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WO2024254329A1 - Triphosphates didésoxynucléotides modifiés par une unité de liaison multivalente et leurs procédés de fabrication et d'utilisation - Google Patents

Triphosphates didésoxynucléotides modifiés par une unité de liaison multivalente et leurs procédés de fabrication et d'utilisation Download PDF

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Publication number
WO2024254329A1
WO2024254329A1 PCT/US2024/032844 US2024032844W WO2024254329A1 WO 2024254329 A1 WO2024254329 A1 WO 2024254329A1 US 2024032844 W US2024032844 W US 2024032844W WO 2024254329 A1 WO2024254329 A1 WO 2024254329A1
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Prior art keywords
compound
hapten
binding
formula
thiol
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WO2024254329A8 (fr
Inventor
Rigoberto PANTOJA
Xiangyuan YANG
Eric Vermaas
Yin Nah TEO
Liu DI
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Illumina Inc
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Illumina Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • the disclosure relates to multivalent binding unit modified dideoxynucleotide triphosphates (ddNTP) and methods of making and using the same.
  • ddNTP dideoxynucleotide triphosphates
  • SNPs Single nucleotide polymorphisms
  • allele specificity can be conferred by a single base extension that incorporates one or four labeled nucleotides. When excited by a laser, the nucleotide label emits a single. The intensity of that signal conveys information about allelic ration at that locus.
  • the compounds and methods of the disclosure utilize multivalent binding unit modified ddNTP to enhance binding unit binding partner binding affinity and thereby improve signal detection. This can beneficially result in decreased nonspecific binding that is mediated by both binding unit binding partner surface and dyes covalently attached to the binding unit binding partner for fluorescence detection. This can also beneficially reduce the amount of binding unit binding partner required in current reagent formulations, which in turn can minimize nonspecific binding and improve COGS. Finally, the compounds and method of the disclosure can beneficially decrease the number of staining rounds required for signal amplification.
  • the binding unit molecule can be haptens and the binding unit binding partner can be hapten binding partners.
  • a multivalent binding unit modified ddNTP can be a compound of formula I:
  • each ARM comprising a binding unit and a PEO- containing linker, each Q is independently selected from, and each Q attaches to 2 or 3 wherein for each Li and L 2 , d is independently selected from 1 to 10, and e is independently selected from 1 to 10, R is independently selected from a functionality resulting from a coupling reaction between one of the following pairs: amine- imidoester, amine-pentafluorophenyl ester, amine-/V-hydroxysuccinimide ester, aminehydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, al
  • each ARM can include wherein for each ARM z is independently selected from 1 to 48 and y is independently selected from 1 to 48 T is independently selected from a functionality resulting from a coupling reaction between one of the following pairs: amine-imidoester, amine-pentafluorophenyl ester, amine-N- /tydroxysuccinimide ester, amine-hydroxymethyl phosphine, carboxyl-carbodiimide, thiol- maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cycl
  • a kit can include a bead array comprising a plurality of beads, each bead having at least one capture oligonucleotide probe attached to a surface thereof; anti-DNP antibodies, each comprising a first fluorescence 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.
  • Figure 1 is a schematic illustration of a method of making a multivalent hapten- modified ddNTP in accordance with the disclosure
  • Figure 2 is a schematic illustration of a method of making a multivalent hapten- modified ddNTP in accordance with the disclosure
  • Figure 3 is a graph showing fluorescence analysis results comparing use of a multivalent hapten modified ddNTP to a commercially available monovalent DNP-ddNTP and a lab-produced monovalent DNP-ddNTP.
  • Figure 4 is a graph showing fluorescence analysis results comparing use of a multivalent hapten modified ddNTP to a commercially available monovalent DNP-ddNTP and a lab-produced monovalent DNP-ddNTP after multiple staining rounds.
  • Figure 5 is a graph showing fluorescence analysis in the green channel.
  • Figure 6 is a schematic illustration of an example assay in accordance with the disclosure.
  • Figure 7A is a graph showing fluorescence analysis in the red channel comparing extension efficiency using a multivalent hapten modified ddNTP to a commercially available monovalent DNP-ddNTP and a lab-produced monovalent DNP-ddNTP.
  • Figure 7B is a graph showing fluorescence analysis in the green channel comparing extension efficiency using a multivalent hapten modified ddNTP to a commercially available monovalent DNP-ddNTP and a lab-produced monovalent DNP-ddNTP.
  • Figure 8 is a graph showing the results in both extension and non-extension control sets comparing extension efficiency using a multivalent hapten modified ddNTP to a commercially available monovalent DNP-ddNTP and a lab-produced monovalent DNP-ddNTP.
  • Figure 9A is a graph showing the fluorescence analysis results of two rounds of staining using increased hapten-modified ddNTP concentrations.
  • Figure 9B is a graph showing the fluorescence analysis results of three rounds of staining using increased hapten-modified ddNTP concentrations.
  • Figure 10 is a graph showing the signal-to-noise ratio for performance testing on the Infinium EX microarray platform for compounds in accordance with the disclosure as compared to standard design ddNTP.
  • Figure 11 is a graph showing call rate and logR deviation metrics for performance testing of compounds in accordance with the disclosure as compared to standard design ddNTPs on the Infinium EX microarray platform.
  • ddNTP dideoxynucleotide triphosphates
  • the compounds of the disclosure can be useful in a variety of applications, including genotyping and multi-omics applications.
  • the binding unit modified ddNTP in accordance with the disclosure include multiple binding units bound or attached to the ddNTP, which has been observed to enhance binding affinity of the fluorescently labeled binding unit binding partner.
  • the binding unit modified ddNTPs in accordance with the disclosure can decrease nonspecific binding. This can beneficially allow for a reduced amount of binding unit binding partner to be used and/or reduce the number of staining rounds required for signal amplification.
  • the binding unit modified ddNTP can be a compound of formula I:
  • each ARM a - Q b - Mc - J -ddNTP, wherein a is at least 2, b is 1 to 3, c is 0 or 1 , each ARM comprising a binding unit and a PEO- containing linker, each Q is independently selected from, each Q attaches to 2 or 3
  • each Li and L 2 are independently selected from wherein for each L 1 and L 2 , d is independently selected from 1 to 10, and e is independently selected from 1 to 10, R is independently selected from a functionality resulting from a coupling reaction between one of the following pairs: amine- imidoester, amine-pentafluorophenyl ester, amine-N-hydroxysuccinimide ester, aminehydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine
  • a can be at least 2 and up to 9.
  • a can be 2, 3, 4, 5, 6, 7, 8, or 9.
  • the binding unit in each ARM can be the same, for example.
  • the binding unit in two or more of the ARMS can be different, alternatively.
  • the compounds of the disclosure are hapten modified dideoxynucleotide triphosphates (ddNTP), which can be useful in genotyping applications to improve signal detection.
  • the hapten modified ddNTP in accordance with the disclosure include multivalent hapten molecules, which has been observed to enhance binding affinity of the fluorescently labeled hapten binding partner.
  • the hapten modified ddNTPs in accordance with the disclosure can decrease nonspecific binding. This can beneficially allow for a reduced amount of hapten binding partner to be used and/or reduce the number of staining rounds required for signal amplification.
  • the hapten modified ddNTP can be a compound of formula IA:
  • each ARM comprising a hapten and a PEO- containing linker, each Q is independently selected from, and each Q attaches to 2 or 3 , wherein for each L 1 and L 2 , d is independently selected from 1 to 10, and e is independently selected from 1 to 10, R is independently selected from a functionality resulting from a coupling reaction between one of the following pairs: amine- imidoester, amine-pentafluorophenyl ester, amine-N-hydroxysuccinimide ester, amine- hydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl
  • a can be at least 2 and up to 9.
  • a can be 2, 3, 4, 5, 6, 7, 8, or 9.
  • the hapten in each ARM can be the same, for example.
  • the hapten in two or more of the ARMS can be different, alternatively.
  • the hapten in each ARM can be independently selected from, for example, digoxigenin (DIG), 3-nitrotyrosine, biotin, alkyl chloride, O-GIcNAc, alkyl guanine, Ni-NTA and 2,4-dinitrophenol (DNP).
  • DIG digoxigenin
  • 3-nitrotyrosine biotin
  • alkyl chloride O-GIcNAc
  • alkyl guanine alkyl guanine
  • Ni-NTA 2,4-dinitrophenol
  • each ARM can be any ARM.
  • each ARM can include In each such ARM, 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 values therebetween and ranges defined by such 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.
  • T is independently selected from a functionality that results from a coupling reaction between one of the following pairs of compounds: amine-imidoester, aminepentafluorophenyl ester, amine-N-hydroxysuccinimide ester, amine-hydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azidephosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, azidenorbornene, or azide-alkyne.
  • T can example
  • each ARM T can be
  • T can be in at least one arm least one other arm.
  • each ARM, z and y can be same and the compound can be a symmetrical compound.
  • the compound can be asymmetrical.
  • two or more of the ARM, y and z can be different and the compound can be asymmetrical.
  • b can be 1 , 2 or 3.
  • c can be 0 or 1 .
  • Other selections for Q when c is 0 are also contemplated herein.
  • Each Li and L2 are independently selected from , wherein for each Li and L 2 , d is independently selected from
  • J is wherein x is from 1 to 48, 1 to 12, 1 to 10, 13 to 24, 35 to 36, or 37 to 48.
  • the ddNTP in the compound of formula I or formula IA can be any one of ddATP, ddTTP, ddllTP, ddGTP, or ddCTP.
  • the compound of formula I or formula IA is a structure as shown in Table A
  • the anti-DNP antibody hybridizes to the DNP, thereby labeling the bead with the first label.
  • the first label can be, for example, a fluorescence label.
  • the label or interaction between the binding unit and the binding unit binding pair can results in a detectable signal such as fluorescence, bioluminescent, or electrical detection.
  • the kit can be useful for screening and/or identifying binding pairs between binding units and binding unit binding partners. For example, a plurality of binding unit bindings partners and/or a plurality of binding units can be included in the kit to screen for binding interactions through use of the kit.
  • a kit for genotyping can include, for example, a bead array comprising a plurality of beads, each bead having at least one capture oligonucleotide probe attached to a surface thereof, a hapten binding partner comprising a first fluorescence label, at least one compound of formula I, and instructions for use.
  • the hapten binding partner containing the first fluorescence label selectively binds to the hapten present on the ARM(s) of the compound of formula I.
  • the hapten binding partner can be an anti-DNP antibody comprising a first fluorescence label.
  • the anti-DNP antibody hybridizes to the DNP, thereby labeling the bead with the first fluorescence label.
  • Binding unit binding partners can be independently selected from antibodies, proteins, oligonucleotides, DNA, RNA, drugs, small molecules, dyes, peptides, and the like.
  • the hapten binding partners can be independently selected from anti-DNP antibodies, avidin, anti-DIG antibodies, WGA, SNAP tags, anti-nitrotyrosine antibodies, and His-Tag.
  • Hapten/Hapten binding partner pairs are identified above, and suitable binding unit binding partners can be selected based on the binding unit present on the compounds 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 binder partner can be a protein, oligo, RNA or DNA.
  • 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 the protein, oligo, RNA, or DNA.
  • the binding unit binding partner can include a label, such as a fluorescence label, bioluminescence label, or charge (for electrical detection), to allow for indication of binding pairs that occur.
  • the compounds and methods of the disclosure can be used, for example, to screen for interactions that may be useful, for example, in drug development.
  • Compounds and methods of the disclosure can utilize binding unit/binding unit partners for various applications.
  • the compounds and methods of the disclosure can be used in genotyping applications, binding pair screening and/or identification, and multi- omics.
  • counting multi-omics can be performed using the compounds of the disclosure in which the binding sets provided by the compounds of the disclosure and the binding unit partner can allow for detection of multiple analytes such as DNA, RNA, and protein binding sites.
  • the kit can include multiple different compounds of formula I and/or formula IA, for example, with different ddNTP portions.
  • the compounds of formula I can include a binding unit-modified ddATP and a binding unit-modified ddUTP, such that a single fluorescence signal from the first fluorescence label will be generated when both A and T are present on the bead.
  • Single detection of A and T as a single channel is commonly used in various genotyping process.
  • Any known bead array technology can be used with the kits of the disclosure.
  • the beads can be an Infinium bead array (Illumina).
  • the kit can include two sets of compounds of formula I and/or formula IA, each set having different binding units for binding to different fluorescently labeled binding unit binding partners.
  • a first set of the compound of formula I can include DNP as the hapten present on the arm, and an anti-DNP antibody with a first fluorescent label can be provided.
  • This first set of compounds can include a two subset of compounds, one subset including ddATP as the ddNTP and the other subset including ddUTP as the ddNTP.
  • the first set of compounds of formula I would result in fluorescently labeling A and T detection with a first fluorescent label.
  • the kit can further include a second set of compounds of formula I, for example, where the hapten present on the arm is biotin and the hapten binding partner is avidin with a second fluorescent label, different from the first fluorescent label.
  • the second set of compounds of formula I can include two subsets, one in which the ddNTP is ddGTP and the other in which the ddNTP is ddCTP.
  • the second set of compounds of formula I would result in fluorescently labeling G and C detection with the second fluorescent label. This can result in two channels of detection, for example, a red (A and T) and a green channel (G and C).
  • kits of the disclosure can include other platforms, such as non-bead platforms, in which the capture oligonucleotide probe is attached to a different substrate.
  • the methods and kits of the disclosure can be useful, for example, with DNA, RNA, and protein sequencing technologies.
  • Process of Using the Compounds of the Disclosure can utilize the compounds for various application, such as identifying binding pairs, phenotyping, genotyping, and in multi-omics applications.
  • a method of using the compounds of the disclosure can include capturing a target oligonucleotide on a substrate or bead array having a capture oligonucleotide. Upon contact with the sample, the capture oligonucleotide binds the target oligonucleotide form the sample, thereby capturing the target oligonucleotide.
  • the substrate having the captured target oligonucleotides are 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 binds to the target capture oligonucleotide as a single base extension thereof.
  • binding unit partners can be labeled such that binding between the binding unit present on the compound of formula I and/or formula IA and the binding unit partner can be identified. Selection of the binding pairs (binding unit and corresponding binding unit binding partners) can be made depending on the information desired to obtain from the process. For example, binding unit pairs can be used in methods of drug development and discovery to identifying potential candidates with a targeted activity. [0050] Compounds of the disclosure can be used, for example, in processes of genotyping using generally the same procedures as conventional ddNTP-hapten molecules.
  • the beads array or other substrate includes a capture oligonucleotide that, upon contact with a sample, captures through complementary binding target oligonucleotides from the sample.
  • the capture oligonucleotide binds to the complementary sequence in the sample, stopping one base before the locus of interest, thereby providing a bead having a captured target complementary sequence.
  • Allele specificity is conferred by a single base extension that includes one of four labeled nucleotides.
  • the labeled nucleotides hybridized to the captured target oligonucleotide having a complementary base thereto, thereby extending the capture oligonucleotide by a single base.
  • At least one of the label nucleotides can be a compound of formula I and/or formula IA.
  • the sample is contacted with one or more fluorescently labeled hapten binding partners.
  • a single hapten binding partner that specifically binds to the hapten present on the compound of formula I and/or formula IA is used.
  • multiple hapten binding partners each having distinct fluorescent labels can be contacted with the sample to bind to the respective hapten present on the labeled nucleotides.
  • the labeled nucleotides can include the compound of formula I for detection of a single nucleotide, while conventional labeled nucleotides can be used for detection of the other nucleotides.
  • the detection can be two-channel detection, whereby A and T bases are detected by a first signal and G and C bases are detected by a second single.
  • the labeled nucleotides comprising ddATP and ddTTP or ddllTP can be the compounds of formula I and/or formula IA.
  • a process of genotyping using a two-signal detection can include contacting a bead array or other substrate with a sample DNA, each bead or surface region of the bead array or other substrate, respectively, includes one or more capture oligonucleotide probes that each bind to a target complementary sequence in the sample, wherein the target complementary sequence binds to the one or more capture oligonucleotide probes stopping at least one base before a locus of interest thereby providing a bead array having captured target complementary sequence bound thereto.
  • the bead array or other substrate having the captured target complementary sequences bound to the capture oligonucleotide probe is then contacted with a composition comprising at least a first hapten modified ddNTPs and a second hapten modified ddNTP under conditions sufficient to hybridize the first and second hapten modified ddNTP to respective ones of the captured target complementary sequences as a single base extension of an associated capture oligonucleotide probe.
  • the first and second haptens are different.
  • One or both of the first and second hapten modified ddNTPs can be a compound of formula I.
  • the method further includes contacting the bead array comprising the hybridized first and second hapten modified ddNTPs with a labeling composition that includes a first hapten binding partner and a second hapten binding partner under conditions sufficient to bind the first hapten binding partner to the first hapten and the second hapten binding partner to the second hapten.
  • the first hapten binding partner comprises a first fluorescence label
  • the second hapten binding partner comprises a second fluorescence label.
  • the first hapten modified ddNTP cm include 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
  • the first hapten modified ddNTP (DNP modified ddNTP) to be a compound of formula I and the biotin modified ddNTP to be monovalent.
  • DNP modified ddNTP a compound of formula I
  • biotin modified ddNTP to be monovalent.
  • ddATP- DNP and ddUTP-DNP are used in genotyping applications to generate a red channel signal. It has been observed that when monovalent ddNTP are utilized, the red channel signal is weaker than a green channel signal resulting from ddGTP-biotin and ddCTP-biotin labels. .
  • all four labeled nucleotides can be compounds of formula I.
  • a first set of compounds of formula can include a first hapten and a second set of compounds of formula I can include a second hapten.
  • the first hapten can be DNP and the second hapten can be biotin.
  • two subsets of compounds of formula I can be further provided, with one set having ddATP as the nucleotide and the other set having ddTTP or ddUTP as the nucleotide.
  • two subsets of compounds of formula I can be provided, with one set having ddGTP and the other subset having ddCTP.
  • the compound of formula I can be provided as four distinct sets, each set having a different nucleotide and different hapten. This can allow, for example, for four distinct fluorescent labels to be applied by a corresponding hapten binding partner.
  • the contacting of the bead array or other substrate with the hapten binding partner can be performed one or more times for each of the distinct hapten binding partners.
  • the staining by contacting can be performed 2 or more times, for example 3 or more times.
  • the contacting step for staining the bead array or other substrate can be performed a reduced number of times as compared when a monovalent hapten labeled nucleotide while maintaining the same or even having improved signal intensity.
  • Use of the multi-valent hapten modified ddNTPs of the disclosure can improve hapten binding partner binding affinity to the hapten, thereby allowing for a reduced amount of hapten binding partner to be use and/or reduce a number of staining or contacting rounds needed for labeling the ddNTP with the fluorescent labeled hapten binding partner.
  • the improve binding efficiency and increased signal to noise ratio resulting therefrom can also allow the bead array to be formed using smaller beads and/or with reduced bead spacing. This can beneficially increase the bead density in the array, allowing or an increased amount of content that can be assayed by the array.
  • alkyl refers to straight chained and branched saturated hydrocarbon groups containing one to thirty carbon atoms, for example, one to twenty carbon atoms, or one to ten carbon atoms.
  • C n means the alkyl group has “n” carbon atoms.
  • C4alkyl refers to an alkyl group that has 4 carbon atoms.
  • Ci ealkyl refers to an alkyl group having a number of carbon atoms encompassing the entire range (i.e. , 1 to 6 carbon atoms), as well as all subgroups (e.g., 1 -5, 2-5, 1 -4, 2-5, 1 , 2, 3, 4, 5, and 6 carbon atoms).
  • alkyl groups include, methyl, ethyl, n-propyl, isopropyl, n-butyl, secbutyl (2-methylpropyl), and t-butyl (1 ,1 -dimethylethyl).
  • an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.
  • alkylene refers to a bivalent saturated aliphatic radical.
  • C n means the alkylene group has "n" carbon atoms.
  • Ci-ealkylene refers to an alkylene group having a number of carbon atoms encompassing the entire range, as well as all subgroups, as previously described for "alkyl” groups.
  • alkene or “alkenyl” is defined identically as “alkyl” except for containing at least one carbon-carbon double bond, and having two to thirty carbon atoms, for example, two to twenty carbon atoms, or two to ten carbon atoms.
  • C n means the alkenyl group has “n” carbon atoms.
  • C4alkenyl refers to an alkenyl group that has 4 carbon atoms.
  • C ⁇ alkenyl refers to an alkenyl group having a number of carbon atoms encompassing the entire range (i.e.
  • alkenyl groups include ethenyl, 1 -propenyl, 2-propenyl, and butenyl.
  • an alkenyl group can be an unsubstituted alkenyl group or a substituted alkenyl group.
  • an alkenyl group can be a cis-alkenyl or trans-alkenyl.
  • alkyne or “alkynyl” is defined identically as “alkyl” except for containing at least one carbon-carbon triple bond, and having two to thirty carbon atoms, for example, two to twenty carbon atoms, or two to ten carbon atoms.
  • C n means the alkynyl group has “n” carbon atoms.
  • C4alkynyl refers to an alkynyl group that has 4 carbon atoms.
  • C 27 alkynyl refers to an alkynyl group having a number of carbon atoms encompassing the entire range (i.e., 2 to 7 carbon atoms), as well as all subgroups (e.g., 2-6, 2- 5, 3-6, 2, 3, 4, 5, 6, and 7 carbon atoms).
  • Specifically contemplated alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, and butynyl.
  • an alkynyl group can be an unsubstituted alkynyl group or a substituted alkynyl group.
  • carbocycle refers to an aromatic or nonaromatic (i.e., fully or partially saturated) ring in which each atom of the ring is carbon.
  • a carbocycle can include, for example, from three to ten carbon atoms, four to eight carbon atoms, or five to six carbon atoms.
  • the term “carbocycle” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is carbocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, aryls, heteroaryls, and/or heterocycles.
  • cycloalkyl specifically refers to a non-aromatic carbocycle.
  • C n means the cycloalkyl group has “n” carbon atoms.
  • C5 cycloalkyl refers to a cycloalkyl group that has 5 carbon atoms in the ring.
  • C5-8 cycloalkyl refers to cycloalkyl groups having a number of carbon atoms encompassing the entire range (i.e., 5 to 10 carbon atoms), as well as all subgroups (e.g., 5-10, 5-9, 5-8, 5-6, 6-8, 7-8, 5-7, 5, 6, 7, 8, 9 and 10 carbon atoms).
  • Nonlimiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unless otherwise indicated, a cycloalkyl group can be an unsubstituted cycloalkyl group or a substituted cycloalkyl group.
  • aryl refers to an aromatic carbocycle, and can be monocyclic or polycyclic (e.g., fused bicyclic and fused tricyclic) carbocyclic aromatic ring systems.
  • aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, phenanthrenyl, biphenylenyl, indanyl, indenyl, anthracenyl, fluorenyl, tetralinyl.
  • an aryl group can be an unsubstituted aryl group or a substituted aryl group.
  • heterocycle is defined similarly as carbocycle, except the ring contains one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur.
  • a heterocycle can be a 3-10 membered aromatic or non-aromatic ring having 1 or 2 heteroatoms selected from N, O, and S.
  • a heterocycle can be a 5-6 membered ring having 1 or 2 ring heteroatoms selected from N, O, and S.
  • Nonlimiting examples of heterocycle groups include piperdine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, oxazepaneyl, thiazole, pyrrole, and pyridine.
  • Heterocyclic groups optionally can be further N-substituted as described herein. Other substituents contemplated for the disclosed rings is provided elsewhere in this disclosure.
  • heteroaryl refers to an aromatic heterocycle, and can be monocyclic or polycyclic (e.g., fused bicyclic and fused tricyclic) aromatic ring systems, wherein one to four-ring atoms are selected from oxygen, nitrogen, or sulfur, and the remaining ring atoms are carbon, said ring system being joined to the remainder of the molecule by any of the ring atoms.
  • heteroaryl groups include, but are not limited to, pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, tetrazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, furanyl, thienyl, quinolinyl, isoquinolinyl, benzoxazolyl, benzimidazolyl, benzofuranyl, benzothiazolyl, triazinyl, triazolyl, purinyl, pyrazinyl, purinyl, indolinyl, phthalzinyl, indazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl, pyridopyridinyl
  • hydroxy or “hydroxyl” as used herein refers to an “-OH” group. Accordingly, a “hydroxyalkyl” refers to an alkyl group substituted with one or more -OH groups.
  • alkoxy or “alkoxyl” refers to a “ — O-alkyl” group.
  • halo is defined as fluoro, chloro, bromo, and iodo. Accordingly, a “haloalkyl” refers to an alkyl group substituted with one or more halo atoms. A “haloalkoxy” refers to an alkoxy group that is substituted with one or more halo atoms.
  • a “substituted” functional group e.g., a substituted alkyl, cycloalkyl, aryl, or heteroaryl is a functional group having at least one hydrogen radical that is substituted with a nonhydrogen radical (i.e., a substituent).
  • non-hydrogen radicals 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, amido, oxy (or oxo), alkoxy, ester, thioester, acyl, carboxyl, cyano, nitro, amino, sulfhydryl, and halo.
  • the substituents can be bound to the same carbon or two or more different carbon atoms.
  • the compounds provided herein can be synthesized using conventional techniques readily available starting materials known to those skilled in the art. In general, the compounds provided herein are conveniently obtained via standard organic chemistry synthesis methods.
  • the synthetic processes disclosed herein can tolerate a wide variety of functional groups; therefore, various substituted starting materials can be used.
  • the processes generally provide the desired final compound at or near the end of the overall process, although it may be desirable in certain instances to further convert the compound to a pharmaceutically acceptable salt thereof.
  • the compounds of the disclosure can be synthesized in line with the examples shown below.
  • the compounds can be prepared by alkylation of the appropriate amine having a carboxyl group, with appropriate protecting groups as necessary.
  • the intermediate can be saponified, for example, to expose a reactive carboxylate. Then, amide coupling between the appropriate amine and the free carboxylate can occur.
  • the amine for the amide coupling noted above can be prepared via known synthetic techniques using appropriate starting materials and protecting groups, as necessary.
  • Single base extension reagent was formulated with compounds in accordance with the disclosure, as well as standard monovalent compounds as controls.
  • a single base extension regent having a compound in accordance with formula I was prepared by admixing single base extension premix (EML premix), SSBs, tTAQ608, and the labeled nucleotides.
  • EML premix single base extension premix
  • SSBs single base extension premix
  • tTAQ608 the labeled nucleotides.
  • G and C (green) channel detection conventional monovalent ddGTP-biotin and ddCTP-biotin labels were used.
  • a and T (red) channel detection following compounds in accordance with the disclosure were used.
  • diEML single base extension premix
  • Figure 1 shows a synthesis scheme for making the ddATP-diDNP
  • Figure 2 shows a synthesis scheme for making ddllTP-diDNP.
  • the ddATP-diDNP and ddllTP-diDNP were prepared by admixing 1 equiv bis-amine linker with 2.2 equiv DNP-NHS dissolved in 0.2M NaHCC>3:DMSO (1 .4 v/v). The admixture was stirred for 8 hours at room temperature. The mixture was purified by HPLC using water and ACN as eluant. The di-DNP-COOH product was then lyophilized. The lyophilized product was then reconstituted in DMF.
  • Controls were run using a convention, commercially available single base extension premix having monovalent labeled nucleotides (referred to herein as pEML), as well as a single base extension premix formulated with monovalent ddNTPs (referred to herein as devEML).
  • the formulated control included the same EML premix, SSBs, and tTAQ608 as used in the diEML, but included as the labeled nucleotides DNP-11 -ddATP, DNP-11 -ddUTP, as well as the same ddGTP-biotin and ddCTP-biotin used in diEML. Structures for DNP-1 1 -ddATP and DNP-11 -ddUTP are shown below.
  • Each of the reagents included a nominal ddNTP concentration of 0.125 pM.
  • the reagents were separately tested using an Infinium bead array (Illumina) with a standard MSA7 workflow (Infinium XT-ST workflow) with DPB purity instead of precipitate. Amplification was preformed using RAM 10 nm input with 18-hour incubation. The sample tested for this genotype testing included a single DNA type - NA11993.
  • Staining with the fluorescent labeled hapten binding partner was performed using the Xstain protocol in Tecan. Three staining rounds were performed and thee signal strength was evaluated with an iScan N392 after each round.
  • Figure 3 illustrates the signal in the red channel for each of the reagent samples tested.
  • the diEML having the compounds in accordance with the disclosure provided a detectible signal even after a single staining round.
  • Figure 3 further illustrates that ddNTPs coupled to PEG linkers with multiple DNP moieties can be incorporated by the tTaq608 polymerase using the same biochemical conditions as the monovalent ddNTP linker DNP species.
  • the increased signal resulting in a third round of staining as compared to the second round of staining was comparable between the diEML and pEML control, and that the diEML had a greater improvement in signal as compared to the devEML control.
  • Figure 5 illustrates that detection in the green channel with the biotin-modified ddGTP and ddCTP was unaffected by the use of the compounds in accordance with the claims.
  • the compounds of the disclosure were also evaluated using synthetic probes.
  • the same single base extension reagents as described in Example 1 were used, including the controls.
  • the example evaluated the extension efficiency of A, T, C, and G nucleotides from a hairpin probe. Both red (A, T), and green (C, G) channels were monitored.
  • Figure 6 is a schematic illustration of the process.
  • Figure 8 illustrates that increased signal was achieved with the A and T extension, in the diEML, utilizing the compound of the disclosure, as compared to the controls.
  • the dashed box in Figure 7 is the non-extension control set and no increase in signal was anticipated in that non-extension control set.
  • the purity of the hapten labeled ddNTPs was > 95% (HPLC).
  • the ddNTPs (standard and in accordance with the disclosure) were prepared in a 1 mM concentration in a formulation that included 10 mM Tris-HCI, 1 mM EDTA and had a pH of 7.6 ⁇ 0.2.
  • EML performance was measured using standard EML reagents in an Infinium EX microarray platform, which included a beadchip with 1 pm beads. Testing was done with decreasing antibody concentration in STX and ATX.
  • Standard EML reagents for testing the standard design ddNTPs included equimolar amounts of the standard design ddNTPS. Testing was performed with increasing concentrations of divalent ddNTPs in accordance with the disclosure. Testing conditions used are shown in the table below:

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Abstract

Les composés de l'invention comprennent des triphosphates didésoxynucléotides modifiés (ddNPT) comprenant de multiples unités de liaison contenant des ARM pour fournir des molécules d'unité de liaison multivalentes.
PCT/US2024/032844 2023-06-06 2024-06-06 Triphosphates didésoxynucléotides modifiés par une unité de liaison multivalente et leurs procédés de fabrication et d'utilisation Pending WO2024254329A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009054922A1 (fr) * 2007-10-19 2009-04-30 The Trustees Of Columbia University In The City Of New York Séquençage d'adn avec des terminateurs réversibles nucléotidiques non fluorescents et des terminateurs nucléotidiques modifiés par un marqueur séparable
WO2021007458A1 (fr) * 2019-07-09 2021-01-14 The Trustees Of Columbia University In The City Of New York Nouveaux analogues nucléotidiques et leurs procédés d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009054922A1 (fr) * 2007-10-19 2009-04-30 The Trustees Of Columbia University In The City Of New York Séquençage d'adn avec des terminateurs réversibles nucléotidiques non fluorescents et des terminateurs nucléotidiques modifiés par un marqueur séparable
WO2021007458A1 (fr) * 2019-07-09 2021-01-14 The Trustees Of Columbia University In The City Of New York Nouveaux analogues nucléotidiques et leurs procédés d'utilisation

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GREENE, T.W.WUTS, P.G. M.: "Protective Groups in Organic Synthesis", 1999, JOHN WILEY & SONS
KUMAR SHIV ET AL: "Synthesis and Energy Transfer Efficiency of FRET Terminators Derived from Different Linkers", NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, vol. 22, no. 5-8, 1 October 2003 (2003-10-01), US, pages 1595 - 1598, XP093203929, ISSN: 1525-7770, DOI: 10.1081/NCN-120023042 *
NAMPALLI SATYAM ET AL: "FLUORESCENCE RESONANCE ENERGY TRANSFER DYE NUCLEOTIDE TERMINATORS: A NEW SYNTHETIC APPROACH FOR HIGH-THROUGHOUT DNA SEQUENCING", NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, vol. 20, no. 4-7, 31 March 2001 (2001-03-31), US, pages 361 - 367, XP093128253, ISSN: 1525-7770, DOI: 10.1081/NCN-100002307 *
RAO T S ET AL: "Four color FRET dye nucleotide terminators for DNA sequencing", NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, TAYLOR & FRANCIS, US, vol. 20, no. 4-7, 1 January 2001 (2001-01-01), pages 673 - 676, XP002244469, ISSN: 1525-7770, DOI: 10.1081/NCN-100002348 *
RAO T S ET AL: "Synthesis of novel piperidinyl linker based energy transfer terminators and their potential use in DNA sequencing", NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, TAYLOR & FRANCIS, US, vol. 24, no. 5-7, 1 January 2005 (2005-01-01), pages 801 - 804, XP009090421, ISSN: 1525-7770, DOI: 10.1081/NCN-200060586 *
SMITH, M. B., MARCH: "J., March' s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure", 2001, JOHN WILEY & SONS

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