Multivalent binding unit modified dideoxynucleotide triphosphates and methods of making and using same

Abstract

The compounds of the present disclosure include a modified dideoxynucleotide triphosphate (ddNPT) comprising a plurality of binding units comprising arms to provide a multivalent binding unit molecule.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 471,425, filed June 6, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates to dideoxynucleotide triphosphates (ddNTPs) modified with multivalent binding units, and methods for their preparation and use. Background Technology

[0003] Single nucleotide polymorphisms (SNPs) are a common form of polymorphism in the human genome. In genotyping, allele specificity is conferred by a single-base extension incorporating one or four labeled nucleotides. When excited by a laser, the nucleotide label emits a signal. The intensity of this signal conveys information about the allele ratio at that locus. Summary of the Invention

[0004] Signal detection defects have been observed in red channel (ddATP-DNP and ddUTP-DNP) detection. Lower signals in the red channel have been observed after three rounds of staining compared to green channel detection. While not intending to be bound by theory, it is believed that some of the differences in signal intensity between the red and green channels may be due to higher nonspecific anti-DNP antibody-dye conjugate binding. This was observed through higher red signals in homozygous green SNPs.

[0005] The compounds and methods disclosed herein utilize ddNTPs modified with multivalent binding units to enhance the binding affinity of the binding unit-binding partner, thereby improving signal detection. This can advantageously lead to a reduction in nonspecific binding mediated by both the surface of the binding unit-binding partner and the dye covalently linked to it, for fluorescence detection. This can also advantageously reduce the amount of binding unit-binding partner required in current reagent formulations, which in turn minimizes nonspecific binding and improves COGS. Finally, the compounds and methods disclosed herein can advantageously reduce the number of staining rounds required for signal amplification. For example, the binding unit molecule can be a hapten, and the binding unit-binding partner can be a hapten-binding partner.

[0006] According to this disclosure, the ddNTP modified with a multivalent binding unit can be a compound of formula I: ARM a -Q b -M c -J-ddNTP, Where a is at least 2, b is 1 to 3, c is 0 or 1, each ARM contains a bonding unit and a PEO-containing connector, and each Q is independently selected from: , or And each Q is connected to 2 or 3 ARMs, and each M is Each L1 and L2 is independently selected For each L1 and L2, d is independently selected from 1 to 10, and e is independently selected from 1 to 10, R is independently selected from the functional groups generated by the coupling reaction between a pair of the following: amine-imino ester, amine-pentafluorophenyl ester, amine-N-hydroxysuccinimide ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-acylhydrazine, aldehyde-alkoxyamine, hydroxy-isocyanate, azido-phosphine, trans-cyclooctene-tetraazine, norbornene-tetraazine, azido-cyclooctynylene, azido-norbornene, or azido-alkyne; J is , where x is from 1 to 48.

[0007] According to this disclosure, each ARM may contain For each ARM, z is independently selected from 1 to 48, and y is independently selected from 1 to 48, and T is independently selected from the functional groups generated by the coupling reaction between a pair of the following: amine-imino ester, amine-pentafluorophenyl ester, amine-N-hydroxysuccinimide ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azido-phosphine, trans-cyclooctene-tetraazine, norbornene-tetraazine, azido-cyclooctyne, azido-norbornene, or azido-alkyne.

[0008] According to this disclosure, a kit may include a bead array containing a plurality of beads, each bead having at least one capture oligonucleotide probe attached to its surface; an anti-DNP antibody, each of which comprises a first fluorescent label; at least one compound of formula I; and instructions for use such that, upon contact, one or more anti-DNP antibodies bind to each ARM of the at least one compound of formula I.

[0009] According to this disclosure, a method for genotyping may include: contacting a bead array with sample DNA, each bead in the bead array containing one or more capture oligonucleotide probes, each of the one or more capture oligonucleotide probes binding to a target complementary sequence in the sample, wherein the target complementary sequence binds to the one or more capture oligonucleotide probes to stop at least one base before the locus of interest, thereby providing a bead array having a captured target complementary sequence; mixing the bead array having the captured target complementary sequence with a composition containing at least a first hapten-modified ddNTP and a second hapten-modified ddNTP, under conditions sufficient to hybridize a first hapten-modified ddNTP with a corresponding captured target complementary sequence in the captured target complementary sequence as a single-base extension of the associated capture oligonucleotide probe, wherein the first hapten and the second hapten-modified ddNTP... The two haptens are different, and the ddNTP modified by the first hapten is a compound of formula I; under the condition that the first hapten binding partner is bound to the first hapten and the second hapten binding partner is bound to the second hapten, the bead array containing the hybridized first hapten-modified ddNTP and the second hapten-modified ddNTP is contacted with a labeled composition containing the first hapten binding partner and the second hapten binding partner, wherein the first hapten binding partner contains a first fluorescent label and the second hapten binding partner contains a second fluorescent label, and the bead array is fluorescently labeled when the first hapten and / or the second hapten is bound to the corresponding first hapten binding partner and / or second hapten binding partner; and after the first hapten binding partner and the second hapten binding partner are bound, the fluorescence signal of the bead array is analyzed. Attached Figure Description

[0010] Figure 1 This is a schematic diagram of a method for preparing multivalent hapten-modified ddNTPs according to this disclosure; Figure 2 This is a schematic diagram of a method for preparing multivalent hapten-modified ddNTPs according to this disclosure; Figure 3 This is a graph showing the fluorescence analysis results of using multivalent hapten-modified ddNTPs, commercially available monovalent DNP-ddNTPs, and laboratory-produced monovalent DNP-ddNTPs.

[0011] Figure 4 This is a graph showing the results of fluorescence analysis comparing the use of multivalent hapten-modified ddNTPs with commercially available monovalent DNP-ddNTPs and laboratory-produced monovalent DNP-ddNTPs after multiple rounds of staining.

[0012] Figure 5 This is a graph showing the fluorescence analysis in the green channel.

[0013] Figure 6 This is a schematic diagram based on an example measurement of this disclosure.

[0014] Figure 7A The graph shows the fluorescence analysis in the red channel, which compares the extension efficiency of ddNTPs modified with multivalent hapten with commercially available monovalent DNP-ddNTPs and laboratory-produced monovalent DNP-ddNTPs.

[0015] Figure 7B This is a graph showing the fluorescence analysis in the green channel, which compares the extension efficiency of ddNTPs modified with multivalent hapten with commercially available monovalent DNP-ddNTPs and laboratory-produced monovalent DNP-ddNTPs.

[0016] Figure 8 The graph shows the results in both the extended control group and the non-extended control group, comparing the extension efficiency of ddNTPs modified with multivalent hapten with commercially available monovalent DNP-ddNTPs and laboratory-produced monovalent DNP-ddNTPs.

[0017] Figure 9A This is a graph showing the fluorescence analysis results of two rounds of staining using increased hapten-modified ddNTP concentrations.

[0018] Figure 9B This is a graph showing the fluorescence analysis results of three rounds of staining using increased hapten-modified ddNTP concentrations.

[0019] Figure 10 This is a graph showing the signal-to-noise ratio of the compound according to this disclosure on the Infinium EX microarray platform compared to the standard ddNTP design.

[0020] Figure 11 This is a graph showing the detection rate and logR deviation measure of the compound according to this disclosure compared to the standard design ddNTP on the Infinium EX microarray platform. Detailed Implementation

[0021] The compounds disclosed herein are dideoxynucleotide triphosphates (ddNTPs) modified with multivalent binding units. These compounds can be used in a variety of applications, including genotyping and multi-omics applications. The ddNTPs modified with binding units according to this disclosure comprise multiple binding units that bind to or are linked to the ddNTP, and their enhanced binding affinity to the fluorescently labeled binding units for the mating body has been observed. The ddNTPs modified with binding units according to this disclosure can reduce nonspecific binding. This can advantageously allow for the use of reduced amounts of binding units to the mating body and / or reduce the number of staining rounds required for signal amplification.

[0022] The ddNTPs with combined unit modification can be compounds of formula I: ARM a -Q b -M c -J-ddNTP, Where a is at least 2, b is 1 to 3, c is 0 or 1, each ARM contains a bonding unit and a PEO-containing connector, and each Q is independently selected from: , or And each Q is connected to 2 or 3 ARMs, and each M is Each L1 and L2 is independently selected For each L1 and L2, d is independently selected from 1 to 10, and e is independently selected from 1 to 10, R is independently selected from the functional groups generated by the coupling reaction between a pair of the following: amine-imino ester, amine-pentafluorophenyl ester, amine-N-hydroxysuccinimide ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-acylhydrazine, aldehyde-alkoxyamine, hydroxy-isocyanate, azido-phosphine, trans-cyclooctene-tetraazine, norbornene-tetraazine, azido-cyclooctynylene, azido-norbornene, or azido-alkyne; J is , where x is from 1 to 48.

[0023] In compounds of formula I, a can be at least 2 and at most 9. For example, a can be 2, 3, 4, 5, 6, 7, 8, or 9.

[0024] For example, the bonding units in each ARM can be the same. Alternatively, the bonding units in two or more ARMs can be different.

[0025] The binding unit can be any one or more of antibodies, proteins, oligonucleotides, DNA, RNA, drugs, small molecules, dyes, peptides, etc.

[0026] Hapten-modified multivalent DDNTPs In all respects, the compounds disclosed herein are hapten-modified dideoxynucleotide triphosphates (ddNTPs) that can be used in genotyping applications to improve signal detection. The hapten-modified ddNTPs of this disclosure comprise multivalent hapten molecules, which have been observed to enhance the binding affinity of fluorescently labeled hapten-binding couplers. The hapten-modified ddNTPs of this disclosure reduce nonspecific binding. This can advantageously allow for the use of reduced amounts of hapten-binding couplers and / or a reduction in the number of staining rounds required for signal amplification.

[0027] Hapten-modified ddNTPs can be compounds of formula IA: ARM a -Q b -M c -J-ddNTP, Where a is at least 2, b is 1 to 3, c is 0 or 1, each ARM contains a hapten and a PEO-containing linker, and each Q is independently selected from: , or And each Q is connected to 2 or 3 ARMs, and each M is Each L1 and L2 is independently selected For each L1 and L2, d is independently selected from 1 to 10, and e is independently selected from 1 to 10, and R is independently selected from the functional groups generated by the coupling reaction between a pair of the following: amine-imino ester, amine-pentafluorophenyl ester, amine- N- Hydroxysuccinimide ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-acylhydrazine, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-phosphine, trans-cyclooctene-tetraazine, norbornene-tetraazine, azide-cyclooctene, azide-norbornene, or azide-alkyne; J is , where x is from 1 to 48.

[0028] In compounds of formula IA, a can be at least 2 and at most 9. For example, a can be 2, 3, 4, 5, 6, 7, 8, or 9.

[0029] For example, the hapten in each ARM can be the same. Alternatively, the hapten in two or more ARMs can be different.

[0030] The hapten in each ARM can be independently selected from, for example, digoxigenin (DIG), 3-nitrotyrosine, biotin, alkyl chloride, O-GlcNAc, alkylguanine, Ni-NTA, and 2,4-dinitrophenol (DNP). The following shows the binding partners of various haptens contained in an ARM that can be part of a compound of formula (I).

[0031]

[0032] In compounds of formula I or formula IA, for example, each ARM may contain For example, each ARM may contain In each of these ARMs, z can be independently selected from 1 to 48, 1 to 12, 1 to 10, 13 to 24, 35 to 36, or 37 to 48, and any value therein and the range defined by these values. In each ARM, y can be independently selected from 1 to 48, 1 to 12, 1 to 10, 13 to 24, 35 to 36, or 37 to 48.

[0033] In each ARM, T is independently selected from the functional group generated by the coupling reaction between one of the following compound pairs: amine-imino ester, amine-pentafluorophenyl ester, amine-N-hydroxysuccinimide ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azido-phosphine, trans-cyclooctene-tetraazine, norbornene-tetraazine, azido-cyclooctyne, azido-norbornene, or azido-alkyne.

[0034] For example, T could be or For example, in each ARM, T can be... For example, in each ARM, T can be... For example, T can be in at least one arm. And in at least one other arm it can be .

[0035] In each ARM, z and y can be the same, and the compound can be a symmetrical compound. Alternatively, in two or more ARMs, y and z can be different, and the compound can be asymmetrical.

[0036] In compounds of formula I or formula IA, b can be 1, 2, or 3.

[0037] In compounds of formula I or IA, c can be 0 or 1. For example, when c is 0, Q can be... When c is 0, this paper also considers other options for Q.

[0038] When it exists, M is Each L1 and L2 is independently selected. For each L1 and L2, d is independently selected from 1 to 48, 1 to 12, 1 to 10, 13 to 24, 35 to 36 or 37 to 48, and e is independently selected from 1 to 48, 1 to 12, 1 to 10, 13 to 24, 35 to 36 or 37 to 48, and R is independently selected from the functional groups generated by the coupling reaction between a pair of the following: amine-imino ester, amine-pentafluorophenyl ester, amine-N-hydroxysuccinimide ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-acylhydrazine, aldehyde-alkoxyamine, hydroxy-isocyanate, azido-phosphine, trans-cyclooctene-tetraazine, norbornene-tetraazine, azido-cyclooctyne, azido-norbornene or azido-alkyne.

[0039] In compounds of formula I or formula IA, J is , where x is 1 to 48, 1 to 12, 1 to 10, 13 to 24, 35 to 36, or 37 to 48.

[0040] In compounds of formula I or IA, the ddNTP can be any of ddATP, ddTTP, ddUTP, ddGTP, or ddCTP.

[0041] In various cases, compounds of formula I or formula IA have the structures shown in Table A.

[0042]

[0043]

[0044]

[0045]

[0046] Reagent test kit The kit may include, for example, a bead array comprising a plurality of beads, each bead having at least one capture oligonucleotide probe attached to its surface; a binding unit binding partner comprising a first label; at least one compound of formula I and / or formula IA; and instructions for use. The binding unit binding partner containing the first label selectively binds binding units present on the ARM of the compound of formula I and / or formula IA. For example, in the case where the hapten is DNP, the hapten binding partner may be an anti-DNP antibody comprising a first fluorescent label. When the anti-DNP antibody is contacted with a compound of formula I, the anti-DNP antibody hybridizes with DNP, thereby labeling the beads with the first label. The first label may be, for example, a fluorescent label. The labeling or interaction between binding units and binding unit binding pairs can produce a detectable signal, such as fluorescence, bioluminescence, or electrodetection. The kit may be used to screen and / or identify binding pairs between binding units and binding unit binding partners. For example, the kit may include a plurality of binding unit binding partners and / or a plurality of binding units to screen binding interactions by using the kit.

[0047] A kit for genotyping may include, for example, a bead array comprising a plurality of beads, each bead having at least one capture oligonucleotide probe attached to its surface; a hapten-binding partner comprising a first fluorescent label; at least one compound of formula I; and instructions for use. The hapten-binding partner comprising the first fluorescent label selectively binds to the hapten present on the ARM of the compound of formula I. For example, in the case where the hapten is DNP, the hapten-binding partner may be an anti-DNP antibody comprising the first fluorescent label. When the anti-DNP antibody is contacted with a compound of formula I, the anti-DNP antibody hybridizes with the DNP, thereby labeling the beads with the first fluorescent label.

[0048] The binding unit binding partner can be independently selected from antibodies, proteins, oligonucleotides, DNA, RNA, drugs, small molecules, dyes, and peptides. For example, the hapten binding partner can be independently selected from anti-DNP antibodies, avidin, anti-DIG antibodies, WGA, SNAP tags, anti-nitrotyrosine antibodies, and His-tags. Hapten / hapten binding partner pairs have been identified above, and suitable binding unit binding partners can be selected based on the binding unit present on the compound of Formula I. The binding unit on the compound of Formula I can be, for example, a small molecule, peptide, or antibody, while the binding unit binding partner can be a protein, oligonucleotide, RNA, or DNA. Alternatively, the binding unit can be a protein, and the binding unit binding partner can be a small molecule, peptide, or antibody to be screened for binding to a protein, oligonucleotide, RNA, or DNA. The binding unit binding partner may include a label, such as a fluorescent label, a bioluminescent label, or a charge (for electrodetection), to allow indication of the binding pair that occurs. The compounds and methods disclosed herein can be used, for example, to screen interactions that can be used for, for example, drug development.

[0049] The compounds and methods disclosed herein can be used for a variety of applications. For example, the compounds and methods disclosed herein can be used in genotyping applications, binding pair screening and / or identification, and multi-omics. For example, the compounds disclosed herein can be used for multi-omics counting, wherein the binding set provided by the compounds and binding pair of the disclosed presents allows for the detection of a variety of analytes, such as DNA, RNA, and protein binding sites.

[0050] The kit may include a variety of different compounds of formula I and / or formula IA, for example, with different ddNTP moieties. For example, a compound of formula I may include ddATP modified with a binding unit and ddUTP modified with a binding unit, such that when both A and T are present on the bead, a single fluorescent signal from a first fluorescent label will be generated. Single detection of A and T as a single channel is commonly used in various genotyping procedures. Any known bead array technology can be used with the kit disclosed herein. For example, the beads may be an Infinium bead array (Illumina).

[0051] For example, the kit may include two groups of compounds of Formula I and / or Formula IA, each group having different binding units for binding different fluorescent labels to the binding partner. For example, the first group of Formula I compounds may include DNP as a hapten present on the arm and may provide an anti-DNP antibody with a first fluorescent label. This first group of compounds may include compounds in two subgroups, one subgroup including ddATP as a ddNTP and the other subgroup including ddUTP as a ddNTP. The first group of Formula I compounds will result in the detection of fluorescently labeled A and T with the first fluorescent label. The kit may also include compounds in a second group of Formula I, for example, where the hapten present on the arm is biotin, and the hapten binding partner is an avidin protein with a second fluorescent label different from the first fluorescent label. The second group of Formula I compounds may include two subgroups, one subgroup in which the ddNTP is ddGTP and the other subgroup in which the ddNTP is ddCTP. The second group of Formula I compounds will result in the detection of fluorescently labeled G and C with the second fluorescent label. This can produce two detection channels, for example, a red channel (A and T) and a green channel (G and C).

[0052] This document also envisions that the kits disclosed herein may include other platforms (such as non-bead platforms) in which oligonucleotide probes are captured and ligated to different substrates. The methods and kits disclosed herein can be used, for example, in DNA, RNA, and protein sequencing technologies.

[0053] The process of using the compounds disclosed herein can enable the compounds to be used in a variety of applications, such as identification of binding pairs, phenotyping, genotyping and multi-omics applications.

[0054] Methods using the compounds of this disclosure may include capturing a target oligonucleotide on a substrate or bead array having a capturing oligonucleotide. Upon contact with a sample, the capturing oligonucleotide binds to the target oligonucleotide from the sample, thereby capturing the target oligonucleotide. The substrate having the captured target oligonucleotide is then contacted with one or more compounds of formula I and / or formula IA, wherein the ddNTP portion of the compound of formula I and / or formula IA serves as its monobase extension to bind the target oligonucleotide. The bead array or substrate is then contacted with one or more binding unit couplers. The binding unit couplers may be labeled such that the binding between the binding unit present on the compound of formula I and / or formula IA and the binding unit coupler can be identified. The selection of the binding pair (binding unit and corresponding binding unit binding coupler) may be based on information desired to be obtained from the process. For example, the binding unit pair may be used in drug development and discovery methods to identify potential candidates with targeting activity.

[0055] The compounds disclosed herein can be used, for example, in genotyping processes using the same procedures typically employed with conventional ddNTP-hapten molecules. Generally, bead arrays or other substrates comprise capturing oligonucleotides that capture target oligonucleotides from the sample via complementary binding upon contact with the sample. The capturing oligonucleotide binds to a complementary sequence in the sample, stopping one base before the locus of interest, thus providing beads with the captured target complementary sequence. Allelic specificity is conferred by a single-base extension comprising one of four labeled nucleotides. The labeled nucleotide hybridizes with the captured target oligonucleotide having its complementary base, thereby extending the capturing oligonucleotide by a single base. At least one of the labeled nucleotides can be a compound of formula I and / or formula IA. After single-base extension using the labeled nucleotide, the sample is contacted with one or more fluorescently labeled hapten-binding conjugates. For single-signal detection, a single hapten-binding conjugate present on a compound that specifically binds to formula I and / or formula IA is used. For multi-signal detection, multiple hapten binding couples, each with different fluorescent labels, can be brought into contact with the sample to bind the corresponding hapten present on the labeled nucleotide.

[0056] In some cases, the labeled nucleotide may include compounds of Formula I for the detection of a single nucleotide, while conventional labeled nucleotides may be used to detect other nucleotides. In some cases, the detection may be a dual-channel detection, whereby A and T bases are detected by a first signal, while G and C bases are detected by a second signal. In such cases, the labeled nucleotides containing ddATP and ddTTP or ddUTP may be compounds of Formula I and / or Formula IA.

[0057] For example, a process for genotyping using dual-signal detection (where a first signal detects A and T, and a second signal detects G and C) may include contacting a bead array or other substrate with sample DNA, each bead or surface region of the bead array or other substrate comprising one or more capture oligonucleotide probes, each of which binds to a target complementary sequence in the sample, wherein the target complementary sequence binds to one or more capture oligonucleotide probes to stop at least one base before the locus of interest, thereby providing a bead array having the captured target complementary sequence bound thereto. The bead array or other substrate having the captured target complementary sequence bound to the capture oligonucleotide probe is then contacted with a composition containing at least one first hapten-modified ddNTP and one second hapten-modified ddNTP, under conditions sufficient to hybridize the first hapten-modified ddNTP with the corresponding captured target complementary sequence in the captured target complementary sequence as a single-base extension of the associated capture oligonucleotide probe, in conditions sufficient to cause hybridization of the first hapten-modified ddNTP and the second hapten-modified ddNTP. The first antigen and the second antigen are different. One or both of the first hapten-modified ddNTP and the second hapten-modified ddNTP can be compounds of Formula I. The method further includes contacting a bead array containing the hybridized first hapten-modified ddNTP and second hapten-modified ddNTP with a labeled composition containing the first hapten-modified and second hapten-modified ddNTPs, under conditions sufficient to bind the first hapten-modified conjugate to the first hapten and the second hapten-modified conjugate to the second hapten, respectively. The first hapten-modified conjugate contains a first fluorescent label, and the second hapten-modified conjugate contains a second fluorescent label. The bead array or other substrate is fluorescently labeled upon binding of the first hapten-modified ddNTP and / or the second hapten-modified ddNTP to the corresponding first hapten-modified conjugate and / or second hapten-modified conjugate. The fluorescence signal generated by the bead array can then be analyzed for genotyping of the sample. For example, in this process, the first hapten-modified ddNTP may include the first hapten-modified ddATP and the first hapten-modified ddUTP, and the second hapten-modified ddNTP may include the second hapten-modified ddGTP and the second hapten-modified ddCTP.

[0058] In some cases, such as when DNP is used as the first hapten and biotin as the second hapten, it may be advantageous to make the first hapten-modified ddNTP (DNP-modified ddNTP) a compound of formula I and the biotin-modified ddNTP monovalent. This, for example, can balance the signals in the red and green channels. Conventionally, ddATP-DNP and ddUTP-DNP are used in genotyping applications to generate red channel signals. It has been observed that when using monovalent ddNTPs, the red channel signal is weaker than the green channel signal generated by ddGTP-biotin and ddCTP-biotin labeling. In some cases, all four labeled nucleotides can be compounds of Formula I. For example, for dual-channel detection, the first set of Formula I compounds may contain a first hapten, and the second set of Formula I compounds may contain a second hapten. For example, the first hapten may be DNP, and the second hapten may be biotin. In the first set, two subsets of Formula I compounds may be provided, one set having ddATP as a nucleotide and the other having ddTTP or ddUTP as a nucleotide. Similarly, in the second set, two subsets of Formula I compounds may be provided, one set having ddGTP and the other having ddCTP. Due to the different sets of Formula I compounds, detection of A or T can be performed by the presence of a first signal generated by the first fluorescent label, while detection of G or C can be performed by the presence of a second signal generated by the second fluorescent label. In yet another embodiment, the compounds of Formula I can be provided as four different groups, each with different nucleotides and different haptens. This allows, for example, four different fluorescent labels to be applied by the corresponding hapten binding coupler.

[0059] In any of the methods disclosed herein, for each of the different hapten-binding partners, contact between the bead array or other substrate and the hapten-binding partner may be performed once or multiple times. For example, contact staining may be performed two or more times, such as three or more times. For example, when the hapten-labeled nucleotide is a compound of Formula I, the contact steps for staining the bead array or other substrate may be performed fewer times compared to monovalent hapten-labeled nucleotides, while maintaining the same or even improved signal intensity.

[0060] Using the multivalent hapten-modified ddNTPs of this disclosure can improve the binding affinity of the hapten-binding partner to the hapten, thereby allowing the use of a reduced amount of hapten-binding partner and / or reducing the number of staining or contact rounds required to label ddNTPs with fluorescently labeled hapten-binding partners.

[0061] When the compounds of this disclosure are used in conjunction with bead arrays, the resulting improved binding efficiency and increased signal-to-noise ratio, in particular, allow for the use of smaller beads and / or the formation of bead arrays with reduced bead spacing. This can advantageously increase the bead density in the array, thereby allowing for or increasing the amount of contents that can be measured by the array.

[0062] Chemical definition As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group containing one to thirty carbon atoms (e.g., one to twenty carbon atoms or one to ten carbon atoms). The term C... n This refers to an alkyl group having "n" carbon atoms. For example, C4 alkyl means an alkyl group with 4 carbon atoms. 1-6 An alkyl group is an alkyl group having a number of carbon atoms covering the entire range (i.e., 1 to 6 carbon atoms) and all subgroups (e.g., 1 to 5, 2 to 5, 1 to 4, 2 to 5, 1, 2, 3, 4, 5, and 6 carbon atoms). Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), and tert-butyl (1,1-dimethylethyl). Unless otherwise stated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.

[0063] As used herein, the term "alkylene" refers to a divalent saturated aliphatic group. The term C n This refers to an alkylene group having "n" carbon atoms. For example, C 1-6 Alkylene refers to an alkylene group having a number of carbon atoms that covers the entire range and all subgroups, as previously described for the "alkyl" group.

[0064] As used herein, the term "olefin" or "alkenyl" is defined in the same way as "alkyl," except that it contains at least one carbon-carbon double bond and has two to thirty carbon atoms, for example, two to twenty carbon atoms, or two to ten carbon atoms. The term C... n This refers to an alkenyl group having "n" carbon atoms. For example, C4 alkenyl refers to an alkenyl group with 4 carbon atoms. 2-7 An alkenyl group is an alkenyl group having a number of carbon atoms covering the entire range (i.e., 2 to 7 carbon atoms) and all subgroups (e.g., 2 to 6, 2 to 5, 3 to 6, 2, 3, 4, 5, 6, and 7 carbon atoms). Particularly considered alkenyl groups include vinyl, 1-propenyl, 2-propenyl, and butenyl. Unless otherwise specified, an alkenyl group can be an unsubstituted alkenyl group or a substituted alkenyl group. Unless otherwise specified, an alkenyl group can be cis-alkenyl or trans-alkenyl.

[0065] As used herein, the terms "alkynyl" or "alkynyl group" are defined in the same way as "alkyl," except that they contain at least one carbon-carbon triple bond and have two to thirty carbon atoms, for example, two to twenty carbon atoms, or two to ten carbon atoms. The term C... n This means that the ynyl group has "n" carbon atoms. For example, C4 ynyl means an ynyl group with 4 carbon atoms. 2-7 An alkynyl group is a group having a number of carbon atoms covering the entire range (i.e., 2 to 7 carbon atoms) and all subgroups (e.g., 2 to 6, 2 to 5, 3 to 6, 2, 3, 4, 5, 6, and 7 carbon atoms). Particularly considered alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, and butynyl. Unless otherwise specified, an alkynyl group can be an unsubstituted or substituted alkynyl group.

[0066] As used herein, the term "carbocyclic ring" refers to an aromatic or non-aromatic (i.e., fully or partially saturated) ring in which each atom of the ring is carbon. A carbocyclic ring may include, for example, three to ten carbon atoms, four to eight carbon atoms, or five to six carbon atoms. As used herein, the term "carbocyclic ring" also includes polycyclic systems having two or more rings, in which two or more carbon atoms are shared by two adjacent rings, wherein at least one of these rings is a carbocyclic ring; for example, other rings may be cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclic.

[0067] As used herein, the term "cycloalkyl" specifically refers to a non-aromatic carbide ring. The term C n This indicates that the cycloalkyl group has "n" carbon atoms. For example, C5 cycloalkyl refers to a cycloalkyl group with 5 carbon atoms in the ring. 5-8 A cycloalkyl group is a cycloalkyl group having a number of carbon atoms covering the entire range (i.e., 5 to 10 carbon atoms) and all subgroups (e.g., 5 to 10, 5 to 9, 5 to 8, 5 to 6, 6 to 8, 7 to 8, 5 to 7, 5, 6, 7, 8, 9, and 10 carbon atoms). Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unless otherwise stated, a cycloalkyl group can be an unsubstituted cycloalkyl group or a substituted cycloalkyl group.

[0068] As used herein, the term "aryl" refers to an aromatic carbon ring, and can be a monocyclic or polycyclic (e.g., fused bicyclic and fused tricyclic) carbocyclic aromatic ring system. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, phenanthryl, biphenylene, indenyl, indenyl, anthracel, fluorenyl, and tetrahydronaphthyl. Unless otherwise stated, an aryl group can be an unsubstituted aryl group or a substituted aryl group.

[0069] As used herein, the term "heterocycle" is defined similarly to that of a carbocyclic ring, except that the ring contains one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur. For example, a heterocycle can be a 3- to 10-membered aromatic or non-aromatic ring having one or two heteroatoms selected from N, O, and S. As another example, a heterocycle can be a 5- to 6-membered ring having one or two cyclic heteroatoms selected from N, O, and S. Non-limiting examples of heterocyclic groups include piperidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, oxonium-nitrogen heterocyclic heptyl groups, thiazoles, pyrroles, and pyridine.

[0070] The carbocyclic and heterocyclic groups can be saturated or partially unsaturated cyclic systems, which are optionally substituted by, for example, one to three groups, independently selected from alkyl, alkoxy, alkylene, OH, C(O)NH2, NH2, oxo(=O), aryl, haloalkyl, haloalkoxy, C(O)-alkyl, SO2alkyl, halogen, OH, NHC 1-3 alkylene-aryl, OC 1-3 alkylene-aryl, C 1-3 Alkylene-aryl and C having one to three heteroatoms selected from N, O and S 3-6 Heterocyclic alkyl groups. As described herein, the heterocyclic groups may optionally be further substituted with N. Other anticipated substituents for the disclosed rings are provided elsewhere in this disclosure.

[0071] As used herein, the term “heteroaryl” refers to an aromatic heterocycle, which can be a monocyclic or polycyclic (e.g., fused bicyclic and fused tricyclic) aromatic ring system, wherein one to four ring atoms are selected from oxygen, nitrogen, or sulfur, and the remaining ring atoms are carbon, and the ring system is bonded to the rest of the molecule by any of the ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrroleyl, pyrazolyl, imidazoleyl, thiazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, furanyl, thiopheneyl, quinolinyl, isoquinolinyl, benzoxazolyl, benzimidazolyl, benzofuranyl, benzothiazolyl, triazinyl, triazolyl, purinyl, pyrazinyl, purinyl, indololinyl, phthalazinyl, indolyl, quinolinyl, isoquinolinyl, cenolinyl, quinazolinyl, naphridinyl, pyridopyridinyl, indolyl, 3H-indolyl, pteridinyl, and quinoxolinyl. Unless otherwise stated, a heteroaryl group may be an unsubstituted heteroaryl group or a substituted heteroaryl group.

[0072] As used herein, the term "hydroxyl" refers to the "-OH" group. Therefore, "hydroxyalkyl" refers to an alkyl group that is substituted with one or more "-OH" groups.

[0073] As used herein, the term "alkoxy" refers to the "-O-alkyl" group.

[0074] As used herein, the term "halogenated" is defined as fluorinated, chlorinated, brominated, and iodinated. Therefore, "halogenated alkyl" refers to an alkyl group substituted with one or more halogen atoms. "Halogenated alkoxy" refers to an alkoxy group substituted with one or more halogen atoms.

[0075] A “substituted” functional group (e.g., a substituted alkyl, cycloalkyl, aryl, or heteroaryl group) is a functional group having at least one hydrogen group substituted by a non-hydrogen group (i.e., a substituent). Examples of non-hydrogen groups (or substituents) include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, ether, aryl, O-alkylene aryl, N-alkylene aryl, alkylene aryl, heteroaryl, heterocycloalkyl, hydroxy, hydroxyalkyl, haloalkoxy, amide, oxy (or oxo), alkoxy, ester, thioester, acyl, carboxyl, cyano, nitro, amino, mercapto, and halogen. When the substituted alkyl group includes more than one non-hydrogen group, the substituent may be attached to the same carbon atom or to two or more different carbon atoms.

[0076] Synthesis of the compounds disclosed herein The compounds described herein can be synthesized using conventional techniques with readily available starting materials known to those skilled in the art. Generally, the compounds described herein can be conveniently obtained using standard organic chemical synthesis methods.

[0077] Although not limited to any one or a few sources, classic texts such as Smith, MB, and March, J.'s *March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure*, 5th ed., John Wiley Sons: New York, 2001; and Greene, TW, and Wuts, PGM's *Protective Groups in Organic Synthesis*, 3rd ed., John Wiley Sons: New York, 1999, are useful and widely recognized reference textbooks in organic synthesis known to those skilled in the art. The following description of synthetic methods is intended to illustrate, but not limit, the general procedures used to prepare the compounds of this disclosure.

[0078] The synthetic processes disclosed herein are tolerant of a wide range of functional groups; therefore, a variety of substituted starting materials can be used. These processes typically provide the desired final compound at or near the end of the process, but in some cases it may be desirable to further convert the compound into its pharmaceutically acceptable salt.

[0079] Generally, the compounds disclosed herein can be synthesized according to the examples shown below. For example, the compounds can be prepared by alkylation of a suitable amine having a carboxyl group, with the use of a suitable protecting group if necessary. The intermediate can be saponified, for example, to expose the reactive carboxylic acid ester. Then, amide coupling between a suitable amine and the free carboxylic acid ester can occur.

[0080] The amines used for the above-mentioned amide coupling can be prepared by known synthetic techniques using appropriate starting materials and protecting groups (if desired).

[0081] Further modifications can be performed, for example, to introduce additional substituents, such as halogenated groups or alkyl groups.

[0082] Example Example 1 Monobasic extension reagents are formulated using compounds according to this disclosure and standard monovalent compounds as controls. Monobasic extension reagents having compounds according to Formula I are prepared by mixing a monobasic extension premix (EML premix), SSB, tTAQ608, and labeled nucleotides. For G and C (green) channel detection, conventional monovalent ddGTP-biotin-labeled and ddCTP-biotin-labeled reagents are used. For A and T (red) channel detection, the following compounds according to this disclosure are used. In the dataset, samples run using extension reagents formulated with compounds of Formula I are referred to as diEML.

[0083]

[0084] Figure 1 A synthetic scheme for preparing ddATP-diDNP is shown, and Figure 2 A synthetic protocol for preparing ddUTP-diDNP is shown. ddATP-diDNP and ddUTP-diDNP were prepared by mixing 1 equivalent of a diamine linker with 2.2 equivalents of DNP-NHS dissolved in 0.2 M NaHCO3:DMSO (1.4 v / v). The admixtures were stirred at room temperature for 8 hours. The mixture was purified by HPLC using water and ACN as eluents. The di-DNP-COOH product was then lyophilized. The lyophilized product was then reconstituted in DMF. 1.5 equivalents of TSTU were added to the mixture, followed by 20 equivalents of DIPEA, and the mixture was stirred at room temperature for 30 minutes. Then, 1 equivalent of PA-ddNTP was added, and the mixture was stirred overnight at room temperature. The resulting product was purified by HPLC using 50 mM TEAA (pH 7.2) and ACN as eluents. The product was then lyophilized.

[0085] A conventional, commercially available monovalently labeled nucleotide monobase extension premix (referred to herein as pEML) and a monovalently labeled ddNTP-formulated monobase extension premix (referred to herein as devEML) were used as controls. Specifically, the controls consisted of the same EML premix, SSB, and tTAQ608 as used in diEML, but included the labeled nucleotides DNP-11-ddATP, DNP-11-ddUTP, and the same ddGTP-biotin and ddCTP-biotin used in diEML. The structures of DNP-11-ddATP and DNP-11-ddUTP are shown below.

[0086]

[0087] Each reagent in the kit includes a nominal ddNTP concentration of 0.125 μM. The reagents were tested individually using an Infinium bead array (Illumina) with a standard MSA7 workflow (Infinium XT-ST workflow), replacing the precipitate with DPB purity. Amplification was performed using RAM at 10 nm input, followed by incubation for 18 hours. The sample tested for this genotype assay included a single DNA type – NA11993.

[0088] Staining of the fluorescently labeled hapten-binding mating bodies was performed using the Xstain protocol in Tecan. Three rounds of staining were performed, and signal intensity was assessed using an iScan N392 after each round.

[0089] Figure 3 The signal in the red channel for each reagent sample tested is illustrated. The diEML containing the compounds according to this disclosure provides a detectable signal even after a single staining cycle. Figure 3 Further examples illustrate the incorporation of ddNTPs coupled to PEG linkers with multiple DNP motifs via tTaq608 polymerase, using the same biochemical conditions as monovalent ddNTP linker DNP substances.

[0090] refer to Figure 4 As can be seen, the signal increase leading to the third round of staining was comparable between diEML and pEML controls compared to the second round of staining, and diEML showed a greater signal improvement compared to the devEML control.

[0091] at last, Figure 5 The detection of biotin-modified ddGTP and ddCTP in the green channel is illustrated, and is not affected by the use of the compounds according to the claims.

[0092] Example 2 The compounds of this disclosure were also evaluated using synthetic probes. The same single-base extension reagents as described in Example 1, including controls, were used. This example evaluated the extension efficiency of A, T, C, and G nucleotides from the hairpin probe. Both the red (A, T) and green (C, G) channels were monitored. Figure 6 This is a schematic diagram of the process.

[0093] refer to Figure 7A and Figure 7B The compounds (divalent DNP formulations) disclosed herein were observed to exhibit higher signal intensity. Non-specific extension was observed by extending the signal in C and G. Nucleotide incorporation efficiency and specificity are affected by template base, pH, and the concentration and stoichiometry of the following components: ddNTP-diDNP, polymerase, and catalytic metal (Mg). 2+ It is anticipated that further optimization of these parameters using the compounds disclosed herein will lead to further improvements in efficiency and specificity.

[0094] Figure 8 An example is shown of signal increase achieved in diEML via A and T extensions using the compounds of this disclosure compared to a control. The dashed box in Figure 7 represents the non-extension control group, in which no signal increase is expected.

[0095] Example 3 The effect of ddNTP concentration on the incorporation rate was evaluated by comparing the performance of reagents with 0.125 μM ddNTP with those with 0.625 μM ddNTP. The reagents were prepared as described in Example 1, except for the concentration.

[0096] refer to Figure 9A After two rounds of staining, it was observed that diEML with an increased concentration of 0.625 μM of the compound according to the invention showed significantly higher intensity in the red channel compared to the produced EML, but not significantly higher than devEML prepared with a higher (0.625 μM) concentration of monovalent ddNTPs. However, as Figure 9B As shown, an increase in signal was observed in samples with higher concentrations of diEML.

[0097] Example 4 Using Infinium EX ® The platform evaluates the compounds disclosed herein. The performance of the compounds (ddATP-diDNP and ddUTP-diDNP) according to this disclosure is compared with that of standard ddNTPs, as shown in the table below.

[0098]

[0099]

[0100] The purity of the hapten-labeled ddNTPs is ≥95% (HPLC). The ddNTPs (standard and according to this disclosure) are prepared at a concentration of 1 mM in a formulation containing 10 mM Tris-HCl, 1 mM EDTA and pH 7.6 ± 0.2.

[0101] EML performance was measured using standard EML reagents on the Infinium EX microarray platform, which includes a bead chip with 1 μm beads. Testing was performed at decreasing antibody concentrations in STX and ATX. The standard EML reagents used to test standard-designed ddNTPs comprised equimolar amounts of standard-designed ddNTPs. According to this disclosure, tests were performed with increasing concentrations of divalent ddNTPs according to this disclosure. The test conditions used are shown in the table below:

[0102] refer to Figure 10 Although a high signal-to-noise ratio (SNR) was observed at p95 for the compounds according to this disclosure, a lower SNR was observed at p50. It is believed that the test conditions can be modified to reduce the SNR at p95. The SNR of the EML reagent includes... Figure 10 Use the middle as a comparison. Reference Figure 11 An increased detection rate was observed with increasing divalent ddNTP concentration, independent of antibody concentration. The Log R bias metric was observed to decrease with increasing divalent ddNTP concentration, independent of antibody concentration. Low Log R bias is beneficial for optimal genotyping on the Infinium EX microarray platform.

[0103] The foregoing description is provided only for the purpose of clearly understanding the present invention and should not be construed as an unnecessary limitation, as modifications within the scope of this disclosure will be readily apparent to those skilled in the art.

[0104] All patents, patent applications, government publications, government regulations, and references cited in this specification are incorporated herein by reference in their entirety. In case of any discrepancy, this specific embodiment and its definitions shall prevail.

[0105] Throughout this specification, when compounds, compositions, methods, and / or processes are described as comprising components, steps, or materials, unless otherwise described, it is contemplated that the compounds, compositions, methods, and / or processes may also comprise, consist substantially of, or consist of any combination of said components or materials. Unless otherwise specifically stated, component concentrations may be expressed as weight concentrations. Consideration is given that combinations of components include homogeneous and / or heterogeneous mixtures, as understood by one of ordinary skill in the art in light of the foregoing disclosure.

Claims

1. A compound of formula I, ARM a -Q b -M c -J-ddNTP, in: a is at least 2. Each ARM contains a bonding unit and a connector with PEO. b is 1 to 3. Each Q is selected independently. , or Each Q is connected to 2 or 3 ARMs. c is 0 or 1 M is Each L1 and L2 is independently selected For each L1 and L2, d is independently selected from 1 to 10, and e is independently selected from 1 to 10, and R is independently selected from the functional groups generated by the coupling reaction between a pair of the following: amine-imino ester, amine-pentafluorophenyl ester, amine- N- Hydroxysuccinimide ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-acylhydrazine, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-phosphine, trans-cyclooctene-tetraazine, norbornene-tetraazine, azide-cyclooctene, azide-norbornene, or azide-alkyne, and J is , where x is from 1 to 48.

2. The compound of claim 1, wherein the binding unit is one or more of an antibody, protein, oligonucleotide, DNA, RNA, drug, small molecule, dye, and peptide.

3. The compound according to claim 1 or 2, wherein each ARM comprises For each ARM, z is independently selected from 1 to 48, and y is independently selected from 1 to 48, and T is independently selected from the functional groups generated by the coupling reaction between a pair of the following: amine-imino ester, amine-pentafluorophenyl ester, amine-N-hydroxysuccinimide ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azido-phosphine, trans-cyclooctene-tetraazine, norbornene-tetraazine, azido-cyclooctyne, azido-norbornene, or azido-alkyne.

4. A compound of formula IA, ARM a -Q b -M c -J-ddNTP, in: a is at least 2. Each ARM contains a hapten and a PEO-containing adapter. b is 1 to 3. Each Q is selected independently. , or Each Q is connected to 2 or 3 ARMs. c is 0 or 1 M is Each L1 and L2 is independently selected For each L1 and L2, d is independently selected from 1 to 10, and e is independently selected from 1 to 10, and R is independently selected from the functional groups generated by the coupling reaction between a pair of the following: amine-imino ester, amine-pentafluorophenyl ester, amine- N- Hydroxysuccinimide ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-acylhydrazine, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-phosphine, trans-cyclooctene-tetraazine, norbornene-tetraazine, azide-cyclooctene, azide-norbornene, or azide-alkyne, and J is , where x is from 1 to 48.

5. The compound according to claim 4, wherein the hapten of each ARM is independently selected from digoxigenin (DIG), 3-nitrotyrosine, biotin, alkyl chloride, O-GlcNAc, alkylguanine, Ni-NTA and 2,4-dinitrophenol (DNP).

6. The compound according to claim 4 or 5, wherein each ARM comprises For each ARM, z is independently selected from 1 to 48, and y is independently selected from 1 to 48, and T is independently selected from the functional groups generated by the coupling reaction between a pair of the following: amine-imino ester, amine-pentafluorophenyl ester, amine-N-hydroxysuccinimide ester, amine-hydroxymethylphosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azido-phosphine, trans-cyclooctene-tetraazine, norbornene-tetraazine, azido-cyclooctyne, azido-norbornene, or azido-alkyne.

7. The compound according to claim 3 or 5, wherein z and y are the same in each ARM.

8. The compound according to claim 3 or 5, wherein z and y are different in at least two ARMs of the ARM.

9. The compound according to claim 3 or 5, wherein T is in each of the ARMs. .

10. The compound according to claim 3 or 5, wherein T is in each ARM. .

11. The compound according to claim 3 or 5, wherein T is in at least one ARM. And T is in at least one other ARM .

12. The compound according to claim 3 or 5, wherein T and...

13. The compound according to any one of the preceding claims, wherein R is .

14. The compound according to any one of the preceding claims, wherein c is 1.

15. The compound according to any one of claims 1 to 13, wherein c is 0 and Q is .

16. The compound according to any one of the preceding claims, wherein the ddNTP is ddATP or ddUTP.

17. The compound according to any one of the preceding claims, wherein the ddNTP is ddGTP or ddCTP.

18. The compound according to any one of the preceding claims, wherein a is 2 to 9.

19. The compound according to claim 4, wherein the compound is .

20. The compound according to claim 4, wherein the compound is, 。 21. The compound according to claim 4, wherein the compound is one or more of the following: Where z and y have the same value on every ARM, Where at least one of z and y has a different value on each ARM, Where z and y have the same value on each ARM, or , where at least one of z and y has a different value on each ARM.

22. A compound of formula I, said compound being selected from... , and .

23. The compound according to claim 4, wherein the compound is or .

24. A kit comprising: The substrate comprises a plurality of capture oligonucleotide probes attached to its surface; Each of the binding units and the binding bodies contains a marker. At least one compound of formula I according to any one of the preceding claims; and Instructions for use, which are for capturing targets from samples on the substrate, binding at least one compound of formula I to the captured target as a monobase extension of the captured oligonucleotide, and binding the binding unit to each ARM of the at least one compound of formula I.

25. The kit of claim 24, wherein the substrate is a bead array and each bed has at least one capture oligonucleotide probe attached to its surface.

26. A kit comprising: A bead array comprising a plurality of beads, each bead having at least one capture oligonucleotide probe attached to its surface; Hapten-binding couplers, each of which contains a first fluorescent label; At least one compound of formula IA according to any one of claims 4 to 23; and Instructions for use, which are for capturing target oligonucleotides from a sample on the bead array, binding a compound of at least one formula IA to the captured target oligonucleotide as a monobase extension of the captured oligonucleotide, and binding one or more hapten-binding mates to each ARM of a compound of at least one formula IA.

27. The kit according to any one of claims 24 to 26, wherein the at least one compound of formula I or formula IA comprises a first compound of formula I or formula IA containing ddATP and a second compound of formula I or formula IA containing ddUTP.

28. The kit according to claim 26 or 27, wherein the hapten binding partner is independently selected from anti-DNP antibody, anti-biotin, anti-DIG antibody, WGA, SNAP tag, anti-nitrotyrosine antibody and His-tag.

29. A method comprising: The substrate is brought into contact with sample DNA. The substrate contains a plurality of capture oligonucleotide probes, each of which binds to a target oligonucleotide in the sample. The target oligonucleotide has a sequence complementary to the capture oligonucleotide probe, such that the target oligonucleotide binds to the one or more capture oligonucleotide probes to stop at least one base before the locus of interest, thereby providing a substrate with the captured target oligonucleotide. Under conditions sufficient to hybridize the first modified ddNTP with one or more of the captured target oligonucleotides as a single-base extension of the associated captured oligonucleotide probe, the substrate having the captured target oligonucleotide is mixed with a composition containing at least the first modified ddNTP, wherein the first modified ddNTP is a compound of formula I according to any one of the preceding claims containing a first binding unit. Under the condition that the first binding unit binding coupler is bound to the first binding unit, the substrate containing the hybridized first modified ddNTP is contacted with a labeling composition containing at least the first binding unit binding coupler, wherein the first binding unit binding coupler contains a first label, and the substrate is labeled when the first binding unit is bound to the first binding unit binding coupler. as well as Analyze the signals associated with the marker.

30. The method of claim 29, wherein the substrate is a bead array, and each bead array contains a capture oligonucleotide attached to its surface.

31. The method of claim 29 or 30, wherein analyzing the signal associated with the tag comprises: The signal was analyzed to identify binding pairs.

32. The method according to any one of claims 29 to 31, wherein the first binding unit comprises an antibody, protein, oligonucleotide, DNA, RNA, drug, small molecule, dye, and peptide.

33. The method according to any one of claims 29 to 32, wherein the first binding unit binds to a partner comprising one or more of an antibody, protein, oligonucleotide, DNA, RNA, drug, small molecule, dye, and peptide.

34. The method of any one of claims 29 to 33, wherein the labeling composition comprises a plurality of binding unit binding mating bodies, each of the plurality of binding unit binding mating bodies comprising a label, and the composition comprising at least the first modified ddNTP comprises a plurality of modified ddNTPs, the plurality of modified ddNTPs comprising a plurality of different binding units, wherein, upon contact of the labeling composition with the substrate, at least a portion of the plurality of binding unit binding mating bodies binds to one of the plurality of different binding units; and analyzing the signal comprises: Identification of the pair.

35. The method according to any one of claims 29 to 34, wherein the marker is a fluorescent marker.

36. A method for genotyping, the method comprising: The bead array is brought into contact with sample DNA, each bead in the bead array containing one or more capture oligonucleotide probes, each of the one or more capture oligonucleotide probes binding to a target complementary sequence in the sample, wherein the target complementary sequence binds the one or more capture oligonucleotide probes to stop at least one base before the locus of interest, thereby providing a bead array with a captured target complementary sequence; Under conditions sufficient to hybridize the first hapten-modified ddNTP and the second hapten-modified ddNTP with the corresponding captured target complementary sequence in the captured target complementary sequence as a single-base extension of the associated captured oligonucleotide probe, an array of beads having the captured target complementary sequence is mixed with a composition comprising at least the first hapten-modified ddNTP and the second hapten-modified ddNTP, wherein the first hapten and the second hapten are different, and the at least the first hapten-modified ddNTP is a compound of formula I according to any one of the preceding claims. Under conditions where a first hapten-binding partner is bound to the first hapten and a second hapten-binding partner is bound to the second hapten, the bead array comprising a ddNTP modified with the hybridized first hapten and a ddNTP modified with the second hapten is contacted with a labeled composition comprising the first hapten-binding partner and the second hapten-binding partner, wherein the first hapten-binding partner comprises a first fluorescent label and the second hapten-binding partner comprises a second fluorescent label, and the bead array is fluorescently labeled when the first hapten and / or the second hapten is bound to the respective first hapten-binding partner and / or second hapten-binding partner; as well as After binding the first hapten-binding coupler and the second hapten-binding coupler, the fluorescence signal of the bead array is analyzed.

37. The method of claim 36, wherein the first hapten-modified ddNTP comprises a first hapten-modified ddATP and a first hapten-modified ddUTP, and the second hapten-modified ddNTP comprises a second hapten-modified ddGTP and a second hapten-modified ddCTP.

38. The method of claim 36 or 37, wherein the method comprises repeatedly contacting the bead array with the marking composition.

39. The method of claim 36, wherein the bead array is fluorescently labeled with fewer repetitions of contacting the bead array with the labeling composition, and at least maintains the same fluorescence signal intensity, compared to contacting the bead array with a contact step comprising a hapten-modified ddNPT having only a single ARM.

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