WO2024059754A1 - Polythiolated linkers for stable nanoparticle suspensions and uses thereof - Google Patents

Abstract

The invention is directed to polythiolated linker compounds comprising dithiolane moieties for attaching labeled biomolecules, such as polynucleotides, to metallic particles for analysis with nanopore techniques. Further disclosed a method of determining a sequence of a nucleic acids comprising translocating through one or more nanopores a labeled polynucleotide comprising polythiolated linker, wherein each nanopore comprises a passage through an insulative layer and an opaque layer.

Description

TITLE POLYTHIOLATED LINKERS FOR STABLE NANOPARTICLE SUSPENSIONS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No.63/375,662, filed September 14, 2022, which is incorporated by reference herein in its entirety. BACKGROUND [0002] Gold nanoparticles have been used in a wide variety of assays because of their optical properties, ease of synthesis and derivatization, and availability in precise shapes and sizes, e.g. Niemeyer, Angew. Chem. Int. Ed., 40: 4128-4158 (2001); Daniel et al, Chem. Rev., 104: 293-346 (2004); Ielo et al, Molecules, 26: 5823 (2021); and the like. Different applications require analytes or reagents to be linked to such particles with different strengths and densities without jeopardizing a colloidal state by an undesired aggregation of particles. [0003] For example, in certain approaches to nanopore-based sequencing of nucleic acids and proteins, e.g. Huber et al, International patent publication WO/2021/217146, it is desirable to have a flexible linker compound for firmly attaching biopolymers with varying physiochemical properties to gold nanoparticles at desired densities, while minimizing the risk of particle aggregation. [0004] In view of the above, such applications of gold and metallic particles would be advanced by the availability of new linkers for attaching a variety of analytes to such particles while minimizing the risk of the particles aggregating. SUMMARY OF THE INVENTION [0005] The invention is directed to polythiolated linker compounds and their use for attaching biomolecules to metal particles, especially gold particles. In some embodiments, biomolecules comprise polynucleotides and are linked to gold particles by polythiolated linkers of the invention. In some embodiments, labeled polynucleotides attached to gold particles by polythiolated linkers of the invention are used in nanopore sequencing. Levine Bagade Han LLP 1 of 21 Docket No.: QNTPZ02800WO [0006] In one aspect, polythiolated linkers of the invention have the general form as illustrated in Fig.1A comprising (i) a polythiolated component having greater than six sulfur atoms capable of forming sulfur-metal bonds, and (ii) a hydrophilic spacer moiety. Such linkers are used to make products of the general form as illustrated in Fig.1C which comprise: (i) a polythiolated component having greater than six sulfur atoms capable of forming sulfur-metal bonds, (ii) a hydrophilic spacer moiety, and (iii) a biomolecule. In another aspect, polythiolated linkers of the invention have the general form as illustrated in Fig.1E, which may also be written as follows: LA-L3 –AA-[( K*)^j, AA^k]- where LA is a lipoic acid moiety, AA is an amino acid (each occurrence of which in the above formula may be the same or different), L₃ is a linking moiety linking the lipoic acid moiety to the N terminal primary amine of AA, K* is a modified lysine of the formula LA- L₄-K, where LA is a lipoic acid moiety, K is lysine and L₄ is a linking moiety linking LA to the side chain amine of the lysine, and the bracketed term –[(K*)^j, AA^k]- is a peptide of j LA-modified lysines (K*’s) and k amino acids in any order, where j is an integer in the range of from 3 to 7, inclusive, and k is an integer in the range of from 0 to 10, inclusive. As illustrated in Fig.1F, linkers of Fig.1E may be attached to biomolecules directly without a hydrophilic spacer. In some embodiments, each AA of the bracketed term is a hydrophilic amino acid. In some embodiments, the first –AA- (that is, the AA outside of the bracketed term) is a lysine. [0007] In some embodiments, the biomolecule is a polynucleotide. In some embodiments of the compounds of Figs.1A and 1B, a polynucleotide may be attached by its 5’ end to the hydrophilic component. In other embodiments of the compounds of Figs. 1A and 1B, a polynucleotide may be attached by its 3’ end to the hydrophilic component. Likewise, in some embodiments of the compounds of Fig.1E, a polynucleotide may be attached by its 5’ end to the polythiolated linker of Fig.1E, and in other embodiments, a polynucleotide may be attached by its 3’ end to such linker. In some embodiments, the polynucleotide may be single stranded and in other embodiments, the polynucleotide may be double stranded. In some embodiments, the biomolecule is a peptide or a protein. In some embodiments of the compounds of Figs.1A and 1B, the peptide or protein component may be attached to the hydrophilic component by its C-terminus. In other such embodiments, the peptide or protein component may be attached to the hydrophilic Levine Bagade Han LLP 2 of 21 Docket No.: QNTPZ02800WO component by its N-terminus. In still other embodiments, the peptide or protein component may be attached to the hydrophilic component by an amino acid side chain. [0008] In another aspect, labeled polynucleotides linked to metallic particles by linker compounds of the invention are used in certain nanopore sequencing techniques. In such applications a method of determining a sequence of a nucleic acids may be carried out with the following steps: (a) translocating through one or more nanopores a labeled polynucleotide, wherein each nanopore comprises a passage through an insulative layer and an opaque layer, the passage through the opaque layer and the nanopore each having a diameter, wherein the labeled polynucleotide is attached to a gold carrier particle having a diameter greater than the diameter of the nanopore through the insulative layer, and wherein the labeled polynucleotide is attached to the gold carrier particle by a polythiolate linker defined by the formula:

wherein n is in the range of from 3 to 7, inclusive; L₁ is a linking moiety; L₂ is a linking moiety; and hydrophilic spacer is any hydrophilic moiety; (b) illuminating the passage from the direction of the opaque layer with a light beam having a wavelength greater than the diameter of the nanopore through the opaque layer, so that an excitation zone of non- propagating light is created within the passage through the opaque layer; (c) digesting the labeled polynucleotide in the nanopore outside of the excitation zone to release labeled nucleotides one at a time at a rate less than the expected time of diffusion of the released labeled nucleotides out of the nanopore into the excitation zone; (d) identifying each released labeled nucleotide by detecting the signal generated by its label as the released labeled nucleotide diffuses out of the nanopore through the excitation zone. [0009] In still another aspect, labeled polynucleotides linked to metallic particles by linker compounds of the invention are used in certain nanopore sequencing techniques. In such applications a method of determining a sequence of a nucleic acids may be carried out with the following steps: (a) translocating through one or more nanopores a labeled Levine Bagade Han LLP 3 of 21 Docket No.: QNTPZ02800WO polynucleotide, wherein each nanopore comprises a passage through an insulative layer and an opaque layer, the passage through the opaque layer and the nanopore each having a diameter, wherein the labeled polynucleotide is attached to a gold carrier particle having a diameter greater than the diameter of the nanopore through the insulative layer, and wherein the labeled polynucleotide is attached to the gold carrier particle by a polythiolate linker defined by the formula:

wherein j is in the range of from 3 to 7, inclusive; AA is an amino acid (which may be the same or different in each occurrence in the above formula); k is in the range of from 0 to 10, inclusive; K is a lysine; L₃ is a linking moiety; L₄ is a linking moiety; and the bracketed moiety is any ordering (i.e., multiset permutation) of the groups –(K-L4-lipoic acid)- and AA; (b) illuminating the passage from the direction of the opaque layer with a light beam having a wavelength greater than the diameter of the nanopore through the opaque layer, so that an excitation zone of non-propagating light is created within the passage through the opaque layer; (c) digesting the labeled polynucleotide in the nanopore outside of the excitation zone to release labeled nucleotides one at a time at a rate less than the expected time of diffusion of the released labeled nucleotides out of the nanopore into the excitation zone; (d) identifying each released labeled nucleotide by detecting the signal generated by its label as the released labeled nucleotide diffuses out of the nanopore through the excitation zone. Levine Bagade Han LLP 4 of 21 Docket No.: QNTPZ02800WO BRIEF DESCRIPTION OF THE DRAWINGS [0010] Figs.1A-1F illustrate the general structure of compounds of the invention. [0011] Figs.2A-2E illustrate the synthesis of an exemplary compound of the invention and its attachment to a gold surface. [0012] Figs.3A-3B illustrate an application of polythiolated compounds of the invention in nanopore sequencing. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS [0013] While the invention is amenable to various modifications and alternative forms, specifics thereof are shown by way of example in the drawings and are described in further detail herein. It should be understood, however, that the intention is not to limit the invention to the particular embodiments described herein. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. [0014] The invention is directed to polythiolated linker compounds for attaching biomolecules, especially polynucleotides or proteins, to metallic particles, especially gold particles. The invention includes applications of such linker compounds in the nanopore sequencing field, as exemplified by the approach disclosed by Huber et al, International patent publication WO/2021/217146, which is incorporated herein by reference. [0015] In some embodiments, polythiolated linker compounds of the invention (100) comprise two components as illustrated in Fig.1A. Namely, a polythiolate moiety having greater than six thiolate groups (102) each capable of forming a bond with a gold surface and a hydrophilic spacer moiety (104). The structure of hydrophilic spacer moiety (104) may vary widely and, in some embodiments, are selected to ensure that the metal particles remain in a colloidal state when biomolecules are attached with linker compounds of the invention. In some embodiments, factors related to such selections include the composition and size of metallic particle, whether the colloid comprises physiological conditions, the type of biomolecule, and the like. Hydrophilic spacer moiety (104) is attached to a biomolecule through linker (106) which may be a bond or a group formed from the reaction of functionalities, such as a primary amine and an NHS ester. In some embodiments, compounds of the invention comprise products of the linker compounds and biomolecules as illustrated in Fig.1C. In some embodiments, as illustrated in Fig.1D, the biomolecule is an oligonucleotide or polynucleotide, which may be attached to the Levine Bagade Han LLP 5 of 21 Docket No.: QNTPZ02800WO hydrophilic moiety by its 5’ end or its 3’ end, and wherein the polythiolate moiety has the formula: wherein

n is in the range of from 3 to 7, inclusive; L1 and L2 are each linking moieties comprising from 2 to 20 carbon atoms and having a molecular weight in the range of 20 to 400 Daltons. [0016] In some embodiments, the hydrophilic spacer may be any hydrophilic moiety that is inert to nanopore sequencing assay conditions. In some embodiments, the hydrophilic spacer comprises polyethylene glycol moieties and has a molecular weight of 3000 Daltons or less. In some embodiments, the hydrophilic spacer comprises polyethylene glycol (PEG) moieties of varying sizes alternating with phosphate moieties, which may be conveniently synthesized using commercially available phosphoramidite monomers. In some embodiments, the hydrophilic spacer comprises hydrophilic functional groups selected from the group consisting of carboxylic acid, sulfonic acid, and ammonium salts and has a molecular weight of less than 3000 Daltons. In still other embodiments, a hydrophilic spacer comprises hydrophilic mercaptoundecanoic acid (MUA), mercapto sulfonic acid (MPS), Sodium 3-mercapto-1-propanesulfonate (3MPS)), hydrophilic polymer, poly(3-dimethylammonium-1-propyne hydrochloride) (PDMPAHCl), and has a molecular weight of 3000 Daltons or less. [0017] In some embodiments, the polythiolate linking compounds of the invention are defined by the formula of Fig.1E, which may also be written as follows: LA-L3-AA-[( K*)^j, AA^k]- where LA is a lipoic acid moiety, AA is an amino acid (each occurrence of which in the above formula may be the same or different), L3 is a linking moiety linking the lipoic acid Levine Bagade Han LLP 6 of 21 Docket No.: QNTPZ02800WO moiety to the N-terminal amine of AA, K* is a modified lysine of the formula LA-L4-K, where LA is a lipoic acid moiety, K is lysine and L4 is a linking moiety linking LA to the side chain amine of the lysine, and the bracketed term –[(K*)^j, AA^k]- is a peptide of j LA- modified lysines (K*’s) and k amino acids in any order (that is, a multiset permutation of the K*’s and the AA’s), where j is an integer in the range of from 3 to 7, inclusive, and k is an integer in the range of from 0 to 10, inclusive. By way of example, if j equals 3 and k equals 1, then the bracketed structure would represent the following 4-mer peptides: -K*- K*-K*-AA-, -K*-K*-AA-K*-, -K*-AA-K*-K*-, and -AA-K*-K*-K*-. By way of another example, if j equals 3 and k equals 2, then the bracketed structure would represent the following 5-mer peptides: -K*-K*-K*-AA-AA-, -K*-AA-AA-K*-K*-, -K*-K*-AA- AA-K*-, -AA-AA- K*-K*-K*-, -AA- K*-K*-K*-AA-, -K*-AA-K*-K*-AA-, -K*-K*-AA- K*-AA-, -AA-K*-K*-AA-K*-, -AA-K*-AA-K*-K*-, and –K*-AA-K*-AA-K*- (where each AA may be the same or different in each peptide). As illustrated in Fig.1F, linkers of Fig.1E may be attached to biomolecules directly without a hydrophilic spacer. L3 and L4 may be the same or different and, in some embodiments, are each comprising from 2 to 20 carbon atoms and having a molecular weight in the range of 20 to 400 Daltons. In some embodiments, each AA within the bracketed term is a hydrophilic amino acid selected from the group consisting of arginine (R), asparagine (N), aspartate (D), glutamine (Q), glutamic acid (E), histidine (H), lysine (K), serine (S) and threonine (T). The number and kind of the amino acids selected may vary widely and, in some embodiments, are selected to ensure that the metal particles remain in a colloidal state when biomolecules are attached with linker compounds of the invention. In some embodiments, AA is a modified amino acid comprising the Sulfonate group (cysteic acid or 3-sulfo-L-alanine), a phosphorus- containing amino acid (L-phosphoserine)or Glutamic acid where backbone coupled to any linker to provide additional desirable passivity to surface. [0018] As noted above, the chemical form of linkers L₁, L₂, L₃ and L₄ may vary widely consistent with not interfering with the functions of the linked components or introducing undesired side reactions. In some embodiments, L₁, L₂, L₃ and L₄ each separately comprises alkyl-, cycloalkyl-, alkyl- aryl-, alkylene-, alkenylene-, aryl-alkylene-, alkynylene-, aryl-alkynylene-, alkoxy-, ether, amide or oligoethyleneglycol linear or branched format, wherein such linkers possibly containing heteroatoms and having a molecular weight within the range of from 20 to 400 Daltons. In some embodiments, such linkers contain from 0 to 8 heteroatoms. In some embodiments, such heteroatoms are selected from the group consisting of oxygen, nitrogen, sulfur and phosphorous. Levine Bagade Han LLP 7 of 21 Docket No.: QNTPZ02800WO Exemplary linkers comprise: Hydrophilic poly(ethylene glycol), Hydrophobic poly(propylene glycol), DithiolalkanearomaticPEG6, Poly(oligo(ethylene glycol) monomethyl ether methacrylate) (POEOMA), Poly(2-alkyl-2-oxazoline)s (PAOXAs) and PAOXA derivatives, such as poly(2-methyl-2-oxazoline)- 4-vinyl pyridine (PMOXA-r- 4VP ), hydroxybenzaldehyde (HBA) groups, zwitterionic moieties, (tertiary amines along with carboxy and/or hydroxyl groups, Sulfobetaine ligand). Nanopore Sequencing [0019] As described more fully in Huber et al (cited above), in some embodiments, the step for identifying a nucleotide sequence of a labeled polynucleotide comprises providing a solid state substrate comprising a cis side and a trans side, the substrate comprising a reaction well that defines a reaction volume and comprises (i) a proximal throughhole extending between the cis side and the trans side of the substrate, (ii) one or more side walls, and (iii) a distal opening. (As used herein, the term “throughhole” is used interchangeably with the term “nanopore”). The solid state substrate further comprises an opaque metal layer that substantially blocks excitation light from penetrating into the reaction volume and from penetrating to the cis side of the substrate. Also provided is a carrier particle comprising a fluorescently labeled polynucleotide strand that is attached to the carrier particle by a linker compound of the invention. The fluorescently labeled polynucleotide strand comprises (i) a proximal end that is attached to the carrier particle, (ii) a distal end that is cleavable by an exonuclease, and (iii) at least one fluorescently labeled nucleotide comprising a fluorescent label. The carrier particle is located on the cis side of the substrate, but does not pass through the throughhole, such that the attached fluorescently labeled polynucleotide strand protrudes through the throughhole so that the distal end of the fluorescently labeled strand is in the reaction volume. The trans side of the substrate is illuminated with excitation light to create a fluorescence excitation zone adjacent to the distal opening of the reaction well. While the substrate is illuminated, the fluorescently labeled polynucleotide strand is reacted with an exonuclease so that mononucleotides are released serially from the distal end of the strand and diffuse through the fluorescence excitation zone, so that fluorescently labeled mononucleotides in the excitation zone emit fluorescent signals. The fluorescent signals are detected as a function of time, whereby a nucleotide sequence is determined from the time order of fluorescent signals detected from the released fluorescently labeled mononucleotides. Levine Bagade Han LLP 8 of 21 Docket No.: QNTPZ02800WO [0020] In some embodiments, the distal opening of the reaction well has a minimum diameter of at least 30 nm. In some embodiments, the distal opening of the reaction well has a minimum diameter of 50 nm to 150 nm. In some embodiments, the one or more walls of the reaction well are not tapered. In some embodiments, the one or more walls of the reaction well are substantially cylindrical. In some embodiments, the opaque metal layer comprises gold or aluminum. In some embodiments, the opaque metal layer has a thickness of 100 nm to 600 nm. In some embodiments, the solid state substrate comprises a plurality of opaque metal layers. In some embodiments, the reaction well has a well depth of at least 200 nm. In some embodiments, the reaction well has a well depth of 200 nm to 1000 nm. In some embodiments, the fluorescently labeled polynucleotide strand in the reaction volume comprises a fluorescently labeled polynucleotide segment containing at least 100 contiguous nucleotides. In some embodiments, the throughhole has a minimum diameter of at least 2 nm. In some embodiments, the throughhole has a minimum diameter of 2 nm to 50 nm. In some embodiments, the substrate comprises a thin membrane layer that contains the proximal throughhole and that has a thickness of between 20 nm and 50 nm. In some embodiments, the thin membrane layer comprises silicon nitride. In some embodiments, the excitation light has a wavelength of 380 nm or greater. In some embodiments, the solid substrate comprises surface portion(s) that define the reaction volume, and the surface portion(s) comprise at least one surface passivation coating. In some embodiments, one or more side walls of the reaction well comprise one or both of a silicon oxide coating and aluminum oxide coating. In some embodiments, the fluorescently labeled polynucleotide strand comprises at least two different kinds of nucleotides, each kind labeled with a distinguishing fluorescent label. [0021] In some embodiments, during said reacting, the carrier particle is maintained next to the proximal throughhole by a voltage bias. In some embodiments, the carrier particle comprises a plurality of fluorescently labeled polynucleotide strands having polynucleotide sequences that are different from each other. In some embodiments, after said reacting, the voltage bias is stopped to allow the carrier particle to move away from the proximal throughhole, so that the remaining fluorescently labeled polynucleotide strand is removed from the reaction volume, and then a voltage bias is applied to move the same or a different carrier particle toward the proximal throughhole so that a new fluorescently labeled polynucleotide strand is delivered into the reaction well for reacting with an exonuclease. Levine Bagade Han LLP 9 of 21 Docket No.: QNTPZ02800WO [0022] In some embodiments, the fluorescently labeled polynucleotide strand in the reaction volume comprises a double-stranded nucleic acid. In some embodiments, the fluorescently labeled polynucleotide strand in the reaction volume comprises a single- stranded nucleic acid. In some embodiments, the carrier particle comprises a plurality of fluorescently labeled polynucleotide strands, which in some embodiments, are single- stranded or double-stranded nucleic acids. [0023] In some embodiments, the solid state substrate comprises a plurality of reaction wells. In some embodiments, the plurality of reaction wells are configured as a one- dimensional or two-dimensional array. [0024] In some aspects, the sequencing method is directed to fluorescence-based analysis of oligonucleotide or polynucleotide barcodes using sequential digestion of fluorescently labeled polynucleotide strands by exonuclease activity. [0025] Fluorescently labeled polynucleotide strands for sequencing may be prepared by any suitable method. Each fluorescently labeled strand may have a proximal end and a distal end. The proximal end is coupled, directly or indirectly, to a carrier particle as described further below. The distal end of the fluorescently labeled strand protrudes away from the carrier particle when the fluorescently labeled strand is coupled to a carrier particle. Each fluorescently labeled strand is capable of being cleaved by an exonuclease, so that mononucleotides, some or all of which comprise fluorescent labels, are released serially (one-by-one) from the distal end of the strand for subsequent detection (discussed further below). [0026] In some embodiments, fluorescently labeled strands may be provided in single- stranded form, for serial cleavage of a distal end of the fluorescently labeled strand by a single-strand-specific exonuclease. In other embodiments, fluorescently labeled strands may be provided in double-stranded form comprising a fluorescently labeled strand, for serial cleavage of a distal end of the fluorescently labeled strand by a double-strand- specific exonuclease. [0027] In some embodiments, a 5’-exonuclease is used. The 5’-exonuclease may be single-strand-specific or double-strand-specific. For a single-strand-specific 5’- exonuclease, the fluorescently labeled strand may be provided in single-stranded form such that the 3’-end is the proximal end coupled to the carrier particle, and the 5’-end is the distal end to be cleaved by the 5’-exonuclease. For a double-strand-specific 5’- exonuclease, all of the features of the immediately preceding sentence apply, except that Levine Bagade Han LLP 10 of 21 Docket No.: QNTPZ02800WO the fluorescently labeled strand is provided in double-stranded form that comprises a complementary strand hybridized to the fluorescently labeled strand. [0028] In some embodiments, a 3’-exonuclease is used. The 3’-exonuclease may be single-strand-specific or double-strand-specific. For a single-strand-specific 3’- exonuclease, the fluorescently labeled strand may be provided in single-stranded form such that the 5’-end is the proximal end coupled to the carrier particle, and the 3’-end is the distal end to be cleaved by the 3’-exonuclease. For a double-strand-specific 3’- exonuclease, all of the features of the immediately preceding sentence apply, except that the fluorescently labeled strand is provided in double-stranded form that comprises a complementary strand hybridized to the fluorescently labeled strand. [0029] The carrier particles have dimensions that are sufficiently large to prevent the carrier particles from moving through the throughholes of the reaction wells into the reaction wells. [0030] In some embodiments, carrier particles have a diameter, or a largest diameter, that is at least 15 nm, or at least 20 nm, or at least 25 nm, or at least 30 nm, or from 15 nm to 100 nm, or from 15 nm to 75 nm, or from 15 nm to 50 nm, or from 20 nm to 50 nm. However, carrier particles having larger or smaller diameters may also be used. As used herein, “nanoparticle” refers to a carrier particle having a diameter, or largest diameter, that is less than 200 nm, or less than 150 nm, or less than 100 nm. [0031] The carrier particles may be charged or uncharged. The carrier particles may have a net neutral charge, a net positive charge, or a net negative charge, based on the net balance of positively charged groups and negatively charged groups on the particles under the pH conditions of the surrounding aqueous medium, which usually comprises an aqueous buffer. Preferably, for control of movement by an electric field (voltage bias), the carrier particles have a net negative charge when they comprise one or more attached fluorescently labeled polynucleotide strands. [0032] In some embodiments, each carrier particle is capable of being moved by an electromagnetic force away from a throughhole of a reaction well, to remove a cleaved fluorescently labeled polynucleotide strand from the throughhole. In preferred embodiments of methods of the present invention, after exonuclease cleavage of a fluorescently labeled polynucleotide strand in a well, the carrier particle is moved away from the well so that the cleaved strand is withdrawn from the well, and then the same or a different carrier particle is moved near the throughhole of the well to deliver the distal end of a second (sometimes called “new”) fluorescently labeled polynucleotide strand through Levine Bagade Han LLP 11 of 21 Docket No.: QNTPZ02800WO the throughhole into the reaction well. To facilitate movement of distal ends of fluorescently labeled polynucleotide strand into and out of the reaction wells by voltage bias or magnetic field, the carrier particles are not covalently coupled to the throughholes. [0033] In some embodiments, the carrier particles comprise spherical particles. In some embodiments, the carrier particles comprise non-spherical particles, having, for example, ellipsoid or irregular shapes. In some embodiments, the carrier particles comprise both spherical particles and non-spherical particles. In some embodiments, the carrier particles are provided as a uniform population of substantially identical carrier particles within a size range (e.g., within plus or minus a standard deviation or coefficient of variation), but the carrier particles are not necessarily identical, provided that they effectively carry and deliver fluorescently labeled polynucleotide strands to the reaction wells. [0034] In some embodiments, the carrier particles are metal particles, such as metal nanoparticles. In some embodiments, the carrier particles are gold nanoparticles. In some embodiments, the carrier particles are silver nanoparticles. [0035] In some aspects of the inventions disclosed herein, a solid state substrate comprises a cis side and a trans side. The substrate comprises a reaction well that defines a reaction volume. The reaction well comprises (i) a proximal throughhole extending between the cis side and the trans side of the substrate, (ii) one or more side walls, and (iii) a distal opening. The solid state substrate further comprises an opaque metal layer that substantially blocks excitation light that is incident on the trans side of the substrate from penetrating into the reaction volume of the reaction well and from penetrating to the cis side of the substrate. [0036] Reaction wells for containing fluorescently labeled polynucleotide strands to be sequenced may have any of a variety of shapes and sizes. For example, although cylindrical wells with circular cross-sections and parallel side walls are suitable, reaction wells may also have elliptical, triangular, square, rectangular, pentagonal, hexagonal, octagonal or other regular or irregular cross-sectional shapes, with parallel or non-parallel side walls. For example, the side walls of reaction wells having any of the foregoing shapes may be parallel, tapered, truncated-conical, or hour-glass shaped. For example, a cylindrical well may be considered to have a single side wall that is inherently parallel with itself. [0037] Reaction wells may have any of a variety of dimensions that may be chosen by the user. The choice of specific dimensions can take into consideration a selected length Levine Bagade Han LLP 12 of 21 Docket No.: QNTPZ02800WO and a minimum diameter of the fluorescently labeled strands that will be sequenced, whether the fluorescently labeled strands are in single- or double-stranded form, and any other relevant considerations. [0038] The depth and minimum diameters of reaction wells are usually selected so that each reaction well can contain (1) a distal end of a fluorescently labeled polynucleotide strand to be sequenced and also (2) an exonuclease molecule that is bound to the distal end of the strand during nucleolytic cleavage of terminal mononucleotides. [0039] As used herein, “minimum diameter” means the shortest diameter of a reaction well or of a throughhole, as applicable. For example, a cylinder has a single diameter, which is the minimum diameter. For a reaction well having a square overhead cross- section that is perpendicular to the depth axis of the reaction well, the minimum diameter is the distance between (and perpendicular to) two opposing walls of the reaction well (the length of a side of the square cross-section), whereas the maximum diameter is the length of a diagonal across the square cross-section. For a reaction well having tapered or other non-parallel walls, the minimum diameter of the reaction well is the shortest dimension in a cross-section of the well. More generally, the distal opening, and at least a portion of the reaction well extending from the distal opening, have a minimum diameter that satisfies requirements (1) and (2) above. Therefore, if the distal opening of a reaction well has a particular minimum diameter, then the minimum diameter of at least a portion of, or all of, the reaction well extending from the distal opening towards the throughhole of the well is equal to or greater than the minimum diameter of the distal opening of a reaction well. [0040] The above description is summarized in Figs.3A and 3B. Oligonucleotides with predetermined sequences (302) bound by their 3’ ends to particle (301) by polythiolate linkers of the invention are combined (306) with sample polynucleotides (304) containing complementary segments to the bound oligonucleotides under annealing conditions so that polynucleotides (304) are captured. Bound oligonucleotides (302) are extended using a polymerase in the presence of labeled nucleoside triphosphates. After removal of the template strand, particle (301) having labeled polynucleotides (310) attached, wherein the different shapes (solid star, solid circle, open circle) represent differently labeled nucleotides. As described above, such loaded particles (301) are forced into contact with nanopore sequencing device (350) comprising the following elements: nanopore (or throughhole) (314), cis surface (312), layer (316) (including an opaque layer) in which reaction chamber (324) is formed. The side opposite throughhole (314) is illuminated with a light that produces non-transmitting light energy in region (326). As a Levine Bagade Han LLP 13 of 21 Docket No.: QNTPZ02800WO sequence of labeled nucleotides are directed through throughhole (314) they are cleaved from the labeled polynucleotide by exonuclease (318) where they are initially released into the proximal (unilluminated) portion of reaction chamber (324), e.g. labeled nucleotide (320). Eventually, each such cleaved labeled nucleotide diffuses (322) out of reaction chamber (324) into illuminated region (326) where it is detected by a characteristic signal (328). Conjugation of Polynucleotides to Carrier Particles [0041] Conjugation of DNA to Gold Nanoparticles. Oligonucleotides functionalized at 3’- or 5’-end with acyclic disulfide group (CH2)n-S-S-(CH2)n-OH (n = 3 or 6), 1, 2 or 3 DTPA (from IDT, Iowa) or (±)-α-lipoic acid are incubated with a 50-fold excess of a reducing agent (tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) in phosphate buffer at pH 7.4 for 45 min. The reduced thiol-functionalized oligonucleotides are purified by desalting with gel filtration columns packed with Sephadex G-25 and milli-Q water as eluent. [0042] Purified oligonucleotides are mixed with gold nanoparticles (10 nm, 15 nm, 50 nm, and 100 nm diameter from Sigma-Aldrich) at various molar ratios (100:1, 300:1, 500:1, 1000:1, 3000:1, 10000:1, 20000:1, etc.) and the pH is adjusted to 4.3 with 50 mM citrate-HCl buffer, or to pH 7.4 or pH 8 with 10 mM sodium phosphate buffer. Tween 20 is added to a final concentration of 0.02%, 0.05% or 0.1%, or instead of Tween 20, SDS is added to a final concentration of 0.01%, 0.025%, or 0.05%. After initial incubation at ambient temperature for 1 to 24 hours, 4 M NaCl is added in small portions up to 1 M total concentration. The reaction mixture is further incubated for 1 to 24 hours. Optionally, to block unreacted surface areas on the gold nanoparticles (also referred to as “back-filling”), a water-soluble oligo(ethylene glycol)-alkylthiol, where oligo(ethylene glycol) is PEG3 or PEG6, and alkyl is (CH₂)₈ – (CH₂)₁₂, is added to the reaction mixture (variable ratios relative to thiol-modified oligonucleotide: 1:1, 4:1, 10:1, 25:1, 100:1) and the reaction mixture is incubated for 15 min to 24 hours. Oligonucleotide-nanoparticle conjugates are harvested by centrifuging and multiple washing with appropriate buffer (e.g.10 mM phosphate buffer pH 8 with 0.02% Tween 20). Nanoparticles are stored at 4 °C in the same washing buffer before further use. [0043] For quantitation of DNA loading, oligonucleotides are released from nanoparticles via etching with KCN / K3[Fe(CN)6] mixture or DTT. Released oligonucleotides are quantified with a fluorometer using SYBR^TM Gold or OliGreen^TM Levine Bagade Han LLP 14 of 21 Docket No.: QNTPZ02800WO (ThermoFisher Scientific) as a staining agent. For internally labeled oligonucleotides that are obtained by primer extension in presence of at least one fluorescently labeled (Cy3, Cy5, Cy7, Alexa Fluor 488 etc.) deoxynucleotide triphosphate (dATP, dCTP, dGTP or dUTP), intrinsic absorbance or fluorescence is used as a readout for quantification after nucleotide release from nanoparticles. Alternatively, reversible hybridization of suitably labeled complementary oligonucleotide is used for quantification. Example 1 Synthesis of Exemplary Polythiolated Linker [0044] An exemplary polythiolated linker compound of the invention was synthesized as illustrated in Figs.2A-2D. Lipoic acid was used to introduce two thiols at a time via amide coupling. For direct conjugation of an oligonucleotide to gold nanoparticles, multiple thiol groups were attached on either the 3’ or 5’ end of the oligonucleotide. Hydrophilic spacers of various lengths were introduced between the polythiolate moiety and the oligonucleotide to facilitate access of enzymes. NHS esters of lipoic acid were prepared by known methods, e.g. Dougan et al, Nucleic Acids Research, 35(11): 3668- 3675 (2007). Primary amine groups for amide coupling were inserted using commercially available amino-modified phosphoramidites (e.g. Clontech or IDT). [0045] As illustrated in Fig.2A, 5’-O-protected nucleoside (200) attached to a controlled pore glass (CPG) support (dN-1caa-CPG, Glen Research) repeatedly is subject to cycles (202) of deprotection and coupling of successive nucleoside phosphoramidites until 5’-O-protected oligonucleotide of SEQ ID NO: 1 (204) is obtained, after which m cycles (206) of deprotection and coupling of successive PEG(N) phosphoramidites (Sp18, Glen Research). The resulting product is again subjected to multiple cycles (208) of deprotection and coupling linker phosphoramidite (UniAmM, Takara) to give product (209)(Fig.2B). Product (209) is deprotected and coupled (210) to a disulfide phosphoramidite (ThioMC6-D), after which the resulting product is deprotected, cleaved from the CPG support and purified (212) to give product (214) (Fig.2C). The free amines of product (214) is then reacted with an NHS-ester of lipoic acid to give thiolated linker compound (215) (Fig.2D). Exemplary values for n are 3, 4, 5, 6 and 7 and exemplary values for m are 1, 2, 3, 4, 5, 6, 7 and 8. Fig.2E illustrates linker compound (215) bound to gold surface (216) wherein sulfurs of five lipoic acid moieties and one sulfur from the terminal disulfide form bonds (222) with gold surface (216). Hydrophilic spacer (220) formed from the Sp 18 moieties links the bound sulfurs to oligonucleotide (218). Levine Bagade Han LLP 15 of 21 Docket No.: QNTPZ02800WO Example 2 Enzymatic Extension of Primers Linked To Gold Particles By Polythiolated Linkers [0046] The double-stranded DNA-AuNP conjugate was prepared by enzymatic extension reaction, covalent incorporation of nucleotides to form the complement of the template by the DNA polymerase. In this experiment, the 5-Lipo-PEG6x3-oligonucleotide conjugated to the gold nanoparticles served as a primer for enzymatic extension reactions. The reaction mixture consisted of reaction buffer (20 mM Tris-HCl, 10 mM (NH4)₂SO₄, 10 mM KCl, 2 mM MgSO4, 0.1% Triton®-X-100, pH 8.8 at 25 °C) and solution of AuNP- primer-to-template ratio of 2:1, 1:1, 1:0.5, etc., The sample was heated to 75 °C for 5 min and incubated at 56 °C for 25 minutes. Following annealing of the template to the primer, the reaction was brought to a total reaction volume of 50 µl by addition of 0.5 µl of 10 mM dATP, 0.5 µl of 10 mM dCTP, 0.5 µl of 10 mM dGTP, 0.5 µl of 10 mM labeled-dUTP, 0.5 µl of 100 % Formamide, and 0.5 µl of DNA polymerase (15.8 µM). The sample was again heated to 56 °C for 5 min and incubated at 72 °C for 1.5 h for the extension. The solution was centrifuged at 14,000 rcf for 30 min to separate the AuNPs from the unreacted reagents. The DNA- AuNPs were washed 7 times with PBS buffer, pH 8.0, containing 0.025% Tween-20. The control experiment in which the primer was not attached to the gold nanoparticle was performed using the same oligonucleotide sequence. Example 3 Oligonucleotides Desorption During Storage [0047] The dissociation of SH-conjugates (desorption of the oligonucleotides) from the Au surface is critical for obtaining accurate sequencing data via fluorescence read-out. To remove the physioabsorbed primers from AuNP surface and reduce the optical background, the conjugates were incubated for 2-4 hours in 20 mM Tris pH 7.5, 1mM DTT, 10 mM MgCl2 at 37 °C. Samples were then centrifuged at 14,000 rcf for 10 min 3 times, with a rinse of 800 μL of PBS buffer pH 8.0, 0.05% Tween-20 in between. Samples were resuspended in the same buffer for analysis by fluorescence spectroscopy. [0048] After the reaction was complete, the characterization of DNA probes immobilized on gold nanoparticles was performed by fluorescence spectroscopy. The intensity of fluorescence associated with the solution free of nanoparticles was used to determine the oligonucleotide amount released from the nanoparticles. Levine Bagade Han LLP 16 of 21 Docket No.: QNTPZ02800WO [0049] In a typical experiment DNA-functionalized gold nanoparticle (8-20 fmol) were incubated at 37° for 15-45 minutes in 20 mM Tris pH 7.5, 10 mM MgCl2. At designated time points, the AuNPs were centrifuged, and the supernatant fluorescence intensity was measured. This fluorescence assay allowed the stability evaluation of these DNA-AuNPs probes. Based on our data, the optical background for 5Lipo-primer was about 12-fold lower than in the case of Lipo-primer (Table 1). Additionally, the optical background was significantly reduced after DTT treatment compared with the untreated samples (Table 2). In summary, we demonstrated that both the 5-Lipo-primer and DTT treatment could contribute to the optical background (ensure that SH- conjugates have covalent character), leading to the overall stability increase of DNA-conjugates. Stable linkage between DNA and AuNPs is important for most applications. Considering the simplicity of this modification/treatment, it might find many applications in nanotechnology and material science. Table1. Primers Fluorescence (RFU) STD

Sample Before treatment After treatment

[0050] This disclosure is not intended to be limited to the scope of the particular forms set forth, but is intended to cover alternatives, modifications, and equivalents of the variations described herein. Further, the scope of the disclosure fully encompasses other variations that may become apparent to those skilled in the art in view of this disclosure. Levine Bagade Han LLP 17 of 21 Docket No.: QNTPZ02800WO

Claims

CLAIMS 1. A compound defined by the formula: Hydrophilic R wherein

n is in the range of from 3 to 7, inclusive; L1 and L2 are each linking moieties comprising from 2 to 8 carbon atoms; hydrophilic spacer is any hydrophilic moiety having a molecular weight of 4000 Daltons or less; and R is a bond or a linking moiety comprising from 1 to 8 carbon atoms.

2. The compound of claim 1 wherein L₁ is –(CH₂)_n-, where n=2-6.

3. The compound of claim 1 wherein L₂ is –(CH₂)_m-C(=O)-O-NH-, where m=2-6.

4. The compound of claim 1 wherein said hydrophilic spacer comprises polyethylene glycol. Levine Bagade Han LLP 18 of 21 Docket No.: QNTPZ02800WO

5. A method of determining a sequence of a nucleic acids comprising the steps of: (a) translocating through one or more nanopores a labeled polynucleotide, wherein each nanopore comprises a passage through an insulative layer and an opaque layer, the passage through the opaque layer and the nanopore each having a diameter, wherein the labeled polynucleotide is attached to a gold carrier particle having a diameter greater than the diameter of the nanopore through the insulative layer, and wherein the labeled polynucleotide is attached to the gold carrier particle by a polythiolate linker defined by the formula:

wherein n is in the range of from 3 to 7, inclusive; L1 and L2 are linking moieties; hydrophilic spacer is any hydrophilic moiety having a molecular weight of less than 4000 Daltons; or by the formula: LA-L₃-AA-[( K*)^j, AA^k]-[5’-oligonucleotide] where LA is a lipoic acid moiety, AA is an amino acid which may be the same or different, L3 is a linking moiety linking the lipoic acid moiety to the N-terminal amine of AA, K* is a modified lysine of the formula LA-L₄-K, where LA is a lipoic acid moiety, K is lysine and L4 is a linking moiety linking LA to the primary amine of K, and the bracketed term – [(K*)^j, AA^k]- is a peptide of j K* amino acids and k AA amino acids in any order, where j is an integer in the range of from 3 to 7, inclusive, and k is an integer in the range of from 0 to 10, inclusive; (b) illuminating the passage from the direction of the opaque layer with a light beam having a wavelength greater than the diameter of the nanopore through the opaque layer, so that an excitation zone of non-propagating light is created within the passage through the opaque layer; Levine Bagade Han LLP 19 of 21 Docket No.: QNTPZ02800WO (c) digesting the labeled polynucleotide in the nanopore outside of the excitation zone to release labeled nucleotides one at a time at a rate less than the expected time of diffusion of the released labeled nucleotides out of the nanopore into the excitation zone; (d) identifying each released labeled nucleotide by detecting the signal generated by its label as the released labeled nucleotide diffuses out of the nanopore through the excitation zone.

6. The method of claim 5 wherein said AA is a hydrophilic amino acid.

7. A compound defined by the formula: LA-L3-AA-[( K*)^j, AA^k]- where LA is a lipoic acid moiety, AA is an amino acid which may be the same or different, L₃ is a linking moiety linking the lipoic acid moiety to the N-terminal amine of AA, K* is a modified lysine of the formula LA-L4-K, where LA is a lipoic acid moiety, K is lysine and L₄ is a linking moiety linking LA to the primary amine of K, and the bracketed term – [(K*)^j, AA^k]- is a peptide of j K* amino acids and k AA amino acids in any order, where j is an integer in the range of from 3 to 7, inclusive, and k is an integer in the range of from 0 to 10, inclusive.

8. The compound of claim 7 wherein L₃ and L₄ may be the same or different and each comprising from 2 to 20 carbon atoms and having a molecular weight in the range of 20 to 400 Daltons.

9. The compound of claim 7 wherein said AA is a hydrophilic amino acid. Levine Bagade Han LLP 20 of 21 Docket No.: QNTPZ02800WO