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WO2025085492A1 - Agents réducteurs boranes - Google Patents

Agents réducteurs boranes Download PDF

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Publication number
WO2025085492A1
WO2025085492A1 PCT/US2024/051528 US2024051528W WO2025085492A1 WO 2025085492 A1 WO2025085492 A1 WO 2025085492A1 US 2024051528 W US2024051528 W US 2024051528W WO 2025085492 A1 WO2025085492 A1 WO 2025085492A1
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Prior art keywords
hydrogen
alkyl
alkoxy
hydroxyalkyl
kit
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English (en)
Inventor
Joel Desharnais
Marta GARBACZ
Yibin Liu
Timothy Francis SUITS
Sarah WALSH
Thomas WILKS
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Exact Sciences Innovation Ltd
Exact Sciences Corp
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Exact Sciences Innovation Ltd
Exact Sciences Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/89—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • borane reducing reagents and in particular borane reducing reagents for use in methylation detection assays that comprise a step of reducing 5- formylcytosine (5fC) and/or 5-carboxylcytosine (5caC) to dihydrouracil (DHU).
  • Methylation is a major epigenetic modification in the mammalian genome that is associated with biological and pathological processes, including as well -accepted biomarkers of various diseases and cancer.
  • other modifications such as oxidation of methylated cytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and/or 5- carboxylcytosine (5caC), and methylation of adenine (A) to N 6 -methyladenine (6-mA), have also been identified as important epigenetic regulators. Therefore, detection of epigenetic modifications, such as methylation, has become critically important for scientific/diagnostic purposes.
  • Methylation is determined, for example, by a whole-genome, base-resolution, and quantitative sequencing method, such as bisulfite sequencing.
  • bisulfite sequencing is damaging to the nucleic acid and expensive; therefore, current methylation sequencing is limited by being low-depth, targeted, or low-resolution and qualitative enrichment-based sequencing.
  • borane reducing reagents and in particular borane reducing reagents for use in methylation detection assays that comprise a step of reducing 5- formylcytosine (5fC) and/or 5-carboxylcytosine (5caC) to dihydrouracil (DHU).
  • Embodiments of the present disclosure include the following.
  • the present invention provides methods for converting 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC) to dihydrouracil (DHU) comprising contacting a nucleic acid sample comprising 5caC and/or 5fC with a borane reducing agent of formula (I): or a salt thereof, wherein:
  • A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclyl, each of which is substituted with R 1 , R 2 , R 3 , R 4 , and R 5 ;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, halo, hydroxy, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 aminoalkyl, C 1 -C 4 mercaptoalkyl, C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl, amidoximyl, aryl, and heterocyclyl; and each X is independently hydrogen or deuterium, wherein the compound is not:
  • the present invention provides methods for converting 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC) to dihydrouracil (DHU) comprising contacting a nucleic acid sample comprising 5caC and/or 5fC with a borane reducing agent of formula (I): or a salt thereof, wherein:
  • A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclyl, each of which is substituted with R 1 , R 2 , R 3 , R 4 , and R 5 ;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, halo, hydroxy, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 aminoalkyl, C 1 -C 4 mercaptoalkyl, C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl, and amidoximyl; and each X is independently hydrogen or deuterium, wherein the compound is not:
  • A is selected from pyridine, imidazole, quinoline, 9,10-dihydroacridine, and imidazo[l,5-a]pyridine.
  • the borane reducing agent is a compound of formula (la):
  • the borane reducing agent is a compound of formula (la): wherein:
  • R 1 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, C 1 -C 4 hydroxyalkyl, C 1 -C 4 mercaptoalkyl, and C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl;
  • R 2 is selected from hydrogen, halo, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, and amidoximyl; wherein R 1 and R 2 are optionally taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring optionally substituted with a hydroxy group;
  • R 3 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 mercaptoalkyl, amidoximyl, and aryl;
  • R 4 is selected from hydrogen, halo, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, and heterocyclyl; wherein R 3 and R 4 are optionally taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring; and
  • R 5 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 hydroxyalkyl.
  • R 1 and R 2 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring.
  • R 3 and R 4 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring.
  • R 1 and R 2 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring, R 3 and R 4 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring.
  • R 1 and R 2 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring substituted with a hydroxy group.
  • R 3 and R 4 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring.
  • R 1 and R 2 are taken together with the carbon atoms to which they are attached to form a 5- or 6- membered ring substituted with a hydroxy group
  • R 3 and R 4 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring.
  • R 1 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, C 1 -C 4 hydroxyalkyl, C 1 -C 4 mercaptoalkyl, and C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl;
  • R 2 is selected from hydrogen, halo, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, and amidoximyl;
  • R 3 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 mercaptoalkyl, and amidoximyl;
  • R 4 is selected from hydrogen, halo, hydroxy, C 1 -C 4 alkyl, and C 1 -C 4 hydroxyalkyl;
  • R 1 is C 1 -C 4 hydroxyalkyl
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, phenyl, and a monocyclic 5- or 6-membered heterocyclyl having one nitrogen atom.
  • R 1 is hydroxymethyl
  • R 2 is hydrogen
  • R 3 is selected from hydrogen, C 1 -C 4 alkyl, and phenyl;
  • R 4 is selected from C 1 -C 4 alkyl, C 1 -C 4 alkoxy, and a monocyclic 5-membered heterocyclyl having one nitrogen atom;
  • R 5 is hydrogen
  • R 1 is C 1 -C 4 hydroxyalkyl
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy.
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, wherein at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is hydroxymethyl.
  • R 1 is selected from hydrogen, methyl, and hydroxymethyl
  • R 2 is selected from hydrogen, methyl, methoxy, and hydroxymethyl
  • R 3 is selected from hydrogen, methyl, methoxy, and hydroxymethyl
  • R 4 is selected from hydrogen and methyl
  • R 5 is hydrogen; wherein at least one of R 1 , R 2 , and R 3 is hydroxymethyl.
  • the borane reducing agent is a compound of formula (lb): or a salt thereof, wherein:
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, aryl, and heterocyclyl; and each X is independently hydrogen or deuterium.
  • the borane reducing agent is a compound of formula (lb): or a salt thereof, wherein:
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy; and each X is independently hydrogen or deuterium.
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, phenyl, and a monocyclic 5-membered heterocyclyl having one nitrogen atom.
  • R 2 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy;
  • R 3 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy;
  • R 4 is selected from hydrogen and C 1 -C 4 alkyl
  • R 5 is hydrogen
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidinyl.
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, and methoxy.
  • R 2 is selected from hydrogen, methyl, and methoxy
  • R 3 is selected from hydrogen, methyl, and methoxy
  • R 4 is selected from hydrogen and methyl
  • R 5 is hydrogen
  • each X is hydrogen.
  • each X is deuterium.
  • the borane reducing agent is selected from: [0031] In some preferred embodiments, the borane reducing agent is selected from: , and salts thereof.
  • the borane reducing agent is selected from:
  • the methods further comprise contacting the nucleic acid sample with an oxidizing agent prior to contacting with the borane reducing agent.
  • the oxidizing agent is a Ten-Eleven Translocation (TET) enzyme.
  • the TET enzyme comprises human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3, Naegleria gulberi TET (AgTET), Coprinopsis cinerea TET (CcTET), or derivatives or analogs thereof.
  • the oxidizing agent comprises a chemical oxidizing agent.
  • the chemical oxidizing agent is selected from the group consisting of manganese oxide (MnCE), potassium perruthenate (KRuO 4 ), Cu(II)/ 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenylphosphoniurn ruthenate, copper salts or complexes of 3 -carbamoyl-2, 2,5,5- tetramethyl-3 -pyrrolin- 1-yloxy (3 -Carbamoyl-PROXYL), copper salts or complexes of 2- azaadamantane-N-oxyl (AZADO), and copper salts or complexes of 9-azabicyclo [3.3.1
  • the methods further comprise adding a blocking group to one or more modified cytosines in the nucleic acid sample.
  • the blocking group is added prior to contacting with the oxidizing agent.
  • the one or more modified cytosines comprises 5hmC.
  • the blocking group comprises a sugar or a uridine diphosphate (UDP)-linked sugar.
  • the blocking group is added after contacting with the oxidizing agent and prior to contacting with the borane reducing agent.
  • the one or more modified cytosines comprises 5caC or 5fC.
  • the blocking group comprises an aldehyde reactive compound.
  • the aldehyde reactive compound comprises a hydroxylamine derivative, a hydrazine derivative, or a hydrazide derivative.
  • adding the blocking group comprises contacting the nucleic acid sample with (i) a coupling agent and (ii) an amine, hydrazine, or hydroxylamine compound.
  • the methods further comprise sequencing the nucleic acid sample after contacting with the borane reducing agent to identify converted cytosine bases.
  • the methods further comprise amplifying the copy number of the nucleic acid sample.
  • the amplifying is performed after contacting the nucleic acid sample with the borane reducing agent.
  • the amplifying is performed before sequencing the nucleic acid sample.
  • the amplifying is performed after contacting the nucleic acid sample with the borane reducing agent and before sequencing the nucleic acid sample.
  • the nucleic acid sample is a DNA sample.
  • the present invention provides a compound selected from:
  • the present invention provides a compound selected from:
  • the present invention provides a compound selected from: and salts thereof.
  • the present invention provides a system or kit for converting 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC) to dihydrouracil (DHU), comprising a borane reducing reagent of formula (I): or a salt thereof, wherein:
  • A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclyl, each of which is substituted with R 1 , R 2 , R 3 , R 4 , and R 5 ;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, halo, hydroxy, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 aminoalkyl, C 1 -C 4 mercaptoalkyl, C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl, amidoximyl, aryl, and heterocyclyl; and each X is independently hydrogen or deuterium, wherein the compound is not:
  • the present invention provides a system or kit for converting 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC) to dihydrouracil (DHU), comprising a borane reducing reagent of formula (I): or a salt thereof, wherein:
  • A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclyl, each of which is substituted with R 1 , R 2 , R 3 , R 4 , and R 5 ;
  • R 3 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 mercaptoalkyl, amidoximyl, and aryl;
  • R 5 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 hydroxyalkyl.
  • R 1 and R 2 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring substituted with a hydroxy group.
  • R 3 and R 4 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring.
  • R 1 and R 2 are taken together with the carbon atoms to which they are attached to form a 5- or 6- membered ring substituted with a hydroxy group
  • R 3 and R 4 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring.
  • R 1 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, C 1 -C 4 hydroxyalkyl, C 1 -C 4 mercaptoalkyl, and C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl;
  • R 2 is selected from hydrogen, halo, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, and amidoximyl;
  • R 3 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 mercaptoalkyl, and amidoximyl;
  • R 4 is selected from hydrogen, halo, hydroxy, C 1 -C 4 alkyl, and C 1 -C 4 hydroxyalkyl;
  • R 5 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 hydroxyalkyl.
  • R 1 is C 1 -C 4 hydroxyalkyl
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, phenyl, and a monocyclic 5- or 6-membered heterocyclyl having one nitrogen atom.
  • R 1 is hydroxymethyl
  • R 2 is hydrogen
  • R 3 is selected from hydrogen, C 1 -C 4 alkyl, and phenyl;
  • R 4 is selected from C 1 -C 4 alkyl, C 1 -C 4 alkoxy, and a monocyclic 5-membered heterocyclyl having one nitrogen atom;
  • R 5 is hydrogen
  • R 1 is C 1 -C 4 hydroxyalkyl
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy.
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, wherein at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is hydroxymethyl.
  • R 1 is selected from hydrogen, methyl, and hydroxymethyl
  • R 2 is selected from hydrogen, methyl, methoxy, and hydroxymethyl
  • R 3 is selected from hydrogen, methyl, methoxy, and hydroxymethyl
  • R 4 is selected from hydrogen and methyl
  • R 5 is hydrogen; wherein at least one of R 1 , R 2 , and R 3 is hydroxymethyl.
  • the borane reducing agent is a compound of formula (lb): or a salt thereof, wherein:
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, aryl, and heterocyclyl; and each X is independently hydrogen or deuterium.
  • the borane reducing agent is a compound of formula (lb): or a salt thereof, wherein:
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy; and each X is independently hydrogen or deuterium.
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, phenyl, and a monocyclic 5-membered heterocyclyl having one nitrogen atom.
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy.
  • R 2 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy;
  • R 3 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy;
  • R 4 is selected from hydrogen and C 1 -C 4 alkyl
  • R 5 is hydrogen
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidinyl.
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, and methoxy.
  • R 2 is selected from hydrogen, methyl, and methoxy
  • R 3 is selected from hydrogen, methyl, and methoxy
  • R 4 is selected from hydrogen and methyl
  • R 5 is hydrogen
  • each X is hydrogen.
  • each X is deuterium.
  • the borane reducing agent is selected from:
  • the borane reducing agent is selected from: and salts thereof.
  • the borane reducing agent is selected from: [0075]
  • the system or kit further comprises an oxidizing reagent.
  • the oxidizing agent is a ten-eleven translocation (TET) enzyme.
  • the TET enzyme comprises human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3, Naegleria gulberi TET (AgTET), Coprinopsis cinerea (CcTET), or derivatives or analogues thereof.
  • the oxidizing agent comprises a chemical oxidizing agent.
  • the chemical oxidizing agent is selected from the group consisting of manganese oxide (MnCL), potassium perruthenate (KRuO 4 ), Cu(II)/ 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenylphosphoniurn ruthenate, copper salts or complexes of 3 -carbamoyl-2, 2,5,5- tetramethyl-3 -pyrrolin- 1-yloxy (3 -Carbamoyl-PROXYL), copper salts or complexes of 2- azaadamantane-N-oxyl (AZADO), and copper salts or complexes of 9-azabicyclo [3.3.1
  • the system or kit further comprises a blocking reagent.
  • the blocking reagent is selected from the group consisting of a sugar, a uridine diphosphate (UDP)-linked sugar, and an aldehyde reactive compound.
  • the aldehyde reactive compound is selected from the group consisting of a hydroxylamine derivative, a hydrazine derivative, and a hydrazide derivative.
  • the blocking reagent is an aldehyde reactive compound selected from the group consisting of a hydroxylamine derivative, a hydrazine derivative, and a hydrazide derivative.
  • the blocking reagent is a sugar or a uridine diphosphate (UDP)-linked sugar, and the system or kit further comprises a glucosyltransferase enzyme.
  • FIG. 1 General GC bias plot comparing the use of either 2-methylpyridine borane complex (“Pic”, solid lines) or 2-hydroxymethylpyridine borane complex (“ESI047”, dashed line) in a TAPS reaction on fully methylated Lambda and partially methylated pUC19 templates.
  • the general GC bias plot represents bias across human genomic DNA that is a component of a library consisting of 92.975% NA12878 (human genomic DNA); 5% methylated Lambda; 2% methylated pUC19; and 0.025% unmethylated 2kb DNA spike-ins.
  • FIG. 2. Lambda GC bias plot comparing the use of either 2-methylpyridine borane complex (“Pic”, solid lines) or 2-hydroxymethylpyridine borane complex (“ESI047”, dashed line) in a TAPS reaction.
  • FIG. 3 Plot comparing library complexity of samples produced by a TAPS reaction with either 2-methylpyridine borane complex (“Pic-KU”, solid line) or 2- hydroxymethylpyridine borane complex (“ESI47-KU”, short dashed line).
  • FIG. 4A-F Graphs showing normalized coverage of selected biomarkers ((A) B3GALT6, (B) ZMYM4, (C) ZDHHC1, (D) TRIO, (E) ODZ2, (F) ZFAND3) in samples produced by a TAPS reaction with either 2-methylpyridine borane complex (“Pic-KU”) or 2- hydroxymethylpyridine borane complex (“ESI47-KU”).
  • biomarkers (A) B3GALT6, (B) ZMYM4, (C) ZDHHC1, (D) TRIO, (E) ODZ2, (F) ZFAND3) in samples produced by a TAPS reaction with either 2-methylpyridine borane complex (“Pic-KU”) or 2- hydroxymethylpyridine borane complex (“ESI47-KU”).
  • FIG. 5 Graph showing post-TAPS amplification yields for TAPS reduction reactions using selected pyridine borane complexes. The black bars represent reactions with pre-extension while the gray bars represent reactions without pre-extension. Mean values from two replicates are presented.
  • FIG. 6 Graph showing lambda conversion rates (%) for TAPS reduction reactions using selected pyridine borane complexes. The black bars represent reactions with pre- extension while the gray bars represent reactions without pre-extension. Mean values from two replicates are presented.
  • FIG. 7 Graph showing false positive rates (%) for TAPS reduction reactions using selected pyridine borane complexes. The black bars represent reactions with pre-extension while the gray bars represent reactions without pre-extension. Mean values from two replicates are presented.
  • FIG. 8 Graph showing lambda GC-dropout for TAPS reduction reactions using selected pyridine borane complexes. The black bars represent reactions with pre-extension while the gray bars represent reactions without pre-extension. Mean values from two replicates are presented.
  • FIG. 9 Graph showing general GC-dropout for TAPS reduction reactions using selected pyridine borane complexes. The black bars represent reactions with pre-extension while the gray bars represent reactions without pre-extension. Mean values from two replicates are presented.
  • FIGs. 10A-G Lambda GC bias plots comparing the use of either 2-methylpyridine borane complex (“picB”, solid line with pre-extension, dotted line without pre-extension) or a test pyridine borane complex (FIG. 10A “ESI 047”; FIG. 10B “ESI 070”; FIG. 10C “ESI 075”; FIG. 10D “ESI 076”; FIG, 10E “ESI 079”; FIG. 10F “ESI 090”; FIG. 10G “ESI 093”; solid circle with pre-extension, open circle without pre-extension) in a TAPS reaction.
  • FIGs. 11 A-G General GC bias plots comparing the use of either 2-methylpyridine borane complex (“picB”, solid line with pre-extension, dotted line without pre-extension) or a test pyridine borane complex (FIG 11 A “ES1047”; FIG 1 IB “ES1070”; FIG 11C “ES1075”; FIG 11D “ES1076”; FIG, 11E“ES1O79”; FIG 1 IF “ES1090”; FIG 11G “ESI 093”; solid circle with pre-extension, open circle without pre-extension) in a TAPS reaction.
  • picB solid line with pre-extension, dotted line without pre-extension
  • FIG 11D ES1076
  • FIG, 11E“ES1O79” FIG 1 IF “ES1090”
  • FIG 11G “ESI 093” solid circle with pre-extension, open circle without pre-extension
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • methylation refers to any and all processes by which methyl group(s) are added to a nucleic acid.
  • methylation may include, but is not limited to, the addition of methyl groups at positions C5 or N4 of cytosine or at the N6 position of adenine.
  • a “methylated nucleotide base” or “methylated nucleotide” refers to the presence of a methyl moiety on a nucleotide base, where the methyl moiety is not present in a recognized canonical nucleotide base.
  • cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide and 5-methylcytosine is a methylated nucleotide.
  • a “methylated nucleic acid” refers to a nucleic acid molecule that contains one or more methylated nucleotides.
  • a nucleic acid molecule containing a methylated cytosine is considered methylated (e.g., the methylation state of the nucleic acid molecule is methylated).
  • a nucleic acid molecule that does not contain any methylated nucleotides is considered unmethylated.
  • nucleic acid or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)).
  • the present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like.
  • the polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced.
  • the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
  • a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No.
  • LNA locked nucleic acid
  • cyclohexenyl nucleic acids see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000), and/or a ribozyme.
  • nucleic acid or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double- stranded, and represent the sense or antisense strand.
  • nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein.
  • a subject or patient may include either adults or juveniles (e.g., children) and may mean any living organism, such as a mammal (e.g., human or non-human).
  • mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs, and the like.
  • non-mammals include, but are not limited to, birds, fish, and the like.
  • the mammal is a human.
  • nucleic acid sample refers to nucleic acid obtained from an organism from the Monera (bacteria), Protista, Fungi, Plantae, and Animalia Kingdoms.
  • the nucleic acid may also be obtained from a virus.
  • Nucleic acid samples may be obtained from a patient or subject, from an environmental sample, or from an organism of interest (e.g., both cellular and circulating cell-free DNA (cfDNA), for instance obtained from tissue (including tissue from the lymph gland, breast, liver, bile ducts, pancreas, mouth, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gall bladder, anus, prostate, endometrium, vagina, ovary, cervix, skin, bladder, kidney, lung, and/or peritoneum), biopsies, a cell, collection of cells, blood, plasma, serum, secretions (such as an organ secretion, vaginal secretions, or gastric secretions), semen (seminal fluid), cerebral spinal fluid (CSF), saliva, mucus, urine, stool, sweat, pancreatic juice, gastric fluid (gastric lavage), ascitic fluid, synovial fluid, pleural fluid (pleural
  • the target nucleic acid may be obtained from a sample that contains diseased tissue or cells, or is suspected of containing diseased tissue or cells (e.g., a sample that is cancerous, or contains cancerous tissue or cells, or is suspected of being cancerous or suspected of containing cancerous tissue or cells).
  • the nucleic acid sample is obtained from a subject that has a disease or disorder (e.g., cancer), is suspected of having the disease or disorder, or is being screened to determine the presence of the disease or disorder.
  • the nucleic acid sample is circulating cell-free DNA (cell-free DNA or cfDNA), for instance DNA found in the blood and is not present within a cell.
  • cfDNA can be isolated from a bodily fluid using methods known in the art.
  • Commercial kits are available for isolation of cfDNA including, for example, the Circulating Nucleic Acid Kit (Qiagen).
  • the nucleic acid sample may result from an enrichment step, including, but not limited to antibody immunoprecipitation, chromatin immunoprecipitation, restriction enzyme digestion-based enrichment, hybridization-based enrichment, or chemical labeling-based enrichment.
  • alkyl refers to a radical of a straight or branched saturated hydrocarbon chain.
  • the alkyl chain can include, e.g., from 1 to 16 carbon atoms (C 1 -C 16 alkyl), 1 to 14 carbon atoms (C 1 -C 14 alkyl), 1 to 12 carbon atoms (C 1 -C 12 alkyl), 1 to 10 carbon atoms (C 1 -C 10 alkyl), 1 to 8 carbon atoms (C 1 -C 8 alkyl), 1 to 6 carbon atoms (C 1 - C 6 alkyl), 1 to 4 carbon atoms (C 1 -C 4 alkyl), 1 to 3 carbon atoms (C 1 -C 3 alkyl), or 1 to 2 carbon atoms (C 1 -C 2 alkyl).
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3 -methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.
  • alkoxy refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
  • Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.
  • alkoxyalkyl refers to an alkyl group, as defined herein, in which at least one hydrogen atom is replaced with an alkoxy group, as defined herein.
  • Representative examples of alkoxyalkyl include, but are not limited to, methoxymethyl and 2-methoxy ethyl.
  • amino refers to a group -N(R)2, wherein each R is independently hydrogen or alkyl (as defined herein). When one R is hydrogen and the other is alkyl, the group may be referred to as “alkylamino,” and when both R are alkyl, the group may be referred to as “dialkylamino.”
  • aminoalkyl refers to an alkyl group, as defined herein, in which at least one hydrogen atom is replaced with an amino group, as defined herein.
  • aryl refers to a radical of a monocyclic, bicyclic, or tricyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 71 electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms (“C 6 -C 14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C 6 aryl,” i.e., phenyl).
  • an aryl group has ten ring carbon atoms (“C 10 aryl,” e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C 14 aryl,” e.g., anthracenyl and phenanthrenyl).
  • halo or halogen refers to fluoro, chloro, bromo, or iodo.
  • haloalkyl refers to an alkyl group, as defined herein, in which at least one hydrogen atom is replaced with a halogen. In some embodiments, each hydrogen atom of the alkyl group is replaced with a halogen.
  • Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2- trifluoroethyl, and 3,3,3-trifluoropropyl.
  • heteroaryl refers to an aromatic group having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic) having one or more ring heteroatoms independently selected from O, N, and S.
  • the aromatic monocyclic rings are five- or six-membered rings containing at least one heteroatom independently selected from O, N, and S (e.g. 1, 2, 3, or 4 heteroatoms independently selected from O, N, and S).
  • the five-membered aromatic monocyclic rings have two double bonds, and the six- membered aromatic monocyclic rings have three double bonds.
  • the bicyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring appended fused to a monocyclic aryl group, as defined herein, or a monocyclic heteroaryl group, as defined herein.
  • the tricyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring fused to two rings independently selected from a monocyclic aryl group, as defined herein, and a monocyclic heteroaryl group as defined herein.
  • monocyclic heteroaryl include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4- thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl.
  • pyridinyl including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl
  • pyrimidinyl pyrazinyl
  • bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzodi oxolyl, benzofuranyl, benzooxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, benzotri azolyl, benzoxadiazolyl, benzoxazolyl, chromenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thi enopyrrolyl, and thi enothienyl.
  • tricyclic heteroaryl include, but are not limited to, dibenzimidazo
  • heterocycle refers to a saturated or partially unsaturated non-aromatic cyclic group having one or more ring heteroatoms independently selected from O, N, and S.
  • the heterocycle can be monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle.
  • the monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from O, N, and S.
  • the three- or four-membered ring contains zero or one double bond, and one heteroatom selected from O, N, and S.
  • the five-membered ring contains zero or one double bond and one, two or three heteroatoms selected from O, N, and S.
  • the six- membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from O, N, and S.
  • the seven- and eight-membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from O, N, and S.
  • the heteroatom in the ring can be oxidized (e.g., if the ring heteroatom is S, it can be oxidized to SO or SO2).
  • monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro
  • the bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocycle group, or a bridged monocyclic heterocycle ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms.
  • bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3 -dihydrobenzothienyl, 2,3- dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2- azabicyclo[2.2.
  • Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms.
  • tricyclic heterocycles include, but are not limited to, octahydro- 2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan,hexahydro-lH-l,4- methanocyclopenta[c]furan, aza-adamantane (l-azatricyclo[3.3.1.1 3 ’ 7 ]decane), and oxa- adamantane (2-oxatricyclo[3.3.1. l 3,7 ]decane).
  • hydroxy refers to an -OH group.
  • hydroxyalkyl refers to an alkyl group, as defined herein, in which at least one hydrogen atom is replaced with a hydroxy group. Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2 -hydroxy ethyl, and 3-hydroxypropyl.
  • mercapto means an -SH group.
  • mercaptoalkyl means an alkyl group, as defined herein, in which at least one hydrogen atom is replaced with a mercapto group.
  • Representative examples of mercaptoalkyl include, but are not limited to, mercaptomethyl, 2-mercaptoethyl, 2-mercaptopropyl, 3 -mercaptopropyl, and 4-mercaptobutyl.
  • any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom.
  • a position is designated as “H” or “hydrogen,” the position is understood to have hydrogen at its natural abundance isotopic composition.
  • D when a position is designated specifically as “D” or “deuterium,” the position is understood to have deuterium at an abundance that is at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% incorporation of deuterium).
  • the position has deuterium at an abundance that is at least 3500 times greater than the natural abundance of deuterium (52.5% deuterium incorporation), at least 4000 times greater than the natural abundance of deuterium (60% deuterium incorporation), at least 4500 times greater than the natural abundance of deuterium (67.5% deuterium incorporation), at least 5000 times greater than the natural abundance of deuterium (75% deuterium), at least 5500 times greater than the natural abundance of deuterium (82.5% deuterium incorporation), at least 6000 times greater than the natural abundance of deuterium (90% deuterium incorporation), at least 6333.3 times greater than the natural abundance of deuterium (95% deuterium incorporation), at least 6466.7 times greater than the natural abundance of deuterium (97% deuterium incorporation), at least 6600 times greater than the natural abundance of deuterium (99% deuterium incorporation), or at least 6633.3 times greater than the natural abundance of deuterium (99.5% deuterium incorporation).
  • Embodiments of the present disclosure provide borane reducing reagents (also referred to herein as borane complexes).
  • the borane reducing reagents may be used in methods for detecting 5-methylcytosine (5mC), 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and/or 5-carboxylcytosine (5caC) in a target nucleic acid sequence.
  • the borane reducing reagents that can be used in the methods disclosed herein include compounds of formula (I): and salts thereof, wherein:
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, halo, hydroxy, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 aminoalkyl, C 1 -C 4 mercaptoalkyl, C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl, amidoximyl, aryl, and heterocyclyl; and each X is independently hydrogen or deuterium, wherein the compound is not:
  • the borane reducing reagents that can be used in the methods disclosed herein include compounds of formula (I): and salts thereof, wherein: A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclyl, each of which is substituted with R 1 , R 2 , R 3 , R 4 , and R 5 ;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, halo, hydroxy, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 aminoalkyl, C 1 -C 4 mercaptoalkyl, C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl, and amidoximyl; and each X is independently hydrogen or deuterium, wherein the compound is not:
  • A is selected from pyridine, imidazole, quinoline, 9,10- dihydroacridine, and imidazo[l,5-a]pyridine. In some embodiments, A is pyridine.
  • the borane reducing agent is a compound of formula (la): wherein:
  • R 1 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, C 1 -C 4 hydroxyalkyl, C 1 -C 4 mercaptoalkyl, and C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl;
  • R 2 is selected from hydrogen, halo, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, and amidoximyl; wherein R 1 and R 2 are optionally taken together with the carbon atoms to which they are attached to form a 5- or 6-membered ring optionally substituted with a hydroxy group;
  • R 3 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 mercaptoalkyl, amidoximyl, and aryl;
  • R 5 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 hydroxyalkyl.
  • R 1 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, Ci- C4 hydroxyalkyl, C 1 -C 4 mercaptoalkyl, and C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl
  • R 2 is selected from hydrogen, halo, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, and amidoximyl
  • R 3 is selected from hydrogen, halo, amino, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 1 -C 4 hydroxyalkyl, C 1 -C 4 haloalkyl, C 1 -C 4 mercaptoalkyl, and amidoximyl
  • R 4 is selected from hydrogen, halo, hydroxy, C 1 -C 4 alkyl, and C 1 -C 4 hydroxyalkyl
  • R 5 is selected from hydrogen
  • R 1 is C 1 -C 4 hydroxyalkyl
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, phenyl, and a monocyclic 5- or 6-membered heterocyclyl having one nitrogen atom.
  • R 1 is hydroxymethyl
  • R 2 is hydrogen
  • R 3 is selected from hydrogen, C 1 -C 4 alkyl, and phenyl
  • R 4 is selected from C 1 -C 4 alkyl, C 1 -C 4 alkoxy, and a monocyclic 5-membered heterocyclyl having one nitrogen atom
  • R 5 is hydrogen.
  • At least one of R 1 , R 2 , R 3 , R 4 , and R 5 is not hydrogen. In some embodiments, at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is not hydrogen or methyl. In some embodiments, at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is C 1 -C 4 hydroxyalkyl.
  • R 1 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 hydroxyalkyl
  • R 2 is selected from hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, and C 1 -C 4 hydroxyalkyl
  • R 3 is selected from hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, and C 1 -C 4 hydroxyalkyl
  • R 4 is selected from hydrogen and C 1 -C 4 alkyl
  • R 5 is hydrogen; wherein at least one of R 1 , R 2 , and R 3 is C 1 -C 4 hydroxyalkyl.
  • R 1 is C 1 -C 4 hydroxyalkyl
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy.
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, wherein at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is hydroxymethyl.
  • R 1 is selected from hydrogen, methyl, and hydroxymethyl
  • R 2 is selected from hydrogen, methyl, methoxy, and hydroxymethyl
  • R 3 is selected from hydrogen, methyl, methoxy, and hydroxymethyl
  • R 4 is selected from hydrogen and methyl
  • R 5 is hydrogen; wherein at least one of R 1 , R 2 , and R 3 is hydroxymethyl.
  • each X is hydrogen. In some embodiments, each X is deuterium.
  • the borane reducing agent is a compound of formula (lb): or a salt thereof, wherein:
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, C 1 -
  • the borane reducing agent is a compound of formula (lb): or a salt thereof, wherein:
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy; and each X is independently hydrogen or deuterium.
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, phenyl, a monocyclic 5-membered heterocyclyl having one nitrogen atom. In some embodiments, R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy.
  • R 2 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy
  • R 3 is selected from hydrogen, C 1 -C 4 alkyl, and C 1 -C 4 alkoxy
  • R 4 is selected from hydrogen and C 1 -C 4 alkyl
  • R 5 is hydrogen.
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidinyl.
  • R 2 is hydrogen; R 3 is selected from hydrogen, C 1 -C 4 alkyl, and phenyl; R 4 is selected from hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, and a monocyclic 5-membered heterocyclyl having one nitrogen atom; and R 5 is hydrogen.
  • R 2 is hydrogen; R 3 is selected from hydrogen, methyl, and phenyl; R 4 is selected from hydrogen, methyl, methoxy, and pyrrolidinyl; and R 5 is hydrogen.
  • R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, and methoxy.
  • R 2 is selected from hydrogen, methyl, and methoxy
  • R 3 is selected from hydrogen, methyl, and methoxy
  • R 4 is selected from hydrogen and methyl
  • R 5 is hydrogen.
  • each X is hydrogen. In some embodiments, each X is deuterium.
  • the compound of formula (I) (or compound of formula (la) or (lb)) is selected from:
  • the compound of formula (I) (or compound of formula (la) or (lb)) is selected from:
  • the compound of formula (I) (or compound of formula (la) or (lb)) is selected from: , .
  • the compound of formula (I) (or compound of formula (la) or (lb)) is selected from:
  • the compound of formula (I) (or compound of formula
  • (la) or (lb)) is: , or a salt thereof.
  • the compound of formula (I) (or compound of formula (la) or (lb)) is: , or a salt thereof.
  • the compound of formula (I) (or compound of formula (la) or (lb)) is:
  • the compound of formula (I) (or compound of formula (la) or (lb)) is: [0149] In some embodiments, the compound of formula (I) (or compound of formula (la) or (lb)) is: , or a salt thereof.
  • the compound of formula (I) (or compound of formula (la) or (lb)) is: , or a salt thereof.
  • Embodiments of the present disclosure provide a bi sulfite-free, base-resolution method for detecting 5-methylcytosine (5mC) and/or 5-hydroxymethylcytosine (5hmC) in a sequence (e.g., TAPS and associated methods TAPS ⁇ and CAPS, referred to collectively as TAPS), including for use with DNA obtained from blood samples (cellular DNA as well as cfDNA) and biopsies.
  • TAPS 5-methylcytosine
  • 5hmC 5-hydroxymethylcytosine
  • TAPS comprises the use of mild enzymatic and chemical reactions to detect 5mC and/or 5hmC directly and quantitatively at base- resolution without affecting unmodified cytosine.
  • the present disclosure also provides methods to detect 5-formylcytosine (5fC) and/or 5-carboxylcytosine (5caC) at base resolution without affecting unmodified cytosine.
  • the methods provided herein provide mapping of 5mC, 5hmC, 5fC and/or 5caC and overcome the disadvantages of previous methods such as bisulfite sequencing.
  • the methods of the present disclosure can include the step of converting the 5mC and 5hmC (or just the 5mC if the 5hmC is blocked) to 5caC and/or 5fC.
  • this step comprises contacting the nucleic acid sample, e.g., DNA or RNA sample, with a Ten Eleven Translocation (TET) enzyme.
  • TET Ten Eleven Translocation
  • the TET enzymes are a family of enzymes that catalyze the transfer of an oxygen molecule to the C5 methyl group on 5mC resulting in the formation of 5-hydroxymethylcytosine (5hmC).
  • TET further catalyzes the oxidation of 5hmC to 5fC and the oxidation of 5fC to form 5caC.
  • TET enzymes useful in the methods of the present disclosure include, but are not limited to, one or more of human TET1, TET2, and TET3; murine TET1, TET2, and TET3; Naegleria gulberi TET (AgTET); Coprinopsis cinerea TET (CcTET); the catalytic domain of mouse TET1 (mTETICD); and derivatives or analogues thereof.
  • Methods of the present disclosure can also include the step of converting the 5caC and/or 5fC in a nucleic acid sample to DHU.
  • this step comprises contacting the nucleic acid sample, e.g., DNA or RNA sample, with a borane reducing agent as described in the preceding section.
  • the step of converting the 5hmC to 5fC comprises oxidizing the 5hmC to 5fC by contacting the nucleic acid sample, e.g., DNA sample, with, for example, manganese oxide (MnCE), potassium perruthenate (KRuO 4 ), Cu(II)/ 2,2,6 ,6- tetramethylpiperidine-l-oxyl (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenylphosphoniurn ruthenate, copper salts or complexes of 3 -carbamoyl-2, 2,5,5- tetramethyl-3 -pyrrolin- 1-yloxy (3 -Carbamoyl-PROXYL), copper salts or complexes of 2- azaadamantane-N-oxyl (AZADO)
  • MnCE manganes
  • the methods of the present disclosure include identifying 5mC in a nucleic acid sample, e.g., DNA sample (targeted DNA or whole-genome), and providing a quantitative measure for the frequency of the 5mC modification at each location where the modification was identified in the nucleic acid sample, e.g., DNA sample.
  • the percentages of the T at each transition location provide a quantitative level of 5mC at each location in the nucleic acid sample, e.g., DNA sample.
  • methods for identifying 5mC can include the use of a blocking group. In other embodiments, methods for identifying 5mC do not require the use of a blocking group.
  • the 5hmC in the sample is blocked so that it is not subject to conversion to 5caC and/or 5fC.
  • the 5hmC in the nucleic acid sample e.g., DNA sample
  • the blocking group is a sugar, including a modified sugar, for example glucose or 6-azide-glucose (6-azido-6-deoxy-D-glucose).
  • the sugar blocking group can be added to the hydroxymethyl group of 5hmC by contacting the nucleic acid sample, e.g., DNA sample, with uridine diphosphate (UDP)-sugar in the presence of one or more glucosyltransferase enzymes.
  • the glucosyltransferase is selected from T4 bacteriophage P-glucosyltransferase ( ⁇ GT), T4 bacteriophage a-glucosyltransf erase (aGT), and derivatives and analogs thereof.
  • ⁇ GT is an enzyme that catalyzes a chemical reaction in which a beta-D-glucosyl (glucose) residue is transferred from UDP-glucose to a 5-hydroxymethylcytosine residue in a nucleic acid.
  • the methods of the present disclosure include identifying 5mC or 5hmC in a nucleic acid sample, e.g., DNA sample (targeted DNA or whole-genome).
  • the method provides a quantitative measure for the frequency of 5mC or 5hmC modifications at each location where the modifications were identified in the nucleic acid sample, e.g., DNA sample.
  • the percentages of the T at each transition location provide a quantitative level of 5mC or 5hmC at each location in the nucleic acid sample, e.g., DNA sample.
  • the present disclosure provides a method for identifying 5mC and identifying 5hmC in a nucleic acid sample, e.g., DNA sample, by performing the method for identifying 5mC on a first nucleic acid sample, e.g., DNA sample, and performing the method for identifying 5mC or 5hmC on a second nucleic acid sample, e.g., DNA sample.
  • the first and second nucleic acid samples e.g., DNA samples
  • the first and second samples may be separate aliquots taken from a sample comprising nucleic acid, e.g., DNA, to be analyzed (e.g., cellular DNA or cfDNA).
  • the method identifies the locations and percentages of 5hmC in the DNA through the comparison of 5mC locations and percentages with the locations and percentages of 5mC or 5hmC (together).
  • the location and frequency of 5hmC modifications in a DNA can be measured directly.
  • the method includes identifying 5caC in a nucleic acid sample, e.g., DNA sample (targeted DNA or whole- genome), and provides a quantitative measure for the frequency of 5caC modification at each location where the modification was identified in the nucleic acid sample, e.g., DNA sample.
  • the percentages of the T at each transition location provide a quantitative level of 5caC at each location in the nucleic acid sample, e.g., DNA sample.
  • methods for identifying 5caC can include the use of a blocking group. In other embodiments, methods for identifying 5caC do not require the use of a blocking group.
  • the identification of 5caC in the nucleic acid sample can occur.
  • adding a blocking group to the 5fC in the nucleic acid sample, e.g., DNA sample comprises contacting the nucleic acid sample, e.g., DNA sample, with an aldehyde reactive compound including, for example, hydroxylamine derivatives, hydrazine derivatives, and hydrazide derivatives.
  • Hydrazide derivatives include - toluenesulfonylhydrazide, N-acylhydrazide, N,N-alkylacylhydrazide, N,N- benzylacylhydrazide, N,N-arylacylhydrazide, N-sulfonylhydrazide, N,N- alkylsulfonylhydrazide, N,N-benzyl sulfonylhydrazide, and N,N-arylsulfonylhydrazide. [0163] Methods for Identifying 5fC.
  • the method includes identifying 5fC in a nucleic acid sample, e.g., DNA sample (targeted DNA or whole- genome), and provides a quantitative measure for the frequency of 5fC modification at each location where the modification was identified in the nucleic acid sample, e.g., DNA sample.
  • the percentages of the T at each transition location provide a quantitative level of 5fC at each location in the nucleic acid sample, e.g., DNA sample.
  • methods for identifying 5fC can include the use of a blocking group. In other embodiments, methods for identifying 5fC do not require the use of a blocking group.
  • adding a blocking group to the 5caC in the nucleic acid sample can be accomplished by (i) contacting the nucleic acid sample, e.g., DNA sample, with a coupling agent, for example a carboxylic acid derivatization reagent like carbodiimide derivatives such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or N,N' -dicyclohexyl carbodiimide (DCC), and (ii) contacting the nucleic acid sample, e.g., DNA sample, with an amine, hydrazine or hydroxylamine compound.
  • a coupling agent for example a carboxylic acid derivatization reagent like carbodiimide derivatives such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or N,N' -dicyclohexyl carbodiimide (DCC)
  • 5caC can be blocked by treating the nucleic acid sample, e.g., DNA sample, with EDC and then benzylamine, ethylamine, or another amine to form an amide that blocks 5caC from conversion to DHU (e.g., by borane reduction).
  • the methods further comprise a sequencing step so that a methylation signature may be obtained.
  • the method includes isolating nucleic acid, such as DNA (e.g., cellular or cfDNA), from a sample; preparing a sequencing library comprising the nucleic acid, e.g., DNA; and performing TAPS on the sequencing library to obtain a methylation signature of the nucleic acid, e.g., DNA.
  • the methylation signature is a whole-genome methylation signature.
  • preparing the sequencing library comprises ligating sequencing adapters to the isolated nucleic acid, e.g., DNA, to facilitate performing a sequencing reaction.
  • Suitable sequencing adapters for massively parallel sequencing technologies may be utilized.
  • the present invention is not limited to any particular sequencing technology.
  • sequencing technologies such as those provided by Illumina or Nanopore may be utilized.
  • suitable sequencing technologies for use in the present invention include, but are not limited to, those described in US Pat. Publ. 20100120098, US Pat. Publ. 20120208705, US Pat. Publ. 20120208724, WO2012/061832, and US Pat. Publ. 2015/0368638, each of which is incorporated herein by reference in its entirety.
  • the adapter comprises one or more sites that can hybridize to a primer.
  • an adapter comprises at least a first primer site.
  • an adapter comprises at least a first primer site and a second primer site.
  • the orientation of the primer sites in such embodiments can be such that a primer hybridizing to the first primer site and a primer hybridizing to the second primer site are in the same orientation, or in different orientations.
  • the primer sequence in the linker can be complementary to a primer used for amplification. In another embodiment, the primer sequence is complementary to a primer used for sequencing.
  • a linker can include a first primer site, a second primer site having a non-amplifiable site disposed therebetween.
  • the non-amplifiable site can be useful to block extension of a polynucleotide strand between the first and second primer sites, wherein the polynucleotide strand hybridizes to one of the primer sites.
  • the non-amplifiable site can also be useful to prevent concatamers. Examples of non-amplifiable sites include a nucleotide analogue, non-nucleotide chemical moiety, amino-acid, peptide, and polypeptide.
  • a non-amplifiable site comprises a nucleotide analogue that does not significantly basepair with A, C, G, or T.
  • Some embodiments include a linker comprising a first primer site, a second primer site having a fragmentation site disposed therebetween.
  • Other embodiments can use a forked or Y-shaped adapter design useful for directional sequencing, for instance as described in U.S. Pat. No. 7,741,463, which is incorporated herein by reference.
  • the adapter may comprise an index or barcode sequence.
  • the adapter may comprise a Unique Molecular Identifier (UMI).
  • UMI Unique Molecular Identifier
  • carrier nucleic acids or a mix of carrier nucleic acids are added to the sequencing library prior to performing TAPS.
  • Carrier nucleic acids can be any specific or non-specific nucleic acid, e.g., DNA, molecules (or nucleic acid derivatives thereof) that enhance one or more aspects of nucleic acid, e.g., DNA, recovery from a sample.
  • nucleic acid e.g., DNA
  • methylation signatures can be useful for understanding basic biological processes and disease pathology as well as for disease detection.
  • methylation signatures/frequencies/markers, etc. can be useful in understanding and studying gene regulation, genomic imprinting, differentiation, development, gene-environment interaction (e.g., smoking, nutrition), aging, numerous diseases and conditions (e.g., auto-immune diseases, cancer, cardiovascular diseases, CNS diseases, congenital diseases, infectious diseases, metabolic diseases and status, NIPT-related testing, etc.), for detecting and diagnosing cancer and other diseases, and for monitoring transplants.
  • diseases and conditions e.g., auto-immune diseases, cancer, cardiovascular diseases, CNS diseases, congenital diseases, infectious diseases, metabolic diseases and status, NIPT-related testing, etc.
  • the method further comprises identifying at least one methylation biomarker from the nucleic acid, e.g., DNA, methylation signature (such as a whole-genome DNA methylation signature) and determining if the methylation biomarker differs from the methylation biomarker in a reference or control sequence.
  • the methylation biomarker comprises a differentially methylated region (DMR).
  • the method further comprises classifying the sample based on the DMR as compared to a reference DMR.
  • the reference DMR corresponds to a non-disease control, or a disease control.
  • the method further comprises identifying at least one methylation biomarker from the nucleic acid, e.g., DNA, methylation signature, and determining a tissue-of-origin corresponding to the methylation biomarker. In some embodiments, the method further comprises classifying the sample based on the tissue- of-origin biomarker.
  • the method further comprises identifying a nucleic acid, e.g., DNA, fragmentation profile, and determining whether the fragmentation profile is indicative of a condition (such as cancer or another disease).
  • nucleic acid, e.g., DNA, fragmentation profile can be determined from TAPS sequencing data (e.g., read pair alignment positions).
  • the method further comprises identifying at least one sequence variant in the nucleic acid sample, e.g., DNA sample, and determining whether the sequence variant is indicative of a condition (such as cancer or another disease).
  • TAPS can also differentiate methylation from C-to-T genetic variants or single nucleotide polymorphisms (SNPs), and therefore, can be used to detect genetic variants.
  • SNPs single nucleotide polymorphisms
  • methylations and C-to-T SNPs can result in different patterns in TAPS. For example, methylations can result in T/G reads in an original top strand/original bottom strand, and A/C reads in strands complementary to these.
  • C-to- T SNPs can result in T/A reads in an original top strand/original bottom strand and strands complementary to these.
  • This further increases the utility of TAPS in providing both methylation information and genetic variants, and therefore mutations, in one experiment and sequencing run.
  • This ability of the TAPS methods disclosed herein provides integration of genomic analysis with epigenetic analysis, and a substantial reduction of sequencing cost by eliminating the need to perform, for example, standard whole genome sequencing (WGS).
  • methods of the present disclosure can include the use of TAPS to generate information pertaining to methylation signatures, methylation biomarkers, DNA fragment profiles, DNA sequence information (e.g., variants), and tissue-of-origin information in a single experiment to diagnose/detect a disease or other condition (e.g., those provided as examples above) in a subject.
  • TAPS to generate information pertaining to methylation signatures, methylation biomarkers, DNA fragment profiles, DNA sequence information (e.g., variants), and tissue-of-origin information in a single experiment to diagnose/detect a disease or other condition (e.g., those provided as examples above) in a subject.
  • TAPS as disclosed herein can be used to generate any combination of methylation signatures, methylation biomarkers, DNA fragment profiles, DNA sequence information (e.g., variants), and tissue-of-origin information to diagnose/detect a disease or other condition (e.g., those provided as examples above) in a subject.
  • a methylation signature can be obtained, and one or more of a methylation biomarker, a DNA fragment profile, DNA sequence information (e.g., variants), and tissue-of-origin information can also be obtained and used to diagnose/detect a disease or other condition (e.g., those provided as examples above) in a subject.
  • the methylation status of a biomarker can be obtained, and one or more of a methylation signature, a DNA fragment profile, DNA sequence information (e.g., variants), and tissue-of-origin information can also be obtained and used to diagnose/detect a disease or other condition (e.g., those provided as examples above) in a subject.
  • a DNA fragmentation profile can be obtained, and one or more of a methylation signature, a methylation biomarker, DNA sequence information (e.g., variants), and tissue-of-origin information can also be obtained and used to diagnose/detect a disease or other condition (e.g., those provided as examples above) in a subject.
  • a DNA sequence variant can be identified, and one or more of a methylation signature, a methylation biomarker, a DNA fragment profile, and tissue-of-origin information can also be obtained and used to diagnose/detect a disease or other condition (e.g., those provided as examples above) in a subject.
  • tissue-of-origin information can be obtained (e.g., from a whole genome DNA methylation signature), and one or more of the methylation signature, a methylation biomarker, a DNA fragment profile, and DNA sequence information (e.g., variants), can also be obtained and used to diagnose/detect a disease or other condition (e.g., those provided as examples above) in a subject.
  • performing TAPS on the sequencing library to obtain the whole-genome methylation signature comprises identifying 5mC modifications in the DNA and providing a quantitative measure for frequency of the 5mC modifications.
  • performing TAPS on the sequencing library to obtain the whole-genome methylation signature comprises identifying 5hmC modifications in the nucleic acid sample, e.g., DNA sample, and providing a quantitative measure for frequency of the 5hmC modifications.
  • performing TAPS on the sequencing library to obtain the whole-genome methylation signature comprises identifying 5caC modifications in the DNA and providing a quantitative measure for frequency of the 5caC modifications.
  • performing TAPS on the sequencing library to obtain the whole-genome methylation signature comprises identifying 5fC modifications in the nucleic acid sample, e.g., DNA sample, and providing a quantitative measure for frequency of the 5fC modifications.
  • the methods described herein can be used to diagnose/detect any type of cancer.
  • Types of cancers that can be detected/diagnosed using the methods of the present disclosure include, but are not limited to, lung cancer, melanoma, colon cancer, colorectal cancer, neuroblastoma, breast cancer, prostate cancer, renal cell cancer, transitional cell carcinoma, cholangiocarcinoma, brain cancer, non-small cell lung cancer, pancreatic cancer, liver cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, thyroid cancer, head and neck cancer, osteosarcoma, hepatocellular carcinoma, carcinoma of unknown primary, ovarian carcinoma, endometrial carcinoma, glioblastoma, Hodgkin lymphoma, and non-Hodgkin lymphomas.
  • types of cancers or metastasizing forms of cancers that can be detected/diagnosed by the methods of the present disclosure include, but are not limited to, carcinoma, sarcoma, lymphoma, germ cell tumor, and blastoma.
  • the cancer is invasive and/or metastatic cancer (e.g., stage II cancer, stage III cancer or stage IV cancer).
  • the cancer is an early-stage cancer (e.g., stage 0 cancer, stage I cancer), and/or is not invasive and/or metastatic cancer.
  • the cancer is not metastatic cancer.
  • the present disclosure provides methods for identifying the location of one or more of 5mC, 5hmC, 5caC and/or 5fC in a nucleic acid quantitatively with base-resolution without affecting the unmodified cytosine.
  • the nucleic acid is DNA.
  • the DNA is cfDNA (e.g., circulating cfDNA).
  • the nucleic acid is RNA.
  • a nucleic acid sample comprises a target nucleic acid that is DNA or a target nucleic acid that is RNA.
  • the methods are applied to a whole genome, and not limited to a specific target nucleic acid.
  • the nucleic acid may be any nucleic acid having cytosine modifications (i.e., 5mC, 5hmC, 5fC, and/or 5caC), including but not limited to DNA fragments and/or genomic DNA.
  • the nucleic acid can be a single nucleic acid molecule in the sample, or may be the entire population of nucleic acid molecules in a sample, or any portion thereof (whole genome or a subset thereof).
  • the nucleic acid can be the native nucleic acid from the source (e.g., cells, tissue samples, etc.) or can pre-converted into a high-throughput sequencing-ready form, for example by fragmentation, repair, and ligation with adapters for sequencing.
  • nucleic acids can comprise a plurality of nucleic acid sequences such that the methods described herein may be used to generate a library of target nucleic acid sequences that can be analyzed individually (e.g., by determining the sequence of individual targets) or in a group (e.g., by high-throughput or next generation sequencing methods).
  • the methods of the present disclosure can also include the step of amplifying the copy number of a modified nucleic acid by methods known in the art.
  • the modified nucleic acid is DNA
  • the copy number can be increased by, for example, PCR, cloning, and primer extension.
  • the copy number of individual target DNAs can be amplified by PCR using primers specific for a particular target DNA sequence.
  • a plurality of different modified target DNA sequences can be amplified by cloning into a DNA vector by standard techniques.
  • the copy number of a plurality of different modified target DNA sequences is increased by PCR to generate a library for next generation sequencing where, e.g., double-stranded adapter DNA has been previously ligated to the sample DNA (or to the modified sample DNA) and PCR is performed using primers complimentary to the adapter DNA.
  • the method comprises the step of detecting the sequence of the modified nucleic acid.
  • the modified target nucleic acid e.g., DNA or RNA
  • DHU acts as a T in DNA replication and sequencing methods.
  • the cytosine modifications can be detected by any direct or indirect method that identifies a C to T transition known in the art. Such methods include sequencing methods such as Sanger sequencing, microarray, and next generation sequencing methods.
  • the C to T transition can also be detected by restriction enzyme analysis where the C to T transition abolishes or introduces a restriction endonuclease recognition sequence.
  • Embodiments of the present disclosure also provide systems or kits for oxidizing a methylated nucleotide (e.g., 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)).
  • a methylated nucleotide e.g., 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)
  • the systems or kits comprise a borane reducing agent as described above.
  • the kits comprise an oxidizing reagent.
  • Suitable enzymatic oxidizing reagents include, but are not limited to, TET family members, for example, TET1, TET2, TET3, CXXC4, an active fragment, derivative, or analogue thereof, or any combination thereof.
  • Suitable chemical oxidizing reagents include, but are not limited to manganese oxide (MnCE), potassium ruthenate (K2RUO4), Cu(II)/ 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenylphosphonium ruthenate, copper salts or complexes of 3- carbamoyl-2, 2,5,5-tetramethyl-3-pyrrolin-l-yloxy (3 -Carbamoyl-PROXYL), copper salts or complexes of 2-azaadamantane-N-oxyl (AZADO), or copper salts or complexes of 9- azabicyclo [3.3.1] nonane N'-oxyl (ABNO).
  • MnCE manganese oxide
  • K2RUO4 potassium rut
  • the systems or kits further comprise a blocking group and/or a glucosyltransferase enzyme.
  • the blocking group is a sugar.
  • the sugar is a naturally-occurring sugar or a modified sugar, for example glucose or a modified glucose.
  • the blocking group functions with UDP linked to a sugar, for example UDP -glucose or UDP linked to a modified glucose in the presence of a glucosyltransferase enzyme, for example, T4 bacteriophage P- glucosyltransferase ( ⁇ GT) and T4 bacteriophage a-glucosyltransferase ( ⁇ GT) and derivatives and analogs thereof.
  • This Example describes the testing of novel functionalized pyridine borane complexes that demonstrate reduced GC bias and DHU amplification bias, as well as increased normalized coverage of biomarkers in sequencing following the TAPS assay.
  • use of borane or deuteri oborane complexes containing a substituted 2- hydroxymethylpyridine moiety in the TAPS procedure reduces DHU amplification bias and improves normalized coverage for biomarkers. This allows for more sequencing reads on highly methylated regions of the genome relevant to methylation detection. This improvement in DHU amplification bias is achieved with comparable conversion and false positive rates to existing reducing agents.
  • the compounds were evaluated in TAPS reduction reactions with a target nucleic acid.
  • the TAPS reduction reaction was then followed by amplification (PCR) and next-generation sequencing.
  • Lambda GC bias for TAPS reduction reactions performed with either a 2- methylpyridine borane complex or 2-hydroxymethylpyridine borane complex was also determined.
  • TAPS reduction reactions using 2-methylpyridine borane complex (“Pic”, solid lines) as the reducing agent produced a GC bias curve with a negative slope, indicating lower coverage in GC-rich regions of the genome.
  • 2- hydroxymethylpyridine borane complex (“ESI047”, dashed lines) produced a GC bias curve with a much flatter slope, indicating greater coverage in GC-rich regions and highly methylated regions compared to pic borane.
  • the Lambda GC bias represents the sequencing bias from the methylated Lambda DNA spike-in to the TAPS reaction where the majority of Cs are methylated. The data was measured on an Illumina NextSeq instrument.
  • Table 3 provides data on the comparison of methylation of fully methylated Lambda following a TAPS reduction reaction with either a 2-methylpyridine borane complex or 2-hydroxymethylpyridine borane complex.
  • the reaction with 2-hydroxymethylpyridine borane complex (“ESI047”) showed a comparable methylation rate to standard conditions with 2-methylpyridine borane complex (“Pic”).
  • the data was measured on an Illumina NextSeq instrument.
  • This Example describes the testing of additional functionalized pyridine borane complexes.
  • TAPS, post-TAPS amplification yields, lambda conversion rates, false positive rates, lambda GC dropout, and general GC dropout were assessed as described above in Example 1.
  • the DNA input for the TAPS reactions comprised a total of 75 ng DNA, specifically 94.8% gDNA NA12878 with the following spike-ins: 100% me-lambda (at 5.0%), 100% me-pUC19 (at 0.2%), and non-me 2 kb amplicon (at 0.025%).
  • TAPS reduction reactions were performed using the additional pyridine borane complexes as reducing agents.
  • FIGs. 5-9 provide the post-TAPS amplification yields, lambda conversion rates, false positive rates, lambda-GC dropout, and general GC-dropout, respectively, obtained using the pyridine borane complexes.
  • the TAPS reduction reactions were performed both with pre-extension (blacks bars) or without pre-extension (gray bars).
  • Pre-extension conditions involved incubating post-reduction DNA in the presence of polymerase mix for 30 minutes at 72°C before PCR, whereas in conditions without pre- extension, post-reduction DNA proceeded to PCR without prior incubation in the presence of polymerase mix. Mean values from two replicates are presented unless otherwise indicated (ND, not determined). Error bars reflect standard deviation values. Performance of the pyridine borane complexes was compared to that of 2-methylpyridine borane complex (“picB”).
  • FIGs 10A-G and 11 A-G provide plots for lambda (FIG. 10) and general (FIG. 11) GC-dropout for the pyridine borane complexes with the highest scores (i.e., the compounds with a score of 4) as indicated in Table 8 (ESI047, ESI070, ESI075, ESI076, ESI079, and ESI093), as well as for ESI090.
  • the additional pyridine borane compounds are compared to pic-borane in reaction with or without pre-extension: solid black line - pic- borane with pre-extension; dotted line - pic-borane without pre-extension; novel compound with pre-extension - solid circles; novel compound without pre-extension - open circles.
  • GC- dropout was decreased for ESI047, ESI070, ESI075, ESI076, ESI079, and ESI093, relative to picB, suggesting that these compounds allow for more efficient amplification of regions with higher GC-content.

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Abstract

La présente invention concerne des agents réducteurs boranes, et en particulier des agents réducteurs boranes destinés à être utilisés dans des dosages de détection de méthylation qui comprennent une étape de réduction de la 5-formylcytosine (5fC) et/ou de la 5-carboxylcytosine (5caC) en dihydrouracile (DHU).
PCT/US2024/051528 2023-10-17 2024-10-16 Agents réducteurs boranes Pending WO2025085492A1 (fr)

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Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US20100120098A1 (en) 2008-10-24 2010-05-13 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
US7741463B2 (en) 2005-11-01 2010-06-22 Illumina Cambridge Limited Method of preparing libraries of template polynucleotides
WO2012061832A1 (fr) 2010-11-05 2012-05-10 Illumina, Inc. Liaison entre des lectures de séquences à l'aide de codes marqueurs appariés
US20120208724A1 (en) 2011-02-10 2012-08-16 Steemers Frank J Linking sequence reads using paired code tags
US20120208705A1 (en) 2011-02-10 2012-08-16 Steemers Frank J Linking sequence reads using paired code tags
US20150368638A1 (en) 2013-03-13 2015-12-24 Illumina, Inc. Methods and compositions for nucleic acid sequencing
WO2019136413A1 (fr) * 2018-01-08 2019-07-11 Ludwig Institute For Cancer Research Ltd Identification par résolution de base sans bisulfite de modifications de cytosine
WO2020056435A1 (fr) 2018-09-11 2020-03-19 Strait Access Technologies Holdings (Pty) Ltd Stent gainé expansible et procédé de fabrication
WO2021000630A1 (fr) 2019-07-02 2021-01-07 华为技术有限公司 Procédé et appareil pour mettre à jour une carte de travail d'un robot mobile, et support de stockage
WO2021051091A1 (fr) 2019-09-13 2021-03-18 Rad Al, Inc. Méthode et système de génération automatique d'une section dans un rapport de radiologie
WO2021161192A1 (fr) * 2020-02-11 2021-08-19 The Chancellor, Masters And Scholars Of The University Of Oxford Séquençage d'acide nucléique à lecture longue cible pour la détermination de modifications de cytosine
WO2022000420A1 (fr) 2020-07-02 2022-01-06 浙江大学 Procédé et système de reconnaissance d'action de corps humain, et dispositif
WO2023007241A2 (fr) * 2021-07-27 2023-02-02 The Chancellor, Masters And Scholars Of The University Of Oxford Compositions et procédés liés au séquençage de pyridine borane assisté par tet pour adn acellulaire
WO2023187012A1 (fr) * 2022-03-31 2023-10-05 Illumina Cambridge Limited Compositions comprenant des complexes amine-borane aqueux et des polynucléotides, et leurs procédés d'utilisation pour détecter la méthylcytosine ou l'hydroxyméthylcytosine

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US7741463B2 (en) 2005-11-01 2010-06-22 Illumina Cambridge Limited Method of preparing libraries of template polynucleotides
US20100120098A1 (en) 2008-10-24 2010-05-13 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
WO2012061832A1 (fr) 2010-11-05 2012-05-10 Illumina, Inc. Liaison entre des lectures de séquences à l'aide de codes marqueurs appariés
US20120208724A1 (en) 2011-02-10 2012-08-16 Steemers Frank J Linking sequence reads using paired code tags
US20120208705A1 (en) 2011-02-10 2012-08-16 Steemers Frank J Linking sequence reads using paired code tags
US20150368638A1 (en) 2013-03-13 2015-12-24 Illumina, Inc. Methods and compositions for nucleic acid sequencing
US20210317519A1 (en) 2018-01-08 2021-10-14 Ludwig Institute For Cancer Research Ltd Bisulfite-free, base-resolution identification of cytosine modifications
WO2019136413A1 (fr) * 2018-01-08 2019-07-11 Ludwig Institute For Cancer Research Ltd Identification par résolution de base sans bisulfite de modifications de cytosine
US20200370114A1 (en) 2018-01-08 2020-11-26 Ludwig Institute For Cancer Research Ltd Bisulfite-free, base-resolution identification of cytosine modifications
WO2020056435A1 (fr) 2018-09-11 2020-03-19 Strait Access Technologies Holdings (Pty) Ltd Stent gainé expansible et procédé de fabrication
WO2021000630A1 (fr) 2019-07-02 2021-01-07 华为技术有限公司 Procédé et appareil pour mettre à jour une carte de travail d'un robot mobile, et support de stockage
WO2021051091A1 (fr) 2019-09-13 2021-03-18 Rad Al, Inc. Méthode et système de génération automatique d'une section dans un rapport de radiologie
WO2021161192A1 (fr) * 2020-02-11 2021-08-19 The Chancellor, Masters And Scholars Of The University Of Oxford Séquençage d'acide nucléique à lecture longue cible pour la détermination de modifications de cytosine
WO2022000420A1 (fr) 2020-07-02 2022-01-06 浙江大学 Procédé et système de reconnaissance d'action de corps humain, et dispositif
WO2023007241A2 (fr) * 2021-07-27 2023-02-02 The Chancellor, Masters And Scholars Of The University Of Oxford Compositions et procédés liés au séquençage de pyridine borane assisté par tet pour adn acellulaire
WO2023187012A1 (fr) * 2022-03-31 2023-10-05 Illumina Cambridge Limited Compositions comprenant des complexes amine-borane aqueux et des polynucléotides, et leurs procédés d'utilisation pour détecter la méthylcytosine ou l'hydroxyméthylcytosine

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ALBERT L. LEHNINGER: "Principles of Biochemistry", 1982, WORTH PUB., pages: 793 - 800
BRAASCHCOREY, BIOCHEMISTRY, vol. 41, no. 14, 2002, pages 4503 - 4510
CARRUTHERS: "Some Modern Methods of Organic Synthesis", 1987, CAMBRIDGE UNIVERSITY PRESS
LAROCK: "Comprehensive Organic Transformations", 2018, JOHN WILEY & SONS, INC.
SMITH: "March's Advanced Organic Chemistry: Reactions, Mechanism, and Structure", 2013, JOHN WILEY & SONS, INC.
SORRELL: "Handbook of Chemistry and Physics", 2006, UNIVERSITY SCIENCE BOOKS
WAHLESTEDT ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 97, 2000, pages 5633 - 5638
WANG, J. AM. CHEM. SOC., vol. 122, 2000, pages 8595 - 8602

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