High yield support

Abstract

This disclosure relates to a support comprising a substrate and a plurality of sample spots on the surface of the support, wherein the sample spots are spatially separated from each other and a single biomolecule is immobilized on a single sample spot, and to a method for fabricating such a support.

Description

[Technical Field]

[0001] explanation This disclosure relates to a support comprising a substrate and a plurality of sample spots on the surface of the support, wherein the sample spots are spatially separated from each other and a single biomolecule is immobilized on a single sample spot, and to a method for fabricating such a support. [Background technology]

[0002] background The sequencing of the human genome or the genomes of other organisms, as well as the determination and comparison of variations in individual sequences, require the provision of sequencing methods that are, firstly, fast and secondly, routine and cost-effective.

[0003] The high demand for cost-effective sequencing has driven the development of high-performance sequencing technologies that parallelize the sequencing process, generating multiple sequences simultaneously. Examples of these sequencing technologies include massively parallel signature sequencing (Lynx Therapeutics), polony sequencing (Life Technologies), 454 pyrosequencing (Roche Diagnostics), illumina sequencing (Solexa Inc.), ligation sequencing (Life Technologies), ion torrent semiconductor sequencing (Life Technologies), and DNA nanoball sequencing (Complete Genomics). These technologies allow for rapid analysis of common sequences within nucleic acid populations. However, among the few sequences in the nucleic acid population being analyzed, for example, mutations present in a small number of cell genomes may be masked by the large number of other sequences present in the population and therefore undetectable.

[0004] To address these issues, several different forms of single-molecule detection processes, such as sequencing, have been developed. For the latter, typically, nucleic acid polymerases and / or nucleases, as well as fluorescently labeled nucleic acids and / or nucleotide building blocks, are used to individually determine the sequence of a single nucleic acid molecule based on the time-dependent change in fluorescence when the nucleotide building block is incorporated into or cleaved from the nucleic acid molecule. Single-molecule sequencing processes and devices adapted to perform such processes are described, for example, in co-owned applications WO 2002 / 097406, WO 2003 / 052137, WO 2006 / 013110, WO 2013 / 131888, WO 2015 / 104245, WO 2017 / 001407, and WO 2018 / 104301.

[0005] In some known processes and apparatuses, molecules of nucleases and / or nuclease synthases, or complexes of such enzyme molecules with nucleic acid molecules, are provided in a form immobilized on a solid support.

[0006] While it is assumed that a single molecule exists on each sample spot, the actual efficiency of the single-molecule immobilization process is not explained.

[0007] In addition to DNA and / or RNA sequencing, there are several additional applications where there is a high demand for single-molecule analysis in high yield.

[0008] The purpose of this disclosure was to provide a support suitable for the analysis of single molecule events, such as the sequencing analysis of a single nucleic acid molecule, including an improved yield of sample spots on which only one biomolecule is immobilized. [Overview of the Initiative]

[0009] Summary of the Invention In the first aspect, this disclosure is: (a) A support comprising a substrate and a plurality of sample spots on the surface of the support, wherein the sample spots are spatially separated from each other and the size of the sample spots is adapted to the size of a particular biomolecule to be immobilized thereon, thereby providing a support to which the sample spots are adapted for immobilizing one single biomolecule on one sample spot, and (b) Incubating the sample spot on the support with the biomolecules under conditions that allow for saturation immobilization of the biomolecules onto the sample spot, where the amount of biomolecules is greater than the amount of available sample spot. This invention relates to a method for preparing supports for analyzing single-molecule events that encompass the reactions of single biomolecules associated with the emission of characteristic electromagnetic radiation, including [specific example of a single biomolecule].

[0010] Further aspects of the present disclosure relate to a support comprising a substrate and a plurality of sample spots on the surface of the support, wherein the sample spots are spatially separated from each other, and biomolecules are immobilized on the sample spots, with single biomolecules immobilized on single sample spots in at least about 50% of the sample spots, and the size of the sample spots is adapted to the size of a particular immobilized biomolecule.

[0011] Further aspects of this disclosure relate to the use of the above-mentioned support for analyzing events encompassing the reaction of a single biomolecule associated with the emission of characteristic electromagnetic radiation at a single sample spot, wherein the event is related to the emission of electromagnetic radiation. In certain embodiments, the event is a single-molecule event.

[0012] Further aspects of this disclosure are: (i) To provide the support wherein a single biomolecule is immobilized on a single sample spot in at least about 50% of the sample spot, and (ii) Analyzing events related to single biomolecules by detecting electromagnetic radiation from individual sample spots. A method for analyzing single-molecule events that include reactions of single biomolecules related to the emission of characteristic electromagnetic radiation is provided. In certain embodiments, the single-molecule event includes sequencing of a single nucleic acid molecule.

[0013] A further aspect of the disclosure is (i) a support comprising a substrate and a plurality of sample spots on a support surface, wherein the sample spots are spatially separated from each other and the sample spots are adapted for immobilization of one single biomolecule on one sample spot, (ii) means for irradiating at least one sample spot on the support with radiation, and (iii) means for analyzing an event associated with a single biomolecule by detecting electromagnetic radiation from individual sample spots, An apparatus for analyzing single-molecule events that include reactions of single biomolecules related to the emission of characteristic electromagnetic radiation is provided. In certain embodiments, the apparatus is adapted for sequencing of a single nucleic acid molecule.

[0014] Items in the statement 1. (c) providing a support comprising a substrate and a plurality of sample spots on a support surface, wherein the sample spots are spatially separated from each other and the sample spots are adapted for immobilization of one single biomolecule on one sample spot, and (d) incubating the sample spots on the support with a biomolecule under conditions that allow for saturation immobilization of the biomolecule to the sample spots A method for preparing a support for analyzing single-molecule events.

[0015] 2. The method of item 1, wherein a single biomolecule is immobilized on a single sample spot in at least about 50% of the sample spots on the support.

[0016] 3. A method according to item 1 or 2, wherein a single biomolecule is immobilized on at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%, and up to 99.99% of the sample spot on the support.

[0017] 4. A method according to any one of the preceding items, wherein the support comprises at least 10, at least 100, at least 1,000, at least 10 4 pieces, at least 10 5 pieces, at least 10 6 pieces, at least 10 7 pieces, or at least 10 8 pieces of sample spots.

[0018] 5. A method according to any one of the preceding items, wherein the sample spots are provided on the support surface such that the distance between adjacent sample spots is from about 1 nm to about 5000 nm, for example, from about 2 nm to about 2000 nm.

[0019] 6. A method according to any one of the preceding items, wherein sample spots having a predetermined size based on the specific biomolecule to be immobilized thereon are provided.

[0020] 7. A method according to item 6, wherein the sample spot has a size of about 5 nm to about 10 nm, particularly about 6 nm to about 8 nm, more particularly about 7 nm, for immobilizing one single biomolecule on one of the sample spots, and the single biomolecule is a polypeptide and / or a nucleic acid molecule.

[0021] 8. A method according to item 7, wherein the biomolecule is a nucleic acid polymerase, particularly DNA or RNA polymerase, and / or a nucleic acid molecule, particularly a DNA or RNA molecule.

[0022] 9. Any one of the preceding items, wherein sample spots having a standard deviation of 20% or less around their average size are provided.

[0023] 10. Any one of the above methods wherein the sample spots are adapted in their surface chemistry to immobilize one single biomolecule on one of the sample spots.

[0024] 11. The method of item 10, wherein the surface of the sample spot contains at least one functional anchor molecule, which includes a portion suitable for direct or indirect attachment of biomolecules.

[0025] 12. The method of item 11, wherein the functional anchor molecule is attached to the surface of the sample spot by a sulfur-containing group, for example, a thiol group.

[0026] 13. The method of item 11 or 12, wherein the sample spot includes a metal surface, particularly an Au or Au / Pd surface.

[0027] 14. The conditions that enable saturated immobilization of the biomolecule onto the sample spot are the following parameters: (i) Concentration of biomolecules; (ii) Incubation time; (iii) Incubation temperature; (iv) Any combination of (i), (ii), and / or (iii) Any one method of the above item, comprising conforming at least one of the following.

[0028] 15. Any one of the above methods, comprising performing the incubation step under conditions that enable saturated immobilization of the biomolecules onto the sample spot, wherein the amount of biomolecules is greater than the amount of available sample spot.

[0029] 16. Method Item 14 in which the amount of biomolecules is at least 10 times greater than the amount of available sample spots.

[0030] 17. Any one of the above methods wherein the support is at least substantially planar. 18. A support structured in any one of the manner described above.

[0031] 19. Any one of the above methods wherein the substrate is optically transparent. 20. Any one of the above methods, wherein the substrate is a material having a refractive index of at least 1.01.

[0032] 21. Any one of the above methods, comprising a non-conductive material, wherein the substrate is selected from the group consisting of, for example, silica, quartz, and glass.

[0033] 22. Any one of the above methods, wherein the substrate has a thickness of approximately 10 μm to approximately 5 mm, and particularly approximately 20 μm to approximately 2 mm.

[0034] 23. Any one method of the item wherein the sample spot comprises at least one conductive material, for example, a pure metal or a combination of metals, for example, an alloy or mixture of several different metals.

[0035] 24. Any one of the above methods, wherein the sample spot comprises a metal capable of forming a bond with a sulfur-containing group, for example, in the form of a thiol or disulfide.

[0036] 25. Any one of the above methods wherein at least a metal has a positive electrochemical potential.

[0037] 26. Any one of the above items, wherein at least one metal is selected from any combination comprising Au, Cu, Ni, Pt, Pd, Rh, Ir, Os, Ru, and at least two of the above metals.

[0038] 27. Any one of the methods described above, wherein at least one sample spot contains at least one metal oxide such as TiO2, NiO, or ITO.

[0039] 28. Any one of the above methods by which a biomolecule is selected from the group consisting of polypeptides, nucleic acids, carbohydrates, and any combination thereof.

[0040] 29. Any one of the above methods, wherein the biomolecule is a nucleic acid polymerase, in particular DNA polymerase or RNA polymerase, or a complex of nucleic acid polymerization molecules, in particular a DNA or RNA polymerization complex containing a nucleic acid polymerase and nucleic acid molecules.

[0041] 30. Any one of the above methods, wherein the biomolecule is a DNA polymerase having a DNA binding cleft, in particular a family A DNA polymerase including but not limited to Klenow, Taq, or T7 DNA polymerase, or any genetically modified version thereof, or a family B polymerase including but not limited to therminator, Phi29, RB-69, or T4 DNA polymerase, or any genetically modified version thereof.

[0042] 31. Any one of the above methods wherein the biomolecule is a gene editing enzyme, in particular a Cas nuclease such as Cas9 nuclease or any genetically modified version thereof, or a gene editing complex comprising a gene editing enzyme and a nucleic acid molecule, such as guide RNA and / or target nucleic acid.

[0043] 32. A support comprising a substrate and a plurality of sample spots on the surface of the support, wherein the sample spots are spatially separated from each other, and biomolecules are immobilized on the sample spots, and single biomolecules are immobilized on single sample spots in at least about 50% of the sample spots. 33. A support for item 32 that can be obtained by any one of the methods described in items 1 to 31.

[0044] 34. Use of support of item 32 or 33 for analyzing single-molecule events. 35. Use of item 34, which involves a single-molecule event, including sequence analysis of a single nucleic acid molecule.

[0045] 36. (i) To provide a support in which a single biomolecule is immobilized on a single sample spot in at least about 50% of the sample spot, and (ii) Analyzing events related to the single biomolecule by detecting electromagnetic radiation from individual sample spots. A method for analyzing single-molecule phenomena, including [specific example].

[0046] 37. A method of item 36, which includes detecting electromagnetic radiation from multiple individual sample spots separately in parallel. 38. A single-molecule event, including a method of item 36 or 37 involving sequence analysis of a single nucleic acid molecule.

[0047] 39. (i) A single biomolecule is immobilized on a single sample spot in at least about 50% of the sample spot, on any one of the supports from items 32 to 33, (ii) means for irradiating at least one sample spot on a support with radiation, (iii) Means for analyzing events related to the single biomolecule by detecting electromagnetic radiation from individual sample spots A device that analyzes single-molecule phenomena, including [specific example].

[0048] 40. Item 39 apparatus adapted for the parallel and separate detection of electromagnetic radiation from multiple individual sample spots. 41. An item 39 or 40 instrument suitable for sequence analysis of a single nucleic acid molecule. [Brief explanation of the drawing]

[0049] [Modes for carrying out the invention]

[0050] Detailed explanation This disclosure relates to a method for preparing a support for analyzing single-molecule phenomena, and to a support that can be obtained by this method. The support comprises a substrate and a plurality of spatially separated sample spots on the surface of the support. In one embodiment, the surface of the support is formed by the substrate and the sample spots. The sample spots are spots on the surface of the support that are suitable for the immobilization of a single biomolecule.

[0051] In one embodiment, the substrate forms a continuous region where the sample spots are distributed. Individual sample spots on the support surface are surrounded, for example, by a substrate different from the sample spot with respect to the material and / or surface. Typically, the substrate is adapted to inhibit and / or block the adhesion of biomolecules such as polypeptides, while the sample spots are adapted to allow the adhesion of desired biomolecules.

[0052] The supports provided by this disclosure include an increase in the yield of sample spots on which a single biomolecule is immobilized. The inventors have identified means by which a single molecule is located on each sample spot, and by which a theoretical yield of 100% (in practice close to 100%, e.g., up to 99.9% or 99.99%) can be achieved.

[0053] In one embodiment, a single biomolecule is immobilized on a single sample spot in a yield of at least about 50% of the sample spot on the support. In a particular embodiment, a single biomolecule is immobilized on a single sample spot in a yield of at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%, and up to 99.99% of the sample spot on the support. The yield of a single biomolecule immobilized on a single sample spot for a particular support can be determined by several spectroscopic and / or microscopic methods. In one embodiment, the determination of the sample spot occupied by a single biomolecule is carried out by fluorescence spectroscopy if the binding molecule is fluorescent or if they are used as a catalyst for a reaction in which a fluorescent product is formed. In a further embodiment, the determination of the sample spot occupied by a single biomolecule is carried out by atomic force microscopy.

[0054] The proportion P of a sample spot occupied by a single biomolecule is calculated as follows: P = n / N (In the formula, n is the number of individual sample spots occupied by a single biomolecule, and N is the total number of sample spots on the support.)

[0055] The term "single biomolecule" encompasses a single molecular entity, such as a polypeptide or a complex composed of multiple individual units; for example, individual molecular entities where the individual units come together to form a functional biological part.

[0056] In one embodiment, the support is substantially planar, i.e., it does not contain any ridges or depressions of about 1000 nm or more or about 100 nm or more. In a further embodiment, the support is a structured support, e.g., about 5 × 10 -24 Liters ~ approximately 1 x 10 -15The support consists of a recess, such as a well, that may have a volume of liters, or a columnar structure with a height of approximately 1 nm to 500 nm. In principle, the support can have any design, as long as a reaction space is formed that allows for the occurrence of a single-molecule event on the sample spot where the single biomolecule is immobilized.

[0057] In some embodiments, the substrate is an optically transparent material, i.e., a material that is substantially transparent to electromagnetic radiation, such as radiation in the visible range and / or the near-infrared range. In some embodiments, the substrate includes a material having an absolute refractive index of at least 1.01, for example, an absolute refractive index of about 1.5 to about 3 in the visible range, or about 1.5 to about 4 in the near-infrared range. In further embodiments, the substrate is an optically opaque material, such as a metal or metalloid, such as silicon.

[0058] In certain embodiments, the substrate includes a non-conductive material. Specific examples include glass, quartz, plastic, metal oxide-based materials, such as glass, silica, or silicon dioxide-based materials such as quartz, or composite materials containing such materials. In further embodiments, the substrate includes a conductive material, such as an optically transparent material such as indium tin oxide. Typically, the substrate has a thickness of approximately 10 μm to 5 mm, and especially approximately 20 μm to 2 mm.

[0059] In certain embodiments, the substrate is coated with a layer of diamond-like carbon and / or amorphous carbon (also referred to as a "carbon film") as described in co-pending application US 63 / 382,624, the contents of which are incorporated herein by reference. The carbon film can be deposited on the substrate by physical vapor deposition, chemical vapor deposition (CVD), or atomic layer deposition (ALD) techniques. The carbon film can form a continuous layer on the support surface excluding the area of the sample spot. In certain embodiments, the carbon film is fluorinated. Fluorine atoms can be introduced by known procedures, for example, by CVD during DLC deposition or by exposure to a fluorine-containing plasma from a fluorocarbon-based gas such as C4F8, CHF3, NF3, or SF6.

[0060] In certain embodiments, the carbon film comprising a fluorinated carbon film has a thickness of from about 0.3 nm to about 200 μm, particularly from about 10 nm to about 100 μm, more particularly from about 1 μm to about 50 μm.

[0061] In certain embodiments, the substrate surface is coated with an organic passivating reagent, such as a polyethylene glycol-containing reagent that inhibits and / or blocks the attachment of biomolecules such as proteins and / or nucleic acids.

[0062] The support surface includes a plurality of sample spots that are spatially separated from each other by the substrate surface. The sample spots are adapted for the attachment of a single biomolecule. In certain embodiments, the support includes a plurality of sample spots, for example, at least 10, at least 100, at least 1,000, at least 10 4 pieces, at least 10 5 pieces, at least 10 6 pieces, at least 10 7 pieces, or at least 10 8 pieces of sample spots. In certain embodiments, the support has a maximum of 10 9It may include one or more sample spots. In one embodiment, the sample spots are arranged on the support surface such that the distance between adjacent sample spots is about 1 nm to about 5000 nm, for example, about 2 nm to about 2000 nm. The distribution of sample spots on the support may be uniform or non-uniform, and may, for example, be clustered in groups.

[0063] In some embodiments, the sample spot comprises or consists of at least one conductive material, for example, a single metal or a combination of metals, for example, an alloy or mixture of several different metals. Suitable examples include a metal that can adhere to a sulfur-containing portion (e.g., in the form of a thiol or disulfide) or a metal that can adhere to a chelate portion (e.g., a polyhistidine tag). In some embodiments, the metal has a positive electrochemical potential. Specific examples of suitable metals include, but are not limited to, Au, Cu, Ni, Pt, Pd, Rh, Ir, Os, Ru, and any combination thereof, including at least two of the aforementioned metals.

[0064] In one embodiment, the sample spot comprises or consists of at least one metal oxide, including a single metal oxide or a combination of metal oxides. Suitable examples include metal oxides adhering to a phosphorus-containing moiety (e.g., in the form of a phosphonic acid or phosphonic acid ester) or metal oxides adhering to a chelate moiety (e.g., a polyhistidine tag). Specific examples of suitable metal oxides include TiO2 and NiO.

[0065] In further embodiments, the sample spot comprises or consists of at least one nonconductive material.

[0066] Sample spots can be prepared by vapor deposition of a vaporized metal on a support covered with a grid mask, which can be manufactured by electron beam lithography or an equivalent technique. The size of the holes in the grid mask may correspond to the size of the spots on the support surface. Alternatively, spots on a support can be prepared by site-specific deposition of nanoparticles, for example, having a size of 2–10 nm, by precision pipetting of particles on the support, particularly on a support having a plane.

[0067] The sample spot may have a size suitable for the attachment of a single biomolecule as defined herein. In certain embodiments, the sample spot has a diameter of about 1 nm to about 30 nm, particularly about 2 nm to about 20 nm.

[0068] In one embodiment, the sample spot is a separated object on the substrate surface. In one embodiment, the sample spot has a lower surface closer to the substrate and an upper surface further away from the substrate, and the distance between the lower and upper surfaces determines the height of the sample spot. In a particular embodiment, the height is about 50 pm to about 500 nm, especially about 100 pm to about 20 nm, and more specifically about 500 pm to about 10 nm, for example, about 2 nm.

[0069] To achieve the desired high yield, the sample spots are fitted to fix one single biomolecule per sample spot. This can be achieved by designing sample spots that can only bind one or fewer biomolecules per spot due to steric reasons (e.g., not enough space for more than exactly one molecule per spot).

[0070] In one embodiment, a sample spot having a predetermined size based on a specific biomolecule is provided thereon. The size of the sample spot, for example, its diameter, is adapted to the size of the specific biomolecule.

[0071] In certain embodiments, the sample spot has a size of about 5 nm to about 10 nm, particularly about 6 nm to about 8 nm, and more particularly about 7 nm, in order to immobilize one single biomolecule, such as a polypeptide and / or nucleic acid molecule, on one sample spot. In those embodiments, the biomolecule is a nucleic acid polymerase, particularly a DNA or RNA polymerase, and / or a nucleic acid molecule, particularly a DNA or RNA molecule.

[0072] In one embodiment, the sample spots have a low standard deviation around their average size, thereby providing a uniform size distribution profile. This can be achieved by modern nanofabrication techniques. In a particular embodiment, the standard deviation of the sample spot sizes around their average size is about 20% or less.

[0073] In one embodiment, the sample spots are adapted by their surface chemistry to immobilize one single biomolecule on one of the sample spots. In a particular embodiment, the sample spots have a coating that provides a limited number of binding sites for biomolecule immobilization, for example, a single binding site for a biomolecule on at least 50% of the sample spots.

[0074] In one embodiment, the surface of the sample spot includes at least one functional anchor molecule having a portion suitable for direct or indirect attachment of biomolecules. The portion suitable for attaching biomolecules may be selected from, for example, biotin or click functional groups. In a particular embodiment, the functional anchor molecule is attached to the surface of the sample spot by a sulfur-containing group, for example, a thiol group. The surface of the metal spot may include, for example, a metal as described herein, particularly an Au or Au / Pd surface.

[0075] In certain embodiments, the sample spot is adapted in size and surface chemistry for the immobilization of a specific single biomolecule. For example, nucleic acid polymerases, particularly DNA or RNA polymerases, can be immobilized on a sample spot having a size of about 6 nm to about 8 nm (more particularly about 7 nm) and a surface (e.g., a metallic surface such as Au and / or Pd) to which an anchor molecule having a sulfur-containing group (e.g., a thiol group) can be attached.

[0076] According to this disclosure, a support comprising a sample spot adapted for the immobilization of a single biomolecule is an incubated support that is incubated with the biomolecule, typically in a suitable liquid medium, under conditions that allow for the saturated immobilization of the biomolecule onto the sample spot.

[0077] Saturated immobilization means that incubation is carried out under saturated conditions with respect to biomolecules, i.e., the binding of biomolecules to each available sample spot on the support is favored.

[0078] In one embodiment, the conditions that enable saturated immobilization of biomolecules on a sample spot are the following parameters: (v) Concentration of biomolecules; (vi) Incubation time; (vii) Incubation temperature; (viii) Any combination of (i), (ii), and / or (iii) This includes adapting, in particular increasing, at least one of the following.

[0079] In one embodiment, incubation is carried out with a high concentration of biomolecules in a liquid culture medium. This provides a means to ensure the binding of a single biomolecule to each spot when the spots are suitable for single-molecule immobilization.

[0080] This represents a significant advantage of the present method compared to prior art methods, which maintained biomolecule concentrations at very strict intervals to optimize the number of sample spots to which single biomolecules were immobilized. In prior art methods, binding follows Poisson probability density, meaning that it is never possible to reach a situation where one biomolecule per sample spot binds to the majority of sample spots. In this innovation, Poisson statistics are avoided, and if binding is performed under saturated conditions, for example by using very high concentrations of biomolecules in the culture medium, up to 100% of spots can have only one biomolecule.

[0081] In one embodiment, conditions that enable saturated immobilization of the biomolecules onto the sample spot include carrying out the incubation step under conditions where the amount of biomolecules is greater than the amount of available sample spot, particularly when the amount of biomolecules is at least twice, at least five times, or at least ten times greater than the amount of available sample spot.

[0082] The sample spots on the support are adapted to the attachment of biomolecules so that a single biomolecule is immobilized on a single sample spot, for example, by covalent or non-covalent bonds. The biomolecules can be selected from polypeptides, nucleic acids, carbohydrates, and any combination thereof, e.g., glycosylated polypeptides or ribonucleoproteins. In some embodiments, the biomolecule is a complex consisting of several individual units, e.g., several polypeptide units, or several polypeptide and nucleic acid units.

[0083] In certain embodiments, the biomolecule is a nucleic acid polymerase, particularly a DNA polymerase or RNA polymerase, or a complex of nucleic acid polymerization molecules, particularly a DNA or RNA polymerization complex comprising a nucleic acid polymerase and a nucleic acid molecule. In certain embodiments, the biomolecule is a DNA polymerase having a DNA-binding cleft, particularly a family A DNA polymerase including, but not limited to, Klenow, Taq, or T7 DNA polymerase, or any genetically modified version thereof, or a family B polymerase including, but not limited to, therminator, Phi29, RB-69, or T4 DNA polymerase, or any genetically modified version thereof. In this context, reference is made to US 7,745,116 B2, the contents of which are incorporated herein by reference.

[0084] In further embodiments, the biomolecule is a nuclease, particularly an exonuclease, or a complex of nuclease-degrading molecules, particularly a complex of DNA or RNA-degrading molecules containing a nuclease and a nucleic acid molecule.

[0085] In another further embodiment, the biomolecule is a gene editing enzyme, in particular a Cas nuclease such as Cas3, Cas9, Cas10, or Cas12 nuclease, or any genetically modified version thereof, e.g., Cas nickase, or a gene editing complex comprising a gene editing enzyme and a nucleic acid molecule, e.g., guide RNA and / or target nucleic acid.

[0086] In one embodiment, the biomolecule includes at least one anchor, for example, a single anchor attached to a single sample spot.

[0087] In some embodiments, biomolecules adhere directly to a sample spot by providing a biomolecule having an anchor suitable for direct covalent or noncovalent bonding to, for example, the inorganic surface of the sample spot. In these embodiments, the anchor may include a reaction product between a thiol group-SH, a substituted thiol group-SR (wherein R is an organic residue, e.g., a C1-C4 alkyl group), a sulfur-containing group such as a disulfide group-SS-, or a phosphorus-containing group, and a metal or metal oxide surface on the sample spot. Alternatively, the anchor may include a reaction product between a silane group and a metal oxide, e.g., a silica surface, or a reaction product between a poly(histidine) tag and a Ni or NiO surface.

[0088] In some embodiments, biomolecules adhere indirectly to a sample spot, for example, by non-covalent bonding to a coating on the sample spot surface, such as an organic coating. In these embodiments, biomolecules can be provided that have attached amino acids such as cysteine, modified phenylalanine, histidine, or glutamine, or attached tags, such as biotin, hapten, poly(histidine) tags, or anchors containing carbohydrate groups, which can form links with complementary portions of the coating attached to the sample spot surface, such as streptavidin, antibodies, lectins, etc.

[0089] In one embodiment, biomolecules can be provided that have bio-orthogonal groups, i.e., groups not present in biomolecules, such as azide groups or alkyne groups, e.g., terminal or strained alkyne groups such as cyclooctyne. Such bio-orthogonal groups can form covalent bonds with complementary bio-orthogonal groups attached to the sample spot surface. In those embodiments, the anchor may include the reaction product of a coupling reaction between two biologically orthogonal reactive groups. In some embodiments, the coupling reaction is a click reaction, for example, a reaction between two click functional groups, such as an azide group and an alkyne group. In some embodiments, the anchor includes a triazole group.

[0090] The supports of this disclosure are suitable for analyzing events occurring on a sample spot, in which the events relate to the emission of electromagnetic radiation from the sample spot. In certain embodiments, the events encompass the reactions of biomolecules associated with the emission of characteristic electromagnetic radiation. In certain embodiments, the events are single-molecule events.

[0091] In certain embodiments, the supports of this disclosure are suitable for analyzing events, for example, single-molecule events occurring on at least one sample spot, and in particular for separately analyzing multiple single-molecule events each occurring on at least one sample spot, and more particularly for separately and in parallel analyzing multiple single-molecule events. In certain embodiments, the single-molecule events include nucleic acid sequencing.

[0092] Further aspects include, (i) To provide a support on which a single biomolecule is immobilized on a single sample spot in at least about 50% of the sample spot, and (ii) Analyzing events related to the single biomolecule by detecting electromagnetic radiation from individual sample spots. This relates to a method for analyzing single-molecule events that encompass the reactions of single biomolecules associated with the emission of characteristic electromagnetic radiation, including [specific example of a single biomolecule]. In certain embodiments, a single-molecule event includes sequence analysis of a single nucleic acid molecule.

[0093] Further aspects include, (i) To provide a support on which a short single-stranded nucleic acid molecule, particularly a DNA or RNA molecule, having a length preferably in the range of 3 to 300 nucleotides, is immobilized on a single sample spot. (ii) Adding a sample containing complementary nucleic acid molecules, particularly complementary DNA or RNA molecules, and (iii) Measuring all hybridization events. This relates to a method for analyzing single-molecule events that encompass the reactions of single biomolecules associated with the emission of characteristic electromagnetic radiation.

[0094] Further aspects include, (i) A support wherein a single biomolecule is immobilized on the single sample spot in at least about 50% of the sample spot, (ii) means for irradiating at least one sample spot on a support with radiation, (iii) Means for analyzing events related to the single biomolecule by detecting electromagnetic radiation from individual sample spots This invention relates to an apparatus for analyzing single-molecule events, including the reactions of single biomolecules associated with the emission of characteristic electromagnetic radiation. In certain embodiments, the device is suitable for sequence analysis of single nucleic acid molecules.

[0095] Methods and apparatus for analyzing single-molecule phenomena are disclosed, for example, in WO 2002 / 097406, WO 2003 / 052137, WO 2006 / 013110, WO 2013 / 131888, WO 2015 / 104245, WO 2017 / 001407, and WO 2018 / 104301, the contents of which are incorporated herein by reference.

[0096] For the analysis of single-molecule events, biomolecules are placed on sample spots on a support. There, they come into contact with a sample solution containing free reaction partners. This defines one or more reaction spaces, particularly those with at least 100, at least 1000, or at least 10000, and up to 106 More than one molecule can be analyzed on a single support, such as a single planar support.

[0097] The nucleic acid molecules whose sequences are determined may be selected from DNA molecules such as genomic DNA fragments, cDNA molecules, plasmids, etc., or from RNA molecules such as mRNA molecules. The nucleic acid molecules may be derived from a genome or expression library generated from a cell or organism, for example, a eukaryotic or prokaryotic cell or organism. This allows for multiple different nucleic acid template molecules, for example, at least 10, 100, 1,000, or 10,000, and up to 100,000, 10 6 pieces, or 10 7 This enables parallel sequencing of one or more different nucleic acid molecules.

[0098] The nucleic acid molecule to be sequenced may be linear or cyclic, for example, a covalently linked cyclic single-stranded nucleic acid molecule. To obtain a cyclic nucleic acid template, a linear nucleic acid molecule may be subjected to a cyclization procedure and optionally a strand separation procedure during sample preparation. Cyclization may be performed by ligation according to known protocols, for example, using DNA or RNA ligase. In some embodiments, an adapter and / or identifier molecule, i.e., a nucleic acid molecule with a known sequence, may be attached to the nucleic acid molecule.

[0099] Sequencing may involve nucleic acid elongation and / or nucleolysis. The sequencing process comprises one or more sequencing cycles.

[0100] Nucleic acid synthase molecules can extend primers annealed to nucleic acid template molecules. Primer extension can be achieved by stepwise incorporating individual nucleotide building blocks into the 3'-end of the growing nucleic acid chain, thereby generating nucleic acid molecules complementary to the sequence of the cyclic nucleic acid template. Nucleic acid synthases are selected from polymerases capable of template-specific nucleic acid polymerization, preferably DNA polymerases and RNA polymerases, including, for example, heat-stable DNA polymerases, as well as natural or modified polymerases.

[0101] Specific examples of suitable DNA polymerases include Taq polymerase, exonuclease-deficient Taq polymerase, E. coli DNA polymerase I, Klenow fragments, reverse transcriptase, Φ29-related polymerases including wild-type Φ29 polymerase, and derivatives of such polymerases such as exonuclease-deficient types, T7 DNA polymerase, T5 DNA polymerase, RB69 polymerase, and others.

[0102] Nuclease molecules can stepwise cleave individual nucleotide building blocks from nucleic acid molecules. Preferably, exonucleases, more preferably single-stranded exonucleases that degrade in the 3'→5' or 5'→3' direction, are used. Particularly preferred exonucleases include 3'→5' exonucleases such as E. coli exonuclease I and E. coli exonuclease III, as well as 5'→3' exonucleases such as T7 exonuclease, E. coli exonuclease II and E. coli exonuclease VIII. Furthermore, the exonuclease activity of various polymerases, such as Klenow fragments, Taq polymerase, or T4 polymerase, may be used.

[0103] The nucleic acid synthase molecule contacts a linear or cyclic nucleic acid template molecule, such as a single-stranded DNA or RNA molecule, and a primer molecule that has annealed to or is annealable to the nucleic acid template molecule. The primer molecule is preferably a single-stranded nucleic acid or nucleic acid analog molecule having a free 3'-end that can be extended by an enzymatic reaction catalyzed by the immobilized nucleic acid synthase molecule. The length of the primer molecule is selected so that it can effectively anneal to the template under the reaction conditions. Typically, the length of the primer molecule is at least 8, at least 10, at least 12, or at least 15 nucleotides, and for example, up to 20, 25, 50, or 100 nucleotides or more. In some embodiments, the primer is resistant to digestion by the nucleic acidase molecule and is stable against degradation, for example, by incorporating nucleotide analog building blocks and / or linkages between nucleotide building blocks. In other embodiments, the primer is sensitive to digestion by the nucleic acidase molecule.

[0104] The primer sequence is selected to effectively anneal to the template molecule under reaction conditions. For example, the primer may be a universal degenerated primer that can statistically anneal to an unknown nucleic acid sequence. In other embodiments, the primer can anneal to a known sequence portion of the nucleic acid template molecule. In this embodiment, a known adapter and / or identifier sequence may be incorporated into the nucleic acid template molecule. The primer may be unlabeled or may contain a fluorescent labeling group.

[0105] Furthermore, the presence of nucleotide building blocks having at least one fluorescent labeling group is required. Preferably, each different nucleotide building block (A, G, C, T / U) contains a different fluorescent labeling group.

[0106] The fluorescent labeling group can be selected from known fluorescent labeling groups used to label biomolecules, particularly nucleic acids, such as fluorescein dyes, rhodamine, oxazine, e.g., Evoblue or Gnothis Blue, phycoerythrin, Cy3, Cy5, IR dyes, or derivatives thereof.

[0107] A nucleotide building block may have (i) a fluorescent labeling group that remains with the building block when it is incorporated into the nucleic acid molecule during primer extension catalyzed by a nucleic acid synthase molecule, and / or (ii) a fluorescent labeling group that is cleaved from the building block when it is incorporated into the nucleic acid molecule during primer extension catalyzed by a nucleic acid synthase molecule. The fluorescent labeling group that remains with the building block is preferably attached to an α-phosphate group, a sugar, and / or a nucleic acid base group.

[0108] In certain embodiments, the fluorescently labeled groups remaining with the building block may have a chain length of, for example, up to 15 carbon atoms, preferably 10 to 12, and may be attached to the nucleic acid base via a linker that optionally contains heteroatoms, such as N, O, or S atoms. The fluorescently labeled groups that are cleaved when the building block is incorporated into the nucleic acid molecule may be attached to the terminal phosphate groups of polyphosphate building blocks, including, but not limited to, hexa-, penta-, tetra-, or triphosphate building blocks, such as the γ-phosphate group of a triphosphate building block. In some embodiments, a building block is selected that contains both (i) fluorescently labeled groups remaining after incorporation and (ii) fluorescently labeled groups that are cleaved during incorporation. In this case, fluorescent groups that can interact with each other by, for example, quenching and / or energy transfer may be selected.

[0109] When nucleic acid molecules are attached to direct sequencing using nuclease molecules, the nucleic acid molecules to be sequenced contain a fluorescent labeling group. On the other hand, when the nucleic acid molecule is used as a template in primer extension, the nucleic acid molecules to be sequenced do not need to contain a fluorescent labeling group.

[0110] The sequencing procedure may include a step of generating a nucleic acid molecule having incorporated nucleotide building blocks in primer extension catalyzed by a nucleic acid synthase molecule, and / or a second step of cleaving individual nucleotide building blocks from the generated nucleic acid molecule, catalyzed by a nuclease molecule. Depending on the type of fluorescent labeling, nucleic acid sequencing may be performed during primer extension and / or degradation.

[0111] Sequencing during primer extension involves the use of nucleotide building blocks having a fluorescent labeling group, which is cleaved from the building block when incorporated into the nucleic acid molecule. In this case, the time-dependent fluorescence change resulting from the cleavage of the fluorescent labeling group from the nucleotide building block can be measured. Sequencing during nucleolysis also involves the use of nucleotide building blocks having a fluorescent labeling group, which remains with the building block when incorporated into the nucleic acid molecule. The gradual cleavage of individual nucleotide building blocks from the nucleic acid molecule causes a time-dependent fluorescence change when the labeled nucleotide building blocks are released from the nucleic acid molecule. In some embodiments, sequencing can also be performed using nucleotide building blocks that have both a fluorescent labeling group that remains with the building block and a fluorescent labeling group that is cleaved from the building block when the building block is incorporated into the nucleic acid molecule, i.e., during extension and degradation. In this embodiment, both fluorescent groups may be the same or different.

[0112] In some embodiments, the method comprises one or more cycles of nucleic acid synthesis and nucleic acid degradation to determine the base sequence of a nucleic acid molecular template. Nucleic acid synthesis involves extending an annealed primer onto a nucleic acid template molecule catalyzed by a nucleic acid synthase molecule, in which a nucleic acid molecule complementary to the sequence of the nucleic acid template is produced. In the next step, the produced nucleic acid molecule is degraded by a nucleic acid degradation enzyme molecule.

[0113] When nucleotide building blocks are incorporated into elongated nucleic acid molecules, a time-dependent change in fluorescence may occur, which can be detected as described above. Preferably, the incorporation of nucleotide building blocks into elongated nucleic acid molecules is associated with a detectable increase in fluorescence, preferably a transient increase in fluorescence. For example, nucleotide building blocks can be used that have a fluorescent labeling group on the portion of the molecule that is cleaved when the building block is incorporated into the primer, for example, on the γ-phosphate group.

[0114] When nucleotide building blocks are cleaved from a synthesized nucleic acid molecule, the time-dependent change in fluorescence may be determined by the interaction between the fluorescently labeled group incorporated into the nucleic acid chain and adjacent groups, such as the chemical groups of the nucleic acid, particularly nucleic acid bases such as G, and / or adjacent fluorescently labeled groups. These interactions result in changes in fluorescence, especially fluorescence intensity, compared to "isolated" forms of fluorescently labeled groups, through quenching processes and / or energy transfer processes. The removal of individual nucleotide building blocks by cleavage alters the overall fluorescence, for example, the fluorescence intensity of the immobilized nucleic acid chain, and this change is a function of the removal of individual nucleotide building blocks by cleavage, i.e., a function of time.

[0115] In one embodiment, the association of labeled nucleotides with biomolecular complexes is detected by measuring the polarization of emitted photons. The polarization of excited-state photons changes due to the rotational motion of the luminescent nucleotide label and can be used to identify free-moving contra-bound labeled nucleotides in the polymerization process.

[0116] The time-dependent changes in fluorescence during this extension and / or degradation can be recorded in parallel for multiple nucleic acid molecules and correlated with the base sequences of individual nucleic acid chains. It is preferable to use a fluorescent labeling group that, when incorporated into the nucleic acid chain, is at least partially quenched, and so on, increases in fluorescence intensity after the nucleotide building block containing the labeling group or adjacent building blocks causing quenching are removed by cleavage.

[0117] During the incorporation and / or removal of individual nucleotide building blocks, changes in the fluorescence intensity of the nucleic acid chain and / or the incorporated or cleaved nucleotide building blocks can be measured by quenching or energy transfer processes. This time-dependent change in fluorescence intensity depends on the base sequence of the nucleic acid chain studied and can therefore be correlated with the sequence.

[0118] The complete sequence of a nucleic acid molecule can be determined by using a mixture of nucleotide building blocks labeled with all four distinct bases, e.g., A, G, C, and T, or combinations of two or three distinct bases. Where appropriate, a "sequence identifier," i.e., a nucleic acid labeled with a known sequence, can also be attached to the nucleic acid strand under study by enzymatic reaction, for example, using a ligase and / or terminal transferase, so that a known fluorescence pattern is obtained first at the start of sequencing, and only thereafter is a fluorescence pattern corresponding to the unknown sequence under study obtained.

[0119] Detection preferably involves irradiating a support with light by a laser or another suitable light source to induce excitation of a fluorescently labeled group. In this regard, one or more laser beams, e.g., a widened laser beam having a cross-section of about 1 to 20 mm, and / or multiple laser beams can be used. Detection preferably involves multipoint fluorescence excitation by laser, e.g., a dot matrix of laser dots generated via diffractive optics (see WO 2002 / 097406) or a quantum well laser.

[0120] The fluorescence emission of multiple nucleic acid strands can be detected in parallel using a detector matrix, for example, an electron detector matrix, such as a CCD camera; a CMOS detector matrix, such as a CMOS camera; or a detector matrix including an avalanche photodiode matrix. Detection can be performed so that fluorescence excitation and detection are carried out in parallel for some or all of the nucleic acid strands being studied. It is preferable to perform detection on fluorescence emitted essentially orthogonally from the support surface, through the reaction space, or through the support body.

[0121] Detection can be performed, for example, by single-molecule detection, such as fluorescence correlation spectroscopy, which involves a very small, preferably confocal, volume element (e.g., 10 -21 ~10 -10l) comprises exposing the sample to excitation light from a laser or another suitable light source, the light of which excites receptors present in the sample volume, the latter emitting fluorescence, the fluorescence emitted from the sample volume being measured by a photodetector, the time evolution of the measured emission correlating with the concentration of the sample, and thus individual molecules in the sample volume can be identified at a suitable high dilution. Details of the procedure and the apparatus used for detection can be found in the disclosure of EP 0 679 251, the contents of which are incorporated herein by reference. Confocal determination of single molecules is further described in Rigler and Mets (Soc. Photo-Opt. Instrum. Eng. 1921 (1993), 239 ff.), and Mets and Rigler (J. Fluoresc. 4 (1994) 259-264), the contents of which are incorporated herein by reference.

[0122] Alternatively, or additionally, detection can also be performed by a time-resolved decay measurement called "time gating," as described, for example, in Rigler et al., "Picosecond Single Photon Fluorescence Spectroscopy of Nucleic Acids" (the contents of which are incorporated herein by reference) in "Ultrafast Phenomena," DH Auston, Ed., Springer 1984. Here, the fluorescent molecule is excited in the measurement volume, and then a detection interval is opened on the photodetector, for example, at time intervals of ≥100 ps. In this way, the background signal generated by the Raman effect can be kept sufficiently low, and a single molecule can be detected in an essentially interference-free manner.

[0123] The methods and apparatus disclosed herein are also suitable for further single-molecule events, namely, the analysis of single biomolecules bound to a select spot for analysis, where there is a high demand for single-molecule analysis in high yield.

[0124] In one embodiment, this disclosure relates to single-molecule analysis of receptor-ligand interactions, for example, encompassing the binding of a receptor protein to a sample spot and subsequently studying its interaction with its ligand, for example, within pharmaceutical development.

[0125] In further embodiments, the disclosure relates to single-molecule analysis of hybridization events, for example, encompassing the attachment and / or immobilization of short single-stranded nucleic acid molecules, e.g., having a length in the range of 3 to 300 nucleotides, e.g., DNA or RNA molecules, followed by the addition of a sample containing complementary nucleic acid molecules and observation of any hybridization events. Application areas may include, for example, the detection of viral RNA / DNA, bacterial DNA / RNA, and the detection of short segments of DNA from cancer cells in the bloodstream.

[0126] Furthermore, this disclosure will be described in detail with reference to the following specific embodiments. Figure 1 shows an embodiment of the prior art. A biomolecule (3) is bound to a sample spot (2) with a diameter of approximately 100 nm on a support (1). The molecules bind probabilistically, and as a result, the surface density of bound molecules follows a Poisson distribution. According to this Poisson distribution, spectra exist for sample spots with no bound biomolecules, sample spots with a single bound biomolecule, and sample spots with two (or more) bound biomolecules. (The parameters of the Poisson distribution depend on several parameters, one of which is the concentration of biomolecules added in the pre-binding step).

[0127] Figures 2A, 2B, and 2C illustrate embodiments of the present disclosure. A (top view) and B (side view): Sample spots (4) on a support (1) having a single diameter of 1–30 nm, particularly 5–10 nm, and more particularly 6–8 nm, have a single biomolecule (3) attached thereto. Due to steric considerations regarding the size and / or surface chemistry of individual spots, only one biomolecule (3) can be bound thereto. By performing the biomolecule immobilization procedure under saturated conditions, a high and (almost) quantitative yield of spots bound to a single biomolecule can be obtained.

[0128] C: A biomolecule (4) having a fluorescent group, for example, an externally labeled group attached to the biomolecule, is depicted. The fluorescent group may have any emission wavelength distribution and a corresponding excitation wavelength distribution. In one embodiment, the fluorescent group is visible for a limited period of time, and in another embodiment, it is visible for a long period of time. In another embodiment, the biomolecule does not require an attached externally labeled group for detection by spectroscopic means, such as Raman spectroscopy.

Claims

1. (a) A support comprising a substrate and a plurality of sample spots on the surface of the support, wherein the sample spots are spatially separated from each other and the size of the sample spots is adapted to the size of a particular biomolecule to be fixed thereon, thereby providing a support to which the sample spots are adapted for fixing one single biomolecule onto one sample spot, and (b) Incubating the sample spot on the support with the biomolecules under conditions that allow for saturation immobilization of the biomolecules onto the sample spot, such that the amount of biomolecules is greater than the amount of available sample spot. A method for preparing a support for analyzing single-molecule events that encompass the reactions of single biomolecules associated with the emission of characteristic electromagnetic radiation, including [specific example of a single biomolecule].

2. The method of claim 1, wherein a single biomolecule is immobilized on the single sample spot on the support in an area of ​​at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%, and up to 99.99% of the sample spot.

3. The support consists of at least 10, at least 100, at least 1,000, and at least 10 ４ at least 10 ５ at least 10 ６ at least 10 ７ one, or at least 10 ８ The method according to any one of the claims, comprising a sample spot.

4. The method according to any one of the claims, wherein the sample spot has a size of about 5 nm to about 10 nm, more particularly about 6 nm to about 8 nm, and more particularly about 7 nm, for immobilizing one single biomolecule.

5. The method according to any one of the claims, wherein the biomolecule is a nucleic acid polymerase, in particular a DNA or RNA polymerase, and / or a nucleic acid molecule, in particular a DNA or RNA molecule.

6. The method according to any one of the claims, wherein the sample spots are adapted in their surface chemistry to immobilize a single biomolecule on one of the sample spots.

7. The method of claim 6, wherein the surface of the sample spot includes at least one functional anchor molecule having a portion suitable for direct or indirect attachment of biomolecules.

8. The method according to claim 7, wherein the functional anchor molecule is attached to the surface of the sample spot by a sulfur-containing group, for example, a thiol group.

9. The method according to claim 7 or 8, wherein the sample spot includes a metal surface, particularly an Au or Au / Pd surface.

10. The conditions that enable saturated immobilization of the biomolecule onto the sample spot are the following parameters: (i) Concentration of biomolecules; (ii) Incubation time; (iii) Incubation temperature; (iv)(i), (ii), and / or any combination of (iii) A method according to any one of the claims, comprising adapting at least one of the following.

11. A support comprising a substrate and a plurality of sample spots on the surface of the support, wherein the sample spots are spatially separated from each other, and biomolecules are immobilized on the sample spots, with single biomolecules immobilized on at least about 50% of the sample spots, and the size of the sample spots is suited to the size of a particular immobilized biomolecule.

12. Use of the support of claim 11 for the analysis of single-molecule events encompassing the reaction of a single biomolecule associated with the emission of characteristic electromagnetic radiation, particularly for the sequence analysis of a single nucleic acid molecule.

13. (i) To provide the support of claim 11, wherein a single biomolecule is immobilized on a single sample spot in at least about 50% of the sample spots, and (ii) Analyzing events related to the single biomolecule by detecting electromagnetic radiation from individual sample spots. A method for analyzing single-molecule events that include the reactions of single biomolecules associated with the emission of characteristic electromagnetic radiation.

14. (i) To provide a support of claim 11, wherein a short single-stranded nucleic acid molecule, particularly a DNA or RNA molecule, having a length preferably in the range of 3 to 300 nucleotides, is immobilized on a single sample spot. (ii) Adding a sample containing complementary nucleic acid molecules, particularly complementary DNA or RNA molecules, and (iii) Measuring all hybridization events. The method according to claim 13, including the method described in claim 13.

15. (i) The support of claim 11, wherein a single biomolecule is immobilized on the single sample spot in at least about 50% of the sample spot, (ii) means for irradiating at least one sample spot on a support with radiation, and (iii) Means for analyzing events related to the single biomolecule by detecting electromagnetic radiation from individual sample spots A device for analyzing single-molecule events, including the reactions of single biomolecules associated with the emission of characteristic electromagnetic radiation.

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