Borane reduction reagent

Abstract

This disclosure provides borane reducing reagents, in particular borane reducing reagents for use in methylation detection assays that include the step of reducing 5-formylcytosine (5fC) and / or 5-carboxylcytosine (5caC) to dihydrouracil (DHU).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest to U.S. Provisional Patent Application No. 63 / 590,928, filed on 17 October 2023, which is incorporated herein by reference in its entirety.

[0002] This disclosure provides borane reducing reagents, in particular borane reducing reagents for use in methylation detection assays that include the step of reducing 5-formylcytosine (5fC) and / or 5-carboxylcytosine (5caC) to dihydrouracil (DHU). [Background technology]

[0003] Methylation, particularly methylation at the 5-position of cytosine, is a major epigenetic modification in the mammalian genome and is associated with biological and pathological processes, including well-established biomarkers for various diseases and cancers. In addition to methylation of cytosine residues, oxidation from methylated cytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and / or 5-carboxylcytosine (5caC), as well as oxidation from adenine (A) to N 6 Other modifications, such as methylation to methyladenine (6-mA), have also been identified as important epigenetic regulators. Therefore, the detection of epigenetic modifications such as methylation has become extremely important for scientific and diagnostic purposes.

[0004] Methylation can be determined, for example, by whole-genome, base-resolution, and quantitative sequencing methods, such as bisulfite sequencing. However, bisulfite sequencing is damaging to nucleic acids and expensive; therefore, current methylation sequencing is limited to low-depth, targeted, or low-resolution and qualitative enrichment-based sequencing. [Overview of the project]

[0005] The present disclosure provides borane reducing reagents, particularly borane reducing reagents for use in methylation detection assays comprising the step of reducing 5-formylcytosine (5fC) and / or 5-carboxylcytosine (5caC) to dihydrouracil (DHU).

[0006] Embodiments of the present disclosure include the following.

[0007] In some preferred embodiments, the present invention provides a method for converting 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC) to dihydrouracil (DHU), which method comprises contacting a nucleic acid sample comprising 5caC and / or 5fC with a borane reducing agent of Formula (I):

Chemical Formula

Chemical Formula

[0008] In some preferred embodiments, the present invention provides a method for converting 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC) to dihydrouracil (DHU), the method comprising converting a nucleic acid sample containing 5caC and / or 5fC to formula (I): [ka] This includes contacting with a borane reducing agent or a salt thereof, in the formula, A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclil, each of which is R 1 , R 2 , R 3 , R 4 , and R 5 It has been replaced with, R 1 , R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, halo, hydroxy, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 aminoalkyl, C1-C4 mercaptoalkyl, C1-C4-alkoxy-C1-C4-alkyl, and amidoximil. Each X is independently either hydrogen or deuterium. The compound in question is [ka] But no.

[0009] In some preferred embodiments, A is selected from pyridine, imidazole, quinoline, 9,10-dihydroacridine, and imidazo[1,5-a]pyridine.

[0010] In some preferred embodiments, the borane reducing agent is a compound of formula (Ia). [ka]

[0011] In some preferred embodiments, the borane reducing agent is of formula (Ia): [ka] It is a compound of the formula, in which, R 1 These are selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl, and C1-C4-alkoxy-C1-C4-alkyl. R 2 This is selected from hydrogen, halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, and amidoximil. Here, R 1 and R 2 They optionally form a 5 or 6-membered ring, optionally substituted with a hydroxyl group, together with the carbon atoms to which they are bonded. R 3 The elements are selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, amidoximil, and aryl. R 4 These are selected from hydrogen, halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, and heterocyclyl. Here, R 3 and R 4 They may optionally form a 5- or 6-membered ring together with the carbon atoms to which they are bonded. R 5 This is selected from hydrogen, C1-C4 alkyl, and C1-C4 hydroxyalkyl.

[0012] In some preferred embodiments, R 1 and R 2 These, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring. In some preferred embodiments, R 3 and R 4These, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring. In some preferred embodiments, R 1 and R 2 These, together with the carbon atoms to which they are bonded, form a 5 or 6-membered ring, R 3 and R 4 These, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring.

[0013] In some preferred embodiments, R 1 and R 2 These, together with the carbon atoms to which they are bonded, form a hydroxyl-substituted 5 or 6-membered ring. In some preferred embodiments, R 3 and R 4 These, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring. In some preferred embodiments, R 1 and R 2 Together with the carbon atoms to which they are bonded, they form a hydroxyl-substituted 5 or 6-membered ring, R 3 and R 4 These, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring.

[0014] In some preferred embodiments, R 1 These are selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl, and C1-C4-alkoxy-C1-C4-alkyl. R 2 This is selected from hydrogen, halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, and amidoximil. R 3 This is selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, and amidoximil. R 4These are selected from hydrogen, halo, hydroxy, C1-C4 alkyl, and C1-C4 hydroxyalkyl. R 5 This is selected from hydrogen, C1-C4 alkyl, and C1-C4 hydroxyalkyl.

[0015] In some preferred embodiments, R 1 It is a C1-C4 hydroxyalkyl group, and R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, a C1-C4 alkyl, a C1-C4 alkoxy, a phenyl, and a monocyclic 5 or 6-membered heterocycline having one nitrogen atom.

[0016] In some preferred embodiments, R 1 It is hydroxymethyl, R 2 That is hydrogen, R 3 However, it is selected from hydrogen, C1-C4 alkyl, and phenyl. R 4 However, these are selected from C1-C4 alkyl, C1-C4 alkoxy, and monocyclic 5-membered heterocyclines having one nitrogen atom. R 5 But it's hydrogen.

[0017] In some preferred embodiments, R 1 It is a C1-C4 hydroxyalkyl group, and R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

[0018] In some preferred embodiments, R 1 , R 2 , R 3 , R 4 , and R 5However, each is independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 1 , R 2 , R 3 , R 4 , and R 5 At least one of them is hydroxymethyl.

[0019] In some preferred embodiments, R 1 However, it is selected from hydrogen, methyl, and hydroxymethyl, R 2 However, it is selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 3 However, it is selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 4 However, selected from hydrogen and methyl, R 5 However, it is hydrogen, Here, R 1 , R 2 , and R 3 At least one of them is hydroxymethyl.

[0020] In some preferred embodiments, the borane reducing agent is of formula (Ib): [ka] It is a compound or salt thereof, in which, R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, aryl, and heterocyclyl. Each X is independently either hydrogen or deuterium.

[0021] In some preferred embodiments, the borane reducing agent is of formula (Ib): [ka] It is a compound or salt thereof, in which, R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy, each X is independently hydrogen or deuterium.

[0022] In some preferred embodiments, R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, phenyl, and monocyclic 5-membered heterocyclyl having one nitrogen atom.

[0023] In some preferred embodiments, R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

[0024] In some preferred embodiments, R 2 is selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy, R 3 is selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy, R 4 is selected from hydrogen and C1-C4 alkyl, R 5 is hydrogen.

[0025] In some preferred embodiments, R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidinyl.

[0026] In some preferred embodiments, R 2 , R 3 , R4 , and R 5 However, each is independently selected from hydrogen, methyl, and methoxy.

[0027] In some preferred embodiments, R 2 However, it is selected from hydrogen, methyl, and methoxy. R 3 However, it is selected from hydrogen, methyl, and methoxy. R 4 However, selected from hydrogen and methyl, R 5 But it's hydrogen.

[0028] In some preferred embodiments, each X is hydrogen.

[0029] In some preferred embodiments, each X is deuterium.

[0030] In some preferred embodiments, the borane reducing agent is selected from the following: [ka] [ka] And their salts.

[0031] In some preferred embodiments, the borane reducing agent is selected from the following: [ka] and their salts.

[0032] In some preferred embodiments, the borane reducing agent is selected from the following: [ka] And their salts.

[0033] In some preferred embodiments, the method further comprises contacting the nucleic acid sample with an oxidizing agent before contacting it with a borane reducing agent. In some preferred embodiments, the oxidizing agent is a Ten-Eleven Translocation (TET) enzyme. In some preferred embodiments, the TET enzyme includes human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria gulberi TET (NgTET), Coprinopsis cinerea TET (CcTET), or derivatives or analogs thereof.

[0034] In some preferred embodiments, the oxidizing agent includes a chemical oxidizing agent. In some preferred embodiments, the chemical oxidizing agent is selected from the group consisting of manganese oxide (MnO2), potassium perthenate (KRuO4), Cu(II) / 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), tetrapropylammonium perthenate (TPAP), tetrabutylammonium perthenate (TBAP), polymer-supported perthenic acid (PSP), tetraphenylphosphonium lutenate, copper salts or complexes of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxy (3-Carbamoyl-PROXYL), copper salts or complexes of 2-azadamantane-N-oxyl (AZADO), and copper salts or complexes of 9-azabicyclo[3.3.1]nonane N'-oxyl (ABNO).

[0035] In some preferred embodiments, the method further includes adding a blocking group to one or more modified cytosines in a nucleic acid sample.

[0036] In some preferred embodiments, the blocking group is added before contact with the oxidizing agent.

[0037] In some preferred embodiments, one or more modified cytosines contain 5hmC.

[0038] In some preferred embodiments, the blocking group comprises a sugar or a uridine diphosphate (UDP)-linked sugar.

[0039] In some preferred embodiments, the blocking group is added after contact with the oxidizing agent and before contact with the borane reducing agent.

[0040] In some preferred embodiments, one or more modified cytosines include 5caC or 5fC.

[0041] In some preferred embodiments, the blocking group comprises an aldehyde-reactive compound. In some preferred embodiments, the aldehyde-reactive compound comprises a hydroxylamine derivative, a hydrazine derivative, or a hydrazide derivative.

[0042] In some preferred embodiments, the addition of a blocking group involves contacting the nucleic acid sample with (i) a coupling agent and (ii) an amine, hydrazine, or hydroxylamine compound.

[0043] In some preferred embodiments, the method further includes sequencing the nucleic acid sample after contact with a borane reducing agent to identify the converted cytosine bases.

[0044] In some preferred embodiments, the method further includes amplifying the copy number of the nucleic acid sample. In some preferred embodiments, the amplification is performed after contact between the nucleic acid sample and the borane reducing agent. In some preferred embodiments, the amplification is performed before sequencing of the nucleic acid sample. In some preferred embodiments, the amplification is performed both after contact between the nucleic acid sample and the borane reducing agent and before sequencing of the nucleic acid sample.

[0045] In some preferred embodiments, the nucleic acid sample is a DNA sample.

[0046] In some preferred embodiments, the present invention provides compounds selected from the following: [ka] [ka] And their salts.

[0047] In some preferred embodiments, the present invention provides compounds selected from the following: [ka]

[0048] In some preferred embodiments, the present invention provides compounds selected from the following: [ka] And their salts.

[0049] In some preferred embodiments, the present invention provides a system or kit for converting 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC) to dihydrouracil (DHU), the system or kit comprising formula (I): [ka] The formula comprises a borane reducing reagent or a salt thereof, A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclil, each of which is R 1 , R 2 , R 3 , R 4 , and R 5 It has been replaced with, R 1 , R 2 , R 3 , R 4 , and R 5Each of these is independently selected from hydrogen, halo, hydroxy, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 aminoalkyl, C1-C4 mercaptoalkyl, C1-C4-alkoxy-C1-C4-alkyl, amidoximil, aryl, and heterocyclyl. Each X is independently either hydrogen or deuterium. The compound in question is [ka] But no.

[0050] In some preferred embodiments, the present invention provides a system or kit for converting 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC) to dihydrouracil (DHU), the system or kit comprising formula (I): [ka] The formula comprises a borane reducing reagent or a salt thereof, A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclil, each of which is R 1 , R 2 , R 3 , R 4 , and R 5 It has been replaced with, R 1 , R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, halo, hydroxy, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 aminoalkyl, C1-C4 mercaptoalkyl, C1-C4-alkoxy-C1-C4-alkyl, and amidoximil. Each X is independently either hydrogen or deuterium. The compound in question is [ka] But no.

[0051] In some preferred embodiments, A is selected from pyridine, imidazole, quinoline, 9,10-dihydroacridine, and imidazo[1,5-a]pyridine.

[0052] In some preferred embodiments, the borane reducing agent is a compound of formula (Ia). [ka]

[0053] In some preferred embodiments, the borane reducing agent is of formula (Ia): [ka] It is a compound of the formula, in which, R 1 These are selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl, and C1-C4-alkoxy-C1-C4-alkyl. R 2 This is selected from hydrogen, halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, and amidoximil. Here, R 1 and R 2 They optionally form a 5 or 6-membered ring, optionally substituted with a hydroxyl group, together with the carbon atoms to which they are bonded. R 3 The elements are selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, amidoximil, and aryl. R 4 These are selected from hydrogen, halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, and heterocyclyl. Here, R3 and R 4 They may optionally form a 5- or 6-membered ring together with the carbon atoms to which they are bonded. R 5 This is selected from hydrogen, C1-C4 alkyl, and C1-C4 hydroxyalkyl.

[0054] In some preferred embodiments, R 1 and R 2 These, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring. In some preferred embodiments, R 3 and R 4 These, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring. In some preferred embodiments, R 1 and R 2 These, together with the carbon atoms to which they are bonded, form a 5 or 6-membered ring, R 3 and R 4 These, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring.

[0055] In some preferred embodiments, R 1 and R 2 These, together with the carbon atoms to which they are bonded, form a hydroxyl-substituted 5 or 6-membered ring. In some preferred embodiments, R 3 and R 4 These, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring. In some preferred embodiments, R 1 and R 2 Together with the carbon atoms to which they are bonded, they form a hydroxyl-substituted 5 or 6-membered ring, R 3 and R 4 These, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring.

[0056] In some preferred embodiments, R 1These are selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl, and C1-C4-alkoxy-C1-C4-alkyl. R 2 This is selected from hydrogen, halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, and amidoximil. R 3 This is selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, and amidoximil. R 4 These are selected from hydrogen, halo, hydroxy, C1-C4 alkyl, and C1-C4 hydroxyalkyl. R 5 This is selected from hydrogen, C1-C4 alkyl, and C1-C4 hydroxyalkyl.

[0057] In some preferred embodiments, R 1 It is a C1-C4 hydroxyalkyl group, and R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, a C1-C4 alkyl, a C1-C4 alkoxy, a phenyl, and a monocyclic 5 or 6-membered heterocycline having one nitrogen atom.

[0058] In some preferred embodiments, R 1 It is hydroxymethyl, R 2 That is hydrogen, R 3 However, it is selected from hydrogen, C1-C4 alkyl, and phenyl. R 4 However, these are selected from C1-C4 alkyl, C1-C4 alkoxy, and monocyclic 5-membered heterocyclines having one nitrogen atom. R 5 But it's hydrogen.

[0059] In some preferred embodiments, R 1 It is a C1-C4 hydroxyalkyl group, and R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

[0060] In some preferred embodiments, R 1 , R 2 , R 3 , R 4 , and R 5 However, each is independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 1 , R 2 , R 3 , R 4 , and R 5 At least one of them is hydroxymethyl.

[0061] In some preferred embodiments, R 1 However, it is selected from hydrogen, methyl, and hydroxymethyl, R 2 However, it is selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 3 However, it is selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 4 However, selected from hydrogen and methyl, R 5 However, it is hydrogen, Here, R 1 , R 2 , and R 3 At least one of them is hydroxymethyl.

[0062] In some preferred embodiments, the borane reducing agent is of formula (Ib): [ka] It is a compound or salt thereof, in which, R2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, aryl, and heterocyclyl. Each X is independently either hydrogen or deuterium.

[0063] In some preferred embodiments, the borane reducing agent is of formula (Ib): [ka] It is a compound or salt thereof, in which, R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy. Each X is independently either hydrogen or deuterium.

[0064] In some preferred embodiments, R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from a monocyclic five-membered heterocycline having a hydrogen, methyl, methoxy, phenyl, and one nitrogen atom.

[0065] In some preferred embodiments, R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

[0066] In some preferred embodiments, R 2 However, it is selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy, R 3 However, it is selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy, R 4However, hydrogen and C1-C4 alkyl are selected, R 5 But it's hydrogen.

[0067] In some preferred embodiments, R 2 , R 3 , R 4 , and R 5 However, each is independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidinyl.

[0068] In some preferred embodiments, R 2 , R 3 , R 4 , and R 5 However, each is independently selected from hydrogen, methyl, and methoxy.

[0069] In some preferred embodiments, R 2 However, it is selected from hydrogen, methyl, and methoxy. R 3 However, it is selected from hydrogen, methyl, and methoxy. R 4 However, selected from hydrogen and methyl, R 5 But it's hydrogen.

[0070] In some preferred embodiments, each X is hydrogen.

[0071] In some preferred embodiments, each X is deuterium.

[0072] In some preferred embodiments, the borane reducing agent is selected from the following: [ka] [ka] And their salts.

[0073] In some preferred embodiments, the borane reducing agent is selected from the following: [ka] And their salts.

[0074] In some preferred embodiments, the borane reducing agent is selected from the following: [ka] And their salts.

[0075] In some preferred embodiments, the system or kit further comprises an oxidizing agent. In some preferred embodiments, the oxidizing agent is a ten-eleven translocation (TET) enzyme. In some preferred embodiments, the TET enzyme includes human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria gulberi TET (NgTET), Coprinopsis cinerea (CcTET), or derivatives or analogs thereof.

[0076] In some preferred embodiments, the oxidizing agent includes a chemical oxidizing agent. In some preferred embodiments, the chemical oxidizing agent is selected from the group consisting of manganese oxide (MnO2), potassium perthenate (KRuO4), Cu(II) / 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), tetrapropylammonium perthenate (TPAP), tetrabutylammonium perthenate (TBAP), polymer-supported perthenic acid (PSP), tetraphenylphosphonium lutenate, copper salts or complexes of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxy (3-Carbamoyl-PROXYL), copper salts or complexes of 2-azadamantane-N-oxyl (AZADO), and copper salts or complexes of 9-azabicyclo[3.3.1]nonane N'-oxyl (ABNO).

[0077] In some preferred embodiments, the system or kit further comprises a blocking reagent. In some preferred embodiments, the blocking reagent is selected from the group consisting of sugars, uridine diphosphate (UDP)-conjugated sugars, and aldehyde-reactive compounds. In some preferred embodiments, the aldehyde-reactive compound is selected from the group consisting of hydroxylamine derivatives, hydrazine derivatives, and hydrazide derivatives. In some preferred embodiments, the blocking reagent is an aldehyde-reactive compound selected from the group consisting of hydroxylamine derivatives, hydrazine derivatives, and hydrazide derivatives. In some preferred embodiments, the blocking reagent is a sugar or a uridine diphosphate (UDP)-conjugated sugar, and the system or kit further comprises a glucosyltransferase enzyme.

[0078] Other aspects and embodiments of this disclosure will become apparent in light of the following detailed description. [Brief explanation of the drawing]

[0079] [Figure 1] Overall GC bias plot comparing the use of either the 2-methylpyridineborane complex ("Pic", solid line) or the 2-hydroxymethylpyridineborane complex ("ESI047", dashed line) in TAPS reactions with fully methylated lambda and partially methylated pUC19 templates. The overall GC bias plot shows bias across human genomic DNA, which is a component of the library consisting of 92.975% NA12878 (human genomic DNA), 5% methylated lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spike-in. [Figure 2] Lambda GC bias plots comparing the use of either the 2-methylpyridineborane complex ("Pic", solid line) or the 2-hydroxymethylpyridineborane complex ("ESI047", dashed line) in the TAPS reaction. [Figure 3]A plot comparing the library complexity of samples produced by TAPS reactions using either the 2-methylpyridineborane complex ("Pic-KU", solid line) or the 2-hydroxymethylpyridineborane complex ("ESI47-KU", short dashed line). [Figure 4] A graph showing the normalized coverage of selected biomarkers ((A) B3GALT6, (B) ZMYM4, (C) ZDHHC1, (D) TRIO, (E) ODZ2, (F) ZFAND3) in samples generated by a TAPS reaction using either a 2-methylpyridineborane complex ("Pic-KU") or a 2-hydroxymethylpyridineborane complex ("ESI47-KU"). [Figure 5] This graph shows the amplification yield after TAPS for TAPS reduction reactions using selected pyridineborane complexes. Black bars indicate reactions with pre-extension, while gray bars indicate reactions without pre-extension. The average value from two replicates is shown. [Figure 6] This graph shows the lambda conversion rate (%) for the TAPS reduction reaction using the selected pyridineborane complex. Black bars indicate reactions with pre-elongation, while gray bars indicate reactions without pre-elongation. The average value from two replicates is shown. [Figure 7] This graph shows the false positive rate (%) for the TAPS reduction reaction using the selected pyridineborane complex. Black bars indicate reactions with pre-elongation, while gray bars indicate reactions without pre-elongation. The average value from two replicates is shown. [Figure 8] A graph showing lambda GC dropout for the TAPS reduction reaction using a selected pyridineborane complex. Black bars indicate reactions with pre-elongation, while gray bars indicate reactions without pre-elongation. The average value from two replicates is shown. [Figure 9] A graph showing the overall GC dropout for the TAPS reduction reaction using the selected pyridineborane complex. Black bars indicate reactions with pre-elongation, while gray bars indicate reactions without pre-elongation. The average value from two replicates is shown. [Figure 10A] Lambda GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-elongation, dotted line indicating no pre-elongation) or the test pyridineborane complex (Figure 10A "ES1047"; Figure 10B "ES1070"; Figure 10C "ES1075"; Figure 10D "ES1076"; Figure 10E "ES1079"; Figure 10F "ES1090"; Figure 10G "ES1093"; filled circles indicating pre-elongation, open circles indicating no pre-elongation) in TAPS reactions. [Figure 10B] Lambda GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-elongation, dotted line indicating no pre-elongation) or the test pyridineborane complex (Figure 10A "ES1047"; Figure 10B "ES1070"; Figure 10C "ES1075"; Figure 10D "ES1076"; Figure 10E "ES1079"; Figure 10F "ES1090"; Figure 10G "ES1093"; filled circles indicating pre-elongation, open circles indicating no pre-elongation) in TAPS reactions. [Figure 10C] Lambda GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-elongation, dotted line indicating no pre-elongation) or the test pyridineborane complex (Figure 10A "ES1047"; Figure 10B "ES1070"; Figure 10C "ES1075"; Figure 10D "ES1076"; Figure 10E "ES1079"; Figure 10F "ES1090"; Figure 10G "ES1093"; filled circles indicating pre-elongation, open circles indicating no pre-elongation) in TAPS reactions. [Figure 10D] Lambda GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-elongation, dotted line indicating no pre-elongation) or the test pyridineborane complex (Figure 10A "ES1047"; Figure 10B "ES1070"; Figure 10C "ES1075"; Figure 10D "ES1076"; Figure 10E "ES1079"; Figure 10F "ES1090"; Figure 10G "ES1093"; filled circles indicating pre-elongation, open circles indicating no pre-elongation) in TAPS reactions. [Figure 10E]Lambda GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-elongation, dotted line indicating no pre-elongation) or the test pyridineborane complex (Figure 10A "ES1047"; Figure 10B "ES1070"; Figure 10C "ES1075"; Figure 10D "ES1076"; Figure 10E "ES1079"; Figure 10F "ES1090"; Figure 10G "ES1093"; filled circles indicating pre-elongation, open circles indicating no pre-elongation) in TAPS reactions. [Figure 10F] Lambda GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-elongation, dotted line indicating no pre-elongation) or the test pyridineborane complex (Figure 10A "ES1047"; Figure 10B "ES1070"; Figure 10C "ES1075"; Figure 10D "ES1076"; Figure 10E "ES1079"; Figure 10F "ES1090"; Figure 10G "ES1093"; filled circles indicating pre-elongation, open circles indicating no pre-elongation) in TAPS reactions. [Figure 10G] Lambda GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-elongation, dotted line indicating no pre-elongation) or the test pyridineborane complex (Figure 10A "ES1047"; Figure 10B "ES1070"; Figure 10C "ES1075"; Figure 10D "ES1076"; Figure 10E "ES1079"; Figure 10F "ES1090"; Figure 10G "ES1093"; filled circles indicating pre-elongation, open circles indicating no pre-elongation) in TAPS reactions. [Figure 11A]Overall GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-extension, dotted line indicating no pre-extension) or the test pyridineborane complex (Figure 11A "ES1047"; Figure 11B "ES1070"; Figure 11C "ES1075"; Figure 11D "ES1076"; Figure 11E "ES1079"; Figure 11F "ES1090"; Figure 11G "ES1093"; filled circles indicating pre-extension, open circles indicating no pre-extension) in TAPS reactions. The overall GC bias plots show bias across human genomic DNA, which is a component of the library consisting of 92.975% NA12878 (human genomic DNA), 5% methylated lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spikein. [Figure 11B] Overall GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-extension, dotted line indicating no pre-extension) or the test pyridineborane complex (Figure 11A "ES1047"; Figure 11B "ES1070"; Figure 11C "ES1075"; Figure 11D "ES1076"; Figure 11E "ES1079"; Figure 11F "ES1090"; Figure 11G "ES1093"; filled circles indicating pre-extension, open circles indicating no pre-extension) in TAPS reactions. The overall GC bias plots show bias across human genomic DNA, which is a component of the library consisting of 92.975% NA12878 (human genomic DNA), 5% methylated lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spikein. [Figure 11C]Overall GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-extension, dotted line indicating no pre-extension) or the test pyridineborane complex (Figure 11A "ES1047"; Figure 11B "ES1070"; Figure 11C "ES1075"; Figure 11D "ES1076"; Figure 11E "ES1079"; Figure 11F "ES1090"; Figure 11G "ES1093"; filled circles indicating pre-extension, open circles indicating no pre-extension) in TAPS reactions. The overall GC bias plots show bias across human genomic DNA, which is a component of the library consisting of 92.975% NA12878 (human genomic DNA), 5% methylated lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spikein. [Figure 11D] Overall GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-extension, dotted line indicating no pre-extension) or the test pyridineborane complex (Figure 11A "ES1047"; Figure 11B "ES1070"; Figure 11C "ES1075"; Figure 11D "ES1076"; Figure 11E "ES1079"; Figure 11F "ES1090"; Figure 11G "ES1093"; filled circles indicating pre-extension, open circles indicating no pre-extension) in TAPS reactions. The overall GC bias plots show bias across human genomic DNA, which is a component of the library consisting of 92.975% NA12878 (human genomic DNA), 5% methylated lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spikein. [Figure 11E]Overall GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-extension, dotted line indicating no pre-extension) or the test pyridineborane complex (Figure 11A "ES1047"; Figure 11B "ES1070"; Figure 11C "ES1075"; Figure 11D "ES1076"; Figure 11E "ES1079"; Figure 11F "ES1090"; Figure 11G "ES1093"; filled circles indicating pre-extension, open circles indicating no pre-extension) in TAPS reactions. The overall GC bias plots show bias across human genomic DNA, which is a component of the library consisting of 92.975% NA12878 (human genomic DNA), 5% methylated lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spikein. [Figure 11F] Overall GC bias plots comparing the use of either the 2-methylpyridineborane complex ("picB", solid line indicating pre-extension, dotted line indicating no pre-extension) or the test pyridineborane complex (Figure 11A "ES1047"; Figure 11B "ES1070"; Figure 11C "ES1075"; Figure 11D "ES1076"; Figure 11E "ES1079"; Figure 11F "ES1090"; Figure 11G "ES1093"; filled circles indicating pre-extension, open circles indicating no pre-extension) in TAPS reactions. The overall GC bias plots show bias across human genomic DNA, which is a component of the library consisting of 92.975% NA12878 (human genomic DNA), 5% methylated lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spikein. [Figure 11G]Overall GC bias plot comparing the use of either 2-methylpyridine borane complex ("picB", solid line indicating pre-extension present, dotted line indicating pre-extension absent) or a test pyridine borane complex (FIG. 11A "ES1047"; FIG. 11B "ES1070"; FIG. 11C "ES1075"; FIG. 11D "ES1076"; FIG. 11E "ES1079"; FIG. 11F "ES1090"; FIG. 11G "ES1093", filled circles indicating pre-extension present, open circles indicating pre-extension absent) in the TAPS reaction. The overall GC bias plot shows bias across human genomic DNA, a component of a library consisting of 92.975% NA12878 (human genomic DNA), 5% methylated lambda, 2% methylated pUC19, and 0.025% unmethylated 2 kb DNA spike-in. Mode for Carrying Out the Invention

[0080] In recent years, TET-assisted sequencing methods that enable detection of methylated bases have been developed. The present disclosure provides borane reducing reagents, particularly borane reducing reagents for use in methylation detection assays comprising the step of reducing 5-formylcytosine (5fC) and / or 5-carboxylcytosine (5caC) to dihydrouracil (DHU). In some preferred embodiments, the assay is a TAPS (Tet-assisted pyridine borane sequencing) assay as described in any of PCT / US2019 / 012627, U.S. Patent Publication No. 20200370114, U.S. Patent Publication No. 20210317519, PCT / IB2020 / 056435, PCT / IB2021 / 000630, PCT / IB2021 / 051091, PCT / IB2022 / 000420, and PCT / US2023 / 075823, each of which is incorporated herein by reference in its entirety.

[0081] Section headings used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0082] 1. Definitions As used herein, the terms "comprise", "include", "having", "has", "can", "contain", and their variants are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising", "consisting of", and "consisting essentially of" the embodiments or elements presented herein, whether explicitly described or not.

[0083] Where a numerical range is disclosed herein, each intervening value with the same precision is expressly contemplated. For example, for the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0084] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of potential ambiguity, definitions provided herein take precedence over any dictionary or external definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0085] As used herein, "methylation" refers to any and all processes by which methyl group(s) are added to a nucleic acid. For example, methylation may include, but is not limited to, the addition of a methyl group at the C5 or N4 position of cytosine, or the N6 position of adenine.

[0086] Therefore, as used herein, “methylated nucleotide base” or “methylated nucleotide” refers to the presence of a methyl moiety on the nucleotide base, which is not present in the recognized canonical nucleotide base. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, while 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Thus, cytosine is not a methylated nucleotide, while 5-methylcytosine is a methylated nucleotide.

[0087] As used herein, “methylated nucleic acid” refers to a nucleic acid molecule containing one or more methylated nucleotides. A nucleic acid molecule containing methylated cytosine is considered methylated (for example, the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered unmethylated.

[0088] As used herein, “nucleic acid” or “nucleic acid sequence” refers to polymers or oligomers of pyrimidine bases 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)). This technique intends any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid components, and any chemical variants thereof (e.g., methylated, hydroxymethylated, or glycosylated bases). The polymers or oligomers may have heterogeneous or homogeneous compositions and may be isolated from natural sources or produced artificially or synthetically. Furthermore, nucleic acids may be DNA or RNA, or mixtures thereof, and may exist permanently or transiently in single-stranded or double-stranded forms, including homo-double-stranded, hetero-double-stranded, and hybrid states. In some embodiments, the nucleic acid or nucleic acid sequence includes, for example, DNA / RNA helices, peptide nucleic acids (PNAs), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 41(14):4503-4510 (2002), and U.S. Patent Nos. 5,034,506), locked nucleic acids (LNAs; see Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97:5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122:8595-8602 (2000)), and / or other types of nucleic acid structures such as ribozymes.Therefore, the terms “nucleic acid” or “nucleic acid sequence” may also include chains containing non-natural nucleotides, modified nucleotides, and / or non-nucleotide constituent units that may perform the same function as natural nucleotides (e.g., “nucleotide analogs”); furthermore, as used herein, the term “nucleic acid sequence” refers to oligonucleotides, nucleotides, or polynucleotides, and their fragments or parts, as well as DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and may represent sense or antisense strands. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used synonymously. These refer to polymeric forms of nucleotides of any length, which are either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0089] "Subject" or "patient" may be human or non-human and may include, for example, a strain or species of animal used as a "model system" for research purposes, such as the mouse model described herein. Similarly, "subject" or "patient" may include either an adult or a young person (e.g., a child) and may mean any living organism such as a mammal (e.g., human or non-human). Examples of mammals include, but are not limited to, any member of the class Mammalia; humans, non-human primates (e.g., chimpanzees and other apes and monkey species); livestock (e.g., cattle, horses, sheep, goats, and pigs); domesticated animals (e.g., rabbits, dogs, and cats); and laboratory animals including rodents (e.g., mice, guinea pigs, etc.). Examples of non-mammals include, but are not limited to, birds, fish, etc. In one embodiment of the methods and compositions provided herein, the mammal is human.

[0090] As used herein, the term “nucleic acid sample” refers to nucleic acids obtained from organisms from Monera (bacteria), Protista, Fungi, Plantae, and Animalia Kingdoms. Nucleic acids may also be obtained from viruses. Nucleic acid samples can be obtained from both cellular DNA and circulating cell-free DNA (cfDNA) from a patient or subject, environmental samples, or the organism of interest (e.g., tissue (including tissue from lymph nodes, breast, liver, bile duct, pancreas, oral cavity, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, prostate, endometrium, vagina, ovaries, cervix, skin, bladder, kidney, lung, and / or peritoneum), biopsy, cells, cell aggregates, blood, plasma, serum, secretions (e.g., organ secretions, vaginal secretions, or gastric secretions), semen (seminal plasma), cerebrospinal fluid (CSF), saliva, mucus, urine, feces, sweat, pancreatic juice, gastric juice (gastric lavage fluid), ascites, synovial fluid, pleural fluid (pleural lavage fluid), pericardial fluid, peritoneal fluid, amniotic fluid, nasal fluid, intraocular fluid, breast milk, or any other bodily fluid containing the desired nucleic acid or cfDNA). In other embodiments, the target nucleic acid may be obtained from a sample containing diseased tissue or cells, or a sample suspected of containing diseased tissue or cells (e.g., a cancerous sample, or a sample containing cancerous tissue or cells, or a sample suspected of being cancerous, or a sample suspected of containing cancerous tissue or cells). In some embodiments, the nucleic acid sample may be obtained from a subject with a disease or disorder (e.g., cancer), a subject suspected of having a disease or disorder, or a subject undergoing screening to determine the presence of a disease or disorder. In some embodiments, the nucleic acid sample may be circulating cell-free DNA (cell-free DNA or cfDNA), e.g., DNA found in blood and not present in cells. As will be understood by those skilled in the art based on this disclosure, cfDNA may be isolated from bodily fluids using methods known in the art. Commercially available kits, such as the Circulating Nucleic Acid Kit (Qiagen), are available for the isolation of cfDNA. The nucleic acid sample may be obtained 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.

[0091] Definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are as defined in Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Specified according to the Ed. (endpaper), specific functional groups are generally defined as described therein. Furthermore, for general principles of organic chemistry, as well as specific functional groups and reactivity, see Sorrell, Organic Chemistry, 2 nd edition,University Science Books,Sausalito,2006;Smith,March's Advanced Organic Chemistry:Reactions,Mechanism,and Structure,7 th Edition,John Wiley & Sons,Inc.,New York,2013;Larock,Comprehensive Organic Transformations,3 rd Edition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd This information is contained in Edition, Cambridge University Press, Cambridge, 1987, and its entirety is incorporated herein by reference.

[0092] As used herein, the term "alkyl" refers to a radical of a straight-chain or branched-chain saturated hydrocarbon chain. An alkyl chain is, for example, a chain with 1 to 16 carbon atoms (C1-C1). 16 Alkyl), 1 to 14 carbon atoms (C1-C 14 Alkyl), 1 to 12 carbon atoms (C1-C 12 Alkyl), 1 to 10 carbon atoms (C1-C 10Alkyl groups may include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, 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.

[0093] As used herein, the term "alkoxy" refers to an alkyl group as defined herein, which is attached to the parent molecule via an oxygen atom. Typical examples of alkoxys include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0094] As used herein, the term "alkoxyalkyl" means an alkyl group as defined herein, wherein at least one hydrogen atom is replaced by an alkoxy group as defined herein. Typical examples of alkoxyalkyls include, but are not limited to, methoxymethyl and 2-methoxyethyl.

[0095] As used herein, the term "amidoximyl" refers to the -C(=NOH)NH2 group.

[0096] As used herein, the term “amino” means a --N(R)² group (wherein each R is independently hydrogen or alkyl group (as defined herein)). If one R is hydrogen and the other is alkyl, the group may be called an “alkylamino” group; if both Rs are alkyl, the group may be called a “dialkylamino” group.

[0097] As used herein, the term "aminoalkyl" means an alkyl group as defined herein, wherein at least one hydrogen atom is substituted with an amino group as defined herein.

[0098] As used herein, the term “aryl” refers to a monocyclic, bicyclic, or tricyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) having 6 to 14 ring carbon atoms and 0 heteroatoms (“C6-C 14 In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl", i.e., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C6 aryl"). 10 "aryl" (e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C"). 14 "Aryl" (e.g., anthracenyl and phenantrenyl).

[0099] As used herein, the term "halo" or "halogen" refers to fluoro, chloro, bromo, or iodine.

[0100] As used herein, the term “haloalkyl” refers to an alkyl group as defined herein, in which at least one hydrogen atom is replaced by a halogen. In some embodiments, each hydrogen atom of the alkyl group is replaced by a halogen. Representative examples of haloalkyls include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, and 3,3,3-trifluoropropyl.

[0101] As used herein, the term “heteroaryl” refers to an aromatic group having a monocyclic (monocyclic) or multicyclic (bicyclic or tricyclic) ring with one or more ring heteroatoms independently selected from O, N, and S. An aromatic monocyclic ring is a five- or six-membered ring having at least one heteroatom independently selected from O, N, and S (for example, one, two, three, or four heteroatoms independently selected from O, N, and S). A five-membered aromatic monocyclic ring has two double bonds, and a six-membered aromatic monocyclic ring has three double bonds. Examples of bicyclic heteroaryl groups include monocyclic aryl groups as defined herein, or monocyclic heteroaryl groups as defined herein, fused with an additional monocyclic heteroaryl ring. Examples of tricyclic heteroaryl groups include monocyclic heteroaryl rings fused with two rings independently selected from monocyclic aryl groups and monocyclic heteroaryl groups as defined herein. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (e.g., pyridine-2-yl, pyridine-3-yl, pyridine-4-yl), pyrimidinyl, pyrazinyl, pyridadinyl, 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. Representative examples of bicyclic heteroaryls include, but are not limited to, benzimidazolyl, benzodioxolyl, benzofuranil, benzoxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, clomenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranil, isoindolyl, isoquinolinyl, naphthilidinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl.Representative examples of tricyclic heteroaryls include, but are not limited to, dibenzofuranyl and dibenzothienyl.

[0102] As used herein, the terms “heterocycle” or “heterocyclic” refer to a saturated or partially unsaturated non-aromatic ring group having one or more ring heteroatoms independently selected from O, N, and S. A heterocycle can be a monocyclic, bicyclic, or tricyclic heterocycle. A monocyclic heterocycle is a 3, 4, 5, 6, 7, or 8-membered ring containing at least one heteroatom independently selected from O, N, and S. A 3 or 4-membered ring contains zero or one double bond, and one heteroatom selected from O, N, and S. A 5-membered ring contains zero or one double bond, and one, two, or three heteroatoms selected from O, N, and S. A 6-membered ring contains zero, one, or two double bonds, and one, two, or three heteroatoms selected from O, N, and S. Seven- and eight-membered rings contain zero, one, two, or three double bonds, as well as one, two, or three heteroatoms selected from O, N, and S. Heteroatoms in the ring can be oxidized (for example, if a ring heteroatom is S, it can be oxidized to SO or SO2). Representative examples of monocyclic heterocycles include azetidinyl, azepanyl, azilidinyl, 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, piperadinyl, and piperidinyl. Examples include, but are not limited to, pyranil, pyrazolinil, pyrazolidinil, pyrrolinil, pyrrolidinil, tetrahydrofuranil, tetrahydropyranil, tetrahydropyridinil, tetrahydrothienyl, thiadiazolinil, thiadiazolidinil, 1,2-thiadinyl, 1,3-thiadinyl, thiazolinil, thiazolidinil, thiomorpholinil, 1,1-dioxidethiomorpholinil (thiomorpholine sulfone), thiopyranil, and trithianil.A bicyclic heterocycle is a monocyclic heterocycle fused with a phenyl group, or a monocyclic heterocycle fused with a monocyclic cycloalkyl group, or a monocyclic heterocycle fused with a monocyclic cycloalkenyl group, or a monocyclic heterocycle fused with a monocyclic heterocycle, or a spiroheterocyclic group, or a bridged monocyclic heterocycle system in which two non-adjacent atoms of the ring are linked by an alkylene bridge with 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge with 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranil, benzothiopyranil, chromanil, 2,3-dihydrobenzofuranil, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hepta-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Examples of tricyclic heterocycles include bicyclic heterocycles fused with a phenyl group, bicyclic heterocycles fused with a monocyclic cycloalkyl group, bicyclic heterocycles fused with a monocyclic cycloalkenyl group, bicyclic heterocycles fused with a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of the bicyclic ring are linked by alkylene bridges with 1, 2, 3, or 4 carbon atoms, or alkenylene bridges with 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, and aza-adamantane(1-azatricyclo[3.3.1.1). 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 Examples include, but are not limited to, the Deccan.

[0103] As used herein, the term "hydroxy" refers to the -OH group.

[0104] As used herein, the term "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-hydroxyethyl, and 3-hydroxypropyl.

[0105] As used herein, the term "mercapto" means a -SH group.

[0106] As used herein, the term "mercaptoalkyl" means an alkyl group, as defined herein, in which at least one hydrogen atom is replaced with a mercapto group. Representative, non-limiting examples of mercaptoalkyl include mercaptomethyl, 2-mercaptoethyl, 2-mercaptopropyl, 3-mercaptopropyl, and 4-mercaptobutyl.

[0107] In the compounds of this disclosure, any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise indicated, where a position is designated as "H" or "hydrogen," that position is understood to have hydrogen in its naturally occurring isotopic composition. Also, unless otherwise indicated, where a position is specifically designated as "D" or "deuterium," that position is understood to have deuterium in an abundance at least 3000 times greater than the naturally occurring abundance of deuterium (i.e., at least 45% deuterium incorporation). In some embodiments, the position 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), The material contains deuterium in an abundance at least 6,000 times greater than the natural abundance of deuterium (90% deuterium incorporation), at least 6,333.3 times greater than the natural abundance of deuterium (95% deuterium incorporation), at least 6,466.7 times greater than the natural abundance of deuterium (97% deuterium incorporation), at least 6,600 times greater than the natural abundance of deuterium (99% deuterium incorporation), or at least 6,633.3 times greater than the natural abundance of deuterium (99.5% deuterium incorporation).

[0108] Preferred methods and materials will be described below, but similar or equivalent methods and materials to those described herein may also be used in the implementation or testing of this disclosure. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. The materials, methods, and examples disclosed herein are illustrative and not intended to limit the scope of this disclosure.

[0109] 2. Borane reducing agent Embodiments of the present disclosure provide borane reducing reagents (also referred to herein as borane complexes). As further detailed below, 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 target nucleic acid sequences.

[0110] In some embodiments, the borane reducing reagent that may be used in the methods disclosed herein is of formula (I): [ka] The compound comprises the and salts thereof, in which, A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclil, each of which is R 1 , R 2 , R 3 , R 4 , and R 5 It has been replaced with, R 1 , R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, halo, hydroxy, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 aminoalkyl, C1-C4 mercaptoalkyl, C1-C4-alkoxy-C1-C4-alkyl, amidoximil, aryl, and heterocyclyl. Each X is independently either hydrogen or deuterium. The compound in question is [ka] But no.

[0111] In some embodiments, the borane reducing reagent that may be used in the methods disclosed herein is of formula (I): [ka] The compound comprises the and salts thereof, in which, A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclil, each of which is R 1 , R 2 , R 3 , R 4 , and R 5 It has been replaced with, R 1 , R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, halo, hydroxy, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 aminoalkyl, C1-C4 mercaptoalkyl, C1-C4-alkoxy-C1-C4-alkyl, and amidoximil. Each X is independently either hydrogen or deuterium. The compound in question is [ka] But no.

[0112] In some embodiments, A is selected from pyridine, imidazole, quinoline, 9,10-dihydroacridine, and imidazo[1,5-a]pyridine. In some embodiments, A is pyridine.

[0113] In some embodiments, the borane reducing agent is a compound of formula (Ia). [ka]

[0114] In some embodiments, the borane reducing agent is given by formula (Ia): [ka] It is a compound of the formula, in which, R 1 These are selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl, and C1-C4-alkoxy-C1-C4-alkyl. R 2 This is selected from hydrogen, halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, and amidoximil. Here, R 1 and R 2 They optionally form a 5 or 6-membered ring, optionally substituted with a hydroxyl group, together with the carbon atoms to which they are bonded. R 3 The elements are selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, amidoximil, and aryl. R 4 These are selected from hydrogen, halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, and heterocyclyl. Here, R 3 and R 4 They may optionally form a 5- or 6-membered ring together with the carbon atoms to which they are bonded.

[0115] R 5 This is selected from hydrogen, C1-C4 alkyl, and C1-C4 hydroxyalkyl.

[0116] In some embodiments, R 1 R is selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl, and C1-C4-alkoxy-C1-C4-alkyl. 2 R is selected from hydrogen, halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, and amidoximil, 3R is selected from hydrogen, halo, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, and amidoximil. 4 R is selected from hydrogen, halo, hydroxy, C1-C4 alkyl, and C1-C4 hydroxyalkyl. 5 This is selected from hydrogen, C1-C4 alkyl, and C1-C4 hydroxyalkyl.

[0117] In some embodiments, R 1 It is a C1-C4 hydroxyalkyl group, and R 2 , R 3 , R 4 , and R 5 Each is independently selected from hydrogen, a C1-C4 alkyl, a C1-C4 alkoxy, a phenyl, and a monocyclic 5 or 6-membered heterocycline having one nitrogen atom. In some embodiments, R 1 It is hydroxymethyl, and R 2 is hydrogen, and R 3 R is selected from hydrogen, C1-C4 alkyl, and phenyl. 4 R is selected from C1-C4 alkyl, C1-C4 alkoxy, and monocyclic 5-membered heterocyclines having one nitrogen atom. 5 It is hydrogen.

[0118] In some embodiments, R 1 , R 2 , R 3 , R 4 , and R 5 At least one of them is not hydrogen. In some embodiments, R 1 , R 2 , R 3 , R 4 , and R 5 At least one of them is not hydrogen or methyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , and R 5At least one of them is a C1-C4 hydroxyalkyl group.

[0119] In some embodiments, R 1 R is selected from hydrogen, C1-C4 alkyl, and C1-C4 hydroxyalkyl, 2 R is selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, and C1-C4 hydroxyalkyl, 3 R is selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, and C1-C4 hydroxyalkyl, 4 R is selected from hydrogen and C1-C4 alkyl groups. 5 is hydrogen, and here, R 1 , R 2 , and R 3 At least one of them is a C1-C4 hydroxyalkyl group.

[0120] In some embodiments, R 1 It is a C1-C4 hydroxyalkyl group, and R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

[0121] In some embodiments, R 1 , R 2 , R 3 , R 4 , and R 5 However, each is independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 1 , R 2 , R 3 , R 4 , and R 5 At least one of them is hydroxymethyl.

[0122] In some embodiments, R 1 R is selected from hydrogen, methyl, and hydroxymethyl. 2 R is selected from hydrogen, methyl, methoxy, and hydroxymethyl.3 R is selected from hydrogen, methyl, methoxy, and hydroxymethyl. 4 R is selected from hydrogen and methyl, 5 is hydrogen, and here, R 1 , R 2 , and R 3 At least one of them is hydroxymethyl.

[0123] In some embodiments, each X is hydrogen. In some embodiments, each X is deuterium.

[0124] In some embodiments, the borane reducing agent is given by formula (Ib): [ka] It is a compound or salt thereof, in which, R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, aryl, and heterocyclyl. Each X is independently either hydrogen or deuterium.

[0125] In some embodiments, the borane reducing agent is given by formula (Ib): [ka] It is a compound or salt thereof, in which, R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy. Each X is independently either hydrogen or deuterium.

[0126] In some embodiments, R 2 , R 3 , R 4 , and R 5Each is independently selected from a monocyclic five-membered heterocycline having hydrogen, methyl, methoxy, phenyl, and one nitrogen atom. In some embodiments, R 2 , R 3 , R 4 , and R 5 Each of these is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

[0127] In some embodiments, R 2 R is selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy. 3 R is selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy. 4 R is selected from hydrogen and C1-C4 alkyl groups. 5 It is hydrogen.

[0128] In some embodiments, R 2 , R 3 , R 4 , and R 5 However, each is independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidinyl. In some embodiments, R 2 is hydrogen, and R 3 R is selected from hydrogen, C1-C4 alkyl, and phenyl. 4 R is selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, and monocyclic 5-membered heterocyclines having one nitrogen atom. 5 is hydrogen. In some embodiments, R 2 is hydrogen, and R 3 R is selected from hydrogen, methyl, and phenyl. 4 R is selected from hydrogen, methyl, methoxy, and pyrrolidinyl. 5 It is hydrogen.

[0129] In some embodiments, R 2 , R 3 , R 4 , and R 5 However, each is independently selected from hydrogen, methyl, and methoxy. In some embodiments, R2 R is selected from hydrogen, methyl, and methoxy. 3 R is selected from hydrogen, methyl, and methoxy. 4 R is selected from hydrogen and methyl, 5 It is hydrogen.

[0130] In some embodiments, each X is hydrogen. In some embodiments, each X is deuterium.

[0131] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is selected from the following: [ka] [ka]

[0132] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is selected from the following: [ka]

[0133] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is selected from the following: [ka] And their salts.

[0134] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is selected from the following: [ka] And their salts.

[0135] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) [ka] or its salt.

[0136] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) [ka] or its salt.

[0137] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) [ka] or its salt.

[0138] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) [ka] or its salt.

[0139] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) [ka] or its salt.

[0140] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) [ka] or its salt.

[0141] 3. Methods for identifying or detecting methylated nucleotide bases Embodiments of the present disclosure provide a bisulfite-free base-resolving method (e.g., TAPS and related methods TAPSβ and CAPS, collectively referred to as TAPS) for detecting 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) in sequences, including for use in blood samples (cellular DNA and cfDNA) and DNA obtained from biopsy materials. As disclosed in PCT / US2019 / 012627, U.S. Patent Publication No. 20200370114, U.S. Patent Publication No. 20210317519, PCT / IB2020 / 056435, PCT / IB2021 / 000630, PCT / IB2021 / 051091, PCT / IB2022 / 000420, and PCT / US2023 / 075823 (each of which is incorporated herein by reference in whole), TAPS involves the use of mild enzymatic and chemical reactions for the direct and quantitative detection of 5mC and / or 5hmC at base resolution without affecting unmodified cytosine. The disclosure also provides a method for detecting 5-formylcytosine (5fC) and / or 5-carboxylcytosine (5caC) at base resolution without affecting unmodified cytosine. Therefore, the method provided herein provides mapping of 5mC, 5hmC, 5fC, and / or 5caC, overcoming the shortcomings of conventional methods such as bisulfite sequencing.

[0142] According to these embodiments, the methods of the present disclosure may include the step of converting 5mC and 5hmC (or 5mC only if 5hmC is blocked) to 5caC and / or 5fC. In some embodiments, this step includes contacting a nucleic acid sample, e.g., a DNA or RNA sample, with a Ten Eleven Translocation (TET) enzyme. TET enzymes are a family of enzymes that catalyze the transfer of an oxygen molecule to a C5 methyl group at 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. Examples of TET enzymes useful in the methods disclosed herein include, but are not limited to, one or more of the following: human TET1, TET2, and TET3; mouse TET1, TET2, and TET3; Naegleria gulberi TET (NgTET); Coprinopsis cinerea TET (CcTET); the catalytic domain of mouse TET1 (mTET1CD); and their derivatives or analogues.

[0143] The method of the present disclosure may also include a step of converting 5caC and / or 5fC in a nucleic acid sample to DHU. In some embodiments, this step includes contacting a nucleic acid sample, for example, a DNA or RNA sample, with a borane reducing agent as described in the preceding section.

[0144] In some embodiments, the step of converting 5hmC to 5fC involves oxidizing a nucleic acid sample, such as a DNA sample, from 5hmC to 5fC by contacting it with, for example, manganese oxide (MnO2), potassium perthenate (KRuO4), Cu(II) / 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), tetrapropylammonium perthenate (TPAP), tetrabutylammonium perthenate (TBAP), polymer-supported perthenic acid (PSP), tetraphenylphosphonium lutenate, a copper salt or complex of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxy (3-Carbamoyl-PROXYL), a copper salt or complex of 2-azadamantane-N-oxyl (AZADO), or a copper salt or complex of 9-azabicyclo[3.3.1]nonane N'-oxyl (ABNO). Subsequently, 5fC in a nucleic acid sample, such as a DNA sample, can be converted to DHU by a method disclosed herein (e.g., a borane reaction).

[0145] Methods for identifying 5mC. In some embodiments, the methods of the present disclosure include identifying 5mC in a nucleic acid sample, e.g., a DNA sample (target DNA or whole genome), and providing a quantitative measure of the frequency of 5mC modifications at each site where modifications are identified in the nucleic acid sample, e.g., a DNA sample. In some embodiments, the percentage of T at each transition site provides a quantitative level of 5mC at each site in the nucleic acid sample, e.g., a DNA sample. According to these embodiments, the methods for identifying 5mC may include the use of blocking groups. In other embodiments, the methods for identifying 5mC do not require the use of blocking groups.

[0146] When identifying 5mC in a nucleic acid sample, such as a DNA sample, without including 5hmC, a blocking group is used to block the 5hmC in the sample from being converted to 5caC and / or 5fC. In some embodiments, 5hmC in a nucleic acid sample, such as a DNA sample, becomes unreactive for subsequent steps by adding a blocking group to 5hmC. In one embodiment, the blocking group is a sugar containing a modified sugar, such as glucose or 6-azido-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, such as a DNA sample, with uridine diphosphate (UDP) sugar in the presence of one or more glucosyltransferase enzymes. In some embodiments, the glucosyltransferase is selected from T4 bacteriophage β-glucosyltransferase (βGT), T4 bacteriophage α-glucosyltransferase (αGT), and their derivatives and analogs. βGT 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 nucleic acids.

[0147] Methods for identifying 5mC and / or 5hmC. In some embodiments, the methods of the present disclosure include identifying 5mC or 5hmC in a nucleic acid sample, e.g., a DNA sample (target DNA or whole genome). In some embodiments, the methods provide a quantitative measure of the frequency of 5mC or 5hmC modifications at each position where modifications are identified in a nucleic acid sample, e.g., a DNA sample. In some embodiments, the percentage of T at each transition site provides a quantitative level of 5mC or 5hmC at each position in a nucleic acid sample, e.g., a DNA sample. In some embodiments, both 5mC and 5hmC are converted to DHU, and the methods for identifying 5mC or 5hmC provide the positions of 5mC and 5hmC but do not distinguish between the two cytosine modifications. The presence of DHU can be detected directly, or the modified DNA can be replicated, e.g., by the methods of the present disclosure in which DHU is converted to T. In some embodiments, the methods for identifying 5hmC include the use of blocking groups. In other embodiments, the methods for identifying 5hmC do not require the use of blocking groups.

[0148] In some embodiments, the Disclosure provides methods for identifying 5mC and 5hmC in a nucleic acid sample, such as a DNA sample, by performing a method for identifying 5mC in a first nucleic acid sample, such as a DNA sample, and a method for identifying 5mC or 5hmC in a second nucleic acid sample, such as a DNA sample. In some embodiments, the first and second nucleic acid samples, such as a DNA sample, are derived from the same nucleic acid sample, such as a DNA sample. For example, the first and second samples may be separate aliquots taken from a sample containing the nucleic acid to be analyzed, such as DNA (e.g., cellular DNA or cfDNA).

[0149] Since 5mC and 5hmC (unblocked) are converted to 5fC and 5caC before conversion to DHU, existing 5fC and 5caC in nucleic acid samples, e.g., DNA samples, may be detected as 5mC and / or 5hmC. However, given that the levels of 5fC and 5caC in genomic DNA are extremely low under normal conditions, this is often acceptable when analyzing methylation and hydroxymethylation in DNA samples. 5fC and 5caC signals can be excluded by protecting 5fC and 5caC from conversion to DHU, for example, by hydroxylamine conjugation and EDC coupling, respectively. In some embodiments, the method identifies the location and proportion of 5hmC in DNA by comparing the location and proportion of 5mC with the location and proportion of 5mC or 5hmC (combined). Alternatively, the location and frequency of 5hmC modifications in DNA can be measured directly.

[0150] In some embodiments, identifying 5fC and / or 5caC provides the location of 5fC and / or 5caC, but does not distinguish between these two cytosine modifications. Rather, in some embodiments, both 5fC and 5caC are converted to DHU, which can be detected by the methods described herein.

[0151] A method for identifying 5caC. In some embodiments, the method comprises identifying 5caC in a DNA sample (target DNA or whole genome) and providing a quantitative measure of the frequency of 5caC modifications at each site in the DNA where modifications are identified. In some embodiments, the percentage of T at each transition site provides a quantitative level of 5caC at each site in the DNA. According to these embodiments, the method for identifying 5caC may include the use of blocking groups. In other embodiments, the method for identifying 5caC does not require the use of blocking groups.

[0152] In some embodiments, when 5fC is blocked (and 5mC and 5hmC are not converted to DHU), 5caC in nucleic acid samples, such as DNA samples, can be identified. In some embodiments, adding a blocking group to 5fC in a nucleic acid sample, such as a DNA sample, involves contacting the nucleic acid sample, such as a DNA sample, with an aldehyde-reactive compound, such as a hydroxylamine derivative, a hydrazine derivative, or a hydrazide derivative. Examples of hydroxylamine derivatives include hydroxylamine (ashydroxylamine), hydroxylamine hydrochloride, hydroxylammonium sulfate, hydroxylamine phosphate, O-methylhydroxylamine, O-hexylhydroxylamine, O-pentylhydroxylamine, and O-benzylhydroxylamine, and in particular, O-ethylhydroxylamine (EtONH2), O-alkylated or O-arylated hydroxylamines, and their acids or salts. Examples of hydrazine derivatives include N-alkylhydrazine, N-arylhydrazine, N-benzylhydrazine, N,N-dialkylhydrazine, N,N-diarylhydrazine, N,N-dibenzylhydrazine, N,N-alkylbenzylhydrazine, N,N-arylbenzylhydrazine, and N,N-alkylarylhydrazine. Examples of hydrazide derivatives include toluenesulfonyl hydrazide, N-acylhydrazide, N,N-alkylacylhydrazide, N,N-benzylacylhydrazide, N,N-arylacylhydrazide, N-sulfonyl hydrazide, N,N-alkylsulfonyl hydrazide, N,N-benzylsulfonyl hydrazide, and N,N-arylsulfonyl hydrazide.

[0153] A method for identifying 5fC. In some embodiments, the method comprises identifying 5fC in a DNA sample (target DNA or whole genome) and providing a quantitative measure of the frequency of 5fC modifications at each site where modifications are identified in the DNA. In some embodiments, the percentage of T at each transition site provides a quantitative level of 5fC at each site in a nucleic acid sample, e.g., a DNA sample. According to these embodiments, the method for identifying 5fC may include the use of blocking groups. In other embodiments, the method for identifying 5fC does not require the use of blocking groups.

[0154] In some embodiments, the addition of a blocking group to 5caC in a nucleic acid sample, such as a DNA sample, can be achieved by (i) contacting the nucleic acid sample, such as a DNA sample, with a coupling agent, such as a carboxylic acid derivatization reagent, such as a carbodiimide derivative such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or N,N'-dicyclohexylcarbodiimide (DCC), and (ii) contacting the nucleic acid sample, such as a DNA sample, with an amine, hydrazine, or hydroxylamine compound. Thus, for example, 5caC can be blocked by treating the nucleic acid sample, such as a DNA sample, with EDC, and then with benzylamine, ethylamine, or another amine to form an amide that blocks the conversion of 5caC to DHU (e.g., by borane reduction).

[0155] In some preferred embodiments, the method further includes the step of sequencing so that a methylation signature can be obtained. In some embodiments, the method includes isolating a nucleic acid, e.g., DNA (e.g., cell or cfDNA) from a sample, preparing a sequencing library containing 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. In some embodiments, the methylation signature is a whole-genome methylation signature.

[0156] In some embodiments, preparing a sequencing library involves ligating a sequencing adapter to isolated nucleic acids, such as DNA, to facilitate the execution of the sequencing reaction. Sequencing adapters suitable for massively parallel sequencing techniques may be utilized. The present invention is not limited to any particular sequencing technique. In some preferred embodiments, sequencing techniques, such as those provided by Illumina or Nanopore, may be utilized. For example, sequencing techniques suitable for use in the present invention include, but are not limited to, those described in U.S. Patent Publication No. 20100120098, U.S. Patent Publication No. 20120208705, U.S. Patent Publication No. 20120208724, WO2012 / 061832, and U.S. Patent Publication No. 2015 / 0368638 (each of which is incorporated herein by reference in whole).

[0157] In some embodiments, the adapter includes one or more sites that can hybridize to a primer. In some embodiments, the adapter includes at least a first primer site. In some embodiments, the adapter includes at least a first primer site and a second primer site. The orientation of the primer sites in such embodiments may be such that the primers hybridizing to the first primer site and the primers hybridizing to the second primer site are oriented in the same direction or in different directions. In one embodiment, the primer sequence in the linker may be complementary to the primers used for amplification. In another embodiment, the primer sequence is complementary to the primers used for sequencing.

[0158] In some embodiments, the linker may include a first primer site and a second primer site (with a non-amplification site positioned between them). The non-amplification site may be useful for blocking the elongation of a polynucleotide chain between the first and second primer sites, causing the polynucleotide chain to hybridize to one of the primer sites. The non-amplification site may also be useful for preventing concatemerization. Examples of non-amplification sites include nucleotide analogs, non-nucleotide chemical moieties, amino acids, peptides, and polypeptides. In some embodiments, the non-amplification site includes nucleotide analogs that do not significantly base-pair with A, C, G, or T.

[0159] Some embodiments include a linker comprising a first primer portion and a second primer portion having a fragmentation portion positioned between them. Other embodiments may utilize fork-type or Y-shaped adapter designs useful for directional sequencing, such as those described herein by reference, for example, U.S. Patent No. 7,741,463.

[0160] In some embodiments, the adapter may include an index sequence or a barcode sequence. In a more preferred embodiment, the adapter may include a unique molecular identifier (UMI).

[0161] In some embodiments, a carrier nucleic acid or a mixture of carrier nucleic acids (e.g., DNA) is added to the sequencing library before performing TAPS. The carrier nucleic acid may be any specific or nonspecific nucleic acid from a sample, e.g., DNA, a molecule (or a nucleic acid derivative thereof) that enhances one or more aspects of DNA recovery.

[0162] Nucleic acids, such as DNA methylation signatures, are intended to be useful for understanding fundamental biological processes and disease pathology, as well as for disease detection. For example, methylation signatures / frequency / markers may be useful for understanding and studying gene regulation, genomic imprinting, differentiation, development, gene-environment interactions (e.g., smoking, nutrition), aging, numerous diseases and conditions (e.g., autoimmune diseases, cancer, cardiovascular diseases, CNS diseases, congenital diseases, infectious diseases, metabolic diseases and conditions, NIPT-related tests, etc.), detection and diagnosis of cancer and other diseases, and monitoring of transplantation. In some embodiments and as described herein, the method further includes identifying at least one methylation biomarker from a nucleic acid, such as a DNA methylation signature (e.g., whole-genome DNA methylation signature), and determining whether the methylation biomarker differs from a methylation biomarker in a reference or control sequence. In some embodiments, the methylation biomarker includes differential methylation regions (DMRs). In some embodiments, the method further includes classifying the samples based on the DMR in comparison to a reference DMR. In some embodiments, the reference DMR corresponds to a non-disease control or a disease control.

[0163] In some embodiments, the method further includes identifying at least one methylation biomarker from a nucleic acid, for example, a DNA methylation signature, and determining the tissue of origin corresponding to the methylation biomarker, as described herein. In some embodiments, the method further includes classifying the sample based on the biomarker of the tissue of origin.

[0164] In some embodiments, the method further includes identifying nucleic acids, e.g., DNA fragmentation profiles, and determining whether the fragmentation profiles are indicators of a pathological condition (e.g., cancer or another disease), as described herein. According to these embodiments, nucleic acids, e.g., DNA fragmentation profiles, can be determined from TAPS sequencing data (e.g., alignment positions of read pairs).

[0165] In some embodiments, the method further includes identifying at least one sequence variant in a nucleic acid sample, e.g., a DNA sample, and determining whether the sequence variant is an indicator of a pathological condition (e.g., cancer or another disease). For example, in some embodiments, TAPS can also distinguish methylation from C-to-T gene variants or single nucleotide polymorphisms (SNPs), and thus this can be used to detect gene variants. In some embodiments, methylation and C-to-T SNPs may result in different patterns in TAPS. For example, methylation may result in T / G reads in the original upper / lower strands and A / C reads in strands complementary to these. In some embodiments, C-to-T SNPs may result in T / A reads in the original upper / lower strands and in strands complementary to these. This further enhances the usefulness of TAPS in obtaining both methylation information and genetic variants, i.e., mutations, in a single experiment and sequencing run. This capability of the TAPS method disclosed herein leads to the integration of genomic and epigenetic analysis, and a significant reduction in sequencing costs, for example, by eliminating the need to perform standard whole-genome sequencing (WGS).

[0166] According to the embodiments described above, the methods of the Disclosure may include the use of TAPS to generate information regarding methylation signatures, methylation biomarkers, DNA fragment profiles, DNA sequence information (e.g., variants), and information regarding the tissue of origin in a single experiment for diagnosing / detecting a disease or other pathological condition (e.g., those provided as examples above) in a subject. As will be understood by those skilled in the art based on the Disclosure, any combination of methylation signatures, methylation biomarkers, DNA fragment profiles, DNA sequence information (e.g., variants), and information regarding the tissue of origin can be generated using the TAPS disclosed herein to diagnose / detect a disease or other pathological condition (e.g., those provided as examples above) in a subject. In some embodiments, a methylation signature can be obtained, and one or more of the methylation biomarkers, DNA fragment profiles, DNA sequence information (e.g., variants), and information regarding the tissue of origin can also be obtained and used to diagnose / detect a disease or other pathological condition (e.g., those provided as examples above) in a subject. In some embodiments, the methylation status of a biomarker can be obtained, along with one or more of the methylation signature, DNA fragment profile, DNA sequence information (e.g., variants), and information on the tissue of origin, which can be used to diagnose / detect a disease or other pathological condition (e.g., those provided as examples above) in a subject. In some embodiments, a DNA fragment profile can be obtained, along with one or more of the methylation signature, methylated biomarker, DNA sequence information (e.g., variants), and information on the tissue of origin, which can be used to diagnose / detect a disease or other pathological condition (e.g., those provided as examples above) in a subject. In some embodiments, DNA sequence variants can be identified, along with one or more of the methylation signature, methylated biomarker, DNA fragment profile, and information on the tissue of origin, which can be used to diagnose / detect a disease or other pathological condition (e.g., those provided as examples above) in a subject.In some embodiments, information about the tissue of origin can be obtained (e.g., from whole-genome DNA methylation signatures), and one or more of the methylation signatures, methylation biomarkers, DNA fragment profiles, and DNA sequence information (e.g., variants) can also be obtained, which can be used to diagnose / detect diseases or other pathological conditions (e.g., those provided as examples above) in a subject.

[0167] In some embodiments, performing TAPS on a sequencing library to obtain a whole-genome methylation signature includes identifying 5mC modifications in DNA and providing a quantitative measure of the frequency of 5mC modifications. In some embodiments, performing TAPS on a sequencing library to obtain a whole-genome methylation signature includes identifying 5hmC modifications in a nucleic acid sample, e.g., a DNA sample, and providing a quantitative measure of the frequency of 5hmC modifications. In some embodiments, performing TAPS on a sequencing library to obtain a whole-genome methylation signature includes identifying 5caC modifications in DNA and providing a quantitative measure of the frequency of 5caC modifications. In some embodiments, performing TAPS on a sequencing library to obtain a whole-genome methylation signature includes identifying 5fC modifications in a nucleic acid sample, e.g., a DNA sample, and providing a quantitative measure of the frequency of 5fC modifications.

[0168] As will be recognized by those skilled in the art based on this disclosure, any type of cancer can be diagnosed / detected using the methods described herein (e.g., TAPS). Types of cancer that can be detected / diagnosed using the methods of this disclosure include, but are not limited to, lung cancer, melanoma, colon cancer, colorectal cancer, neuroblastoma, breast cancer, prostate cancer, renal cell carcinoma, transitional cell carcinoma, cholangiocarcinoma, brain cancer, non-small cell lung cancer, pancreatic cancer, liver cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, thyroid cancer, head and neck cancer, osteosarcoma, hepatocellular carcinoma, carcinoma of unknown primary origin, ovarian cancer, endometrial cancer, glioblastoma, Hodgkin lymphoma, and non-Hodgkin lymphoma. In some embodiments, types of cancer or metastatic forms of cancer that can be detected / diagnosed by the methods of this disclosure include, but are not limited to, carcinomas, sarcomas, lymphomas, germ cell tumors, and blastomas. In some embodiments, the cancer is invasive and / or metastatic cancer (e.g., stage II, stage III, or stage IV cancer). In some embodiments, the cancer is an early-stage cancer (e.g., stage 0 cancer, stage 1 cancer) and / or not an invasive cancer and / or metastatic cancer. In some embodiments, the cancer is not a metastatic cancer.

[0169] According to these embodiments, the disclosure provides a method for quantitatively identifying one or more positions among 5mC, 5hmC, 5caC, and / or 5fC in nucleic acids by base resolution without affecting unmodified cytosine. In some embodiments, the nucleic acid is DNA. In some embodiments, the DNA is cfDNA (e.g., circulating cfDNA). In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid sample includes a target nucleic acid that is DNA or a target nucleic acid that is RNA. In some embodiments, the method is applicable to the whole genome and is not limited to a specific target nucleic acid.

[0170] The nucleic acid can 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 a sample, or an entire population of nucleic acid molecules in a sample, or any part thereof (whole genome or a subset thereof). The nucleic acid may be native nucleic acid from a source (e.g., cells, tissue samples, etc.), or it may be pre-converted into a form suitable for high-throughput sequencing by, for example, fragmentation, repair, and ligation with adapters for sequencing. Therefore, the nucleic acid may comprise multiple nucleic acid sequences so that a library of target nucleic acid sequences can be generated using the methods herein, which can be analyzed individually (e.g., by determining the sequences of individual targets) or in groups (e.g., by high-throughput or next-generation sequencing methods).

[0171] The methods of this disclosure may also include a step of amplifying the copy number of a modified nucleic acid by methods known in the art. When the modified nucleic acid is DNA, its copy number can be increased, for example, by PCR, cloning, and primer extension. The copy number of individual target DNAs can be amplified by PCR using primers specific to a particular target DNA sequence. Alternatively, multiple different modified target DNA sequences can be amplified by cloning them into a DNA vector using standard techniques. In some embodiments, the copy numbers of multiple different modified target DNA sequences are increased by PCR to generate a library for next-generation sequencing, for example, in which double-stranded adapter DNA is pre-ligated to the sample DNA (or modified sample DNA), and PCR is performed using primers complementary to the adapter DNA.

[0172] In some embodiments, the method includes the step of detecting the sequence of a modified nucleic acid. In TAPS, the modified target nucleic acid, e.g., DNA or RNA, contains one or more DHUs (5mC, 5hmC, 5fC, and 5caC) at the positions where they were present in the unmodified target nucleic acid, e.g., DNA or RNA. DHUs act as T in DNA replication and sequencing methods. Thus, cytosine modification can be detected by any direct or indirect method known in the art to identify the transition from C to T. Such methods include sequencing methods such as Sanger sequencing, microarray sequencing, and next-generation sequencing. The transition from C to T can also be detected by restriction enzyme analysis, in which the transition from C to T excludes or introduces restriction endonuclease recognition sequences.

[0173] 4. System or kit Embodiments of this disclosure also provide systems or kits for oxidizing methylated nucleotides (e.g., 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)). In some embodiments, the system or kit comprises the borane reducing agent described above. In some more preferred embodiments, the kit further comprises an oxidizing reagent. Suitable enzymatic oxidizing reagents include, but are not limited to, members of the TET family, e.g., TET1, TET2, TET3, CXXC4, active fragments, derivatives or analogs thereof, or any combination thereof. In some embodiments, the TET enzyme is selected from human TET1, TET2, and TET3; mouse TET1, TET2, and TET3; Naegleria gulberi TET (NgTET); Coprinopsis cinerea TET, active fragments (e.g., the catalytic domain of mouse TET1 (mTET1CD)), derivatives or analogs thereof. Suitable chemical oxidation reagents are selected from the group consisting of manganese oxide (MnO2), potassium ruthenate ((K2RuO4), Cu(II) / 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenic acid (PSP), tetraphenylphosphonium lutenate, copper salts or complexes of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxy (3-Carbamoyl-PROXYL), copper salts or complexes of 2-azadamantane-N-oxyl (AZADO), or copper salts or complexes of 9-azabicyclo[3.3.1]nonane N'-oxyl (ABNO).

[0174] In some embodiments, the TET family dioxygenases have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more sequence identity with any or all of the TET family dioxygenases listed above. In some embodiments, the TET family dioxygenases exhibit characteristic activity of the TET family proteins.

[0175] In some embodiments, the system or kit further comprises a blocking group and / or a glycosyltransferase enzyme. In some embodiments, the blocking group is a sugar. In some embodiments, the sugar is a naturally occurring sugar or a modified sugar, e.g., glucose or modified glucose. In some embodiments, the blocking group functions with a sugar-linked UDP, e.g., UDP-glucose, or a modified glucose-linked UDP, in the presence of a glucosyltransferase enzyme, e.g., T4 bacteriophage β-glucosyltransferase (βGT) and T4 bacteriophage α-glucosyltransferase (αGT), as well as their derivatives and analogues.

[0176] Such systems or kits may also be used for the detection and identification of methylated nucleotides and may include additional components necessary for that purpose. The systems or kits may further include sequencing reagents for detecting methylated nucleotides (e.g., primers, probes, nucleotides, buffers, control nucleic acid sequences, polymerases, etc.), restriction endonucleases, etc. The concepts, kits, and methods described herein may be carried out using any system or instrument, including any manual, automated, or semi-automated system for sequencing reactions.

[0177] The system or kit may include instructions for use in any of the methods described herein. Instructions included in a kit may be affixed to the packaging or included as accompanying documentation in the package. Instructions may be provided in writing or in print, but are not limited thereto. Any medium capable of storing and communicating such instructions to an end user is contemplated in this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. As used herein, the term “instructions” may include the address of an internet site providing such instructions.

[0178] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging. The kit may optionally provide additional components such as buffers and interpretation information. Typically, the kit includes a container and a label or accompanying documentation(s) on or accompanying the container. In some embodiments, the disclosure provides a manufactured product comprising the contents of the kit. [Examples]

[0179] 4. Examples The following are embodiments of the present invention and should not be construed as limitations.

[0180] Example 1 This example describes the testing of a novel functionalized pyridineborane complex that demonstrates reduced GC bias and DHU amplification bias, as well as increased normalized coverage of biomarkers, in sequencing after a TAPS assay. In particular, using a borane or deuterated borane complex containing a 2-hydroxymethylpyridine moiety substituted in the TAPS procedure reduces DHU amplification bias and improves normalized coverage of biomarkers. This results in a greater number of sequencing reads in highly methylated regions of the genome relevant to methylation detection. This improvement in DHU amplification bias is achieved with conversion rates and false-positive rates comparable to those of existing reducing agents.

[0181] Materials and methods Many pyridineborane complexes were synthesized for use in the experiments described in this example. Unless otherwise noted, the following compounds were used in the experiments below: 2-methylpyridineborane complex ("picborane"); 2-methylpyridine deuterated borane complex ("ESI021"); 2-hydroxymethylpyridineborane complex ("ESI047"); 2-hydroxymethylpyridine deuterated borane complex ("ESI050"); 5-hydroxymethyl-2-methylpyridineborane complex ("ESI051"); 3,4-dimethoxy-2-hydroxymethylpyridineborane complex ("ESI052"). [ka] The compounds were evaluated in a TAPS reduction reaction using target nucleic acids. Subsequently, amplification (PCR) and next-generation sequencing were performed following the TAPS reduction reaction.

[0182] result Summary of methylation rates of borane and deuterated borane complexes functionalized with methyl groups at various positions. Numerous different borane and deuterated borane complexes were synthesized and then used in TAPS reduction reactions with fully methylated lambda DNA spike in. Methylation rates were determined using an Illumina NextSeq instrument. The results are shown in Table 1. These data indicate that boranes complexed with pyridine functionalized with methyl groups at R1, and / or R2, and / or R3, and / or R4 yield high levels of conversion in TAPS, and generally, the false-positive rate is comparable to that of 2-methylpyridineborane complexes. Furthermore, the data indicate that borane complexes with pyridine ligands functionalized with hydroxymethyl groups at R1, R2, R3, or R4 are reducing agents with high levels of conversion in TAPS. Methylation indicates the detection of C→T conversion in in-house produced fully methylated lambda DNA spike in. The data presented are from multiple different experiments, but a positive control (Pic borane, 2-methylpyridine borane complex) was included in all tests to provide a basis for comparison. [Table 1]

[0183] Comparison of overall GC bias using 2-methylpyridineborane complex or 2-hydroxymethylpyridineborane complex with different nucleic acid samples. TAPS reduction was performed on a partially methylated DNA sample consisting of 92.975% NA12878, 5% methylated lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spikein, using either the 2-methylpyridineborane complex or the 2-hydroxymethylpyridineborane complex. Therefore, the majority of cytosine in this sample is unmethylated. Regarding Figure 1, the TAPS reduction reaction using the 2-methylpyridineborane complex ("Pic", solid line) as the reducing agent resulted in a GC bias curve with a negative slope, indicating lower coverage in GC-rich regions of the genome. The use of the 2-hydroxymethylpyridineborane complex ("ESI047", dashed line) resulted in a GC bias curve with a much flatter slope, indicating greater coverage in GC-rich regions compared to pic borane. The data was measured using an Illumina NextSeq instrument.

[0184] Lambda GC bias was also determined for TAPS reduction reactions performed using either the 2-methylpyridineborane complex or the 2-hydroxymethylpyridineborane complex. Regarding Figure 2, TAPS reduction reactions using the 2-methylpyridineborane complex ("Pic," solid line) as the reducing agent resulted in a GC bias curve with a negative slope, indicating lower coverage in GC-rich regions of the genome. The use of the 2-hydroxymethylpyridineborane complex ("ESI047," dashed line) resulted in a GC bias curve with a much flatter slope, indicating greater coverage in GC-rich and highly methylated regions compared to pic borane. Lambda GC bias represents sequencing bias from methylated lambda DNA spike-in for TAPS reactions where a large portion of C is methylated. Data were measured using an Illumina NextSeq instrument.

[0185] Comparison of methylation rates in TAPS reactions using 2-methylpyridineborane complex or 2-hydroxymethylpyridine complex. The methylation rate of pUC19 spike ins with approximately 20% methylated cytosine residues was assayed by TAPS using either the 2-methylpyridineborane complex or the 2-hydroxymethylpyridineborane complex. Data are shown in Table 2. Reactions with the 2-hydroxymethylpyridineborane complex ("ESI047") showed increased methylation rates (9.9 or 11) compared to standard conditions using the 2-methylpyridineborane complex ("Pic", 7.3 or 7.4). Methylation indicates the detection of C→T conversion in in-house generated pUC19 DNA spike ins with approximately 20% methylation. The 20:80 pUC19 spike ins with methylation rates are intended to simulate "real-world" patient samples where the majority of isolated DNA is not completely methylated. Data were measured using an Illumina NextSeq instrument.

[0186] Table 3 provides data comparing the methylation of fully methylated lambda after the TAPS reduction reaction using either the 2-methylpyridineborane complex or the 2-hydroxymethylpyridineborane complex. The reaction with the 2-hydroxymethylpyridineborane complex ("ESI047") showed a methylation rate comparable to that under standard conditions using the 2-methylpyridineborane complex ("Pic"). Data were measured using an Illumina NextSeq instrument.

[0187] In summary, these results indicate that the 2-hydroxymethylpyridineborane complex is superior to the 2-methylpyridineborane complex in TAPS reactions using templates designed to simulate real-world conditions. [Table 2] [Table 3]

[0188] The complexity of whole-genome sequencing libraries. The library complexity of samples treated with either the 2-methylpyridineborane complex or the 2-hydroxymethylpyridineborane complex was analyzed by whole-genome sequencing using an Illumina NovaSeq instrument. The data plot is shown in Figure 3. The complexity of both samples treated with 2-hydroxymethylpyridineborane ("ESI47-KU", short dashed line, also referred to as ESI047 throughout this application) and samples treated with 2-methylpyridineborane ("Pic-KU", solid line) was between that of the bisulfite-treated controls with 0% methylation (Zymo-bisulfite-0%meth, dotted line) and 100% methylation (Zymo-bisulfite-100%meth, dashed line), indicating that the complexity of TAPS-treated samples using 2-hydroxymethylpyridineborane was equivalent to that of both the bisulfite-treated samples and the TAPS-treated samples using 2-methylpyridineborane. Library complexity refers to the number of unique DNA fragments present in the library. Libraries with fewer duplicate reads due to PCR amplification are said to have higher complexity and better reflect the composition of the original DNA sample. PCR amplification is inherently a redundant process, and therefore the complexity of the experimental sample will be lower than in the theoretical case where there is zero duplication ("theoretical - no duplication," long dashed line). However, these data still show that libraries treated with 2-hydroxymethylpyridineborane have high complexity.

[0189] Normalized coverage of biomarkers in whole-genome sequencing. Normalized coverage of selected biomarkers in samples treated with 2-methylpyridineborane complex or 2-hydroxymethylpyridineborane complex was investigated using an Illumina NovaSeq instrument. Normalized coverage is defined as the absolute coverage of a region divided by the mean absolute coverage of the library. The data are provided in Figure 4. For five of the six hypermethylated biomarkers analyzed, samples treated with 2-hydroxymethylpyridineborane showed higher normalized coverage than samples treated with 2-methylpyridineborane alone. These data suggest that the use of 2-hydroxymethylpyridineborane as a reducing agent in TAPS results in a library that allows for improved amplification of hypermethylated regions associated with the detection of conditions such as cancer or other diseases.

[0190] Lambda methylation rate GC dropout and false positive rate detected in TAPS samples after treatment with different borane reducing agents. The methylation rate of fully methylated lambda DNA spike in was determined for further borane reducing reagents. The data are shown in Table 5. These data indicate that pyridine deuterated borane complexes detect similar levels of methylation as pyridine borane analogs (see Pic (97.5%; 2-methylpyridine borane complex) and ESI021 (97.6%; 2-methylpyridine deuterated borane complex); ESI047 (93.1%; 2-hydroxymethylpyridine complex) and ESI050 (92.4%; 2-hydroxymethylpyridine deuterated borane complex)). These data also show that complexes other than the 2-methylpyridineborane complex can achieve methylation rates of over 96% (e.g., ESI051 (5-hydroxymethyl-2-methylpyridineborane complex) and ESI052 (3,4-dimethoxy-2-hydroxymethylpyridineborane complex)).

[0191] The false-positive rates in TAPS reactions using different borane complexes were also analyzed. The data are shown in Table 5. These data indicate that the 2-methylpyridine deuterated borane complex ("ESI021") and the 2-hydroxymethylpyridine borane complex ("ESI047") induce similar false-positive rates to the 2-methylpyridine borane complex ("Pic"). The data were measured using an Illumina NextSeq instrument.

[0192] Next, the lambda GC dropout rate was measured after TAPS reactions with various borane complexes. The data are shown in Table 5. These data show that borane complexes with 2-hydroxymethylpyridine exhibit a lower GC dropout rate than complexes without the 2-hydroxymethylpyridine ligand, indicating greater coverage of GC-rich regions of the genome. GC dropout is a metric for the degree of sequencing bias in a sample, where samples with greater GC bias have correspondingly higher GC dropout. Therefore, lambda GC dropout indicates sequencing bias from methylated lambda DNA spike-in for TAPS reactions where the majority of C is methylated. Measurements were performed using an Illumina NextSeq instrument.

[0193] Next, the overall GC dropout rate was measured after TAPS reactions with various borane complexes. The data are shown in Table 5. These data indicate that borane complexes with 2-hydroxymethylpyridine ligand exhibited a lower GC dropout rate than complexes without 2-hydroxymethylpyridine ligand, indicating greater coverage of GC-rich and highly methylated regions of the genome. The overall GC dropout represents the sum of biases from different DNA components during the reaction: 92.975% NA12878, 5% methylated lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spike-in. Data were measured using an Illumina NextSeq instrument. [Table 4] Comparison of pyridineborane complexes with and without hydroxymethyl groups. The overall GC dropout rates for 2-methylpyridineborane and 2-hydroxymethylpyridineborane were determined by TAPS. The data are shown in Table 6. These data indicate that samples treated with 2-hydroxymethylpyridineborane have substantially lower levels of GC dropout compared to samples treated with 2-methylpyridineborane, and that GC-rich region coverage is higher when using 2-hydroxymethylpyridineborane. [Table 5]

[0194] Conclusion: These data suggest that the main advantages of using borane or deuterated borane complexes containing substituted 2-hydroxymethylpyridine over 2-methylpyridine borane complexes are 1) increased coverage in highly methylated regions in both human and fully methylated lambda, including biomarkers, and 2) higher methylation in partially methylated pUC19 spike ins, demonstrating superior performance in "real-world" samples with a much lower percentage of methylated DNA.

[0195] Example 2 This example describes the testing of further functionalized pyridineborane complexes. In this example, further pyridineborane complexes shown in Table 7 below, as well as picB, ESI046, and ESI047, were synthesized and evaluated. [Table 6]

[0196] Unless otherwise stated, TAPS, post-TAPS amplification yield, lambda conversion rate, false positive rate, lambda GC dropout, and overall GC dropout were evaluated as described in Example 1 above. The DNA input for the TAPS reaction contained a total of 75 ng of DNA, specifically 94.8% gDNA NA12878 with the following spike-ins: 100% me-lambda (5.0%), 100% me-pUC19 (0.2%), and non-me 2kb amplicon (0.025%).

[0197] result The TAPS reduction reaction was carried out using additional pyridineborane complexes as reducing agents. Figures 5–9 show the amplification yield, lambda conversion rate, false positive rate, lambda GC dropout, and overall GC dropout after TAPS using the pyridineborane complex, respectively. The TAPS reduction reaction was carried out both with and without pre-extension (black bars). The pre-extension condition involved incubating the reduced DNA at 72°C for 30 minutes in the presence of polymerase mix before PCR, while the non-pre-extension condition involved performing PCR on the reduced DNA without prior incubation in the presence of polymerase mix. Unless otherwise indicated, the mean values ​​shown are obtained from two replicates (ND: undetermined). Error bars reflect the standard deviation. The performance of the pyridineborane complex was compared with that of the 2-methylpyridineborane complex ("picB").

[0198] The results of these experiments are summarized in Table 8. Specifically, pyridineborane complexes were ranked based on whether they met the following criteria: (1) their use did not result in a dramatic decrease in pre-extension amplification yield, (2) their use with pre-extension resulted in a lambda conversion rate of over 96%, (3) their use with pre-extension resulted in a false-positive rate of less than 1.2%, and (4) their use with pre-extension resulted in a lambda GC dropout rate of less than 8.21. The following pyridineborane complexes met all four of these criteria: ESI047, ESI070, ESI075, ESI076, ESI079, and ESI093. [Table 7]

[0199] Figures 10A-G and 11A-G provide plots of lambda (Figure 10) and overall (Figure 11) GC dropout for the pyridineborane complexes (ESI047, ESI070, ESI075, ESI076, ESI079, and ESI093) with the highest scores (i.e., compounds with a score of 4) shown in Table 8, as well as for ESI090. In these plots, further pyridineborane compounds are compared to pic-borane in reactions with or without pre-extension: solid black lines - pic-borane with pre-extension; dotted lines - pic-borane without pre-extension; novel compounds with pre-extension - filled circles; novel compounds without pre-extension - open circles. For ESI047, ESI070, ESI075, ESI076, ESI079, and ESI093, GC dropout was reduced compared to picB, suggesting that these compounds enable more efficient amplification in regions with higher GC content.

[0200] In describing the present invention, numerous references, including patents and various publications, are cited and discussed. Such citations and descriptions are provided solely to clarify the description of the present invention and do not constitute prior art of the present invention as described herein. All references cited and described herein are incorporated herein by reference in their entirety.

Claims

1. A method for converting 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC) to dihydrouracil (DHU), wherein a nucleic acid sample containing 5caC and / or 5fC is given formula (I): 【Chemistry 1】 This includes contacting with a borane reducing agent or a salt thereof, in the formula, A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclil, each of which is R 1 , R 2 , R 3 , R 4 , and R 5 It has been replaced with, 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, amidoxymil, aryl, and heterocyclyl Each X is independently either hydrogen or deuterium. The aforementioned compound, 【Chemistry 2】 However, the aforementioned method is not the same.

2. The method according to claim 1, wherein A is selected from pyridine, imidazole, quinoline, 9,10-dihydroacridine, and imidazo[1,5-a]pyridine.

3. The borane reducing agent is given by formula (Ia): 【Transformation 3】 It is a compound of the formula, in which, R 1 is 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 - Selected from alkyl groups, R 2 is hydrogen, halo, hydroxyl, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy, C 1 -C 4 Selected from hydroxyalkyl and amidoximol, Here, R 1 and R 2 These may, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring optionally substituted with a hydroxyl group. R 3 is 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 Selected from mercaptoalkyl, amidoximil, and aryl, R 4 is hydrogen, halo, hydroxyl, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy, C 1 -C 4 Selected from hydroxyalkyl and heterocyclyl, Here, R 3 and R 4 These may, together with the carbon atoms to which they are bonded, form a five- or six-membered ring. R 5 is hydrogen, C 1 -C 4 Alkyl and C 1 -C 4 The method according to claim 1 or claim 2, wherein the hydroxyalkyl group is selected from the hydroxyalkyl group.

4. R 1 is 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 - Selected from alkyl groups, R 2 is hydrogen, halo, hydroxyl, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy, C 1 -C 4 Selected from hydroxyalkyl and amidoximol, 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 amidoxime, R 4 is hydrogen, halo, hydroxyl, C 1 -C 4 Alkyl and C 1 -C 4 Selected from hydroxyalkyl groups, R 5 is hydrogen, C 1 -C 4 Alkyl and C 1 -C 4 The method according to claim 3, selected from hydroxyalkyl groups.

5. R 1 C 1 -C 4 It is a hydroxyalkyl and R 2 , R 3 , R 4 , and R 5 However, each independently, hydrogen and C 1 -C 4 Alkyl, C 1 -C 4 The method according to any one of claims 1 to 4, selected from alkoxy, phenyl, and monocyclic 5 or 6-membered heterocyclines having one nitrogen atom.

6. R 1 It is hydroxymethyl, R 2 That is hydrogen, R 3 However, hydrogen, C 1 -C 4 Selected from alkyl and phenyl, R 4 However, C 1 -C 4 Alkyl, C 1 -C 4 Selected from alkoxys and monocyclic five-membered heterocyclines having one nitrogen atom, R 5 The method according to any one of claims 1 to 4, wherein the substance is hydrogen.

7. R 1 C 1 -C 4 It is a hydroxyalkyl and R 2 , R 3 , R 4 , and R 5 However, each independently, hydrogen and C 1 -C 4 Alkyl and C 1 -C 4 The method according to any one of claims 1 to 4, selected from alkoxys.

8. R 1 , R 2 , R 3 , R 4 , and R 5 However, each is independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 1 , R 2 , R 3 , R 4 , and R 5 The method according to any one of claims 1 to 4, wherein at least one of them is hydroxymethyl.

9. R 1 However, it is selected from hydrogen, methyl, and hydroxymethyl, R 2 However, it is selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 3 However, it is selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 4 However, selected from hydrogen and methyl, R 5 However, it is hydrogen, Here, R 1 , R 2 , and R 3 The method according to any one of claims 1 to 4, wherein at least one of them is hydroxymethyl.

10. The borane reducing agent is given by formula (Ib): 【Chemistry 4】 It is a compound or salt thereof, in which, R 2 , R 3 , R 4 , and R 5 These are, independently, hydrogen and C 1 -C 4 Alkyl, C 1 -C 4 Selected from alkoxy, aryl, and heterocyclyl, The method according to any one of claims 1 to 4, wherein each X is independently hydrogen or deuterium.

11. R 2 , R 3 , R 4 , and R 5 The method according to claim 10, wherein each is independently selected from a monocyclic five-membered heterocycline having a hydrogen atom, a methyl atom, a methoxy atom, a phenyl atom, and one nitrogen atom.

12. R 2 , R 3 , R 4 , and R 5 However, each independently, hydrogen and C 1 -C 4 Alkyl and C 1 -C 4 The method according to claim 11, wherein the alkoxy is selected.

13. R 2 However, hydrogen, C 1 -C 4 Alkyl and C 1 -C 4 Selected from alkoxy, R 3 However, hydrogen, C 1 -C 4 Alkyl and C 1 -C 4 Selected from alkoxy, R 4 However, hydrogen and C 1 -C 4 Selected from alkyl groups, R 5 The method according to claim 10, wherein the hydrogen is...

14. R 2 , R 3 , R 4 , and R 5 The method according to claim 10, wherein each is independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidinyl.

15. R 2 , R 3 , R 4 , and R 5 The method according to claim 10, wherein each is independently selected from hydrogen, methyl, and methoxy.

16. R 2 However, it is selected from hydrogen, methyl, and methoxy. R 3 However, it is selected from hydrogen, methyl, and methoxy. R 4 However, selected from hydrogen and methyl, R 5 The method according to claim 15, wherein the hydrogen is...

17. The method according to any one of claims 1 to 16, wherein each X is hydrogen.

18. The method according to any one of claims 1 to 16, wherein each X is deuterium.

19. The borane reducing agent is 【Chemistry 5-1】 【Chemistry 5-2】 The method according to claim 1, as well as a selection from those salts.

20. The borane reducing agent is 【Transformation 6】 The method according to claim 1, wherein a selection is made from salts thereof.

21. The method according to any one of claims 1 to 20, further comprising contacting the nucleic acid sample with an oxidizing agent before contacting it with the borane reducing agent.

22. The method according to claim 21, wherein the oxidizing agent is Ten-Eleven Translocation (TET) enzyme.

23. The method according to claim 22, wherein the TET enzyme comprises human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria gulberi TET (NgTET), Coprinopsis cinerea TET (CcTET), or derivatives or analogs thereof.

24. The method according to claim 21, wherein the oxidizing agent includes a chemical oxidizing agent.

25. The aforementioned chemical oxidizing agent is manganese oxide (MnO 2 ), potassium perruthenate (KRuO 4 The method according to claim 24, selected from the group consisting of ), Cu(II) / 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenic acid (PSP), tetraphenylphosphonium lutenate, copper salt or complex of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxy (3-Carbamoyl-PROXYL), copper salt or complex of 2-azaadamantane-N-oxyl (AZADO), and copper salt or complex of 9-azabicyclo[3.3.1]nonane N'-oxyl (ABNO).

26. The method according to any one of claims 21 to 25, further comprising adding a blocking group to one or more modified cytosines in the nucleic acid sample.

27. The method according to claim 26, wherein the blocking group is added before contact with the oxidizing agent.

28. The method according to claim 27, wherein one or more of the modified cytosines contain 5hmC.

29. The method according to claim 28, wherein the blocking group comprises a sugar or a uridine diphosphate (UDP)-linked sugar.

30. The method according to claim 26, wherein the blocking group is added after contact with the oxidizing agent and before contact with the borane reducing agent.

31. The method according to claim 30, wherein the one or more modified cytosines include 5caC or 5fC.

32. The method according to claim 31, wherein the blocking group comprises an aldehyde-reactive compound, or the addition of the blocking group comprises contacting the nucleic acid sample with (i) a coupling agent and (ii) an amine, hydrazine, or hydroxylamine compound.

33. The method according to any one of claims 1 to 32, further comprising sequencing the nucleic acid sample after contact with the borane reducing agent to identify the converted cytosine bases.

34. The method according to claim 33, further comprising amplifying the copy number of the nucleic acid sample, wherein the amplification is optionally performed after contact between the nucleic acid sample and the borane reducing agent and before sequencing of the nucleic acid sample.

35. The method according to any one of claims 1 to 34, wherein the nucleic acid sample is a DNA sample.

36. A compound selected from the following: 【Chemistry 7-1】 【Chemistry 7-2】

37. A system or kit for converting 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC) to dihydrouracil (DHU), wherein formula (I): 【Transformation 8】 The formula comprises a borane reducing reagent or a salt thereof, A is a monocyclic, bicyclic, or tricyclic heteroaryl, or a monocyclic, bicyclic, or tricyclic heterocyclil, each of which is R 1 , R 2 , R 3 , R 4 , and R 5 It has been replaced with, R 1 , R 2 , R 3 , R 4 , and R 5 These are, independently, hydrogen, halo, hydroxyl, amino, and 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 - Selected from alkyl, amidoximol, aryl, and heterocyclyl, Each X is independently either hydrogen or deuterium. The aforementioned compound, 【Chemistry 9】 However, the aforementioned system or kit.

38. The system or kit according to claim 37, wherein A is selected from pyridine, imidazole, quinoline, 9,10-dihydroacridine, and imidazo[1,5-a]pyridine.

39. The borane reducing agent is given by formula (Ia): 【Chemistry 10】 It is a compound of the formula, in which, R 1 is 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 - Selected from alkyl groups, R 2 is hydrogen, halo, hydroxyl, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy, C 1 -C 4 Selected from hydroxyalkyl and amidoximol, Here, R 1 and R 2 These may, together with the carbon atoms to which they are bonded, form a 5- or 6-membered ring optionally substituted with a hydroxyl group. R 3 is 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 Selected from mercaptoalkyl, amidoximil, and aryl, R 4 is hydrogen, halo, hydroxyl, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy, C 1 -C 4 Selected from hydroxyalkyl and heterocyclyl, Here, R 3 and R 4 These may, together with the carbon atoms to which they are bonded, form a five- or six-membered ring. R 5 is hydrogen, C 1 -C 4 Alkyl and C 1 -C 4 A system or kit according to claim 37 or claim 38, selected from hydroxyalkyl groups.

40. R 1 is 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 - Selected from alkyl groups, R 2 is hydrogen, halo, hydroxyl, C 1 -C 4 Alkyl, C 1 -C 4 Alkoxy, C 1 -C 4 Selected from hydroxyalkyl and amidoximol, R 3 is 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 Selected from mercaptoalkyl and amidoximil, R 4 is hydrogen, halo, hydroxyl, C 1 -C 4 Alkyl and C 1 -C 4 Selected from hydroxyalkyl groups, R 5 is hydrogen, C 1 -C 4 Alkyl and C 1 -C 4 The system or kit according to claim 39, selected from hydroxyalkyl groups.

41. R 1 C 1 -C 4 It is a hydroxyalkyl and R 2 , R 3 , R 4 , and R 5 However, each independently, hydrogen and C 1 -C 4 Alkyl, C 1 -C 4 A system or kit according to any one of claims 37 to 40, selected from alkoxy, phenyl, and monocyclic 5 or 6-membered heterocyclines having one nitrogen atom.

42. R 1 It is hydroxymethyl, R 2 That is hydrogen, R 3 However, hydrogen, C 1 -C 4 Selected from alkyl and phenyl, R 4 However, C 1 -C 4 Alkyl, C 1 -C 4 Selected from alkoxys and monocyclic five-membered heterocyclines having one nitrogen atom, R 5 The system or kit according to any one of claims 37 to 40, wherein the hydrogen is...

43. R 1 C 1 -C 4 It is a hydroxyalkyl and R 2 , R 3 , R 4 , and R 5 However, each independently, hydrogen and C 1 -C 4 Alkyl and C 1 -C 4 A system or kit according to any one of claims 37 to 40, selected from alkoxys.

44. R 1 , R 2 , R 3 , R 4 , and R 5 However, each is independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 1 , R 2 , R 3 , R 4 , and R 5 The system or kit according to any one of claims 37 to 40, wherein at least one of them is hydroxymethyl.

45. R 1 However, it is selected from hydrogen, methyl, and hydroxymethyl, R 2 However, it is selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 3 However, it is selected from hydrogen, methyl, methoxy, and hydroxymethyl, R 4 However, selected from hydrogen and methyl, R 5 However, it is hydrogen, Here, R 1 , R 2 , and R 3 The system or kit according to any one of claims 37 to 40, wherein at least one of them is hydroxymethyl.

46. The borane reducing agent is given by formula (Ib): 【Chemistry 11】 It is a compound or salt thereof, in which, R 2 , R 3 , R 4 , and R 5 These are, independently, hydrogen and C 1 -C 4 Alkyl, C 1 -C 4 Selected from alkoxy, aryl, and heterocyclyl, The system or kit according to any one of claims 37 to 40, wherein each X is independently hydrogen or deuterium.

47. R 2 , R 3 , R 4 , and R 5 The system or kit according to claim 46, wherein each is independently selected from a monocyclic five-membered heterocycline having a hydrogen atom, a methyl atom, a methoxy atom, a phenyl atom, and one nitrogen atom.

48. R 2 , R 3 , R 4 , and R 5 However, each independently, hydrogen and C 1 -C 4 Alkyl and C 1 -C 4 A system or kit according to claim 47, selected from alkoxys.

49. R 2 However, hydrogen, C 1 -C 4 Alkyl and C 1 -C 4 Selected from alkoxy, R 3 However, hydrogen, C 1 -C 4 Alkyl and C 1 -C 4 Selected from alkoxy, R 4 However, hydrogen and C 1 -C 4 Selected from alkyl groups, R 5 The system or kit according to claim 46, wherein the hydrogen is hydrogen.

50. R 2 , R 3 , R 4 , and R 5 The system or kit according to claim 46, wherein each is independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidinyl.

51. R 2 , R 3 , R 4 , and R 5 The system or kit according to claim 46, wherein each is independently selected from hydrogen, methyl, and methoxy.

52. R 2 However, it is selected from hydrogen, methyl, and methoxy. R 3 However, it is selected from hydrogen, methyl, and methoxy. R 4 However, selected from hydrogen and methyl, R 5 The system or kit according to claim 51, wherein the hydrogen is hydrogen.

53. The system or kit according to any one of claims 37 to 52, wherein each X is hydrogen.

54. The system or kit according to any one of claims 37 to 52, wherein each X is deuterium.

55. The borane reducing agent is 【Chemistry 12-1】 【Chemistry 12-2】 The system or kit according to claim 37, further comprising salts selected from those salts.

56. The borane reducing agent is 【Chemistry 13】 The system or kit according to claim 37, further comprising salts selected from those salts.

57. The system or kit according to any one of claims 37 to 56, further comprising an oxidizing reagent.

58. The system or kit according to claim 57, wherein the oxidizing agent is a ten-eleven transmission (TET) enzyme.

59. The system or kit according to claim 58, wherein the TET enzyme comprises human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria gulberi TET (NgTET), Coprinopsis cinerea (CcTET), or derivatives or analogs thereof.

60. The system or kit according to claim 57, wherein the oxidizing agent comprises a chemical oxidizing agent.

61. The aforementioned chemical oxidizing agent is manganese oxide (MnO 2 ), potassium perruthenate (KRuO 4 The system or kit according to claim 60, selected from the group consisting of ), Cu(II) / 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), tetrapropylammonium perthenate (TPAP), tetrabutylammonium perthenate (TBAP), polymer-supported perthenic acid (PSP), tetraphenylphosphonium lutenate, copper salt or complex of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxy (3-Carbamoyl-PROXYL), copper salt or complex of 2-azaadamantane-N-oxyl (AZADO), and copper salt or complex of 9-azabicyclo[3.3.1]nonane N'-oxyl (ABNO).

62. The system or kit according to any one of claims 37 to 61, further comprising a blocking reagent.

63. The system or kit according to claim 62, wherein the blocking reagent is selected from the group consisting of sugars, uridine diphosphate (UDP)-conjugated sugars, and aldehyde-reactive compounds.

64. The system or kit according to claim 62 or 63, wherein the blocking reagent is an aldehyde-reactive compound selected from the group consisting of hydroxylamine derivatives, hydrazine derivatives, and hydrazide derivatives.

65. The system or kit according to claim 62 or 63, wherein the blocking reagent is a sugar or a uridine diphosphate (UDP)-linked sugar, and the system or kit further comprises a glucosyltransferase enzyme.

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