Borane reducing reagent

Abstract

This disclosure provides a borane reducing agent, and particularly a borane reducing agent for use in a methylation detection assay that includes the step of reducing 5-formylcytosine (5fC) and / or 5-carboxycytosine (5caC) to dihydrouracil (DHU).

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 590,928, filed October 17, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure provides a borane reducing agent, and particularly a borane reducing agent for use in a methylation detection assay that includes the step of reducing 5-formylcytosine (5fC) and / or 5-carboxycytosine (5caC) to dihydrouracil (DHU). Background Technology

[0004] Methylation, particularly methylation at position 5 of cytosine, is a major epigenetic modification in the mammalian genome associated with biological and pathological processes, including as a recognized biomarker for various diseases and cancers. Besides methylation of cytosine residues, other modifications, such as the oxidation of methylated cytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and / or 5-carboxycytosine (5caC), and the methylation of adenine (A) to N, are also significant. 6- Methyladenine (6-mA) has also been identified as an important epigenetic regulator. Therefore, the detection of epigenetic modifications such as methylation has become crucial for scientific / diagnostic purposes.

[0005] Methylation can be determined, for example, through 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 by low-depth, targeted, or low-resolution sequencing and qualitative enrichment-based sequencing. Summary of the Invention

[0006] This disclosure provides a borane reducing agent, and particularly a borane reducing agent for use in a methylation detection assay that includes the step of reducing 5-formylcytosine (5fC) and / or 5-carboxycytosine (5caC) to dihydrouracil (DHU).

[0007] The implementation scheme disclosed herein includes the following.

[0008] In some preferred embodiments, the present invention provides a method for converting 5-carboxycytosine (5caC) and / or 5-formylcytosine (5fC) into dihydrouracil (DHU), the method comprising reacting a nucleic acid sample containing 5caC and / or 5fC with a borane reducing agent of formula (I):

[0009] (I)

[0010] or its salt in contact, wherein:

[0011] A is a monocyclic, bicyclic, or tricyclic heteroaryl group, or a monocyclic, bicyclic, or tricyclic heterocyclic group, each of which is decomposed by R. 1 R 2 R 3 R 4 and R 5 replace;

[0012] R 1 R 2 R 3 R 4 and R 5 Each group is independently selected from hydrogen, halogenated, hydroxyl, 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, amidoximyl, aryl, and heterocyclic groups; and

[0013] Each X is independently either hydrogen or deuterium.

[0014] The compound in question is not:

[0015] or .

[0016] In some preferred embodiments, the present invention provides a method for converting 5-carboxycytosine (5caC) and / or 5-formylcytosine (5fC) into dihydrouracil (DHU), the method comprising reacting a nucleic acid sample containing 5caC and / or 5fC with a borane reducing agent of formula (I):

[0017] (I)

[0018] or its salt in contact, wherein:

[0019] A is a monocyclic, bicyclic, or tricyclic heteroaryl group, or a monocyclic, bicyclic, or tricyclic heterocyclic group, each of which is decomposed by R. 1 R 2 R 3 R 4 and R 5 replace;

[0020] R 1 R 2 R 3 R 4 and R 5Each is independently selected from hydrogen, halogroup, hydroxyl group, amino group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group, C1-C4 haloalkyl group, C1-C4 aminoalkyl group, C1-C4 mercaptoalkyl group, C1-C4-alkoxy-C1-C4-alkyl group, and geminoxamic group; and

[0021] Each X is independently either hydrogen or deuterium.

[0022] The compound in question is not:

[0023] or .

[0024] In some preferred embodiments, A is selected from pyridine, imidazole, quinoline, 9,10-dihydroacridine, and imidazo[1,5-a]pyridine.

[0025] In some preferred embodiments, the borane reducing agent is a compound of formula (Ia):

[0026] (Ia).

[0027] In some preferred embodiments, the borane reducing agent is a compound of formula (Ia):

[0028] (Ia)

[0029] in:

[0030] R 1 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl and C1-C4-alkoxy-C1-C4-alkyl;

[0031] R 2 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and amylopime group;

[0032] Where R 1 and R 2 Optionally, together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings that are optionally substituted with hydroxyl groups;

[0033] R 3 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, geminosine oxime and aryl;

[0034] R 4 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and heterocyclic group;

[0035] Where R 3 and R 4 Optionally, they form 5-membered or 6-membered rings together with the carbon atoms to which they are attached; and

[0036] R 5 It is selected from hydrogen, C1-C4 alkyl and C1-C4 hydroxyalkyl.

[0037] In some preferred embodiments, R 1 and R 2 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings. In some preferred embodiments, R 3 and R 4 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings. In some preferred embodiments, R 1 and R 2 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings, R 3 and R 4 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings.

[0038] In some preferred embodiments, R 1 and R 2 Together with the carbon atoms to which they are attached, they form 5- or 6-membered rings substituted with hydroxyl groups. In some preferred embodiments, R 3 and R 4 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings. In some preferred embodiments, R 1 and R 2 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings substituted with hydroxyl groups, R 3 and R 4 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings.

[0039] In some preferred implementation schemes:

[0040] R 1 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl and C1-C4-alkoxy-C1-C4-alkyl;

[0041] R 2 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and amylopime group;

[0042] R 3Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl and geminoxamic groups;

[0043] R 4 Selected from hydrogen, halogroup, hydroxyl, C1-C4 alkyl, and C1-C4 hydroxyalkyl; and

[0044] R 5 It is selected from hydrogen, C1-C4 alkyl and C1-C4 hydroxyalkyl.

[0045] In some preferred 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, C1-C4 alkyl, C1-C4 alkoxy, phenyl, and monocyclic 5- or 6-membered heterocyclic groups having one nitrogen atom.

[0046] In some preferred implementation schemes:

[0047] R 1 It is hydroxymethyl;

[0048] R 2 It is hydrogen;

[0049] R 3 Selected from hydrogen, C1-C4 alkyl and phenyl;

[0050] R 4 Selected from C1-C4 alkyl, C1-C4 alkoxy, and monocyclic 5-membered heterocyclic groups having one nitrogen atom; and

[0051] R 5 It is hydrogen.

[0052] In some preferred 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, C1-C4 alkyl, and C1-C4 alkoxy.

[0053] In some preferred embodiments, R 1 R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, wherein R 1 R 2 R 3 R4 and R 5 At least one of them is hydroxymethyl.

[0054] In some preferred implementation schemes:

[0055] R 1 Selected from hydrogen, methyl, and hydroxymethyl;

[0056] R 2 Selected from hydrogen, methyl, methoxy, and hydroxymethyl;

[0057] R 3 Selected from hydrogen, methyl, methoxy, and hydroxymethyl;

[0058] R 4 Selected from hydrogen and methyl; and

[0059] R 5 It is hydrogen;

[0060] Where R 1 R 2 and R 3 At least one of them is hydroxymethyl.

[0061] In some preferred embodiments, the borane reducing agent is a compound of formula (Ib):

[0062] (Ib)

[0063] or its salt, wherein:

[0064] R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, aryl, and heterocyclic groups; and

[0065] Each X is independently either hydrogen or deuterium.

[0066] In some preferred embodiments, the borane reducing agent is a compound of formula (Ib):

[0067] (Ib)

[0068] or its salt, wherein:

[0069] R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy; and

[0070] Each X is independently either hydrogen or deuterium.

[0071] In some preferred embodiments, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, phenyl, and monocyclic 5-membered heterocyclic groups having one nitrogen atom.

[0072] In some preferred embodiments, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

[0073] In some preferred implementation schemes:

[0074] R 2 Selected from hydrogen, C1-C4 alkyl and C1-C4 alkoxy;

[0075] R 3 Selected from hydrogen, C1-C4 alkyl and C1-C4 alkoxy;

[0076] R 4 Selected from hydrogen and C1-C4 alkyl groups; and

[0077] R 5 It is hydrogen.

[0078] In some preferred embodiments, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidine.

[0079] In some preferred embodiments, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, and methoxy.

[0080] In some preferred implementation schemes:

[0081] R 2 Selected from hydrogen, methyl, and methoxy;

[0082] R 3 Selected from hydrogen, methyl, and methoxy;

[0083] R 4 Selected from hydrogen and methyl; and

[0084] R 5 It is hydrogen.

[0085] In some preferred embodiments, each X is hydrogen.

[0086] In some preferred embodiments, each X is tritium.

[0087] In some preferred embodiments, the borane reducing agent is selected from:

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] and

[0096] And their salts.

[0097] In some preferred embodiments, the borane reducing agent is selected from:

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] And their salts.

[0104] In some preferred embodiments, the borane reducing agent is selected from:

[0105] and And their salts.

[0106] In some preferred embodiments, the method further includes contacting the nucleic acid sample with an oxidizing agent prior to contact with the borane reducing agent. In some preferred embodiments, the oxidizing agent is a deca-eicosyltransfer (TET) enzyme. In some preferred embodiments, the TET enzyme includes human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria gulberiTET (NgTET), Coprinopsis cinerea TET (CcTET), or derivatives or analogs thereof.

[0107] In some preferred embodiments, the oxidant includes a chemical oxidant. In some preferred embodiments, the chemical oxidant is selected from the group consisting of: manganese oxide (MnO2), potassium perruthenate (KRuO4), Cu(II) / 2,2,6,6-tetramethylpiperidin-1-oxy (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenylphosphoniurn ruthenate, copper salts or complexes of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrololin-1-yloxy (3-Carbamoyl-PROXYL), copper salts or complexes of 2-azaadamantane-N-oxy (AZADO), and copper salts or complexes of 9-azabicyclo[3.3.1]nonane-N′-oxy (ABNO).

[0108] In some preferred embodiments, the method further includes adding a blocking group to one or more modified cytosines in the nucleic acid sample.

[0109] In some preferred embodiments, a blocking group is added before contact with the oxidant.

[0110] In some preferred embodiments, the one or more modified cytosines comprise 5 hmC.

[0111] In some preferred embodiments, the blocking group includes sugars or sugars linked by uridine diphosphate (UDP).

[0112] In some preferred embodiments, a blocking group is added after contact with the oxidant and before contact with the borane reducing agent.

[0113] In some preferred embodiments, the one or more modified cytosines include 5caC or 5fC.

[0114] In some preferred embodiments, the blocking group includes an aldehyde-reactive compound. In some preferred embodiments, the aldehyde-reactive compound includes a hydroxylamine derivative, a hydrazine derivative, or an acylhydrazine derivative.

[0115] In some preferred embodiments, adding a blocking group includes contacting the nucleic acid sample with (i) a coupling agent and (ii) an amine, hydrazine, or hydroxylamine compound.

[0116] In some preferred embodiments, the method further includes sequencing the nucleic acid sample after contact with a borane reducing agent to identify the transformed cytosine bases.

[0117] In some preferred embodiments, the method further includes amplifying the copy number of the nucleic acid sample. In some preferred embodiments, amplification is performed after the nucleic acid sample has been contacted with a borane reducing agent. In some preferred embodiments, amplification is performed before sequencing the nucleic acid sample. In some preferred embodiments, amplification is performed after contacting the nucleic acid sample with a borane reducing agent and before sequencing the nucleic acid sample.

[0118] In some preferred embodiments, the nucleic acid sample is a DNA sample.

[0119] In some preferred embodiments, the present invention provides compounds selected from the following:

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] and

[0128] And their salts.

[0129] In some preferred embodiments, the present invention provides compounds selected from the following:

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] .

[0136] In some preferred embodiments, the present invention provides compounds selected from the following:

[0137]

[0138] And their salts.

[0139] In some preferred embodiments, the present invention provides a system or kit for converting 5-carboxycytosine (5caC) and / or 5-formylcytosine (5fC) into dihydrouracil (DHU), the system or kit comprising a borane reducing agent of formula (I):

[0140] (I)

[0141] or its salt, wherein:

[0142] A is a monocyclic, bicyclic, or tricyclic heteroaryl group, or a monocyclic, bicyclic, or tricyclic heterocyclic group, each of which is decomposed by R. 1 R 2 R 3 R 4 and R 5 replace;

[0143] R 1 R 2 R 3 R 4 and R 5 Each group is independently selected from hydrogen, halogenated, hydroxyl, 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, amidoximyl, aryl, and heterocyclic groups; and

[0144] Each X is independently either hydrogen or deuterium.

[0145] The compound in question is not:

[0146] or .

[0147] In some preferred embodiments, the present invention provides a system or kit for converting 5-carboxycytosine (5caC) and / or 5-formylcytosine (5fC) into dihydrouracil (DHU), the system or kit comprising a borane reducing agent of formula (I):

[0148] (I)

[0149] or its salt, wherein:

[0150] A is a monocyclic, bicyclic, or tricyclic heteroaryl group, or a monocyclic, bicyclic, or tricyclic heterocyclic group, each of which is decomposed by R. 1 R 2 R 3 R 4 and R 5 replace;

[0151] R 1 R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, halogroup, hydroxyl group, amino group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group, C1-C4 haloalkyl group, C1-C4 aminoalkyl group, C1-C4 mercaptoalkyl group, C1-C4-alkoxy-C1-C4-alkyl group, and geminoxamic group; and

[0152] Each X is independently either hydrogen or deuterium.

[0153] The compound in question is not:

[0154] or .

[0155] In some preferred embodiments, A is selected from pyridine, imidazole, quinoline, 9,10-dihydroacridine, and imidazo[1,5-a]pyridine.

[0156] In some preferred embodiments, the borane reducing agent is a compound of formula (Ia):

[0157] (Ia).

[0158] In some preferred embodiments, the borane reducing agent is a compound of formula (Ia):

[0159] (Ia)

[0160] in:

[0161] R 1Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl and C1-C4-alkoxy-C1-C4-alkyl;

[0162] R 2 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and amylopime group;

[0163] Where R 1 and R 2 Optionally, together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings that are optionally substituted with hydroxyl groups;

[0164] R 3 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, geminosine oxime and aryl;

[0165] R 4 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and heterocyclic group;

[0166] Where R 3 and R 4 Optionally, they form 5-membered or 6-membered rings together with the carbon atoms to which they are attached; and

[0167] R 5 It is selected from hydrogen, C1-C4 alkyl and C1-C4 hydroxyalkyl.

[0168] In some preferred embodiments, R 1 and R 2 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings. In some preferred embodiments, R 3 and R 4 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings. In some preferred embodiments, R 1 and R 2 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings, R 3 and R 4 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings.

[0169] In some preferred embodiments, R 1 and R 2 Together with the carbon atoms to which they are attached, they form 5- or 6-membered rings substituted with hydroxyl groups. In some preferred embodiments, R 3 and R 4Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings. In some preferred embodiments, R 1 and R 2 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings substituted with hydroxyl groups, R 3 and R 4 Together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings.

[0170] In some preferred implementation schemes:

[0171] R 1 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl and C1-C4-alkoxy-C1-C4-alkyl;

[0172] R 2 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and amylopime group;

[0173] R 3 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl and geminoxamic groups;

[0174] R 4 Selected from hydrogen, halogroup, hydroxyl, C1-C4 alkyl, and C1-C4 hydroxyalkyl; and

[0175] R 5 It is selected from hydrogen, C1-C4 alkyl and C1-C4 hydroxyalkyl.

[0176] In some preferred 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, C1-C4 alkyl, C1-C4 alkoxy, phenyl, and monocyclic 5- or 6-membered heterocyclic groups having one nitrogen atom.

[0177] In some preferred implementation schemes:

[0178] R 1 It is hydroxymethyl;

[0179] R 2 It is hydrogen;

[0180] R 3 Selected from hydrogen, C1-C4 alkyl and phenyl;

[0181] R 4Selected from C1-C4 alkyl, C1-C4 alkoxy, and monocyclic 5-membered heterocyclic groups having one nitrogen atom; and

[0182] R 5 It is hydrogen.

[0183] In some preferred 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, C1-C4 alkyl, and C1-C4 alkoxy.

[0184] In some preferred embodiments, R 1 R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, wherein R 1 R 2 R 3 R 4 and R 5 At least one of them is hydroxymethyl.

[0185] In some preferred implementation schemes:

[0186] R 1 Selected from hydrogen, methyl, and hydroxymethyl;

[0187] R 2 Selected from hydrogen, methyl, methoxy, and hydroxymethyl;

[0188] R 3 Selected from hydrogen, methyl, methoxy, and hydroxymethyl;

[0189] R 4 Selected from hydrogen and methyl; and

[0190] R 5 It is hydrogen;

[0191] Where R 1 R 2 and R 3 At least one of them is hydroxymethyl.

[0192] In some preferred embodiments, the borane reducing agent is a compound of formula (Ib):

[0193] (Ib)

[0194] or its salt, wherein:

[0195] R 2 R3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, aryl, and heterocyclic groups; and

[0196] Each X is independently either hydrogen or deuterium.

[0197] In some preferred embodiments, the borane reducing agent is a compound of formula (Ib):

[0198] (Ib)

[0199] or its salt, wherein:

[0200] R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy; and

[0201] Each X is independently either hydrogen or deuterium.

[0202] In some preferred embodiments, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, phenyl, and monocyclic 5-membered heterocyclic groups having one nitrogen atom.

[0203] In some preferred embodiments, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

[0204] In some preferred implementation schemes:

[0205] R 2 Selected from hydrogen, C1-C4 alkyl and C1-C4 alkoxy;

[0206] R 3 Selected from hydrogen, C1-C4 alkyl and C1-C4 alkoxy;

[0207] R 4 Selected from hydrogen and C1-C4 alkyl groups; and

[0208] R 5 It is hydrogen.

[0209] In some preferred embodiments, R 2 R 3 R 4 and R 5Each is independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidine.

[0210] In some preferred embodiments, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, and methoxy.

[0211] In some preferred implementation schemes:

[0212] R 2 Selected from hydrogen, methyl, and methoxy;

[0213] R 3 Selected from hydrogen, methyl, and methoxy;

[0214] R 4 Selected from hydrogen and methyl; and

[0215] R 5 It is hydrogen.

[0216] In some preferred embodiments, each X is hydrogen.

[0217] In some preferred embodiments, each X is tritium.

[0218] In some preferred embodiments, the borane reducing agent is selected from:

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225] and

[0226] And their salts.

[0227] In some preferred embodiments, the borane reducing agent is selected from:

[0228]

[0229]

[0230]

[0231]

[0232]

[0233] And their salts.

[0234] In some preferred embodiments, the borane reducing agent is selected from:

[0235] and And their salts.

[0236] In some preferred embodiments, the system or kit further includes an oxidizing agent. In some preferred embodiments, the oxidizing agent is a deca-eicosyltransfer (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), Coprinus comatus (CcTET), or derivatives or analogs thereof.

[0237] In some preferred embodiments, the oxidant includes a chemical oxidant. In some preferred embodiments, the chemical oxidant is selected from the group consisting of: manganese oxide (MnO2), potassium perruthenate (KRuO4), Cu(II) / 2,2,6,6-tetramethylpiperidin-1-oxy (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenylphosphoniurn ruthenate, copper salts or complexes of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrololin-1-yloxy (3-Carbamoyl-PROXYL), copper salts or complexes of 2-azaadamantane-N-oxy (AZADO), and copper salts or complexes of 9-azabicyclo[3.3.1]nonane-N′-oxy (ABNO).

[0238] In some preferred embodiments, the system or kit further includes a blocking agent. In some preferred embodiments, the blocking agent is selected from the group consisting of sugars, uridine diphosphate (UDP)-linked 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 acylhydrazine derivatives. In some preferred embodiments, the blocking agent is an aldehyde-reactive compound selected from the group consisting of hydroxylamine derivatives, hydrazine derivatives, and acylhydrazine derivatives. In some preferred embodiments, the blocking agent is a sugar or a uridine diphosphate (UDP)-linked sugar, and the system or kit further includes a glucosyltransferase.

[0239] Other aspects and implementations of this disclosure will become apparent from the following detailed description. Attached Figure Description

[0240] Figure 1 A total GC preference plot comparing the use of the 2-methylpyridineborane complex (“Pic”, solid line) or the 2-hydroxymethylpyridineborane complex (“ESI047”, dashed line) in TAPS reactions on templates with fully methylated Lambda and partially methylated pUC19. The total GC preference plot represents the preference of the entire human genomic DNA component as a library consisting of: 92.975% NA12878 (human genomic DNA), 5% methylated Lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spike-in.

[0241] Figure 2 Lambda GC preference plot comparing the use of 2-methylpyridineborane complex (“Pic”, solid line) or 2-hydroxymethylpyridineborane complex (“ESI047”, dashed line) in the TAPS reaction.

[0242] Figure 3 A graph comparing the library complexity of samples generated by TAPS reactions using either the 2-methylpyridineborane complex (“Pic-KU”, solid line) or the 2-hydroxymethylpyridineborane complex (“ESI47-KU”, dashed line).

[0243] Figure 4A -F. 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 TAPS reactions using 2-methylpyridineborane complex (“Pic-KU”) or 2-hydroxymethylpyridineborane complex (“ESI47-KU”).

[0244] Figure 5 The graph shows the post-TAPS amplification yield of TAPS reduction reactions using selected pyridineborane complexes. Black bars represent reactions with pre-extension, while gray bars represent reactions without pre-extension. The average of two replicates is presented.

[0245] Figure 6A graph showing the lambda conversion (%) of TAPS reduction reactions using selected pyridineborane complexes is presented. Black bars represent reactions with pre-extension, while gray bars represent reactions without pre-extension. The average of two replicates is presented.

[0246] Figure 7 A graph showing the false positive rate (%) of TAPS reduction reactions using selected pyridineborane complexes is presented. Black bars represent reactions with pre-extension, while gray bars represent reactions without pre-extension. The average of two replicates is presented.

[0247] Figure 8 A graph showing the lambda GC loss for TAPS reduction reactions using selected pyridineborane complexes is presented. Black bars represent reactions with pre-extension, while gray bars represent reactions without pre-extension. The average of two replicates is presented.

[0248] Figure 9 A graph showing the overall GC loss for TAPS reduction reactions using selected pyridineborane complexes is presented. Black bars represent reactions with pre-extension, while gray bars represent reactions without pre-extension. The average of two replicates is presented.

[0249] Figure 10A -G. For 2-methylpyridineborane complexes (“picB”, solid lines indicate pre-extension, dashed lines indicate no pre-extension) or test pyridineborane complexes ( Figure 10A “ES1047”; Figure 10B “ES1070”; Figure 10C “ES1075”; Figure 10D “ES1076”; Figure 10E “ES1079”; Figure 10F “ES1090”; Figure 10G The Lambda GC preference plot was used to compare the application of “ES1093” (solid circles indicate pre-extended, hollow circles indicate no pre-extended) in the TAPS reaction.

[0250] Figure 11A -G. For 2-methylpyridineborane complexes (“picB”, solid lines indicate pre-extension, dashed lines indicate no pre-extension) or test pyridineborane complexes ( Figure 11A “ES1047”; Figure 11B “ES1070”; Figure 11C “ES1075”; Figure 11D “ES1076”; Figure 11E “ES1079”; Figure 11F “ES1090”; Figure 11GThe overall GC preference map ("ES1093"; solid circles indicate pre-extension, hollow circles indicate no pre-extension) is used in the TAPS reaction for comparison. The overall GC preference map represents the preference of the entire library as a component of human genomic DNA, which consists of: 92.975% NA12878 (human genomic DNA), 5% methylated Lambda, 2% methylated pUC19, and 0.025% unmethylated 2kb DNA spikes. Detailed Implementation

[0251] Recently, TET-assisted sequencing methods that allow the detection of methylated bases have been developed. This disclosure provides borane reducing reagents, and particularly borane reducing reagents for use in methylation detection assays that include the step of reducing 5-formylcytosine (5fC) and / or 5-carboxycytosine (5caC) to dihydrouracil (DHU). In some preferred embodiments, the assay is a TAPS (Tet-assisted pyridineborane sequencing) assay as described in any of PCT / US2019 / 012627, US Patent Publication 20200370114, US Patent Publication 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.

[0252] The chapter titles used in this chapter and all publicly available content herein are for organizational purposes only and are not intended to be restrictive.

[0253] 1. Definition

[0254] The terms “comprising,” “including,” “having,” “has,” “may,” “containing,” and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. Unless explicitly specified otherwise in the context, the singular forms “a” and “the” include plural referents. This disclosure also contemplates other embodiments, whether explicitly stated or not, that “comprising embodiments or elements presented herein,” “consisting of embodiments or elements presented herein,” and “consisting substantially of embodiments or elements presented herein.”

[0255] Regarding the description of numerical ranges in this article, each intermediate value with the same precision is explicitly considered. For example, for the range of 6 to 9, the numbers 7 and 8 are considered 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 explicitly considered.

[0256] Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be unambiguous; however, in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition. Furthermore, unless the context otherwise requires, singular terms shall include plural forms and plural terms shall include singular forms.

[0257] As used herein, “methylation” refers to any and all processes that add one or more methyl groups to nucleic acids. For example, methylation may include, but is not limited to, adding a methyl group at the C5 or N4 position of cytosine or the N6 position of adenine.

[0258] Therefore, as used herein, "methylated nucleotide base" or "methylated nucleotide" refers to a nucleotide base containing a methyl moiety that is not present in a recognized canonical nucleotide base. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine does contain a methyl moiety at the 5-position of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide, while 5-methylcytosine is.

[0259] As used herein, "methylated nucleic acid" refers to a nucleic acid molecule containing one or more methylated nucleotides. Nucleic acid molecules containing methylated cytosine are considered methylated (e.g., the methylation state of the nucleic acid molecule is methylated). Nucleic acid molecules containing no methylated nucleotides are considered unmethylated.

[0260] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases (preferably cytosine, thymine, and uracil, and adenine and guanine, respectively) (see Albert L. Lehninger, Principles of Biochemistry, 793-800 (Worth Pub. 1982)). This technique considers any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component and any chemical variant thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. Polymers or oligomers may be heterogeneous or homogeneous in composition and may be isolated from naturally occurring sources or produced artificially or synthetically. Furthermore, nucleic acids may be DNA or RNA, or mixtures thereof, and may exist permanently or transitionally in single-stranded or double-stranded form (including homoduplex, heteroduplex, and hybrid states). In some implementations, the nucleic acid or nucleic acid sequence contains other types of nucleic acid structures, such as, for example, DNA / RNA helices, peptide nucleic acids (PNAs), morpholinonucleotides (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002) and U.S. Patent No. 5,034,506), locked nucleic acids (LNAs; see Wahllestedt et al., Proc. Natl. Acad. Sci. USA, 97: 5633-5638 (2000)), cyclohexenylnucleotides (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)) and / or ribozymes. Therefore, the term "nucleic acid" or "nucleic acid sequence" may also encompass chains containing non-natural nucleotides, modified nucleotides, and / or non-nucleotide building blocks (such as "nucleotide analogs") that can exhibit the same function as natural nucleotides; furthermore, as used herein, the term "nucleic acid sequence" refers to oligonucleotides, nucleotides, or polynucleotides, as well as fragments or portions thereof, and DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded, and denotes sense or antisense strands. The terms "nucleic acid," "polynucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably. They refer to polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides, or analogs thereof) of any length.

[0261] "Subject" or "patient" can be human or non-human and can include, for example, animal strains or species used as a "model system" (such as a mouse model as described herein) for research purposes. Similarly, subject or patient can include adults or adolescents (such as children) and can refer to any living organism, such as mammals (such as humans or non-humans). Examples of mammals include, but are not limited to, any member of the mammal class: humans, non-human primates such as chimpanzees and other ape and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs. 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 a human.

[0262] As used in this article, the term "nucleic acid sample" refers to nucleic acids obtained from organisms in the prokaryotes (bacteria), protists, fungi, plants, and animals. Nucleic acids can also be obtained from viruses. Nucleic acid samples can be obtained from patients or subjects, from environmental samples, or from organisms of interest (e.g., cell and circulating cell-free DNA (cfDNA), such as from tissues (including tissues from lymph nodes, breasts, liver, bile ducts, pancreas, oral cavity, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, prostate, endometrium, vagina, ovary, cervix, skin, bladder, kidney, lung and / or peritoneum), biopsy tissues, cells, cell aggregates, blood, plasma, serum, secretions (such as organ secretions, vaginal secretions or gastric secretions), sperm (sperm), 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, optic nerve 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 samples containing diseased tissue or cells, or suspected of containing diseased tissue or cells (e.g., cancerous, or containing cancerous tissue or cells, or suspected of being cancerous, or suspected of containing cancerous tissue or cells). In some embodiments, the nucleic acid sample is obtained from a subject who has a disease or condition (e.g., cancer), is suspected of having a disease or condition, or is undergoing screening to determine the presence of a disease or condition. In some embodiments, the nucleic acid sample is circulating cell-free DNA (cell-free DNA or cfDNA), such as DNA found in blood and not present in cells. As those skilled in the art will recognize based on this disclosure, cfDNA can be isolated from bodily fluids using methods known in the art. Commercial kits for isolating cfDNA are available, including, for example, circulating nucleic acid kits (Qiagen). DNA samples may be produced by enrichment steps, including but not limited to antibody immunoprecipitation, chromatin immunoprecipitation, restriction enzyme digestion-based enrichment, hybridization-based enrichment, or chemical labeling-based enrichment.

[0263] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS edition, Handbook of Chemistry and Physics, 75th edition, inner cover, and specific functional groups are generally defined as described therein. In addition, the following references describe the general principles of organic chemistry as well as specific functional parts and reactivity: Sorrell, Organic Chemistry, 2nd ed., University Science Books, Sausalito, 2006; Smith, March's Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7th ed., John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3rd ed., John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd ed., Cambridge University Press, Cambridge, 1987; the entire contents of each of these references are incorporated herein by reference.

[0264] As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon chain group. Alkyl chains can contain, for example, 1 to 16 carbon atoms (C1-C2). 16 Alkyl groups, 1 to 14 carbon atoms (C1-C4) 14 Alkyl groups, 1 to 12 carbon atoms (C1-C2) 12 Alkyl groups, 1 to 10 carbon atoms (C1-C5) 10 Alkyl groups are alkyl groups with 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), 1 to 4 carbon atoms (C1-C4 alkyl), 1 to 3 carbon atoms (C1-C3 alkyl), and 1 to 2 carbon atoms (C1-C2 alkyl). Representative examples of alkyl groups 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 dodecyl.

[0265] As used herein, the term "alkoxy" refers to an alkyl group as defined herein, which is attached to a portion of a parent molecule by an oxygen atom. Representative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0266] As used herein, the term "alkoxyalkyl" refers to an alkyl group as defined herein, wherein at least one hydrogen atom is replaced by an alkoxy group as defined herein. Representative examples of alkoxyalkyl groups include, but are not limited to, methoxymethyl and 2-methoxyethyl.

[0267] As used in this article, the term "mercapto-oxime" refers to the group -C(=NOH)NH2.

[0268] As used herein, the term "amino" refers to the group -N(R)2, where each R is independently hydrogen or alkyl (as defined herein). When one R is hydrogen and the other R is alkyl, the group may be called an "alkylamino"; and when both Rs are alkyl, the group may be called a "dialkylamino".

[0269] As used herein, the term "aminoalkyl" refers to an alkyl group as defined herein, wherein at least one hydrogen atom is replaced by an amino group as defined herein.

[0270] As used herein, the term "aryl" refers to a group ("C6-C") in a monocyclic, bicyclic, or tricyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a ring array) having 6-14 ring carbon atoms and zero heteroatoms. 14 Aryl group (“C6 aryl”). 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”, i.e., phenyl). 10 "Aryl", such as naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has fourteen cyclic carbon atoms ("C"). 14 "Aryl", namely anthracene and phenanthrene.

[0271] As used in this article, the term "halogen" or "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0272] As used herein, the term "haloalkyl" refers to an alkyl group as defined herein, wherein 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 haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, and 3,3,3-trifluoropropyl.

[0273] As used herein, the term "heteroaryl" refers to an aromatic group having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic) having one or more cyclic heteroatoms independently selected from O, N, and S. An aromatic monocyclic ring is a five-membered or six-membered ring containing at least one heteroatom independently selected from O, N, and S (e.g., 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 are monocyclic heteroaryl rings fused with a monocyclic aryl or monocyclic heteroaryl ring as defined herein. Examples of tricyclic heteroaryl are monocyclic heteroaryl rings fused with two rings independently selected from a monocyclic aryl or monocyclic heteroaryl ring as defined herein. Representative examples of monocyclic heteroaryl groups include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrroleyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thiophenyl, furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl. Representative examples of bicyclic heteroaryl groups include, but are not limited to, benzimidazolyl, benzodioxacyclopentenyl, benzofuranyl, benzooxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothiophene, benzotriazolyl, benzooxadiazolyl, benzooxazolyl, chromenyl, imidazopyridine, imidazothiazolyl, indazole, indolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, naphridinyl, purine, pyridinimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazopyridyl, thiazopyrimidinyl, thienopyrroleyl, and thienothienophene. Representative examples of tricyclic heteroaryl groups include, but are not limited to, dibenzofuranyl and dibenzothiophene.

[0274] As used herein, the term "heterocyclic" or "heterocyclic" refers to a saturated or partially unsaturated non-aromatic cyclic group having one or more cyclic heteroatoms independently selected from O, N, and S. Heterocyclic rings can be monocyclic, bicyclic, or tricyclic. Monocyclic heterocyclic rings are three-, four-, five-, six-, seven-, or eight-membered rings containing at least one heteroatom independently selected from O, N, and S. Three- or four-membered rings contain zero or one double bond and one heteroatom selected from O, N, and S. Five-membered rings contain zero or one double bond and one, two, or three heteroatoms selected from O, N, and S. Six-membered rings contain 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 and one, two, or three heteroatoms selected from O, N, and S. Heteratoms in the ring can be oxidized (e.g., if the cyclic heteroatom is S, it can be oxidized to SO or SO2). Representative examples of monocyclic heterocycles include, but are not limited to: aziridine, aziridine-heptidine, aziridine-propidine, diaziridine-heptidine, 1,3-dioxane, 1,3-dioxapentidine, 1,3-dithiopentanyl, 1,3-dithiohexane, imidazolinyl, imidazolinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, isoxazolinyl, morpholinyl, oxadiazolinyl, oxadiazolinyl, oxazolin ... Alzolyl, oxetyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolyl, pyrrolinyl, pyrrolyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothiophenyl, thiadiazolinyl, thiadiazolinyl, 1,2-thiazinyl, 1,3-thiazinyl, thiazolinyl, thiazolinyl, thiomorpholinyl, 1,1-thiomorpholinyl dioxide (thiomorpholinone), thiopyranyl, and trithiaalkyl. Bicyclic heterocycles are monocyclic heterocycles fused to a phenyl group, or monocyclic heterocycles fused to a monocyclic cycloalkyl group, or monocyclic heterocycles fused to a monocyclic cycloalkenyl group, or monocyclic heterocycles fused to a monocyclic heterocycle, or spirocyclic groups, or bridged monocyclic heterocycle ring systems, wherein two non-adjacent atoms of the ring are connected by an alkylene bridge of 1, 2, 3, or 4 carbon atoms or an alkenyl bridge of 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothiophenyl, 2,3-dihydroisoquinolinyl, 2-azaspiro[3.3]heptane-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindololinyl, octahydrocyclopentano[c]pyrroleyl, octahydropyrrolopyridyl, and tetrahydroisoquinolinyl.Examples of tricyclic heterocycles are bicyclic heterocycles fused with phenyl groups, or bicyclic heterocycles fused with monocyclic cycloalkyl groups, or bicyclic heterocycles fused with monocyclic cycloalkenyl groups, or bicyclic heterocycles fused with monocyclic heterocycles, wherein two non-adjacent atoms of the bicyclic ring are connected by an alkylene bridge of 1, 2, 3, or 4 carbon atoms or an alkenyl bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentadiene, hexahydro-2H-2,5-bridged methylenecyclopentano[b]furan, hexahydro-1H-1,4-bridged methylenecyclopentano[c]furan, and aza-adamantane (1-azatricyclic [3.3.1.1]). 3,7 [Decane] and oxa-adamantane (2-oxatricyclo[3.3.1.1]) 3,7 ] Decane).

[0275] As used in this article, the term "hydroxyl group" refers to the -OH group.

[0276] As used herein, the term "hydroxyalkyl" refers to an alkyl group as defined herein, in which at least one hydrogen atom is replaced by a hydroxyl group. Representative examples of hydroxyalkyl groups include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, and 3-hydroxypropyl.

[0277] As used in this article, the term "thiol" refers to the -SH group.

[0278] As used herein, the term “mercaptoalkyl” means an alkyl group, as defined herein, in which at least one hydrogen atom is replaced by a thiol group. Representative examples of mercaptoalkyl groups include, but are not limited to, mercaptomethyl, 2-mercaptoethyl, 2-mercaptopropyl, 3-mercaptopropyl, and 4-mercaptobutyl.

[0279] In the compounds disclosed herein, any atom not explicitly designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise stated, when a position is designated as “H” or “hydrogen”, that position should be understood to have hydrogen at its natural abundance isotopic composition. Furthermore, unless otherwise stated, when a position is explicitly designated as “D” or “deuterium”, that position should be understood to have deuterium at an abundance at least 3000 times that of tritium (0.015%) (i.e., at least 45% deuterium doped). In some embodiments, this position has an abundance of at least 3500 times (52.5% deuterium doping), at least 4000 times (60% deuterium doping), at least 4500 times (67.5% deuterium doping), at least 5000 times (75% deuterium doping), or at least 5500 times (82.5% deuterium doping) of natural deuterium abundance. The abundance of deuterium is at least 6000 times that of naturally occurring deuterium (with 90% deuterium added), at least 6333.3 times that of naturally occurring deuterium (with 95% deuterium added), at least 6466.7 times that of naturally occurring deuterium (with 97% deuterium added), at least 6600 times that of naturally occurring deuterium (with 99% deuterium added), or at least 6633.3 times that of naturally occurring deuterium (with 99.5% deuterium added).

[0280] While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0281] 2. Borane reducing agent

[0282] The embodiments disclosed herein provide a borane reducing agent (also referred to herein as a borane complex). As described in more detail below, the borane reducing agent can be used in methods for detecting 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and / or 5-carboxycytosine (5caC) in target nucleic acid sequences.

[0283] In some embodiments, the borane reducing agent that can be used in the methods disclosed herein includes compounds of formula (I):

[0284] (I)

[0285] and its salts, of which:

[0286] A is a monocyclic, bicyclic, or tricyclic heteroaryl group, or a monocyclic, bicyclic, or tricyclic heterocyclic group, each of which is decomposed by R. 1 R 2 R3 R 4 and R 5 replace;

[0287] R 1 R 2 R 3 R 4 and R 5 Each group is independently selected from hydrogen, halogroup, hydroxyl group, amino group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group, C1-C4 haloalkyl group, C1-C4 aminoalkyl group, C1-C4 mercaptoalkyl group, C1-C4-alkoxy-C1-C4-alkyl group, geminoaminooxime group, aryl group, and heterocyclic group; and

[0288] Each X is independently either hydrogen or deuterium.

[0289] The compound in question is not:

[0290] or .

[0291] In some embodiments, the borane reducing agent that can be used in the methods disclosed herein includes compounds of formula (I):

[0292] (I)

[0293] and its salts, of which:

[0294] A is a monocyclic, bicyclic, or tricyclic heteroaryl group, or a monocyclic, bicyclic, or tricyclic heterocyclic group, each of which is decomposed by R. 1 R 2 R 3 R 4 and R 5 replace;

[0295] R 1 R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, halogroup, hydroxyl group, amino group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group, C1-C4 haloalkyl group, C1-C4 aminoalkyl group, C1-C4 mercaptoalkyl group, C1-C4-alkoxy-C1-C4-alkyl group, and geminoxamic group; and

[0296] Each X is independently either hydrogen or deuterium.

[0297] The compound in question is not:

[0298] or .

[0299] 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.

[0300] In some implementations, the borane reducing agent is a compound of formula (Ia):

[0301] (Ia).

[0302] In some implementations, the borane reducing agent is a compound of formula (Ia):

[0303] (Ia)

[0304] in:

[0305] R 1 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl and C1-C4-alkoxy-C1-C4-alkyl;

[0306] R 2 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and amylopime group;

[0307] Where R 1 and R 2 Optionally, together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings that are optionally substituted with hydroxyl groups;

[0308] R 3 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, geminosine oxime and aryl;

[0309] R 4 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and heterocyclic group;

[0310] Where R 3 and R 4 Optionally, they form 5-membered or 6-membered rings together with the carbon atoms to which they are attached; and

[0311] R 5 It is selected from hydrogen, C1-C4 alkyl and C1-C4 hydroxyalkyl.

[0312] In some implementation schemes: R 1 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl, and C1-C4-alkoxy-C1-C4-alkyl; R2 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group, and geminoxamic group; R 3 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, and geminoxamic; R 4 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group and C1-C4 hydroxyalkyl group; and R 5 It is selected from hydrogen, C1-C4 alkyl and C1-C4 hydroxyalkyl.

[0313] In some implementation schemes, 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, C1-C4 alkyl, C1-C4 alkoxy, phenyl, and monocyclic 5- or 6-membered heterocyclic groups having one nitrogen atom. In some embodiments: R 1 It is hydroxymethyl; R 2 It is hydrogen; R 3 Selected from hydrogen, C1-C4 alkyl and phenyl; R 4 Selected from C1-C4 alkyl, C1-C4 alkoxy, and monocyclic 5-membered heterocyclic groups having one nitrogen atom; and R 5 It is hydrogen.

[0314] In some implementation schemes, R 1 R 2 R 3 R 4 and R 5 At least one of them is not hydrogen. In some implementations, 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 5 At least one of them is a C1-C4 hydroxyalkyl group.

[0315] In some implementation schemes: R 1 Selected from hydrogen, C1-C4 alkyl, and C1-C4 hydroxyalkyl; R 2 Selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, and C1-C4 hydroxyalkyl; R 3 Selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, and C1-C4 hydroxyalkyl; R4 Selected from hydrogen and C1-C4 alkyl; and R 5 It is hydrogen; where R 1 R 2 and R 3 At least one of them is a C1-C4 hydroxyalkyl group.

[0316] In some implementation schemes, 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, C1-C4 alkyl, and C1-C4 alkoxy.

[0317] In some implementation schemes, R 1 R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, and hydroxymethyl, wherein R 1 R 2 R 3 R 4 and R 5 At least one of them is hydroxymethyl.

[0318] In some implementation schemes: R 1 Selected from hydrogen, methyl, and hydroxymethyl; R 2 Selected from hydrogen, methyl, methoxy, and hydroxymethyl; R 3 Selected from hydrogen, methyl, methoxy, and hydroxymethyl; R 4 Selected from hydrogen and methyl; and R 5 It is hydrogen; where R 1 R 2 and R 3 At least one of them is hydroxymethyl.

[0319] In some implementations, each X is hydrogen. In some implementations, each X is tritium.

[0320] In some implementations, the borane reducing agent is a compound of formula (Ib):

[0321] (Ib)

[0322] or its salt, wherein:

[0323] R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, aryl, and heterocyclic groups; and

[0324] Each X is independently either hydrogen or deuterium.

[0325] In some implementations, the borane reducing agent is a compound of formula (Ib):

[0326] (Ib)

[0327] or its salt, wherein:

[0328] R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy; and

[0329] Each X is independently either hydrogen or deuterium.

[0330] In some implementation schemes, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, phenyl, or a monocyclic 5-membered heterocyclic group having one nitrogen atom. In some embodiments, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

[0331] In some implementation schemes: R 2 Selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy; R 3 Selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy; R 4 Selected from hydrogen and C1-C4 alkyl; and R 5 It is hydrogen.

[0332] In some implementation schemes, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidine. In some embodiments: R 2 It is hydrogen; R 3 Selected from hydrogen, C1-C4 alkyl and phenyl; R 4 Selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, and monocyclic 5-membered heterocyclic groups having one nitrogen atom; and R 5 It is hydrogen. In some implementations: R 2 It is hydrogen; R 3 Selected from hydrogen, methyl, and phenyl; R 4 Selected from hydrogen, methyl, methoxy, and pyrrolidinyl; and R5 It is hydrogen.

[0333] In some implementation schemes, R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, and methoxy. In some embodiments: R 2 Selected from hydrogen, methyl, and methoxy; R 3 Selected from hydrogen, methyl, and methoxy; R 4 Selected from hydrogen and methyl; and R 5 It is hydrogen.

[0334] In some implementations, each X is hydrogen. In some implementations, each X is tritium.

[0335] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is selected from:

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343] and

[0344] .

[0345] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is selected from:

[0346]

[0347]

[0348]

[0349]

[0350]

[0351] .

[0352] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is selected from:

[0353] , , , , and And their salts.

[0354] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is selected from:

[0355] and And their salts.

[0356] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is:

[0357] Or its salt.

[0358] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is:

[0359] Or its salt.

[0360] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is:

[0361] Or its salt.

[0362] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is:

[0363] Or its salt.

[0364] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is:

[0365] Or its salt.

[0366] In some embodiments, the compound of formula (I) (or the compound of formula (Ia) or (Ib)) is:

[0367] Or its salt.

[0368] 3. Methods for identifying or detecting methylated nucleotide bases

[0369] Embodiments of this disclosure provide sulfite-free, base-resolution methods (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 methods for use with DNA obtained from blood samples (cellular DNA and cfDNA) and biopsy tissues. As disclosed in PCT / US2019 / 012627, US Patent Publication 20200370114, US Patent Publication 20210317519, PCT / IB2020 / 056435, PCT / IB2021 / 000630, PCT / IB2021 / 051091, PCT / IB2022 / 000420, and PCT / US2023 / 075823 (each of these documents is incorporated herein by reference in its entirety), TAPS includes the direct quantification of 5mC and / or 5hmC at base resolution using mild enzymatic and chemical reactions without affecting unmodified cytosine. This disclosure also provides methods for the base resolution detection of 5-formylcytosine (5fC) and / or 5-carboxycytosine (5caC) without affecting unmodified cytosine. Therefore, the method presented in this paper provides mapping of 5mC, 5hmC, 5fC and / or 5caC, and overcomes the shortcomings of previous methods (such as bisulfite sequencing).

[0370] According to these embodiments, the methods of this disclosure may include the step of converting 5mC and 5hmC (or 5mC only if 5hmC is blocked) into 5caC and / or 5fC. In some embodiments, this step includes contacting a nucleic acid sample (such as a DNA or RNA sample) with a deca-11 translocation (TET) enzyme. TET enzymes are a family of enzymes that catalyze the transfer of oxygen molecules to the C5 methyl group on 5mC, resulting in the formation of 5-hydroxymethylcytosine (5hmC). TET also catalyzes the oxidation of 5hmC to 5fC and the oxidation of 5fC to 5caC. TET enzymes that can be used in the methods of this disclosure include one or more of the following: human TET1, TET2, and TET3; mouse TET1, TET2, and TET3; Naegleria gulberi TET (NgTET); Coprinus comatus TET (CcTET); the catalytic domain of mouse TET1 (mTET1CD); and derivatives or analogs thereof.

[0371] The method disclosed herein 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 the nucleic acid sample (such as a DNA or RNA sample) with a borane reducing agent as described in the preceding section.

[0372] In some preferred embodiments, the step of converting 5hmC to 5fC includes oxidizing 5hmC to 5fC by contacting a nucleic acid sample (such as a DNA sample) with substances such as: manganese oxide (MnO2), potassium perruthenate (KRuO4), Cu(II) / 2,2,6,6-tetramethylpiperidin-1-oxy (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), and tetraphenylphosphonium perruthenate. The copper salts or complexes of ruthenate, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolino-1-yloxy (3-Carbamoyl-PROXYL), 2-azaadamantane-N-oxy (AZADO), or 9-azabicyclo[3.3.1]nonane-N′-oxy (ABNO) can be used to convert 5fC in nucleic acid samples (such as DNA samples) to DHU by methods disclosed herein (such as via borane reaction).

[0373] Methods for identifying 5mC. In some embodiments, the methods of this disclosure include identifying 5mC in a nucleic acid sample such as a DNA sample (targeted DNA or whole genome), and providing a quantitative measure of the frequency of the modification at each site in which the 5mC modification is identified in the nucleic acid sample such as the 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 (such as the DNA sample). According to these embodiments, the methods for identifying 5mC may include the use of a blocking group. In other embodiments, the methods for identifying 5mC do not require the use of a blocking group.

[0374] When a blocking group is used to identify 5mC in a nucleic acid sample (such as a DNA sample) that does not contain 5hmC, the 5hmC in the sample is blocked, and therefore it will not undergo conversion to 5caC and / or 5fC. In some embodiments, by adding a blocking group to 5hmC, the 5hmC in the nucleic acid sample (such as a DNA sample) becomes unreactive to subsequent steps. In one embodiment, the blocking group is a sugar, including modified sugars 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 a uridine diphosphate (UDP) sugar in the presence of one or more glucosyltransferases. In some embodiments, the glucosyltransferase is selected from T4 phage β-glucosyltransferase (βGT), T4 phage α-glucosyltransferase (αGT), and their derivatives and analogs. βGT is an enzyme that catalyzes the chemical reaction in which β-D-glucose residues are transferred from UDP-glucose to 5-hydroxymethylcytosine residues in nucleic acids.

[0375] Methods for identifying 5mC and / or 5hmC. In some embodiments, the methods of this disclosure include identifying 5mC or 5hmC in a nucleic acid sample such as a DNA sample (targeted DNA or whole genome). In some embodiments, the method provides a quantitative measurement of the frequency of the modification at each site in a nucleic acid sample (such as a DNA sample) where a 5mC or 5hmC modification is identified. In some embodiments, the percentage of T at each transition site provides a quantitative level of 5mC or 5hmC at each site in a nucleic acid sample (such as a DNA sample). In some embodiments, both 5mC and 5hmC are converted to DHU, and the methods for identifying 5mC or 5hmC provide the locations 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, for example, by the methods of this disclosure, where DHU is converted to T. In some embodiments, the methods for identifying 5hmC include the use of a blocking group. In other embodiments, the methods for identifying 5hmC do not require the use of a blocking group.

[0376] In some embodiments, this disclosure provides methods for identifying 5mC and 5hmC in nucleic acid samples (such as DNA samples), the method being performed by applying a method for identifying 5mC to a first nucleic acid sample (such as a DNA sample) and applying a method for identifying either 5mC or 5hmC to a second nucleic acid sample (such as a DNA sample). In some embodiments, the first and second nucleic acid samples (such as DNA samples) are derived from the same nucleic acid sample (such as DNA sample). For example, the first and second samples may be separate aliquots obtained from a sample containing the nucleic acid to be analyzed, such as DNA (e.g., cellular DNA or cfDNA).

[0377] Since 5mC and 5hmC (which are not blocked) are converted to 5fC and 5caC before being converted to DHU, any 5fC and 5caC present in a nucleic acid sample (such as a DNA sample) can 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 generally acceptable when analyzing methylation and hydroxymethylation in DNA samples. The 5fC and 5caC signals can be eliminated by protecting 5fC and 5caC from conversion to DHU, for example, through hydroxylamine conjugation and EDC conjugation. In some embodiments, the method identifies the location and percentage of 5hmC in DNA by comparing the location and percentage of 5mC with the location and percentage of 5mC or 5hmC (together). Alternatively, the location and frequency of 5hmC modifications in DNA can be measured directly.

[0378] 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. Instead, in some embodiments, both 5fC and 5caC are converted into DHU that can be detected by the methods described herein.

[0379] Methods for identifying 5caC. In some embodiments, the method includes identifying 5caC in a nucleic acid sample such as a DNA sample (targeted DNA or whole genome) and providing a quantitative measurement of the frequency of the modification at each site in the nucleic acid sample such as the DNA sample where a 5caC modification is identified. In some embodiments, the T percentage at each transition site provides a quantitative level of 5caC at each site in the nucleic acid sample (such as the DNA sample). According to these embodiments, the method for identifying 5caC may include the use of a blocking group. In other embodiments, the method for identifying 5caC does not require the use of a blocking group.

[0380] In some embodiments, the identification of 5caC in a nucleic acid sample (such as a DNA sample) can be performed when 5fC is blocked (and 5mC and 5hmC do not convert to DHU). 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, which includes, for example, hydroxylamine derivatives, hydrazine derivatives, and acylhydrazine derivatives. Hydroxylamine derivatives include, but are not limited to, ashydroxylamine; hydroxylamine hydrochloride; hydroxylamine sulfate; hydroxylamine phosphate; O-methylhydroxylamine; O-hexylhydroxylamine; O-pentylhydroxylamine; O-benzylhydroxylamine; and particularly O-ethylhydroxylamine (EtONH2), O-alkylated or O-arylated hydroxylamines, their acids or salts. Hydrazine derivatives include N-alkylhydrazines, N-arylhydrazines, N-benzylhydrazines, N,N-dialkylhydrazines, N,N-diarylhydrazines, N,N-dibenzylhydrazines, N,N-alkylbenzylhydrazines, N,N-arylbenzylhydrazines, and N,N-alkylarylhydrazines. Acylhydrazine derivatives include toluenesulfonylhydrazines, N-acylhydrazines, N,N-alkylacylhydrazines, N,N-benzylacylhydrazines, N,N-arylacylhydrazines, N-sulfonylhydrazines, N,N-alkylsulfonylhydrazines, N,N-benzylsulfonylhydrazines, and N,N-arylsulfonylhydrazines.

[0381] Methods for identifying 5fC. In some embodiments, the method includes identifying 5fC in a nucleic acid sample such as a DNA sample (targeted DNA or whole genome) and providing a quantitative measurement of the frequency of the modification at each site in the nucleic acid sample such as the DNA sample where a 5fC modification is identified. In some embodiments, the T percentage at each transition site provides a quantitative level of 5fC at each site in the nucleic acid sample (such as the DNA sample). According to these embodiments, the method for identifying 5fC may include the use of a blocking group. In other embodiments, the method for identifying 5fC does not require the use of a blocking group.

[0382] In some embodiments, adding a blocking group to 5caC in a nucleic acid sample (such as a DNA sample) can be accomplished by: (i) contacting the nucleic acid sample (such as a DNA sample) with a coupling agent, such as a carboxylic acid derivatizing agent like a carbodiimide derivative, such as 1-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 followed by benzylamine, ethylamine, or another amine to form an amide that prevents the conversion of 5caC to DHU (e.g., by borane reduction).

[0383] In some preferred embodiments, the method further includes a sequencing step to obtain a methylation signature. In some embodiments, the method includes isolating a nucleic acid such as DNA (e.g., cellular DNA or cfDNA) from a sample; preparing a sequencing library containing the nucleic acid such as DNA; and performing TAPS on the sequencing library to obtain a methylation signature of the nucleic acid such as DNA. In some embodiments, the methylation signature is a genome-wide methylation signature.

[0384] In some embodiments, preparing a sequencing library involves ligating a sequencing adaptor to isolated nucleic acid, such as DNA, to facilitate a sequencing reaction. Suitable sequencing adaptors for massively parallel sequencing technologies can be utilized. This invention is not limited to any particular sequencing technology. In some preferred embodiments, sequencing technologies such as those provided by Illumina or Nanopore can be utilized. For example, suitable sequencing technologies for use in this invention include, but are not limited to, those described in U.S. Patent Publication 20100120098, U.S. Patent Publication 20120208705, U.S. Patent Publication 20120208724, WO2012 / 061832, and U.S. Patent Publication 2015 / 0368638, each of which is incorporated herein by reference in its entirety.

[0385] In some embodiments, the adaptor contains one or more sites capable of hybridizing with primers. In some embodiments, the adaptor contains at least a first primer site. In some embodiments, the adaptor contains at least a first primer site and a second primer site. The orientation of the primer sites in such embodiments can be such that primers hybridizing with the first primer site and primers hybridizing with the second primer site are in the same orientation or different orientations. In one embodiment, the primer sequence in the adaptor can be complementary to the primers used for amplification. In another embodiment, the primer sequence is complementary to the primers used for sequencing.

[0386] In some embodiments, the adapter may include a first primer site, a second primer site, and a non-amplifiable site between them. The non-amplifiable site can be used to block the elongation of a polynucleotide chain between the first and second primer sites, wherein the polynucleotide chain hybridizes with one of the primer sites. The non-amplifiable site can also be used to prevent tandem coupling. Examples of non-amplifiable sites include nucleotide analogs, non-nucleotide chemical moieties, amino acids, peptides, and polypeptides. In some embodiments, the non-amplifiable site includes a nucleotide analog that does not significantly pair with A, C, G, or T bases.

[0387] Some embodiments include a connector comprising a first primer site, a second primer site, and a fragmentation site between them. Other embodiments may use fork-shaped or Y-shaped adaptor designs suitable for directed sequencing, such as those described in U.S. Patent No. 7,741,463, which is incorporated herein by reference.

[0388] In some implementations, the linker may include an index or barcode sequence. In a further preferred implementation, the linker may include a unique molecular identifier (UMI).

[0389] In some implementations, a carrier nucleic acid or a mixture of carrier nucleic acids (such as DNA) is added to the sequencing library prior to TAPS. The carrier nucleic acid can be any specific or non-specific nucleic acid molecule (such as DNA) (or a nucleic acid derivative thereof) that enhances one or more aspects of the recovery of nucleic acids such as DNA from the sample.

[0390] Nucleic acid (e.g., DNA) methylation signatures are used to understand fundamental biological processes and disease pathology, as well as for disease detection. For example, methylation signatures / frequencies / markers can be used to understand and study gene regulation, genomic imprinting, differentiation, development, gene-environment interactions (e.g., smoking, nutrition), aging, various diseases and disorders (e.g., autoimmune diseases, cancer, cardiovascular diseases, CNS diseases, congenital diseases, infectious diseases, metabolic diseases and states, NIPT-related tests, etc.), for the detection and diagnosis of cancer and other diseases, and for monitoring grafts. In some embodiments and as described herein, the method also includes identifying at least one methylation biomarker from a nucleic acid (e.g., DNA) methylation signature (such as a 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 comprises a differentially methylated region (DMR). In some embodiments, the method also includes classifying the sample based on the DMR compared to a reference DMR. In some embodiments, the reference DMR corresponds to a non-disease control or a disease control.

[0391] In some embodiments, and as described herein, the method further includes identifying at least one methylation biomarker from a nucleic acid (such as DNA) methylation signature and determining the tissue of origin corresponding to the methylation biomarker. In some embodiments, the method further includes classifying the sample based on the tissue of origin biomarker.

[0392] In some implementations and as described herein, the method also includes identifying a fragmentation profile of a nucleic acid (such as DNA) and determining whether the fragmentation profile indicates a disease (such as cancer or another disease). According to these implementations, a fragmentation profile of a nucleic acid (such as DNA) can be determined from TAPS sequencing data (e.g., read alignment positions).

[0393] In some embodiments, the method further includes identifying at least one sequence variant in a nucleic acid sample (such as a DNA sample) and determining whether the sequence variant indicates a disease (such as cancer or another disease). For example, in some embodiments, TAPS can also distinguish methylation from C-to-T genetic variants or single nucleotide polymorphisms (SNPs) and can therefore be used to detect genetic variants. In some embodiments, methylation and C-to-T SNPs can result in different patterns in TAPS. For example, methylation can result in T / G reads in the original top / bottom strand and A / C reads in the complementary strands. In some embodiments, C-to-T SNPs can result in T / A reads in the original top / bottom strand and the complementary strands. This further increases the utility of TAPS in providing both methylation information and genetic variants and thus mutation information in a single experiment and sequencing run. This capability of the TAPS method disclosed herein provides integration of genomic analysis and epigenetic analysis and significantly reduces sequencing costs by eliminating the need to perform, for example, standard whole-genome sequencing (WGS).

[0394] According to the above embodiments, the methods of this disclosure may include using TAPS in a single experiment to generate information related to methylation signatures, methylation biomarkers, DNA fragment profiles, DNA sequence information (such as variants), and tissue of origin information to diagnose / detect a subject's disease or other condition (e.g., those provided in the examples above). As will be recognized by those skilled in the art based on this disclosure, TAPS as disclosed herein can be used to generate any combination of methylation signatures, methylation biomarkers, DNA fragment profiles, DNA sequence information (such as variants), and tissue of origin information to diagnose / detect a subject's disease or other condition (e.g., those provided in the examples above). In some embodiments, a methylation signature may be obtained, and one or more of methylation biomarkers, DNA fragment profiles, DNA sequence information (such as variants), and tissue of origin information may also be obtained and used to diagnose / detect a subject's disease or other condition (e.g., those provided in the examples above). In some embodiments, the methylation status of a biomarker may be obtained, and one or more of methylation signatures, DNA fragment profiles, DNA sequence information (such as variants), and tissue of origin information may also be obtained and used to diagnose / detect a subject's disease or other condition (e.g., those provided in the examples above). In some embodiments, DNA fragmentation profiles may be obtained, and one or more of methylation signatures, methylation biomarkers, DNA sequence information (such as variants), and tissue of origin information may also be obtained and used to diagnose / detect diseases or other conditions in the subject (e.g., those provided in the examples above). In some embodiments, DNA sequence variants may be identified, and one or more of methylation signatures, methylation biomarkers, DNA fragmentation profiles, and tissue of origin information may also be obtained and used to diagnose / detect diseases or other conditions in the subject (e.g., those provided in the examples above). In some embodiments, tissue of origin information may be obtained (e.g., from whole-genome DNA methylation signatures), and one or more of methylation signatures, methylation biomarkers, DNA fragmentation profiles, and DNA sequence information (such as variants) may also be obtained and used to diagnose / detect diseases or other conditions in the subject (e.g., those provided in the examples above).

[0395] In some embodiments, performing TAPS on a sequencing library to obtain a genome-wide methylation signature includes identifying 5mC modifications in DNA and providing a quantitative measurement of the frequency of 5mC modifications. In some embodiments, performing TAPS on a sequencing library to obtain a genome-wide methylation signature includes identifying 5hmC modifications in a nucleic acid sample (such as a DNA sample) and providing a quantitative measurement of the frequency of 5hmC modifications. In some embodiments, performing TAPS on a sequencing library to obtain a genome-wide methylation signature includes identifying 5caC modifications in DNA and providing a quantitative measurement of the frequency of 5caC modifications. In some embodiments, performing TAPS on a sequencing library to obtain a genome-wide methylation signature includes identifying 5fC modifications in a nucleic acid sample (such as a DNA sample) and providing a quantitative measurement of the frequency of 5fC modifications.

[0396] Based on this disclosure, those skilled in the art will recognize that the methods described herein (e.g., TAPS) can be used to diagnose / detect any type of cancer. 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, stomach cancer, bladder cancer, esophageal cancer, mesothelioma, thyroid cancer, head and neck cancer, osteosarcoma, hepatocellular carcinoma, cancer of unknown primary origin, ovarian cancer, endometrial cancer, glioblastoma, Hodgkin lymphoma, and non-Hodgkin lymphomas. In some embodiments, types of cancer or metastatic forms of cancer that can be detected / diagnosed using the methods of this disclosure include, but are not limited to, carcinoma, sarcoma, lymphoma, germ cell tumors, and blastoma. In some embodiments, the cancer is invasive and / or metastatic (e.g., stage II, stage III, or stage IV cancer). In some implementations, the cancer is an early-stage cancer (e.g., stage 0, stage I) and / or not an invasive and / or metastatic cancer. In some implementations, the cancer is not a metastatic cancer.

[0397] According to these embodiments, this disclosure provides methods for quantitatively identifying one or more of the following positions in nucleic acids at base resolution: 5mC, 5hmC, 5caC, and / or 5fC, 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 contains a target nucleic acid as DNA or as RNA. In some embodiments, the method is applied to the whole genome and is not limited to a specific target nucleic acid.

[0398] The nucleic acid can be any nucleic acid with cytosine modifications (i.e., 5mC, 5hmC, 5fC, and / or 5caC), including but not limited to DNA fragments and / or genomic DNA. The nucleic acid can be a single nucleic acid molecule in the sample, or it can be an entire population of nucleic acid molecules in the sample or any part thereof (the whole genome or a subset thereof). The nucleic acid can be a native nucleic acid from a source (e.g., cells, tissue samples, etc.) or can be pre-converted to a high-throughput sequencing-ready form, for example, through fragmentation, repair, and adapter binding for sequencing. Therefore, the nucleic acid can contain multiple nucleic acid sequences, making the methods described herein applicable for generating target nucleic acid sequence libraries that can be analyzed individually (e.g., by identifying the sequence of a single target) or in groups (e.g., by high-throughput or next-generation sequencing methods).

[0399] The methods disclosed herein may also include the step of amplifying the copy number of the modified nucleic acid using methods known in the art. When the modified nucleic acid is DNA, the copy number can be increased by, for example, PCR, cloning, and primer extension. The copy number of a single target DNA 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 into a DNA vector using standard techniques. In some embodiments, the copy number of multiple different modified target DNA sequences is increased by PCR to produce a library for next-generation sequencing, wherein, for example, double-stranded adaptor DNA has been pre-conjugated to sample DNA (or conjugated to modified sample DNA), and PCR is performed using primers complementary to the adaptor DNA.

[0400] In some embodiments, the method includes the step of detecting the sequence of the modified nucleic acid. Regarding TAPS, the modified target nucleic acid (such as DNA or RNA) contains DHU at one or more of the following positions in the unmodified target nucleic acid (such as DNA or RNA): 5mC, 5hmC, 5fC, and 5caC. DHU acts as a T in DNA replication and sequencing methods. Therefore, cytosine modification can be detected by any direct or indirect method known in the art for identifying C-to-T transitions. Such methods include sequencing methods such as Sanger sequencing, microarrays, and next-generation sequencing. C-to-T transitions can also be detected by restriction enzyme assays, where the C-to-T transition abolishes or introduces a restriction endonuclease recognition sequence.

[0401] 4. System or reagent kit

[0402] Embodiments of this disclosure also provide systems or kits for oxidizing methylated nucleotides, such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In some embodiments, the system or kit includes a borane reducing agent as described above. In some further preferred embodiments, the kit includes an oxidizing agent. Suitable enzymatic oxidizing agents include, but are not limited to, members of the TET family, such as TET1, TET2, TET3, CXXC4, their active fragments, derivatives, or analogs, 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); Coprinus comatus TET (CcTET); their active fragments (such as the catalytic domain of mouse TET1 (mTET1CD)), derivatives, or analogs. Suitable chemical oxidizing agents include, but are not limited to, manganese oxide (MnO2), potassium ruthenate (K2RuO4), Cu(II) / 2,2,6,6-tetramethylpiperidin-1-oxy (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenylphosphoniurn ruthenate, copper salts or complexes of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrololin-1-oxy (3-Carbamoyl-PROXYL), copper salts or complexes of 2-azaadamantane-N-oxy (AZADO), or copper salts or complexes of 9-azabicyclo[3.3.1]nonane-N′-oxy (ABNO).

[0403] 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 higher sequence identity with any or all of the TET family dioxygenases listed above. In some embodiments, the TET family dioxygenases exhibit the characteristic activities of the TET protein family.

[0404] In some embodiments, the system or kit further includes a blocking group and / or a glucosyltransferase. In some embodiments, the blocking group is a sugar. In some embodiments, the sugar is a naturally occurring sugar or a modified sugar, such as glucose or modified glucose. In some embodiments, the blocking group functions to link to a UDP-glycosyltransferase (UDP-glucose) or to a modified glucose in the presence of a glucosyltransferase, such as T4 phage β-glucosyltransferase (βGT) and T4 phage α-glucosyltransferase (αGT), and their derivatives and analogs.

[0405] Such systems or kits can also be used to detect and identify methylated nucleotides and contain additional components required for the detection and identification of methylated nucleotides. Systems or kits may also include sequencing reagents (e.g., primers, probes, nucleotides, buffers, control nucleic acid sequences, polymerases, etc.), restriction endonucleases, etc., for the detection of methylated nucleotides. The concepts, kits, and methods described herein can be implemented on any system or instrument, including any manual, automated, or semi-automated system used for sequencing reactions.

[0406] The system or kit may include instructions for use in any of the methods described herein. Instructions included in the kit may be affixed to packaging materials or may be included as a packaging insert. Instructions may be written or printed material, but are not limited to this. This disclosure contemplates any medium capable of storing such instructions and communicating them to the end user. Such media include, but are not limited to, electronic storage media (such as disks, magnetic tapes, cassette tapes, chips), optical media (such as CD ROMs), etc. As used herein, the term "instructions" may include the address of the website where the instructions are provided.

[0407] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. The kits may optionally include additional components such as buffer solutions and explanatory information. Typically, the kits include a container and a label or one or more packaging inserts on or associated with the container. In some embodiments, this disclosure provides an article of manufacture containing the contents of the kits described above.

[0408] 4. Example

[0409] The following are embodiments of the present invention and should not be construed as limiting.

[0410] Example 1

[0411] This embodiment describes the testing of novel functionalized pyridine borane complexes that exhibit reduced GC and DHU amplification bias and increased normalized coverage of biomarkers in sequencing following TAPS assays. Specifically, the use of borane or deuterated borane complexes containing a substituted 2-hydroxymethylpyridine moiety in the TAPS procedure reduces DHU amplification bias and improves normalized coverage of biomarkers. This enables the acquisition of more sequencing reads of highly methylated regions in the genome that are associated with methylation detection. The improvement in DHU amplification bias is achieved with conversion rates and false positive rates comparable to existing reducing agents.

[0412] Materials and methods

[0413] Numerous pyridineborane complexes were synthesized for use in the experiments described in this embodiment. Unless otherwise stated, the following compounds were used in the experiments described below: 2-methylpyridineborane complex (“picborane”); 2-methylpyridinedeuteratedborane complex (“ESI021”); 2-hydroxymethylpyridineborane complex (“ESI047”); 2-hydroxymethylpyridinedeuteratedborane complex (“ESI050”); 5-hydroxymethyl-2-methylpyridineborane complex (“ESI051”); 3,4-dimethoxy-2-hydroxymethylpyridineborane complex (“ESI052”)

[0414]

[0415] These compounds were evaluated in a TAPS reduction reaction using the target nucleic acid. Amplification (PCR) and next-generation sequencing were then performed following the TAPS reduction reaction.

[0416] result

[0417] An overview of the methylation rates of borane and deuterated borane complexes functionalized with methyl groups at different positions. Many different borane and deuterated borane complexes were synthesized and then used for TAPS reduction of fully methylated Lambda DNA spikes. Methylation rates were determined on an Illumina NextSeq instrument. The results are presented in Table 1. These data indicate that boranes complexed with pyridines functionalized with methyl groups at R1 and / or R2 and / or R3 and / or R4 produce high conversion levels in TAPS, typically with false positive rates comparable to 2-methylpyridineborane complexes. Furthermore, the data show that borane complexes containing pyridine ligands functionalized with hydroxymethyl groups at R1, R2, R3, or R4 are reducing agents with high conversion levels in TAPS. Methylation represents the detection of C->T conversion in internally produced fully methylated Lambda DNA spikes. Although the data presented came from multiple different experiments, all runs included a positive control (PicBorane, a 2-methylpyridineborane complex) to provide a basis for comparison.

[0418] Table 1: TAPS performance of selected synthesized borane and deuterated borane complexes

[0419]

[0420] A comparison of overall GC preference when using 2-methylpyridineborane complexes or 2-hydroxymethylpyridineborane complexes on 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 spikes using either 2-methylpyridineborane complexes or 2-hydroxymethylpyridineborane complexes. Therefore, most of the cytosine in this sample was unmethylated. Reference Figure 1 TAPS reduction using the 2-methylpyridineborane complex (“Pic”, solid line) as the reducing agent produced a GC preference curve with a negative slope, indicating low coverage of CG-rich regions in the genome. The use of the 2-hydroxymethylpyridineborane complex (“ESI047”, dashed line) produced a GC preference curve with a much gentler slope, indicating higher coverage of GC-rich regions compared to pic borane. Data were measured on an Illumina NextSeq instrument.

[0421] The Lambda GC preference for TAPS reduction reactions using either the 2-methylpyridineborane complex or the 2-hydroxymethylpyridineborane complex was also determined. (Reference) Figure 2 TAPS reduction reactions using the 2-methylpyridineborane complex (“Pic”, solid line) as a reducing agent produced a GC preference curve with a negative slope, indicating low coverage of GC-rich regions in the genome. The use of the 2-hydroxymethylpyridineborane complex (“ESI047”, dashed line) produced a GC preference curve with a much flatter slope, indicating higher coverage of GC-rich and highly methylated regions compared to pic borane. Lambda GC preference represents the sequencing preference of methylated Lambda DNA spikes in TAPS reactions where most C atoms are methylated. Data were measured on an Illumina NextSeq instrument.

[0422] Comparison of methylation rates in TAPS reactions using 2-methylpyridineborane complex or 2-hydroxymethylpyridine complex. The methylation rate of pUC19 spikes with approximately 20% methylated cytosine residues was determined using TAPS reactions with either 2-methylpyridineborane complex or 2-hydroxymethylpyridineborane complex. Data are presented in Table 2. Reactions with the 2-hydroxymethylpyridineborane complex (“ESI047”) showed increased methylation rates (9.9 or 11) compared to standard conditions with the 2-methylpyridineborane complex (“Pic”, 7.3 or 7.4). Methylation represents the detection of C->T conversion in internally produced pUC19 DNA spikes containing approximately 20% methylation. The 20:80 pUC19 spikes were intended to simulate “real-world” patient samples where most of the isolated DNA was not fully methylated. Data were measured on an Illumina NextSeq instrument.

[0423] Table 3 provides comparative data on the methylation of fully methylated Lambda following TAPS reduction with or without the 2-methylpyridineborane complex. The reaction with the 2-hydroxymethylpyridineborane complex (“ESI047”) showed methylation rates comparable to those under standard conditions with the 2-methylpyridineborane complex (“Pic”). Data were measured on an Illumina NextSeq instrument.

[0424] 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.

[0425] Table 2. Methylation of pUC19 after TAPS reduction reaction using 2-methylpyridineborane complex or 2-hydroxymethylpyridine complex

[0426]

[0427] Table 3. Methylation of Lambda after TAPS reduction reaction using 2-methylpyridineborane complex or 2-hydroxymethylpyridine complex

[0428]

[0429] Whole-genome sequencing library complexity. The library complexity of samples treated with 2-methylpyridineborane complex or 2-hydroxymethylpyridineborane complex was analyzed by whole-genome sequencing performed on an Illumina NovaSeq instrument. Figure 3Data plots are provided. The complexity of samples treated with 2-hydroxymethylpyridineborane (“ESI47-KU”, short dashed line, also referred to as ESI047 throughout this application) and 2-methylpyridineborane (“Pic-KU”, solid line) both fall between the 0% (Zymo – bisulfite – 0% methyl, dotted line) and 100% methylated (Zymo – bisulfite – 100% methyl, dashed line) bisulfite-treated controls, indicating that samples treated with 2-hydroxymethylpyridineborane via TAPS have comparable complexity to both bisulfite-treated samples and samples treated with 2-methylpyridineborane via TAPS. Library complexity refers to the number of unique DNA fragments present in a library. Libraries with fewer repetitive reads produced by PCR amplification are considered more complex and better reflect the composition of the original DNA sample. PCR amplification is an inherently repetitive process; therefore, the complexity of experimental samples is lower than the theoretical case of zero replicates (“theoretical – no replicates”, long dashed line). However, these data indicate that the libraries treated with 2-hydroxymethylpyridineborane remain highly complex.

[0430] Normalized coverage of biomarkers in whole-genome sequencing. The normalized coverage of selected biomarkers in samples treated with 2-methylpyridineborane or 2-hydroxymethylpyridineborane complexes was examined using an Illumina NovaSeq instrument. Normalized coverage was defined as the absolute coverage of a region divided by the mean absolute coverage of the library. Data are presented in Table 4. Samples treated with 2-hydroxymethylpyridineborane showed normalized coverage greater than or equal to that of five of the six analyzed highly methylated biomarkers. These data suggest that the use of 2-hydroxymethylpyridineborane as a reducing agent in TAPS produces libraries that enhance the amplification of highly methylated regions associated with the detection of diseases such as cancer or another condition.

[0431] Lambda methylation rate, GC loss, and false positive rate were detected in TAPS samples after treatment with different borane reducing agents. The methylation rate of fully methylated Lambda DNA spikes was determined with additional borane reducing agents. Data are presented in Table 5. These data indicate that the methylation levels detected in the pyridine deuterated borane complexes are similar to those of their pyridine borane analogs (cf. 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 indicate that complexes other than the 2-methylpyridine borane complex can achieve methylation rates greater than 96% (e.g., ESI051 (5-hydroxymethyl-2-methylpyridine borane complex); and ESI052 (3,4-dimethoxy-2-hydroxymethylpyridine borane complex)).

[0432] The false positive rates in TAPS reactions using different borane complexes were also analyzed. The data are presented in Table 5. These data indicate that the false positive rates induced by the 2-methylpyridine deuterated borane complex (“ESI021”) and the 2-hydroxymethylpyridine borane complex (“ESI047”) are similar to those induced by the 2-methylpyridine borane complex (“Pic”). The data were measured on an Illumina NextSeq instrument.

[0433] Next, the GC loss rate of Lambda DNA after TAPS reactions with different borane complexes was measured. The data are presented in Table 5. These data show that borane complexes containing 2-hydroxymethylpyridine exhibit a lower GC loss rate than those without the 2-hydroxymethylpyridine ligand, indicating higher coverage of GC-rich regions in the genome. GC loss is an indicator of sequencing bias in a sample; therefore, samples with higher GC bias have correspondingly higher GC loss. Lambda GC loss thus represents the sequencing bias of methylated Lambda DNA spikes in TAPS reactions where most C atoms are methylated. Measurements were performed on an Illumina NextSeq instrument.

[0434] Next, the overall GC loss rate after TAPS reactions with different borane complexes was measured. The data are presented in Table 5. These data show that borane complexes characterized by the 2-hydroxymethylpyridine ligand exhibit a lower GC loss rate than complexes without the 2-hydroxymethylpyridine ligand, indicating greater coverage of GC-rich and highly methylated regions in the genome. The overall GC loss represents the sum of the preferences for the following different DNA components in the reaction: 92.975% NA12878; 5% methylated Lambda; 2% methylated pUC19; and 0.025% unmethylated 2kb DNA spikes. The data were measured on an Illumina NextSeq instrument.

[0435] Table 5. Lambda methylation, false positive rate, and GC loss after TAPS reduction with different borane complexes.

[0436]

[0437] Comparison of pyridineborane complexes with and without hydroxymethyl groups. The overall GC loss rates of 2-methylpyridineborane and 2-hydroxymethylpyridineborane were determined in TAPS. The data are presented in Table 6. These data indicate that the samples treated with 2-hydroxymethylpyridineborane had significantly lower GC loss levels compared to those treated with 2-methylpyridineborane, suggesting greater coverage of GC-rich regions when using 2-hydroxymethylpyridineborane.

[0438] Table 6. Overall GC loss rate of TAPS samples with high conversion rates after treatment with the novel reducing agent

[0439]

[0440] Conclusions. These data suggest that the main advantages of using borane or deuterated borane complexes containing substituted 2-hydroxymethylpyridine, compared to 2-methylpyridine borane complexes, are: 1) increased coverage of highly methylated regions relevant to methylation detection (including biomarker detection) in both human and fully methylated Lambda; and 2) higher methylation in partially methylated pUC19 spikes, indicating better performance in “real-world” samples with much lower methylated DNA fractions.

[0441] Example 2

[0442] This embodiment describes the testing of additional functionalized pyridineborane complexes. In this embodiment, additional pyridineborane complexes shown in Table 7 below, as well as picB, ESI046, and ESI047, were synthesized and evaluated.

[0443] Table 7. Synthesized Borane and Deuterated Borane Complexes

[0444]

[0445] Unless otherwise stated, TAPS, post-TAPS amplification yield, lambda conversion rate, false positive rate, lambda GC loss, and total GC loss were evaluated as described in Example 1 above. The DNA input for the TAPS reaction comprised a total of 75 ng of DNA, specifically 94.8% gDNA NA12878, and the following spikes: 100% me-lambda (5.0%), 100% me-pUC19 (0.2%), and non-me 2 kb amplicons (0.025%).

[0446] result

[0447] These additional pyridineborane complexes were used as reducing agents for the TAPS reduction reaction. Figure 5-9 The amplification yield, lambda conversion rate, false positive rate, lambda-GC loss, and total GC loss after TAPS using the pyridine borane complex are presented separately. TAPS reduction reactions were performed with and without pre-extension (black bars). Pre-extension involved incubating the reduced DNA at 72°C for 30 minutes in the presence of the polymerase mixture before PCR; under the no-pre-extension condition, the reduced DNA was incubated without pre-extension in the presence of the polymerase mixture before PCR. Unless otherwise specified, the mean of two replicates (ND, not determined) is given. Error bars reflect standard deviation values. The performance of the pyridine borane complex was compared with that of the 2-methylpyridine borane complex (“picB”).

[0448] The results of these experiments are summarized in Table 8. Specifically, the pyridine borane complexes were ranked according to whether they met the following criteria: (1) their use did not result in a significant decrease in pre-extension amplification yield; (2) their use with pre-extension resulted in a lambda conversion greater than 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 loss rate of less than 8.21%. The following pyridine borane complexes met all four criteria: ESI047, ESI070, ESI075, ESI076, ESI079, and ESI093.

[0449] Table 8. Performance Ranking of Novel Boranes

[0450]

[0451] Figure 10A -G and Figure 11A-G provides GC loss plots for the pyridine borane complexes with the highest scores (i.e., compounds with a score of 4) (ESI047, ESI070, ESI075, ESI076, ESI079, and ESI093) as indicated in Table 8, as well as for ESI090 (Figure 10) and the overall plot (Figure 11). In these plots, these additional pyridine borane compounds are compared to pic-boranes in reactions with or without pre-extension: solid black lines – pic-boranes with pre-extension; dashed lines – pic-boranes without pre-extension; novel compounds with pre-extension – solid circles; novel compounds without pre-extension – hollow circles. The reduced GC loss compared to picB for ESI047, ESI070, ESI075, ESI076, ESI079, and ESI093 indicates that these compounds enable more efficient amplification of regions with higher GC content.

[0452] Numerous references, including patents and various publications, have been cited and discussed in this specification. These references and discussions are provided solely to clarify the description of the invention and not to acknowledge that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

1. A method for converting 5-carboxycytosine (5caC) and / or 5-formylcytosine (5fC) into dihydrouracil (DHU), comprising reacting a nucleic acid sample containing 5caC and / or 5fC with a borane reducing agent of formula (I): (I) or its salt in contact, wherein: A is a monocyclic, bicyclic, or tricyclic heteroaryl group, or a monocyclic, bicyclic, or tricyclic heterocyclic group, each of which is decomposed by R. 1 R 2 R 3 R 4 and R 5 replace; R 1 R 2 R 3 R 4 and R 5 Each group is independently selected from hydrogen, halogroup, hydroxyl group, amino group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group, C1-C4 haloalkyl group, C1-C4 aminoalkyl group, C1-C4 mercaptoalkyl group, C1-C4-alkoxy-C1-C4-alkyl group, geminoaminooxime group, aryl group, and heterocyclic group; and Each X is independently either hydrogen or deuterium. The compound in question is not: or .

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

3. The method of claim 1 or claim 2, wherein the borane reducing agent is a compound of formula (Ia): (him) in: R 1 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl and C1-C4-alkoxy-C1-C4-alkyl; R 2 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and amylopime group; Where R 1 and R 2 Optionally, together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings that are optionally substituted with hydroxyl groups; R 3 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, geminosine oxime and aryl; R 4 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and heterocyclic group; Where R 3 and R 4 Optionally, they form 5-membered or 6-membered rings together with the carbon atoms to which they are attached; and R 5 It is selected from hydrogen, C1-C4 alkyl and C1-C4 hydroxyalkyl.

4. The method of claim 3, wherein: R 1 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl and C1-C4-alkoxy-C1-C4-alkyl; R 2 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and amylopime group; R 3 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl and geminoxamic groups; R 4 Selected from hydrogen, halogroup, hydroxyl, C1-C4 alkyl, and C1-C4 hydroxyalkyl; and R 5 It is selected from hydrogen, C1-C4 alkyl and C1-C4 hydroxyalkyl.

5. The method according to any one of claims 1-4, wherein 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, C1-C4 alkyl, C1-C4 alkoxy, phenyl, and monocyclic 5- or 6-membered heterocyclic groups having one nitrogen atom.

6. The method according to any one of claims 1-4, wherein R 1 It is hydroxymethyl; R 2 It is hydrogen; R 3 Selected from hydrogen, C1-C4 alkyl and phenyl; R 4 Selected from C1-C4 alkyl, C1-C4 alkoxy, and monocyclic 5-membered heterocyclic groups having one nitrogen atom; and R 5 It is hydrogen.

7. The method according to any one of claims 1-4, wherein 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, C1-C4 alkyl, and C1-C4 alkoxy.

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

9. The method according to any one of claims 1-4, wherein: R 1 Selected from hydrogen, methyl, and hydroxymethyl; R 2 Selected from hydrogen, methyl, methoxy, and hydroxymethyl; R 3 Selected from hydrogen, methyl, methoxy, and hydroxymethyl; R 4 Selected from hydrogen and methyl; and R 5 It is hydrogen; Where R 1 R 2 and R 3 At least one of them is hydroxymethyl.

10. The method according to any one of claims 1-4, wherein the borane reducing agent is a compound of formula (Ib): (One) Or its salt, wherein: R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, aryl, and heterocyclic groups; and Each X is independently either hydrogen or deuterium.

11. The method of claim 10, wherein R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, phenyl and monocyclic 5-membered heterocyclic groups having one nitrogen atom.

12. The method of claim 11, wherein R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

13. The method of claim 10, wherein: R 2 Selected from hydrogen, C1-C4 alkyl and C1-C4 alkoxy; R 3 Selected from hydrogen, C1-C4 alkyl and C1-C4 alkoxy; R 4 Selected from hydrogen and C1-C4 alkyl groups; and R 5 It is hydrogen.

14. The method of claim 10, wherein R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidine.

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

16. The method of claim 15, wherein: R 2 Selected from hydrogen, methyl, and methoxy; R 3 Selected from hydrogen, methyl, and methoxy; R 4 Selected from hydrogen and methyl; and R 5 It is hydrogen.

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

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

19. The method of claim 1, wherein the borane reducing agent is selected from: and And their salts.

20. The method of claim 1, wherein the borane reducing agent is selected from: And their salts.

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

22. The method of claim 21, wherein the oxidant is a deca-eicosyltransferase (TET).

23. The method of claim 22, wherein the TET enzyme comprises human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria gulberi TET (NgTET), Coprinus comatus TET (CcTET), or derivatives or analogs thereof.

24. The method of claim 21, wherein the oxidant comprises a chemical oxidant.

25. The method of claim 24, wherein the chemical oxidant is selected from the group consisting of: manganese oxide (MnO2), potassium perruthenate (KRuO4), Cu(II) / 2,2,6,6-tetramethylpiperidin-1-oxy (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenyl ruthenate, copper salts or complexes of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolino-1-oxy (3-Carbamoyl-PROXYL), copper salts or complexes of 2-azaadamantane-N-oxy (AZADO), and copper salts or complexes of 9-azabicyclo[3.3.1]nonane-N′-oxy (ABNO).

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

27. The method of claim 26, wherein the blocking group is added prior to contact with the oxidant.

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

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

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

31. The method of claim 30, wherein the one or more modified cytosines comprise 5caC or 5fC.

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

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

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

36. A compound selected from: and 。 37. A system or kit for converting 5-carboxycytosine (5caC) and / or 5-formylcytosine (5fC) to dihydrouracil (DHU), comprising a borane reducing agent of formula (I): (I) Or its salt, wherein: A is a monocyclic, bicyclic, or tricyclic heteroaryl group, or a monocyclic, bicyclic, or tricyclic heterocyclic group, each of which is decomposed by R. 1 R 2 R 3 R 4 and R 5 replace; R 1 R 2 R 3 R 4 and R 5 Each group is independently selected from hydrogen, halogroup, hydroxyl group, amino group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group, C1-C4 haloalkyl group, C1-C4 aminoalkyl group, C1-C4 mercaptoalkyl group, C1-C4-alkoxy-C1-C4-alkyl group, geminoaminooxime group, aryl group, and heterocyclic group; and Each X is independently either hydrogen or deuterium. The compound in question is not: or .

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

39. The system or kit of claim 37 or claim 38, wherein the borane reducing agent is a compound of formula (Ia): (him) in: R 1 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl and C1-C4-alkoxy-C1-C4-alkyl; R 2 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and amylopime group; Where R 1 and R 2 Optionally, together with the carbon atoms to which they are attached, they form 5-membered or 6-membered rings that are optionally substituted with hydroxyl groups; R 3 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl, geminosine oxime and aryl; R 4 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and heterocyclic group; Where R 3 and R 4 Optionally, they form 5-membered or 6-membered rings together with the carbon atoms to which they are attached; and R 5 It is selected from hydrogen, C1-C4 alkyl and C1-C4 hydroxyalkyl.

40. The system or kit of claim 39, wherein: R 1 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 mercaptoalkyl and C1-C4-alkoxy-C1-C4-alkyl; R 2 Selected from hydrogen, halogroup, hydroxyl group, C1-C4 alkyl group, C1-C4 alkoxy group, C1-C4 hydroxyalkyl group and amylopime group; R 3 Selected from hydrogen, halogroup, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 mercaptoalkyl and geminoxamic groups; R 4 Selected from hydrogen, halogroup, hydroxyl, C1-C4 alkyl, and C1-C4 hydroxyalkyl; and R 5 It is selected from hydrogen, C1-C4 alkyl and C1-C4 hydroxyalkyl.

41. The system or kit according to any one of claims 37-40, wherein 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, C1-C4 alkyl, C1-C4 alkoxy, phenyl, and monocyclic 5- or 6-membered heterocyclic groups having one nitrogen atom.

42. The system or kit according to any one of claims 37-40, wherein R 1 It is hydroxymethyl; R 2 It is hydrogen; R 3 Selected from hydrogen, C1-C4 alkyl and phenyl; R 4 Selected from C1-C4 alkyl, C1-C4 alkoxy, and monocyclic 5-membered heterocyclic groups having one nitrogen atom; and R 5 It is hydrogen.

43. The system or kit according to any one of claims 37-40, wherein 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, C1-C4 alkyl, and C1-C4 alkoxy.

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

45. The system or kit according to any one of claims 37-40, wherein: R 1 Selected from hydrogen, methyl, and hydroxymethyl; R 2 Selected from hydrogen, methyl, methoxy, and hydroxymethyl; R 3 Selected from hydrogen, methyl, methoxy, and hydroxymethyl; R 4 Selected from hydrogen and methyl; and R 5 It is hydrogen; Where R 1 R 2 and R 3 At least one of them is hydroxymethyl.

46. ​​The system or kit according to any one of claims 37-40, wherein the borane reducing agent is a compound of formula (Ib): (One) Or its salt, wherein: R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, C1-C4 alkoxy, aryl, and heterocyclic groups; and Each X is independently either hydrogen or deuterium.

47. The system or kit of claim 46, wherein R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, phenyl and monocyclic 5-membered heterocyclic groups having one nitrogen atom.

48. The system or kit of claim 47, wherein R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, C1-C4 alkyl, and C1-C4 alkoxy.

49. The system or kit of claim 46, wherein: R 2 Selected from hydrogen, C1-C4 alkyl and C1-C4 alkoxy; R 3 Selected from hydrogen, C1-C4 alkyl and C1-C4 alkoxy; R 4 Selected from hydrogen and C1-C4 alkyl groups; and R 5 It is hydrogen.

50. The system or kit of claim 46, wherein R 2 R 3 R 4 and R 5 Each is independently selected from hydrogen, methyl, methoxy, phenyl, and pyrrolidine.

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

52. The system or kit of claim 51, wherein: R 2 Selected from hydrogen, methyl, and methoxy; R 3 Selected from hydrogen, methyl, and methoxy; R 4 Selected from hydrogen and methyl; and R 5 It is hydrogen.

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

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

55. The system or kit of claim 37, wherein the borane reducing agent is selected from: and And their salts.

56. The system or kit of claim 37, wherein the borane reducing agent is selected from: and And their salts.

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

58. The system or kit of claim 57, wherein the oxidant is a deca-eicosyltransferase (TET) enzyme.

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

60. The system or kit of claim 57, wherein the oxidant comprises a chemical oxidant.

61. The system or kit of claim 60, wherein the chemical oxidant is selected from the group consisting of: manganese oxide (MnO2), potassium perruthenate (KRuO4), Cu(II) / 2,2,6,6-tetramethylpiperidin-1-oxy (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenyl ruthenate, copper salts or complexes of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolino-1-oxy (3-Carbamoyl-PROXYL), copper salts or complexes of 2-azaadamantane-N-oxy (AZADO), and copper salts or complexes of 9-azabicyclo[3.3.1]nonane-N′-oxy (ABNO).

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

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

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

65. The system or kit of claim 62 or claim 63, wherein the blocking agent is a sugar or a uridine diphosphate (UDP) linked sugar, and the system or kit further comprises a glucosyltransferase.

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