in the second plurality of accessory oligonucleotides is lead 2 primer P5. The activated patterned substrate, i.e., after masking and treatment to form wetting and non-wetting nanodomains, may then be successively immersed in the first polymer, the second polymer, and the third polymer to form three-domain nanoparticles by entanglement and UV-induced crosslinking. Thus, in this non-limiting example, the first polymer is dedicated to lead 1, the third polymer is dedicated to lead 2, and the second polymer is used to seed and capture oligos from the template library.After seeding, the clustering step can be initiated by using a standard cross-linking Examp between the surface primers P5 and P7 of the first and third polymers. Seeding, clustering, and paired-end read sequencing are described in more detail below.

[0048] In some embodiments, the first polymer and the third polymer are pre-functionalized with a first plurality of accessory nucleotides and a second plurality of accessory nucleotides, respectively.

[0049] A non-exclusive, non-limiting list of examples of complementary binding partners (eg, moieties or structures) is provided in Table 1.

[0050] [Table 1-1] TIFF2025532733000003.tif72170

[0051] [Table 1-2]

[0052] [Table 1-3]

[0053] Any of the foregoing may be added to or included in a first, second, or third polymer disclosed herein for connection to a template polynucleotide or accessory oligonucleotide, and the template polynucleotide or accessory oligonucleotide may contain or be modified to contain the aforementioned pair of complementary moieties or structures for attachment. As a non-limiting example, the accessory site of the first polymer may contain an azide moiety as one type of attachment site for the first plurality of accessory oligonucleotides. Correspondingly, the first plurality of accessory oligonucleotides may contain a chemical moiety suitable for forming a covalent bond to the azide attachment site (e.g., an alkyne, phosphine, cyclooctene, or norbornene moiety). Alternatively, the first plurality of accessory oligonucleotides may contain an azide attachment site, and the accessory attachment site of the first polymer may contain a chemical moiety suitable for forming a covalent bond thereto (e.g., an alkyne, phosphine, cyclooctene, or norbornene moiety).

[0054] As another non-limiting example, the accessory site of the third polymer can include an amine moiety as one type of attachment site for the second plurality of accessory oligonucleotides. Correspondingly, the second plurality of accessory oligonucleotides can include a chemical moiety suitable for forming a covalent bond to the amine attachment site (e.g., NHS, imidoester, pentofluorophenylester, hydroxymethylphosphine, or carboxylic acid moiety). Alternatively, the second plurality of accessory oligonucleotides can include an amine attachment site, and the accessory attachment site of the third polymer can include a chemical moiety suitable for forming a covalent bond thereto (e.g., NHS, imidoester, pentofluorophenylester, hydroxymethylphosphine, or carboxylic acid moiety).

[0055] As will be understood by those skilled in the art, the foregoing example can be reversed, with the attachment site of the first polymer comprising an amine site and the first accessory oligonucleotide comprising a site suitable for attachment thereto (e.g., an NHS, imidoester, pentofluorophenylester, hydroxymethylphosphine, or carboxylic acid site), or vice versa, and the third polymer comprising an azide site and the second plurality of accessory oligonucleotides comprising a site suitable for attachment thereto (e.g., an alkyne, phosphine, cyclooctene, or norbornene site), or vice versa.

[0056] Similarly, an accessory site for a polymer (first polymer or third polymer) may include thiol attachment sites for a plurality of accessory oligonucleotides (first accessory oligonucleotide or second accessory oligonucleotide), and the corresponding accessory oligonucleotides (first or second) may include a moiety for forming a covalent bond to the corresponding polymer, such as, for example, a maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, or vinyl sulfone, or vice versa. In another example, an accessory site for a polymer (first polymer or third polymer) may include aldehyde attachment sites for a plurality of accessory oligonucleotides (first accessory oligonucleotide or second accessory oligonucleotide), and the corresponding accessory oligonucleotides (first or second) may include a moiety for forming a covalent bond to the corresponding polymer, such as a hydrazide or alkoxyamine, or vice versa. In another example, an accessory moiety for a polymer (first polymer or third polymer) may include a hydroxyl, trans-cyclooctene, norbornene, or aldehyde or ketone linking moiety for a plurality of accessory oligonucleotides (first accessory oligonucleotide or second accessory oligonucleotide), and the corresponding accessory oligonucleotide (first or second) may include a moiety for forming a covalent bond to the corresponding polymer, such as an isocyanate, a tetrazine (e.g., benzyl-methyltetrazine or benzyltetrazine), or an alkoxyamine, respectively, to form a covalent bond as presented in Table 1, or vice versa.

[0057] All of the above examples are equally applicable to a single template site of a second polymer. For example, the second template site may contain one of the aforementioned pairs of chemical moieties, and the anchor oligonucleotide or template polynucleotide may contain the other of the aforementioned pairs of chemical moieties, and a portion of the anchor oligonucleotide or template polynucleotide may form a covalent bond with a corresponding portion of the second polymer, thereby forming a covalent bond between the template anchor oligonucleotide or template polynucleotide and the second polymer. The template anchor oligonucleotide may contain a sequence of nucleotides complementary to the sequence of nucleotides of the template polynucleotide so that the template anchor oligonucleotide hybridizes to the template polynucleotide.

[0058] As one skilled in the art will appreciate, the types of attachment sites on the first, second, and third polymers, and their corresponding moieties on the accessory oligonucleotide, template anchor oligonucleotide, or template polynucleotide, can be selected to selectively attach a first plurality of accessory oligonucleotides to the first polymer, a second plurality of accessory oligonucleotides to the third polymer, and a template anchor oligonucleotide or template polynucleotide to the second polymer. That is, selecting from among different pairs of attachment moieties such as those shown in Table 1 allows for the selection of mutually orthogonal attachment chemistries, for example, preventing, minimizing, or eliminating attachment of the template anchor oligonucleotide or template polynucleotide to the first or third polymer, the attachment of the first accessory oligonucleotide to the second or third polymer, the attachment of the second accessory oligonucleotide to the first or second polymer, or any combination of two or more of the foregoing.

[0059] Thus, any suitable bioconjugation method for adding or forming a bond between such pairs of complementary moieties or structures can be used. The modified nucleotide may be a commercially available holding example of one or the other of such pairs of examples of complementary moieties or structures, and methods for including one or more such examples of moieties or structures, or for connecting or including them in polymers, nucleotides, or polynucleotides, are also known. Further diversification can be achieved through the use of bifunctional linker molecules having at one end a moiety or structure from one complementary pair of binding partners listed in Table 1 and a moiety or structure from another complementary pair of binding partners listed in Table 1, connected by a polymer, such as, but not limited to, polyethylene glycol or other polymer. To provide a moiety or structure for binding between any of such aforementioned features, a polymer, template polynucleotide, or accessory, or oligo- or polypeptide moiety or structure connecting to any of the aforementioned features can be attached to either end of such a linker, effectively replacing the initial moiety or structure with another linker, i.e., the moiety or structure present at the other end of the linker. Examples of suitable bioconjugation methods for attaching or forming bonds between such pairs of complementary moieties or structures include those described in WO 2021 / 133768, which is incorporated herein by reference in its entirety. Thus, for example, if a first polymer includes an azide moiety as an accessory moiety, the accessory moiety may be contacted with a bifunctional linker having a moiety at one end for covalently binding to the azide moiety (e.g., an alkyne, phosphine, cyclooctene, or norbornene moiety) and a different moiety at one end for forming connections with multiple first accessory oligonucleotides (e.g., without excluding or limiting the foregoing, a thiol, maleimide, amide, or any other of the foregoing examples), thereby converting the first type of attachment moiety of the first polymer from an azide to the other type of attachment moiety on the other end of the bifunctional linker.The same can be achieved by contacting the attachment site of the second polymer or the attachment site of the third polymer with a bifunctional linker, including, without excluding or limiting, all of the combinations and permutations of the foregoing.

[0060] In some embodiments, nanoparticles may undergo reorganization such that the shape of the nanoparticles changes. Nanoparticle reorganization can occur at room temperature, upon cooling, or upon heating. For example, the first polymer and the third polymer each comprise a first domain and a second domain, with the first domain spatially separated from the second domain. Non-limiting examples of the first domain or the second domain include N-isopropylacrylamide (NIPAM), polyacrylamide (PAM), and the like. In another embodiment, the first domain and the second domain are NIPAM and PAM, respectively. It is understood that reorganization results in particles with two distinct domains containing both reverse and forward chains of the same clone. To undergo reorganization, the polymers should not be miscible; in other words, they should phase separate, as, for example, oil and water do. Phase separation can be achieved by modifying the side chains of the polymers. In one example, the first polymer may be a hydrophilic polymer disclosed herein, and the third polymer may comprise the same or a different hydrophilic polymer as disclosed herein, except that a lipophilic chemical moiety may be substituted in place of the side chain of the hydrophilic polymer, making the third polymer a lipophilic polymer. In another example, the third polymer may be a lipophilic polymer disclosed herein, and the first polymer may comprise the same or a different lipophilic polymer as disclosed herein, except that a hydrophilic chemical moiety may be substituted in place of the side chain of the lipophilic polymer, making the first polymer a hydrophilic polymer. In another example, the first polymer and the third polymer may each comprise any given polymer backbone (regardless of whether the polymer comprises a lipophilic or hydrophilic polymer), but a hydrophilic chemical moiety may be substituted in place of the side chain of the first polymer, making it a hydrophilic polymer, and a lipophilic chemical moiety may be substituted in place of the side chain of the third polymer, making it a lipophilic polymer.

[0061] Another embodiment relates to a method of making the nanoparticles disclosed herein. Figure 3 shows a flow diagram of a method of making such nanoparticles on a surface of a substrate. The method includes coating wettable nano-domains on a surface with a soluble polymer to form soluble nano-domains, coating the soluble nano-domains with a first polymer, coating the first polymer with a second polymer, coating the second polymer with a third polymer, and solubilizing the soluble polymer, wherein the first polymer comprises a first plurality of accessory oligonucleotides, the second polymer comprises a single template site for binding to a template polynucleotide, and the third layer comprises a second plurality of accessory oligonucleotides.

[0062] Another aspect relates to nanolithography-based techniques for preparing substrates onto which the nanoparticles disclosed herein are fabricated. In certain embodiments, the substrate may be a silica-based substrate, such as glass, fused silica, and other silica-containing materials. In other embodiments, the silica-based substrate may also be silicon, silicon dioxide, silicon nitride, or silicon hydride. In yet other embodiments, the substrate may comprise a plastic material, such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). In yet other embodiments, the substrate may be metallic. A non-limiting example of a metallic substrate is gold. In further embodiments, the substrate surface comprises a metal oxide. Non-limiting examples of metal oxides include tantalum oxide or titanium oxide.

[0063] Acrylamides, enones, or acrylates may also be utilized as substrate materials. Other substrate materials may include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramics, polyimides, quartz, resins, aryl azides, polymers, and copolymers. The foregoing list is intended to illustrate, but not limit, the present application.

[0064] In some embodiments, the substrate and / or substrate surface may be quartz, hi some other embodiments, the substrate and / or substrate surface may be a semiconductor, i.e., GaAs or ITO.

[0065] The substrate can comprise a single material or multiple different materials. The substrate can be a composite or laminate. The substrate can be flat, circular, textured, or patterned. The pattern can be formed, for example, by metal pads forming features on a non-metallic surface, as described, for example, in U.S. Patent Application Publication No. 13 / 661,524, which is incorporated herein by reference. Another useful patterned surface is one having well features formed thereon, as described, for example, in U.S. Patent Application Publication No. 13 / 787,396, U.S. Patent Application Publication No. 2011 / 0172118(A1), or U.S. Patent No. 7,622,294 (each of which is incorporated herein by reference). For embodiments using a patterned substrate, the gel can selectively connect to the pattern features (e.g., the gel can connect to the metal pads, or the gel can connect to the interior of the wells), or the gel can connect uniformly throughout both the pattern features and the interstitial areas.

[0066] For purposes herein, the term "masking" refers to the process of placing a temporary protective layer over an area to physically block access. The term "unmasking" refers to the opposite, i.e., the process of removing the protective layer to expose the area. The term "masked" refers to the presence of a temporary protective layer, and the term "unmasked" refers to the absence of a protective layer.

[0067] In some embodiments, a template can be used as a temporary mask to selectively treat predetermined nanodomains on a substrate to alter their surface energy and therefore their wettability in different solvents. For example, a template mask may be applied to a substrate so that some areas of the substrate are masked and some other areas are not. The unmasked areas are then treated to alter their surface energy. Non-limiting examples of treatments to alter surface energy include exposing the unmasked areas to UV light and plasma ashing treatments. Non-limiting examples of masks that can be used include photolithography masks, block copolymer lithography, or nanoimprinted substrates with removable sacrificial resin gaps.

[0068] In one embodiment, the method includes coating wettable nano-domains on a surface with a soluble polymer to form soluble nano-domains; coating the soluble nano-domains with a first polymer; coating the first polymer with a second polymer, where the second layer includes a single template site for binding to a template polynucleotide; coating the second polymer with a third polymer; solubilizing the soluble polymer; attaching a first plurality of accessory oligonucleotides to the first layer; and attaching a second plurality of accessory oligonucleotides to the third layer.

[0069] 1A and 1B illustrate a method for fabricating nanoparticles according to an embodiment of the present disclosure. As shown in FIG. 1A, in one embodiment, a mask is applied to a fluorosilanized surface, and a subsequent treatment process creates wettable and non-wetable nanodomains on the surface. Non-limiting examples of masks include photomasks, sacrificial patterned resins, and the like. For example, photolithography masks, block copolymer lithography, or nanoimprinted substrates with removable sacrificial resin gaps can be used to mask the fluorosilanized surface. Non-limiting examples of fluorosilanized surfaces include glass, Si, TaO, and the like. x and the like. Non-limiting examples of treatment processes include deep ultraviolet light, plasma, and the like. For example, exposure to UV light, particularly lower wavelengths, and plasma ashing processes can be used to alter the surface energy of the fluorosilanized surface. In non-limiting examples, the wavelength of the UV light can range from about 100 to about 500 nm, including any and all subranges, such as about 100 to about 370 nm, about 250 to about 500 nm, and about 250 to about 370 nm. Additionally, the wavelength can depend on the activation moiety. Portions of the fluorosilanized surface covered by the mask become non-wettable voids, while portions of the fluorosilanized surface exposed to the treatment process become wettable nanodomains.

[0070] As shown in FIG. 1B, different polymers are sequentially dip-coated onto the wettable nano-domains. A soluble polymer 101 may be applied to the wettable domains, followed by a first polymer 102, a second polymer 103, and a third polymer 104. The soluble polymers may then be solubilized to yield nanoparticles with multiple functional groups. In a non-limiting example, the nanoparticles are multiphase particles composed of functionalized polyacrylamide, or orthogonally reactive polymers, etc.

[0071] The monomers used to form the first polymer 102 may include first accessory moieties that are attachment sites for a plurality of first oligonucleotides, or may include a plurality of first oligonucleotides. The plurality of first oligonucleotides may be attached to the monomers of the first polymer 102 before polymerization of the first polymer 102, after polymerization of the first polymer 102 but before solubilization of the dissolvable layer 101, or after solubilization of the dissolvable layer 101.

[0072] The monomers used to form third polymer 104 may include second accessory moieties that are attachment sites for a plurality of second oligonucleotides, or may include a plurality of second oligonucleotides. The plurality of second oligonucleotides may be attached to the monomers of third polymer 104 before polymerization of third polymer 104, after polymerization of third polymer 104 but before solubilization of dissolvable layer 101, or after solubilization of dissolvable layer 101.

[0073] The second polymer 103 may include a single site for attachment of a template polynucleotide, a single site for attachment of a template anchor oligonucleotide, or a single template anchor oligonucleotide. The single site for attachment of a template polynucleotide, a single site for attachment of a template anchor oligonucleotide, or a single template anchor oligonucleotide may be added to the second polymer 103 during polymerization of the second polymer 103, after polymerization of the second polymer 103 but before solubilization, or after solubilization.

[0074] Although lithographic methods allow for a wide range of particle sizes relative to the horizontal template base, from about 20 nm to about micrometers, the lateral dimensions of the nanoparticles depend on the dip-coating parameters and may be on the order of about 20 nm to about 2000 nm, including any and all subranges therein. In certain embodiments, the lateral dimensions of the nanoparticles may be about 100 to about 400 nm, including any and all subranges therein, such as about 200 to about 250 nm, about 200 to about 400 nm, about 200 to about 350 nm, about 200 to about 300 nm, about 200 to about 300 nm, about 100 to about 250 nm, or about 150 to about 250 nm. In other embodiments, the lateral dimensions of the nanoparticles may be about 200, 210, 220, 230, 240, or 250 nm.

[0075] The geometry and composition of nanoparticles can be precisely controlled using the methods described herein. Non-limiting examples of nanoparticle geometries include spheres, rods, cylinders, rectangular, triangular, or polygonal prisms, pyramids, disks, rings, cones, and the like. In some embodiments, nanoparticles may be configured to have a disk shape. In other embodiments, nanoparticles may have a uniform shape. The shape of the nanoparticles may be determined by the wettable nanodomains formed on the fluorosilanized surface.

[0076] In yet another embodiment, the shape of the nanoparticles may be reconfigured after they are formed. Figure 6 illustrates the reconfiguration of nanoparticles described herein by heating. In some embodiments, non-spherical nanoparticles may be heated to a temperature above the glass transition temperature of the polymer from which they are made, resulting in spherical particles.

[0077] In some embodiments, the polymer layer may be crosslinked by coating a formulation of monomers mixed with a crosslinker or photoinitiator, followed by exposing the substrate to a suitable light source, such as a 365 nm UV LED.

[0078] After the nanoparticles are released from the fluorosilanized surface, the surface template may be reused, opening this method to large-scale production of such nanoparticles. For example, the surface template may be reused 2, 5, 10, 20, 50, or more times.

[0079] In one embodiment, the substrate comprises a nanowell. In another embodiment, the substrate can be silanized TiO2 or fluoro-silane TiO2. In yet another embodiment, the attaching comprises hybridizing a forward primer or a reverse primer to the oligonucleotide attached to the substrate.

[0080] In some embodiments, one or both of the attachment of the first plurality of accessory oligonucleotides to the first layer and the attachment of the second plurality of accessory oligonucleotides to the third layer occurs before solubilizing the soluble polymer. In other embodiments, the soluble polymer can be a sulfonate, a sugar, or a phenol. In yet other embodiments, the soluble polymer can be poly(sodium 4-styrenesulfonate) or poly-4-vinylphenol.

[0081] Another aspect relates to a method of making nanoparticles, the method comprising forming nanoparticles within a nanowell and releasing the nanoparticles from the nanowell. As shown in Figure 4, the forming comprises polymerizing a first polymer within the nanowell, the first polymer comprising a first plurality of accessory oligonucleotides; polymerizing a second polymer on the first polymer, the second polymer comprising a single template site for binding to a template polynucleotide; and polymerizing a third polymer on the second polymer, the second polymer comprising a second plurality of accessory oligonucleotides. In some embodiments, releasing the nanoparticles from the nanowell comprises mechanically releasing the nanoparticles. Mechanically releasing the nanoparticles can include, for example, "squeezing" the nanoparticles from the nanowell. In another embodiment, the layer is flexible so that it can be rolled to release the particles.

[0082] In another embodiment, the method further comprises forming a soluble layer within the nanowell prior to forming the nanoparticles within the nanowell. The soluble polymer is polymerized within the nanowell before the first polymer is polymerized, as shown in Figure 5. In yet another embodiment, releasing the nanoparticles from the nanowell comprises solubilizing the soluble layer.

[0083] In a non-limiting example, a substrate surface is prepared for forming the nanoparticles disclosed herein. When a smooth, homogeneous surface (e.g., a substrate surface as described herein) is contacted with a liquid (e.g., a polymer solution as described herein), the liquid may fully or partially wet the surface and form a finite equilibrium contact angle with the surface. The equilibrium contact angle (θ E ) is Young's relation cosθ E =(γ SV -γ SL ) / γ LV and the solid-vapor force acting on the three-phase contact line (γ SV or surface energy), solid-liquid (γ SL ), and liquid vapor (γ LV The contact angle when a liquid advances from a smooth surface is called the advancing contact angle (θ A ), and the contact angle when a liquid recedes from a smooth surface is called the receding contact angle (θ R A perfectly wettable substrate surface has a θ equal to 0. R , and when it is pulled through a liquid (e.g., dip coated), the substrate is coated with a liquid film of finite thickness, the thickness of which is controlled by the rate at which the substrate is pulled through the liquid. A partially wettable substrate surface has a θ R>0, and when it is pulled by a liquid, the liquid film is unstable and de-wets the surface, leaving the surface dry for velocities below a certain value. Thus, when a patterned substrate surface having wetting nano-domains and non-wetting gaps between the wetting nano-domains is pulled by a liquid (e.g., a liquid), only the wetting nano-domains remain coated by the liquid, and the non-wetting gaps between the wetting nano-domains remain uncoated.

[0084] In one example, the wettable nano-domains (or those with high surface energy) may have receding contact angles of about 5° or less, or about 4° or less, or about 3° or less, or about 2° or less, or about 1° or less, or about 0°, with both non-polar and polar liquids, reflecting surface regions that are wettable or substantially wettable by a variety of polar and non-polar materials applied thereto. In another example, the non-wettable gaps (or those with low surface energy) between the wettable nano-domains of a surface may have receding contact angles of about 10° or more, or about 15° or more, or about 20° or more, or about 30° or more, or about 40° or more, or about 50° or more, or about 60° or more, or about 70° or more, or about 80° or more, or about 90° or more, with both non-polar and polar liquids, reflecting surface regions that are non-wettable or substantially non-wettable by a variety of polar and non-polar materials applied thereto.

[0085] In a non-limiting example, a substrate with low surface energy, such as a fluorosilanized surface or a titanium dioxide surface, can be masked (as described above) and the unmasked areas treated to alter the surface chemistry. Substrates with low surface energy have finite receding contact angles with both aqueous and organic liquids and can be patterned with high surface energy to create wettable nanodomains. When the patterned surface is immersed and withdrawn from the polymer solution, the solution wets the wettable nanodomains and self-assembles within them while receding from the nonwettable gaps with low surface energy. The feature size of the mask corresponds to the final lateral size of the nanoparticles. Thus, the unmasked, treated areas of the surface become wettable nanodomains, while the masked areas remain nonwettable nanodomains. The mask can be removed from the substrate surface without affecting the surface energy modification of the wettable nanodomains. The wettable nanodomains can be coated with a soluble polymer, which can be solubilized to release the nanoparticles from the substrate after they are formed. In some embodiments, the soluble polymer may be a sulfonate, a sugar, or a phenol. Non-limiting examples of soluble polymers include poly(sodium 4-styrenesulfonate) and poly-4-vinylphenol.

[0086] A first polymer comprising a first plurality of accessory oligonucleotides is coated onto a soluble polymer. The first plurality of accessory oligonucleotides is connected to the first polymer via a first chemistry or structure / moiety. A second polymer comprising a single template site for binding a single template polynucleotide is coated onto the first polymer. The single template polynucleotide is connected to a single template site on the second polymer via a second chemistry or structure / moiety. A third polymer comprising a second plurality of accessory oligonucleotides is coated onto the second polymer. The second plurality of accessory oligonucleotides is connected to the third polymer via a third chemistry or structure / moiety. The linkages between the first plurality of accessory nucleotides, the second plurality of accessory nucleotides, and the template polynucleotide are orthogonal in that the first, second, and third chemistries or structures / moieties are different from each other and do not cross-react. The connection means are described in Table 1 above.

[0087] The nanoparticles may be released from the substrate surface by solubilizing a soluble polymer. The nanoparticles may be used in an SBS sequencing system, as described in more detail below.

[0088] Another aspect relates to a method for amplifying a template polynucleotide, as shown in Figure 2. The method includes attaching nanoparticles disclosed herein to a substrate and amplifying the template polynucleotide using a polymerase. In one embodiment, the substrate comprises a nanowell. In another embodiment, the substrate can be silanized TiO2 or fluorosilane TiO2. Non-limiting examples of suitable substrates include glass, NIL resin, laminates, TaOx, and the like. The substrate can be patterned or continuous. In a non-limiting example, the substrate can be a NIL having a patterned surface and a feature size of 200-250 nm. In yet another embodiment, the attaching includes hybridizing a forward primer or a reverse primer to the oligonucleotide attached to the substrate.

[0089] It should also be understood that amplification and / or sequencing of a polynucleotide strand (e.g., a forward strand or a reverse strand) may not necessarily produce an exact copy of the strand or an exact copy of the reverse complement of the strand. This is because, for various reasons, errors can be introduced into the amplification and / or sequencing process, which can introduce defects (e.g., incorrect bases) into the polynucleotide sequence of bases. For example, up to 1 defect in 1 million, 10 defects in 1 million, or 1 defect in 1 million may be introduced into the sequenced or amplified strand. Thus, a cluster of forward or reverse strands 121, 123 may not contain an exact copy of each strand in the cluster, but may contain substantially the same duplication of each strand in the cluster.

[0090] Cluster amplification is an approach for amplifying polynucleotides, for example, for use in gene sequencing. Target polynucleotides are captured by primers (e.g., P5 and P7 primers) linked to the surface of a substrate within a flow cell, forming "seeds" at random locations on the surface. Amplification cycles can be performed to form clusters on the surface around each seed. The clusters contain copies of the seed polynucleotide and complementary copies. In some situations, the substrate can be patterned to define regions bound to different clusters, such as wells that can be filled with each cluster.

[0091] Various known amplification techniques can be used, including, but not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), or random primer amplification (RPA). In some examples, amplification can be performed in solution, for example, if the amplification site can contain amplicons in a volume having a desired capacity. Preferably, amplification techniques used in the disclosed methods under kinetic exclusion conditions are performed on a solid phase. For example, one or more primers used in amplification can be attached to a solid phase at the amplification site. In the example of PCR, one or both primers used in amplification can be attached to a solid phase. Formats utilizing two surface-attached primers are often referred to as bridge amplification, because the double-stranded amplicon forms a bridge-like structure between the two surface-attached primers that flank the copied template sequence. Examples of reagents and conditions that can be used for bridge amplification are described, for example, in U.S. Pat. No. 5,641,658, U.S. Patent Application Publication No. 2002 / 0055100, U.S. Pat. No. 7,115,400, U.S. Patent Application Publication No. 2004 / 0096853, U.S. Patent Application Publication No. 2004 / 0002090, U.S. Patent Application Publication No. 2007 / 0128624, and U.S. Patent Application Publication No. 2008 / 0009420, each of which is incorporated herein in its entirety.

[0092] In one embodiment, the principle of size exclusion is used to prevent individual template polynucleotides from being seeded too close to each other, thereby promoting adjacent / mixed clusters. By associating each individual template polynucleotide with a nanoparticle of sufficient spatial dimension, the template polynucleotides can be attached to the surface of the substrate sufficiently far from each other to reduce the formation of polyclonal clusters and increase the formation of monoclonal clusters. In another embodiment, the second polymer contains a single template site for binding the template polynucleotide. The second polymer can have only one single site for binding the template polynucleotide. Thus, only one template polynucleotide can be attached to a nanoparticle, such that attachment of the template polynucleotide to the second polymer prevents attachment of a second template polynucleotide to the same nanoparticle, with the attached template polynucleotide occupying its single template polynucleotide binding site. Attachment of only a single template polynucleotide per nanoparticle results in a spatial distribution of the template polynucleotides attached to such nanoparticles relative to each other, directly or indirectly due to the size of the attached nanoparticle, reducing the formation of polyclonal clusters.

[0093] In an example of preparing a polynucleotide chain for sequencing, a first adaptor and a different second adaptor are often added to the end of the polynucleotide chain to form what is known as a DNA library. The adaptors are complementary to forward and reverse primers, such as oligonucleotide fragments (oligos), and are immobilized by their 5' ends in the nanowells of a flow cell. Thus, the DNA library to be sequenced can be amplified on a solid support, where the seeds hybridize to the forward and reverse primers to form DNA clusters.

[0094] The forward and reverse primers contain chemical cleavage sites so that the forward or reverse strands can be cleaved and removed independently. Sequencing of the forward and reverse strands may be performed sequentially by first removing the reverse strands, blocking their 3' ends, and sequencing the forward strand to obtain read 1, and then, after the cluster is reamplified, removing the forward strands, blocking their 3' ends, and sequencing the reverse strand to obtain read 2.

[0095] In this example, because the synthesis of the forward and reverse strands occurs sequentially, this process can be very time-consuming. Additionally, the larger the nanowells (e.g., for larger clusters or multiple clusters), the greater the probability that polyclonality (i.e., more than one type of strand is initially seeded into the nanowell and then simultaneously amplified into a polyclonal cluster) can occur. Furthermore, the closer the clusters are to each other, the greater the probability that crosstalk (i.e., light emitted from one cluster enters the light guide of another cluster and is recorded on an unrelated photodetector) can occur.

[0096] Simultaneous paired-end sequencing allows users to simultaneously sequence both the forward and reverse complementary strands of a cluster. In addition, the nanoparticles of the present disclosure allow for physical separation of forward and reverse reads, reducing the possibility of polyclonality and crosstalk between adjacent forward and reverse strand clusters, and allowing for simultaneous sequencing of forward and reverse reads. Examples of methods for simultaneous paired-end sequencing of template polynucleotides are disclosed, for example, in U.S. Patent No. 11,124,824, the entire contents of which are incorporated herein by reference.

[0097] Paired-end sequencing involves two reads from the two ends of a fragment. Paired-end reads are used to resolve ambiguous alignments. Paired-end sequencing allows users to select the length of the insert (or fragment to be sequenced) and sequence either end of the insert, generating high-quality, alignable sequence data. Because the distance between each paired read is known, alignment algorithms can use this information to more accurately map reads over repetitive regions. This results in better alignment of reads, especially across repetitive regions of the genome that are difficult to sequence. Paired-end sequencing can detect rearrangements, including insertions and deletions (indels) and inversions. Methods for fragmenting a target nucleic acid sample (e.g., a genomic DNA sample), ligating primers to form paired-end reads, and reading the sequence from the ends of the fragments are known and may be performed, for example, as described in U.S. Pat. No. 7,754,429, U.S. Pat. No. 8,017,335, and U.S. Pat. No. 8,192,930, each of which is incorporated herein by reference.

[0098] In paired-end read sequencing, each of the first primer sets includes a non-cleavable first primer and a cleavable second primer, and each of the second primer sets includes a cleavable first primer and a non-cleavable second primer. The non-cleavable first primer and the cleavable second primer are an oligo pair, and the non-cleavable first primer is a forward amplification primer and the cleavable second primer is a reverse amplification primer, or the cleavable second primer is a forward amplification primer and the non-cleavable first primer is a reverse amplification primer. In each example of the first primer set, the cleavable second primer includes a cleavage site, while the non-cleavable first primer does not include a cleavage site.

[0099] The cleavable first primer and the non-cleavable second primer are also an oligo pair, for example, the cleavable first primer is a forward amplification primer and the non-cleavable second primer is a reverse amplification primer, or the non-cleavable second primer is a forward amplification primer and the cleavable first primer is a reverse amplification primer. It should be understood that the non-cleavable first primer of the first primer set and the cleavable first primer of the second primer set have the same nucleotide sequence (e.g., both are forward amplification primers or both are reverse amplification primers), except that the cleavable primer has a cleavage site incorporated within the nucleotide sequence or within a linker connected to the nucleotide sequence. It should be understood that if the first primer is a forward amplification primer, the second primer is a reverse primer, and vice versa. Examples of non-cleavable primers include P5 and P7 primers, which are used on the surface of commercially available flow cells sold by Illumina Inc., for example, for sequencing on HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, ISEQ™, GENOME ANALYZER™, and other instrument platforms. P5 and P7 primers have universal sequences for capture and / or amplification purposes.

[0100] In some examples, the nanoparticles disclosed herein can be used for simultaneous paired-end sequencing. Orthogonal chemistry for binding a first type of primer on one side of the nanoparticle and a second type of primer on the opposing side of the nanoparticle can facilitate physical separation of the template polynucleotide and its complementary copies on the opposing side after clustering. For example, the two sides of the nanoparticle can contain forward and reverse primers for clustered amplification of the template polynucleotide, with only a single site for connection for the template polynucleotide contained in the layer between them on the opposing side. On one side, the forward primer can be cleavable from the nanoparticle (e.g., by containing a modified nucleotide such as a modified uracil) and the reverse primer can be non-cleavable; on the opposing side, the reverse primer can be cleavable and the forward primer can be non-cleavable, or vice versa. For example, on one side, the non-cleavable first primer can be P7 and the cleavable (e.g., uracil-modified) second primer can be P5U; on the other side, the cleavable (e.g., uracil-modified) first primer can be P7U and the non-cleavable second primer can be P5. As described above, the chemistry of the first and second primers is orthogonal, allowing amplification across both sets (e.g., P7 / P5U and P7U / P5) and cleavage of some of the generated template strands, leaving the same (forward or reverse) template strand within a specific region. This allows for simultaneous, distinguishable Read 1 and Read 2 signals. After amplification, amplicons attached to the nanoparticles by the cleavable primers can be cleaved from the nanoparticles, leaving only forward amplicons attached to one side of the nanoparticle and reverse amplicons attached to the opposite side of the nanoparticle, or vice versa. Simultaneous reading from the forward and reverse strands can thereby be facilitated by reducing the physical overlap of the fluorescent signals emitted from amplicons attached to each opposing face (e.g., by physically separating the emitted fluorescent signals).

[0101] In this disclosure and claims, where present, the use of order indicators, e.g., (a), (b), (c), etc., should be understood as not conveying any particular order or sequence (except to the extent such order or sequence is explicitly indicated). For example, where there are three steps labeled (i), (ii), and (iii), it should be understood that these steps may be performed in any order (or even simultaneously, unless otherwise prohibited) unless otherwise indicated. For example, if step (ii) involves manipulating an element produced in step (i), step (ii) can be viewed as occurring at some time after step (i). Similarly, if step (i) involves manipulating an element produced in step (ii), the reverse should be understood.

[0102] It should be understood that certain aspects, modes, embodiments, variations, and features of the present disclosure are described below at various levels of detail to provide a substantial understanding of the present technology. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term "including" and other forms is not limiting. The use of the term "having" and other forms is not limiting. When used in this disclosure, whether in a transitional phrase or in the body of a claim, the terms "comprise" and "comprising" should be interpreted as having an open-ended meaning. That is, these terms should be interpreted as synonymous with the phrases "having at least" or "including at least."

[0103] It should also be understood that the use of "to," e.g., "a valve for switching between two flow paths," may be interchangeable with phrases such as "configured to," e.g., "a valve configured to switch between two flow paths."

[0104] The terms "substantially," "approximately," "about," "relatively," or other such similar terms, which may be used throughout this disclosure, including the claims, are used to describe and account for small variations from a reference or parameter, such as due to variations in processing. Such small variations include zero-point variations from a reference or parameter. For example, variations can refer to ±10% or less, such as ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0105] It is further understood that certain features described herein, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0106] In this disclosure, reference is made to the accompanying drawings which form a part hereof, and which show, by way of illustration, specific embodiments which may be practiced. These embodiments are described in detail to enable those skilled in the art to practice the disclosure, it being understood that other embodiments may be utilized and structural, logical, and electrical changes may be made without departing from the scope of the disclosure.

[0107] The terms "connected," "contact," and / or "coupled" encompass a variety of arrangements and assemblies, including, but not limited to, (1) directly joining one component to another without any intervening components between them (i.e., the components are in direct physical contact) and (2) joining one component to another with one or more components between them, provided that a component that is "connected," "in contact," or "coupled" to another component is in some operative communication (e.g., electrical, fluid, physical, optical, etc.) with the other component (optionally with the presence of one or more additional components between them). Components that are in direct physical contact with each other may or may not be in electrical and / or fluid contact with each other. Furthermore, two components that are electrically connected, electrically coupled, optically connected, optically coupled, fluidly connected, or fluidly coupled may or may not be in direct physical contact, and one or more other components may be located between the two connected components. [Example]

[0108] The following examples are intended to illustrate, but not to limit, the scope of the present disclosure, which is set forth in the appended claims.

[0109] FIG. 7 is a workflow scheme showing the creation and detection of polymer particles fabricated using a nanoimprint lithography working stamp.

[0110] A Riverstone fluorinated working stamp was filled with fluorescently tagged azapa-co-acrylamide (PAZAM) polymer by spin-coating an aqueous solution of the polymer onto it (0.25 wt % with 5% ethanol). The working stamp was gently wiped to remove any interstitial polymer, and the remaining material in the nanowells was then cured at 60°C for 1 hour. The fluorescent confocal image of the working stamp below shows that the physical barriers imposed by the working stamp features facilitate gravity-mediated segregation of the polymer particles into the desired templated nanofeatures.

[0111] While preferred embodiments have been shown and described in detail herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the subject matter disclosed herein, and therefore are deemed to be within the scope of the present invention as defined in the following claims.

Claims

1. A nanoparticle, a first layer comprising a first polymer and a first plurality of accessory sites; a second layer comprising a second polymer comprising a single template site for binding to a template polynucleotide; a third layer comprising a third polymer and a second plurality of accessory sites; The second layer is between the first layer and the third layer.

2. The nanoparticle of claim 1 , wherein the first polymer is a hydrophilic polymer.

3. 3. The nanoparticle of claim 2, wherein the hydrophilic polymer is selected from natural polyacrylamide, polyethyleneimine, polypeptides, polysaccharides, polyvinyl alcohol, polyacrylic acid, polyallylamine, polystyrene sulfonate, or polyoxazoline.

4. The nanoparticles according to any one of claims 1 to 3, wherein the third polymer is a lipophilic polymer.

5. 5. The nanoparticle of claim 4, wherein the lipophilic polymer is selected from isopropylacrylamide, acrylic, epoxy, polyethylene, polystyrene, polyvinyl, polymethylsulfonate, polyurethane, and fluorinated polymers.

6. The first polymer and the third polymer are each independently selected from the group consisting of poly(vinylidene fluoride), polystyrene, epoxy polymer, (meth)acrylate polymer, polydimethylsiloxane, SiO ２ 6. The nanoparticle of any one of claims 1 to 5, wherein the containing polymer is selected from poly(lactic acid-co-glycolic acid) polymers, perfluorinated polymers, azapa-co-acrylamide polymers (PAZNAM), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) polymers, poly(o-nitrobenzyl-masked acrylamide-co-acrylamide) copolymers, poly(benzopyrone-masked acrylamide-co-acrylamide) copolymers, poly(aminotriazole-acrylamide-co-acrylamide) copolymers, poly(thiotriazole-acrylamide-co-acrylamide) copolymers, poly(alkenyltriazole-acrylamide-co-acrylamide) copolymers, and thiol / ene crosslinkable monomer mixtures.

7. The nanoparticles of any one of claims 1 to 6, wherein the first polymer is polyacrylamide and the third polymer is isopropylacrylamide.

8. The nanoparticle of any one of claims 1 to 7, wherein the second polymer is a copolymer of the first polymer and the third polymer.

9. The nanoparticles according to any one of claims 1 to 8, wherein the second polymer is a hydrophilic polymer or a lipophilic polymer.

10. The nanoparticles according to any one of claims 1 to 9, wherein the second polymer is a methacrylate.

11. The nanoparticle of any one of claims 1 to 10, further comprising one or both of a first plurality of accessory oligonucleotides attached to the first plurality of accessory moieties and a second plurality of accessory oligonucleotides attached to the second plurality of accessory moieties.

12. 12. The nanoparticle of claim 11, wherein one or both of the first plurality of accessory oligonucleotides and the second plurality of accessory oligonucleotides comprise one or both of a forward primer and a reverse primer, and the sequence of the forward primer and the sequence of the reverse primer enable amplification of the template polynucleotide by a polymerase.

13. The nanoparticles of claim 12, wherein the forward primers of the first plurality of accessory oligonucleotides and the reverse primers of the second plurality of accessory oligonucleotides, or the reverse primers of the first plurality of accessory oligonucleotides and the forward primers of the second plurality of accessory oligonucleotides, are cleavable, and the other primers of the first and second plurality of accessory oligonucleotides are non-cleavable.

14. the single template site A nanoparticle according to any one of claims 1 to 13, comprising a type of connection site for a template anchor oligonucleotide or said template anchor oligonucleotide.

15. 1. A method comprising:

15. A method comprising: attaching the nanoparticles of any one of claims 1 to 14 to a substrate; and amplifying the template polynucleotide using a polymerase.

16. The method of claim 15 , wherein the substrate comprises a nanowell.

17. The substrate is a silanized TiO ２ or fluoro-silane TiO ２ The method according to claim 15 or 16, wherein

18. 18. The method of any one of claims 15 to 17, wherein attaching comprises hybridizing a forward primer or a reverse primer to an oligonucleotide attached to the substrate.

19. 1. A method of making nanoparticles, comprising: coating wettable nano-domains on a substrate surface with a soluble polymer, wherein the wettable nano-domains are separated by non-wettable interstices; coating the soluble polymer with a first polymer, the first polymer comprising a first plurality of accessory moieties; coating the first polymer with a second polymer, the second polymer comprising a single template site; coating the second polymer with a third polymer, the third polymer comprising a second plurality of accessory moieties; solubilizing the soluble polymer to release the nanoparticles.

20. (i) the first plurality of accessory sites comprises a first plurality of accessory oligonucleotides; and 20. The method of claim 19, wherein (ii) the second plurality of accessory sites comprises one or both of a second plurality of accessory oligonucleotides.

21. (i) the first plurality of accessory sites includes a first type of connection site for a first plurality of accessory oligonucleotides but does not include one for a second plurality of accessory oligonucleotides; and (ii) the second plurality of accessory sites comprises a second type of connection site for the second plurality of accessory oligonucleotides but does not comprise any for the first plurality of accessory oligonucleotides, or both.

22. 22. The method of claim 21, further comprising, after solubilizing, one or more of: attaching the first plurality of accessory oligonucleotides to the first plurality of accessory moieties; and attaching the second plurality of accessory oligonucleotides to the second plurality of accessory moieties.

23. the single template site a type of attachment site for a template anchor oligonucleotide, the method further comprising attaching the template anchor oligonucleotide to the single template site; or The method of any one of claims 19 to 22, comprising said template anchor oligonucleotide.

24. 24. The method of any one of claims 19 to 23, wherein the soluble polymer is a sulfonate, a sugar, or a phenol.

25. 25. The method of claim 24, wherein the soluble polymer is poly(sodium 4-styrenesulfonate) or poly-4-vinylphenol.

26. 1. A method comprising: forming nanoparticles within the nanowells, the forming comprising: polymerizing a first polymer, the first polymer comprising a first plurality of accessory moieties; polymerizing a second polymer onto the first polymer, the second polymer comprising a single template site; polymerizing a third polymer onto the second polymer, the third polymer comprising a second plurality of accessory moieties.

27. (i) the first plurality of accessory sites comprises a first plurality of accessory oligonucleotides; and 27. The method of claim 26, wherein (ii) the second plurality of accessory moieties comprises one or both of a second plurality of accessory oligonucleotides.

28. (i) the first plurality of accessory sites includes a first type of connection site for a first plurality of accessory oligonucleotides but does not include one for a second plurality of accessory oligonucleotides; and (ii) the second plurality of accessory sites comprises a second type of connection site for the second plurality of accessory oligonucleotides but does not comprise any for the first plurality of accessory oligonucleotides, or both.

29. the single template site a type of attachment site for a template anchor oligonucleotide, the method further comprising attaching the template anchor oligonucleotide to the single template site; or The method of any one of claims 26 to 28, comprising said template anchor oligonucleotide.

30. 30. The method of any one of claims 26 to 29, further comprising releasing the nanoparticles from the nanowells, wherein releasing comprises mechanically releasing the nanoparticles.

31. 27. The method of claim 26, further comprising polymerizing a soluble polymer before polymerizing the first polymer.

32. 32. The method of claim 31 , further comprising releasing the nanoparticles from the nanowells, wherein releasing comprises solubilizing the soluble polymer.

33. 33. The method of claim 31 or 32, wherein the soluble polymer is a sulfonate, a sugar, or a phenol.

34. 34. The method of claim 33, wherein the soluble polymer is poly(sodium 4-styrenesulfonate) or poly-4-vinylphenol.

35. The method of any one of claims 19 to 34, wherein the first polymer is a hydrophilic polymer.

36. 36. The method of claim 35, wherein the hydrophilic polymer is selected from natural polyacrylamide, polyethyleneimine, polypeptide, polysaccharide, polyvinyl alcohol, polyacrylic acid, polyallylamine, polystyrene sulfonate, or polyoxazoline.

37. The method of any one of claims 19 to 36, wherein the second polymer is a copolymer of the first polymer and the third polymer.

38. The method of any one of claims 19 to 37, wherein the second polymer is a hydrophilic polymer or a lipophilic polymer.

39. The method of any one of claims 19 to 38, wherein the second polymer comprises a methacrylate.

40. 40. The method of any one of claims 19 to 39, wherein the third polymer is a lipophilic polymer.

41. 41. The method of claim 40, wherein the lipophilic polymer is selected from isopropylacrylamide, acrylic, epoxy, polyethylene, polystyrene, polyvinyl, polymethylsulfonate, polyurethane, and fluorinated polymers.

42. The first polymer and the third polymer are each independently selected from the group consisting of poly(vinylidene fluoride), polystyrene, epoxy polymer, (meth)acrylate polymer, polydimethylsiloxane, SiO ２ 35. The method of any one of claims 19 to 34, wherein the containing polymer is selected from poly(lactic acid-co-glycolic acid) polymers, perfluorinated polymers, azapa-co-acrylamide polymers (PAZNAM), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) polymers, poly(o-nitrobenzyl-masked acrylamide-co-acrylamide) copolymers, poly(benzopyrone-masked acrylamide-co-acrylamide) copolymers, poly(aminotriazole-acrylamide-co-acrylamide) copolymers, poly(thiotriazole-acrylamide-co-acrylamide) copolymers, poly(alkenyltriazole-acrylamide-co-acrylamide) copolymers, and thiol / ene crosslinkable monomer mixtures.

43. 43. The method of any one of claims 19 to 42, further comprising attaching a single template polynucleotide to the single template site on the second polymer.

44. 44. The method of any one of claims 19 to 43, wherein the first plurality of accessory oligonucleotides and the second plurality of accessory oligonucleotides comprise a forward primer and a reverse primer, respectively, or a reverse primer and a forward primer, respectively, and the sequence of the forward primer and the sequence of the reverse primer allow amplification of the template polynucleotide by a polymerase.

45. 1. A method of forming nanoparticles, comprising: coating wettable nano-domains of a substrate with a soluble polymer, wherein the wettable nano-domains are separated by non-wettable interstices; coating the soluble polymer with a first polymer comprising a first plurality of accessory moieties; coating the first polymer with a second polymer comprising a single template site for binding to a template polynucleotide; coating the second polymer with a third polymer comprising a second plurality of accessory moieties; attaching a first plurality of accessory oligonucleotides to said first plurality of accessory sites; attaching a second plurality of accessory oligonucleotides to said second plurality of accessory sites; solubilizing the soluble polymer.

46. connecting a first plurality of accessory oligonucleotides to said first plurality of accessory sites; prior to coating the soluble polymer with the first polymer; after coating the soluble polymer with the first polymer and before solubilizing; or 46. ​​The method of claim 45, which occurs after said solubilizing.

47. connecting a second plurality of accessory oligonucleotides to said second plurality of accessory sites; before coating the second polymer with the third polymer; 57. The method of claim 45 or 56, occurring after coating the second polymer with the third polymer and before or after the solubilizing.

48. 48. The method of any one of claims 45 to 47, wherein the soluble polymer is a sulfonate, a sugar, or a phenol.

49. 49. The method of claim 48, wherein the soluble polymer is poly(sodium 4-styrenesulfonate) or poly-4-vinylphenol.

50. 50. The method of any one of claims 45 to 49, wherein the first polymer is a hydrophilic polymer.

51. 51. The method of claim 50, wherein the hydrophilic polymer is selected from natural polyacrylamide, polyethyleneimine, polypeptide, polysaccharide, polyvinyl alcohol, polyacrylic acid, polyallylamine, polystyrene sulfonate, or polyoxazoline.

52. 52. The method of any one of claims 45 to 51, wherein the second polymer is a copolymer of the first polymer and the third polymer.

53. 52. The method of any one of claims 45 to 51, wherein the second polymer is a hydrophilic polymer or a lipophilic polymer.

54. The method of any one of claims 45 to 51, wherein the second polymer comprises a methacrylate.

55. 52. The method of any one of claims 45 to 51, wherein the third polymer is a lipophilic polymer.

56. 56. The method of claim 55, wherein the lipophilic polymer is selected from isopropylacrylamide, acrylic, epoxy, polyethylene, polystyrene, polyvinyl, polymethylsulfonate, polyurethane, and fluorinated polymers.

57. The first polymer and the third polymer are each independently selected from the group consisting of poly(vinylidene fluoride), polystyrene, epoxy polymer, (meth)acrylate polymer, polydimethylsiloxane, SiO ２ 52. The method of any one of claims 45 to 51, wherein the containing polymer is selected from poly(lactic acid-co-glycolic acid) polymers, perfluorinated polymers, azapa-co-acrylamide polymers (PAZNAM), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) polymers, poly(o-nitrobenzyl-masked acrylamide-co-acrylamide) copolymers, poly(benzopyrone-masked acrylamide-co-acrylamide) copolymers, poly(aminotriazole-acrylamide-co-acrylamide) copolymers, poly(thiotriazole-acrylamide-co-acrylamide) copolymers, poly(alkenyltriazole-acrylamide-co-acrylamide) copolymers, and thiol / ene crosslinkable monomer mixtures.

58. 58. The method of any one of claims 45 to 57, further comprising attaching a single template polynucleotide to the single template site on the second polymer.

59. 59. The method of any one of claims 45-58, wherein the first plurality of accessory oligonucleotides and the second plurality of accessory oligonucleotides comprise a forward primer and a reverse primer, respectively, or a reverse primer and a forward primer, respectively, and the sequence of the forward primer and the sequence of the reverse primer allow amplification of the template polynucleotide by a polymerase.

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