LABELLING OF BIOMOLECULES

Abstract

A method of reversibly labeling a biomolecule including providing a linker molecule (11) having a first functional group (LG) with a reactive center, a second functional group (FG) with a reactive center, and a cleavable, e.g. hydrolyzable, moiety (A-B-C). The method further includes forming a covalent bond between the biomolecule (10) and the reactive center of the first functional group (LG), forming a covalent bond between a first label (L1) and the reactive center of the second functional group (FG), cleaving the cleavable moiety (A-B-C), e.g. hydrolyzing the hydrolyzable moiety, of the linker molecule (11) to remove the first label (L1) and to form a third functional group (W) with a reactive center, and forming a covalent bond between a further molecule and the reactive center of the third functional group (W) to reform the cleavable moiety, e.g. hydrolyzable moiety (A-B-C).

Claims

1.-26. (canceled)

27. A method of reversibly labeling a biomolecule, the method comprising the steps of: a. providing a linker molecule, the linker molecule comprising a first functional group comprising a reactive center, a second functional group comprising a reactive center, and a cleavable moiety; b. forming a covalent bond between the biomolecule and the reactive center of the first functional group; c. forming a covalent bond between a first label and the reactive center of the second functional group; d. cleaving the cleavable moiety of the linker molecule to remove the first label and to form a third functional group comprising a reactive center; and e. forming a covalent bond between a further molecule and the reactive center of the third functional group to reform the cleavable moiety.

28. The method according to claim 27, wherein the further molecule comprises a fourth functional group, and wherein the method further comprises step f. forming a covalent bond between a second label and a reactive center of the fourth functional group.

29. The method according to claim 28, wherein the method further comprises step g. cleaving the cleavable moiety to remove the second label and to reform the third functional group.

30. The method according to claim 29, wherein the method further comprises step h. forming a covalent bond between a further molecule and the third functional group to reform the cleavable moiety, wherein the further molecule of step e. is optionally the same species as the further molecule of step h.

31. The method according to claim 27, wherein the further molecule comprises a second label.

32. The method according to claim 27, wherein the linker molecule comprises the following general formula: ##STR00030## wherein FG represents the second functional group comprising a reactive center; Z represents a non-reactive group of an aliphatic linkage or an aromatic linkage; A-B-C together represent the cleavable moiety; Y represents a non-reactive group of an aliphatic linkage or an aromatic linkage; LG represents a first functional group comprising a reactive center.

33. The method according to claim 32, wherein Z represents a polyether chain.

34. The method according claim 27, wherein the cleavable moiety A-B-C represents one of the following moieties: ##STR00031## ##STR00032## wherein R.sup.x represents a hydrogen atom, a deuterium atom, an aliphatic linkage, or an aromatic linkage.

35. The method according to claim 27, wherein the biomolecule is a polynucleotide.

36. The method according to claim 27, wherein step b. forming a covalent bond between the biomolecule and the reactive center of the first functional group further comprises the step of providing a catalyst.

37. The method according to claim 36, wherein the catalyst is an enzyme.

38. The method according to claim 27, wherein the linker molecule is an analogue of an S-adenosyl-1-methionine cofactor and has the following general formula: ##STR00033## wherein FG represents the second functional group; Z represents a non-reactive group of an aliphatic linkage or an aromatic linkage; A-B-C represent the cleavable moiety; Y represents a non-reactive group of an aliphatic linkage or an aromatic linkage; U represents an unsaturated bond comprising at least one of an alkene, an alkyne, an aryl group, a carbon atom comprising a carbonyl group, and a sulfur atom comprising one or two S═O bonds; k represents an integer of 1 or 2; and W.sup.− is a counter ion.

39. The method according to claim 38, wherein the linker molecule comprises an unsaturated moiety U.

40. The method according to claim 38, wherein the linker molecule has one of the following general formulas: ##STR00034## wherein the cleavable moiety is a Schiff base moiety comprising C═N—X—C-Q; p represents a number between 1 to 15; Q represents an oxygen atom, two hydrogen atoms, or one or more deuterium atoms independently bonded to the carbon center; X represents an oxygen atom or a nitrogen atom; Z represents a non-reactive group of an aliphatic linkage or an aromatic linkage; k represents an integer of 1 or 2; and FG represents the second functional group or ##STR00035## wherein the cleavable moiety is a Schiff base moiety comprising —C═N—N—C═O; p represents a number between 1 to 15; q represents a number between 1 to 15; k represents an integer of 1 or 2; and FG represents the second functional group. or ##STR00036## wherein the cleavable moiety is a Schiff base moiety comprising —C═N—O—; p represents a number between 1 to 15; wherein q represents a number between 1 to 15; k represents an integer of 1 or 2; and FG represents the second functional group.

41. The method according to claim 32, wherein the first functional group LG represents a halogen for reaction with an amino acid, a peptide, or a protein and/or the second functional group FG is an azide moiety.

42. The method according to claim 27, wherein forming a covalent bond between a first label and the reactive center of the second functional group comprises a reaction of an azide moiety with an alkyne moiety to form a carbon-nitrogen covalent bond.

43. The method according to claim 27, wherein cleaving the cleavable moiety of the linker molecule to remove a label and to form a third functional group comprises treatment with hydroxylamine.

44. The method according to claim 28, wherein at least one of the first label and the second label comprises at least one of a fluorescent molecule, a radioactive species, and a biological molecule.

45. The method according to claim 27, wherein forming a covalent bond between a further molecule and the third functional group in step e., to reform the cleavable moiety further comprises a reaction of an NH.sub.2 moiety with an aldehyde moiety on the further molecule to form a carbon-nitrogen double bond.

46. A method of reversibly labeling a polynucleotide molecule, the method comprising the steps of: a. providing a linker molecule (Compound A) having the following general formula: ##STR00037## wherein R represents a transferable group; FG represents the second functional group; Z represents a non-reactive group of an aliphatic linkage or an aromatic linkage; A-B-C represent the cleavable moiety; Y represents a non-reactive group of an aliphatic linkage or an aromatic linkage; U represents an unsaturated bond selected from the group consisting of an alkene, an alkyne, an aryl group, a carbon atom comprising a carbonyl group, and a sulfur atom comprising one or two S═O bonds; k represents an integer of 1 or 2; and b. forming a covalent bond between the polynucleotide molecule and the R group of Compound A using a DNA methyltransferase enzyme which is capable of using Compound A as a cofactor and under conditions that allow for the transfer of the R group of Compound A onto the polynucleotide molecule; c. forming a covalent bond between a first label and the second functional group FG of Compound A; d. hydrolyzing the cleavable moiety or hydrolyzable moiety of the linker molecule to remove the first label and to form an O-substituted hydroxylamine or an N-substituted hydrazone; e. forming a covalent bond between an aldehyde moiety of a further molecule and the O-substituted hydroxylamine or the N-substituted hydrazone to reform a Schiff base moiety.

Description

[0128] Embodiments of the invention will now be described by way of example only with reference to the accompanying drawings in which:

[0129] FIG. 1 is a schematic representation of the reversible and rewritable modification of a biomolecule according to an embodiment of the invention;

[0130] FIG. 2A is a schematic representation of linker molecules according to an embodiment of the invention;

[0131] FIG. 2B is a schematic representation of the reversible and rewritable modification of a biomolecule according to a further embodiment of the invention;

[0132] FIG. 3 is a reaction scheme for the formation of a precursor to the linker molecule containing a hydrazone Schiff base 21A;

[0133] FIG. 4 is a reaction scheme for the formation of a precursor to the linker molecule containing an oxime Schiff base 21B;

[0134] FIG. 5 is a reaction scheme for the formation of S-adenosyl-1-methionine cofactor analogues;

[0135] FIG. 6A is a schematic representation of a restriction assay;

[0136] FIG. 6B displays analysis of enzymatic DNA labelling by gel electrophoresis;

[0137] FIG. 7 displays analytical HPLC chromatographs following rewriting the original functionality of DNA;

[0138] FIG. 8 is a schematic representation of the dual functionalisation of DNA; and

[0139] FIG. 9 is a reaction scheme for the formation of a further linker molecule.

[0140] Referring now to FIG. 1, there is shown a schematic representation 1 of the reversible and rewritable modification of biomolecule 10 according to an embodiment of the invention.

[0141] There is shown a biomolecule 10 and a linker molecule 11. The linker molecule ii comprises a first functional group LG comprising a reactive centre, a second functional group FG comprising a reactive centre, a hydrolysable moiety (e.g. a Schiff base moiety) A-B-C, non-reactive groups Y and Z, and an unsaturated bond U. There is also shown a first label L1 and a second label L2.

[0142] The method of reversibly labelling the biomolecule 10 comprises the following steps, which are labelled on the schematic representation: [0143] a) providing a linker molecule 11, the linker molecule 11 comprising a first functional group LG comprising a reactive centre, a second functional group FG comprising a reactive centre, and a hydrolysable moiety, e.g. a Schiff base moiety, A-B-C; [0144] b) forming a covalent bond between the biomolecule 10 and the reactive centre of the first functional group LG; [0145] c) forming a covalent bond between the first label L1 and the reactive centre of the second functional group FG; [0146] d) hydrolysing the hydrolysable moiety A-B-C of the linker molecule 11 to remove the first label L1 and to form a third functional group W comprising a reactive centre; [0147] e) forming a covalent bond between a further molecule (not shown) and the reactive centre of the third functional group W to reform the hydrolysable moiety A-B-C.

[0148] Advantageously, the covalent bond formed between the reactive centre of the first functional group LG of linker molecule 11 and the biomolecule 10 adds chemical functionality to the biomolecule 10 in the form of the further functional group FG and the hydrolysable moiety A-B-C.

[0149] The method of the invention may further comprise the optional step i. of forming a covalent bond between a further molecule comprising a label, e.g. the second label L2, and the reactive centre of the third functional group W, to reform the hydrolysable moiety A-B-C.

[0150] In embodiments, step c. may be performed before step b.

[0151] Referring first to FIG. 2A, there is shown two linker molecule structures; 21A and 21B.

[0152] Referring also to FIG. 2B, there is shown a schematic representation 2 of the reversible and rewritable modification of biomolecule 20, according to a further embodiment of the invention.

[0153] There is shown a biomolecule 20 and the linker molecule 21. The linker molecule 21 may be either of those shown in FIG. 2A. The linker molecules 21A, 21B each comprise a first functional group LG, a second functional group FG, and a hydrolysable moiety A-B-C.

[0154] There is also shown a first label L1′ and a second label L2′.

[0155] In this embodiment, the biomolecule 20 is a DNA molecule and the linker molecule 21 is an analogue of the S-adenosyl-1-methionine cofactor.

[0156] The first functional group LG of the linker molecule 21A, 21B is S-adenosyl-I-homocysteine, the second functional group FG of the linker molecule is an azide moiety, the hydrolysable moiety is one of an hydrazone (Linker 21A) or an oxime (Linker 21B).

[0157] In this embodiment, hydrolysis of the hydrolysable moiety (e.g. Schiff base) A-B-C further comprises treatment with hydroxylamine, for example in an ammonium acetate buffer solution, e.g. at pH 4. The hydrolysable moiety A-B-C is hydrolysed to provide a third functional group W comprising a reactive centre; an N-substituted hydrazone (wherein the Schiff base is a hydroxylamine) or an O-substituted hydroxylamine (wherein the Schiff base is an oxime).

[0158] The further molecule 22 for use in step e. to form a covalent bond with the NH.sub.2 group of the N-substituted hydrazone (21A) or an O-substituted hydroxylamine (21B) to reform the hydrazone (21A) or the oxime (21B) Schiff base moiety is also shown in FIG. 2A.

[0159] In this embodiment, the functional group of the first label L1′ and the second label L2′ are both an alkyne moiety, which forms a C—N covalent bond with the reactive centre of the second functional group FG (an azide moiety) via a click chemistry reaction.

[0160] In this embodiment, step b. of the method 2 further comprises providing a catalyst, for example, an enzyme, e.g. a DNA methyltransferase enzyme capable of transferring an alkyl group from a S-adenosyl-1-methionine cofactor analogue.

[0161] The reversible and rewritable modification of DNA molecule 20 comprises the following steps, which are labelled on the schematic representation: [0162] a) providing a linker molecule 21A or 21B [0163] the linker molecule 21A or 21B comprising a S-Adenosyl-I-homocysteine moiety comprising a CH.sub.2-substituted trivalent sulphonium ion moiety LG, an azide moiety FG, and a hydrazone (21A) or an oxime (21B) or Schiff base moiety A-B-C; [0164] b) site-selective MTase-directed writing of DNA molecule 20 [0165] forming a covalent bond between the DNA molecule 20 (on one or more of a cytosine C5, cytosine N4 or adenosine N6) and the carbon of the CH.sub.2 group of the linker molecule 21A or 21B; [0166] c) modification of DNA molecule 20 via azide-alkyne cycloaddition [0167] forming a C—N covalent bond between the alkyne moiety of the first label L1′ and the azide of the linker molecule 21A or 21B; [0168] d) erasing introduced functionality via dynamic exchange [0169] hydrolysing the hydrazone (21A) or oxime (21B) Schiff base moiety of the linker molecule 21A or 21B to form N-substituted hydrazone (21A) or an O-substituted hydroxylamine (21B). [0170] e) re-writing the intermediate DNA molecule via Schiff-base formation [0171] forming a covalent bond between a further molecule (not shown) and the NH.sub.2 group of the N-substituted hydrazone (21A) or an O-substituted hydroxylamine (21B) to reform the hydrazone (21A) or the oxime (21B) Schiff base moiety.

[0172] Optionally, Step i. may involve further functionalising the DNA intermediate via standard conjugation techniques.

[0173] The further molecule, e.g. that of step e. or that of step i.; comprises an aldehyde moiety for reaction with the NH.sub.2 functionality of the third functional group to reform the Schiff base.

[0174] To further exemplify the invention, reference is also made to the following non-limiting Examples:

Synthesis of Precursor 1 for Use in the Synthesis of Linker 21A

[0175] Referring now to FIG. 3, there is shown a reaction scheme for the formation of a precursor 1 to the linker molecule containing a hydrazone Schiff base 21A. The precursor 1 was synthesised using the following protocol. [0176] 1.1 Synthesis of 8-hydroxyoct-6-ynoic acid 7. [0177] A solution of 6-heptynoic acid (2 g, 15.87 mmol) was made in dry THF (42 ml) under argon, to this HMPA (34.9 mmol, 6.13 ml) was added and the solution was cooled to −78° C. To this nBuLi (1.6 M in hexanes, 34.9 mmol, 21.8 ml) was added dropwise whilst maintaining the temperature below −60° C. The solution was then warmed to −40° C. and stirred for 1 hour. After 1 hour paraformaldehyde (1.47 g, 47.6 mmol) was added via powder funnel under an argon flow. The reaction mixture was then warmed to 45° C. for 4 hours. After reaction, the mixture was quenched with 1 M HCl to pH 4-5 and extracted with EtOAc. The solvent was then dried and the EtOAc was removed by rotary evaporation giving the crude product. Purification was completed using flash column chromatography (silica gel, Hex:EtOAc, 6:4): Yield=68%, Rf=0.27 (Hex:EtOAc, 6:4); .sup.1H NMR (300 MHz, DMSO-d6) δ 12.03 (s, 1H), 5.03 (s, 1H), 4.02 (d, J=2.6 Hz, 2H), 2.29-2.14 (m, 4H), 1.63-1.50 (m, 2H), 1.50-1.39 (m, 2H); MS: m/z [M−H]=155.46. [0178] 1.2 Synthesis of tert-butyl 2-(8-hydroxyoct-6-ynoyl)hydrazine-1-carboxylate 8 [0179] 8-hydroxyoct-6-ynoic acid 7 (1.35 g, 8.65 mmol) and tert-butyl carbazate (1.4 g, 10.38 mmol) were dissolved in 2:1 THF:H2O (13.5:6.75 ml). To this EDC.HCl (1.87 g, 9.52 mmol) was added slowly over 15 minutes. The mixture was left to stir for 3 hours and then extracted with EtOAc. The organic layer was washed with 0.1 M HCl, water and brine and then the organic layer is collected, dried over anhydrous sodium sulfate and the and the solvent was removed under reduced pressure yielding the product as a white solid: Yield=63%; .sup.1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 8.66 (s, 1H), 5.04 (t, J=5.9 Hz, 1H), 4.02 (dt, J=5.9, 2.2 Hz, 2H), 2.19 (tt, J=7.1, 2.2 Hz, 2H), 2.06 (t, J=7.2 Hz, 2H), 1.58 (p, J=7.3 Hz, 2H), 1.50-1.32 (m, 12H); .sup.13C NMR (101 MHz, DMSO) δ 172.01, 84.36, 80.94, 79.42, 49.59, 33.06, 28.53, 28.08, 24.70, 18.24; MS: m/z [M+Na]=294.15. [0180] 1.3 Synthesis of tert-butyl 2-(8-bromooct-6-ynoyl)hydrazine-1-carboxylate 1 [0181] A solution of tert-butyl 2-(8-hydroxyoct-6-ynoyl)hydrazine-1-carboxylate 8 (300 mg, 1.11 mmol) was made in dry DCM (3.33 ml) and cooled on ice. Triphenylphosphine (437 mg, 1.67 mmol) was added and left to dissolve, once dissolved tetrabromomethane (552 mg, 1.67 mmol) was added slowly. The reaction was then brought to room temperature and left to stir for 1 hour. After reaction the solvent was removed under reduced pressure and the crude mixture was purified by flash column chromatography (silica gel Hex:EtOAc, 7:3): Yield=55%; Rf=0.15 (Hex:EtOAc 7:3); .sup.1H NMR (300 MHz, DMSO-d6) δ 9.48 (s, .sup.1H), 8.67 (s, 1H), 4.21 (t, J=2.3 Hz, 2H), 2.27 (tt, J=6.9, 3.4 Hz, 2H), 2.06 (t, J=7.4 Hz, 2H), 1.65-1.31 (m, 13H); .sup.13C NMR (101 MHz, DMSO) δ 171.4, 155.2, 87.7, 78.9, 76.3, 54.9, 39.5, 32.5, 28.0, 27.3, 24.1, 17.9, 17.2; MS: m/z [M+Na]=355/357.08.

Synthesis of Precursor 4 for Use in the Synthesis of Linker 21B

[0182] Referring now to FIG. 4, there is shown a reaction scheme for the formation of a precursor 4 to the linker molecule containing an oxime Schiff base 21B. The precursor 4 was synthesised using the following protocol. [0183] 2.1 Synthesis of 7-Bromo-hept-1-yne 9 [0184] A solution of 6-heptyn-1-ol (5 g, 44.6 mmol) was made in dry DCM (60 ml) and cooled on ice. To this triphenylphosphine (17.6 g, 67 mmol) was added, upon complete dissolution tetrabromomethane (22.2 g, 67 mmol) was added slowly. The reaction mixture was brought to room temperature and stirred for 1 hr. After completion, the solvent was removed under reduced pressure. Hexane was added to the crude forming a white suspension. The hexanefraction was filtered, collected and then the solvent was removed. An oily residue remained which was purified by flash column chromatography with hexane: Yield=91%, Rf=0.45 (hexane); %); vmax(neat)/cm-1 540 (C—Br); .sup.1H NMR (300 MHz, DMSO-d6) δ 3.53 (t, J=6.7 Hz, 2H), 2.75 (t, J=2.7 Hz, 1H), 2.23-2.10 (m, 2H), 1.89-1.74 (m, 2H), 1.50-1.43 (m, 4H). [0185] 2.2 Synthesis of 8-bromooct-2-yn-1-ol 10 [0186] A solution of 7-bromohept-1-yne 9 (20.56 mmol, 3600 mg) was made in Dry THF (12.3 ml) and cooled to −78° C. under Argon. To this a solution of nBuLi in hexanes (1.6 M, 13 ml) was added dropwise, whilst maintaining the temperature below −60° C. The reaction mixture was then warmed to 0° C. in an ice bath at which point paraformaldehyde (1718 mg, 55.5 mmol) was added under a flow of Argon and stirred for 30 minutes. The mixture was then warmed to room temperature and left to stir, the temperature was maintained below 30° C. until the exothermic reaction had stopped. The mixture was then heated to 45° C. for 2 hrs. Once complete the reaction was extracted with ether and sat. NH.sub.4Cl. The organic layer was collected and the solvents were removed under reduced pressure to yield the crude product as an oil. Once dry, purification was completed by flash column chromatography (silica gel, Hexane: Ethyl Acetate, 9:1). The product was then collected as a colourless oil: Yield=55%, Rf=0.15 (Hex: EtOAc 9:1), .sup.1H NMR (300 MHz, DMSO-d6) δ 5.04 (t, J=5.7 Hz, 1H), 4.03 (dt, J=5.5, 2.1 Hz, 2H), 3.54 (t, J=6.7 Hz, 2H), 2.20 (m, 2H), 1.88-1.75 (m, 2H), 1.52-1.40 (m, 4H). [0187] 2.3 Synthesis of tert-butyl ((8-hydroxyoct-6-yn-1-yl)oxy)carbamate 11 [0188] To a solution of N-Boc Hydroxyl amine (890 mg, 6.55 mmol) in DMF (4.3 ml) 8-bromooct-2-yn-1-ol (10) (1200 mg, 5.85 mmol) and 1,8-Diazabicyclo[5.4.0]undec-7-ene (1000 mg, 6.55 mmol) was added. The solution was stirred at 50° C. for 20 hrs. Once complete, the reaction was extracted with DCM and 15% citric acid solution. The organic phases were dried and collected and the solvent was removed under reduced pressure. A colourless oil was collected as the crude product. This was further purified by flash column chromatography (silica gel, Hexane: Ethyl Acetate, 8:2). The product was collected as a colourless oil: Yield=73%, Rf=0.27; .sup.1H NMR (300 MHz, DMSO-d6) δ 9.91 (s, 1H), 5.03 (t, J=5.9 Hz, 1H), 4.02 (dt, J=5.9, 2.2 Hz, 2H), 3.66 (t, J=6.2 Hz, 2H), 2.17 (tt, J=6.7, 1.7 Hz, 2H), 1.40 (m, 15H); MS: m/z [M+H]=258.2. [0189] 2.4 Synthesis of tert-butyl ((8-bromooct-6-yn-1-yl)oxy)carbamate 4 [0190] A solution of tert-butyl((8-hydroxyoct-6-yn-1-yl)oxy)carbamate 11 (1 g, 3.89 mmol) was made in dry DCM (5.2 ml) and cooled on ice. To this triphenylphosphine (1.53 g, 67 mmol) was added. Upon complete dissolution tetrabromomethane (1.94 g, 67 mmol) was added slowly. The reaction mixture was brought to room temperature and allowed to stir for 1 hr. After completion, the solvent was removed under reduced pressure. Purification was completed using flash column chromatography (silica gel, Hexane: Ethyl Acetate, 8:2): Yield=67%, Rf 0.52 (Hex:EtOAc, 8:2); vmax(neat)/cm-1 1712 (C═O), 607 (C—Br); .sup.1H NMR (300 MHz, DMSO-d6) δ 9.90 (s, 1H), 4.21 (t, J=2.4 Hz, 2H), 3.66 (t, J=6.2 Hz, 2H), 2.25 (tt, J=6.9, 2.4 Hz, 2H), 1.40 (m, 15H); .sup.13C NMR (101 MHz, DMSO) δ 156.04, 87.85, 79.37, 76.22, 75.05, 39.52, 28.05, 27.64, 27.04, 24.76, 18.06, 17.25; MS: m/z [M+Na]=342.35/344.35, [M-.sup.tBuOH]=246.38/248.38.

Example of a Linker Molecule for the Enzymatic Labelling of a Polynucleotide: MTase-Directed Labelling of a Polynucleotide

Synthesis of Linker Molecules 21A, 21B

[0191] Referring now to FIG. 5, there is shown a reaction scheme 5A for the formation the linker molecule containing a hydrazone Schiff base 21A from precursor 1. There is also shown a reaction scheme 5B for the formation the linker molecule containing an oxime Schiff base 21B from precursor 4.

3) Synthesis of S-adenosyl-1-methionine Cofactor Analogues 21A, 21B

[0192] 3.1 General coupling procedure [0193] Precursors 1, 4 were prepared and reacted with S-adenosyl-L-homocysteine under acidic conditions to give reversible and rewritable Boc-protected AdoMet derivatives. [0194] A solution of S-adenosyl-1-homocysteine (15 mg, 0.04 mmol) was made in a 1:1 mixture of formic and acetic acid (300 μl). Precursor 1 or 4 (tert-butyl 2-(8-bromooct-6-ynoyl)hydrazone-1-carboxylate or tert-butyl ((8-bromooct-6-yn-1-yl)oxy)carbamate) (1.2 mmol, 30 equivs) was then added dropwise, on ice. The reaction mixture was warmed to 35° C. and left to stir overnight. After overnight stirring the reaction mixture was extracted with diethyl ether and the aqueous layer was collected and dried by lyophilisation: MS: m/z [M+H]=638 (2), [M+H]=624 (5). [0195] 3.2 Cofactor Deprotection [0196] The AdoMet analogues were deprotected under acidic conditions to reveal the hydrazone or alkoxyamine moieties. [0197] The crude product was dissolved in TFA (400 μl) and left stir for 2 hrs at room temperature. After reaction the acid was removed under a flow of argon. [0198] 3.3 Cofactor Purification [0199] Any excess precursor was removed by purification. [0200] Both diastereomers of the deprotected cofactors could be separated by HPLC, a separation which was not possible at later stages. [0201] The crude reaction mixture was then dissolved in water (2 ml). Purification of AdoMet analogues was performed by preparative reversed-phase HPLC (ACE 5 C-18 25×2.12 cm) eluting with 20 mM Ammonium Acetate pH 5.5 Water (A)/MeOH (B) gradient at a flow rate of 10 ml/min. Gradient system: 30 mins 3-30% B, 30-97% B over 30 mins, hold at 97% B for 5 minutes, stop programme. Retention times: Hydrazide iso. 1=17.51 mins, iso. 2=18.73 mins, hydroxylamine iso. 1=25.47 mins, iso. 2=28.24 mins: MS: m/z [M+H]=538 (2), [M+H]=524 (5). [0202] The deprotected AdoMet derivatives slowly degrade, in particular following freeze-drying, via multiple pathways, giving additional peaks at higher retention times. [0203] 3.4 Aldehyde coupling [0204] To mitigate against degradation the AdoMet derivatives were reacted with a commercially available benzaldehyde immediately after purification by HPLC in order to minimise side reactions due to the nucleophilic nature of the hydrazone and alkoxyamine moieties. [0205] To the collected HPLC fractions Ald-PEG3-N3 (1.2 equivs) was added and rolled for 30 mins at room temperature. The fractions were then dried by lyophilsation. Once dry the solids were dissolved in 100 μl 0.1% Acetic Acid and stored at −20° C. Concentrations were determined by UV absorption analysis with E260=15.400 dm.sup.−3 mol.sup.−1 cm.sup.−1: MS: m/z [M+H]=867 (3), [M+H]=856 (6). [0206] The resulting linker molecules 21A and 21B contain reactive terminal azides that can be readily conjugated to a range if functional groups, while condensation of the aldehyde with the hydrazone or alkoxyamine incorporates a dynamic functionality, that can be reversibly functionalised. [0207] A slight excess of aldehyde (1.2 equivs) was employed to ensure full functionalisation of the deprotected intermediate. [0208] No degradation of the freeze-dried AdoMet derivatives 21A and 21B was observed.
MTase-Directed Labelling of a Polynucleotide with 21A and 21B

[0209] A restriction assay was used to demonstrate the activity of the MTases with the linker molecules 21A and 21B.

[0210] M.Taql (an N6-adenine DNA MTase) was incubated at 50° C. for 1 hour with a linker molecule 21A, 21B and plasmid pUC19, which has four recognition sites (TCGA) for the enzyme, see FIG. 6A.

[0211] Successful transfer of the functional group by M.Taql results in protection of the plasmid from restriction digestion by R.Taql, an endonuclease with the same target site as M.Taql.

[0212] Referring now to FIG. 7B, there is shown gel electrophoresis of pUC19 following enzymatic treatment with M.Taql and/or R.Taql in the presence or absence of AdoMet (375 μl) or linker 21B.

[0213] In the absence of M.Taql-mediated alkylation (FIG. 6B, lanes 4, 8 and 12), pUC19 is cut into fragments, of which the largest three can be identified by gel electrophoresis. M.Taql-mediated alkylation with AdoMet (FIG. 6B, lane 10) or linker 21B (FIG. 6B, lanes 1-3 and 5-7) results in partial to full protection from restriction by R.Taql, with mainly open circular and supercoiled plasmid DNA being observed by gel electrophoresis. Neither isomer interferes with the ability of R.Taql to digest plasmid DNA (FIG. 6B, lanes 4 and 8).

[0214] Controls were run in the absence of AdoMet (FIG. 6B, lanes 11 and 12), in the absence of M.Taql (FIG. 6B, lanes 4, 8 and 12) and in the absence of R.Taql (FIG. 6B, lane 9) and showed labelling to be successful.

[0215] To test the labelling efficiency of each isomer, a cofactor dilution series was run to highlight any differences in affinity with the enzyme, with the second fraction having higher activity. The preferential isomer, diastereomer II, was carried forward for future experiments. Similar effects were seen with linker 21A. Both linker molecules 21A and 21B have the potential to be employed for the dynamic labelling of biomolecules.

[0216] M.Mpel is a cytosine-C5 MTase which targets the CpG dinucleotide. pUC19 was incubated with mutant M.Mpel (Q136A, N347A) and linker 21B before restriction enzyme R.Haell, which targets a subset of the CpG dinucleotides, was added. Efficient transalkylation of plasmid DNA with M.Mpel was also observed.

[0217] Evidence of the ability of MTases to alkylate DNA with linker 21A, 21B was achieved by targeting a 14 base pair oligonucleotide with one copy of M.Taql sequence (TCGA) for transalkylation. Labelling of the oligo was monitored directly using HPLC. Analysis was performed above the melting temperature of the DNA so that both strands could be clearly identified in the chromatogram. A clear shift in the retention time was seen upon labelling with linker 21A, 21B when compared to the retention times of the unmodified DNA. The shift was observed for both peaks, demonstrating that M.Taql was able to label both strands as a consequence of the palindromic nature of the sequence MTase recognizes. The shift was proportional to the size and nature of the linker transferred, with the AdoMet methylation resulting in a small shift in retention time and the oxime derivate 21B giving the biggest shift. The presence of a small amount of erased oligo DNA was observed and is likely due to hydrolysis under the HPLC conditions. Analysis of the individual peaks was carried out using MS which confirmed labelling was successful and the nature of the sidechain functionality introduced following incubation.

Reversible Enzymatic Labelling Protocol

[0218] The following protocols may be used in accordance with the embodiment shown in FIGS. 2A and 2B. [0219] 1) Enzymatic Labelling (Step b of Method of the Invention) [0220] For each sample a solution of oligo biomolecule (120 μl, 10 μM), buffer (40 μl, 10× NEB cutsmart buffer), M.Taql (45 μl), water (189 μl) and linker molecule (6 μl, 20 mM) was made. Samples were incubated at 50° C. for 1.5 hrs. After incubation proteinase K (2.5 μl) was added and the samples were incubated at 50° C. for a further 1 hr. The samples were then purified using the Qiagen(RTM) Qiaquick nucleotide clean up kit and eluted into 50 μl water and their concentration was measured by shimadzu biospec-nano. Samples not to be reversed were taken and stored in the fridge until HPLC analysis. [0221] 2) Schiff Base Reversal (Step c of Method of the Invention) [0222] To the hydrazone labelled DNA, a solution of H.sub.2NOH.HCl in water (10 μl, 10 equivs) was added. The pH of the solution was then adjusted using 100 mM Ammonium Acetate Buffer (pH 4.0, 7 μl). The samples were then incubated at 50° C. for 1.5 hrs and then stored in the fridge until analysis. [0223] 3) HPLC Labelling Analysis [0224] Purification of labelled oligonucleotides was performed by analytical reversed-phase HPLC (Phenomenex, Gemini, 5 μm, C18, 110 Å) eluting with a 0.1 M Triethyl amine acetate buffer, pH 7.0 (A)/MeCN (B) gradient, at a flow rate of 1 ml/min 60° C. Gradient system A: 5-18% B over 25 mins, to 100% 5 mins, hold at 100% 10 mins, lower to 5% for 5 mins. System B: 5-31% B over 50 mins, to 100% 10 mins, hold at 100% 5 mins, lower to 5% for 10 mins. For unlabelled and methylated oligonucleotides gradient A was used and for all remaining samples system B was used. Fractions were collected and analysed by mass spectrometry. [0225] Under these conditions the hydrazone labelled oligo DNA melts and both DNA strands can be observed independently. Over 85% of the functional linker was cleaved with the new HPLC peak shifted to lower retention time, following the loss of the potentially hydrophobic aldehyde. [0226] The oxime labelled oligo DNA under these conditions remains intact, consistent with the higher stability of this type of Schiff base. [0227] 4) Rewriting the original functionality via Schiff base formation [0228] The hydrazide functionalized oligo DNA (FIG. 7, line A) was incubated in the presence of an excess of aldehyde 5 (FIG. 7). A clear shift in the retention time of the main peaks associated with oligo DNA was observed (FIG. 7, line B). Chromatographs obtained following incubation of oligo DNA with M.Taql and linker 21A (FIG. 7, line C) show good overlap of the peaks associated with azide-functionalized DNA at ˜6.2 and 6.4 mins, and a similar ratio of this peak to that of the free hydrazide (at 3.8 and 4.0 mins). The presence of a small amount of hydrazide functionalized oligo DNA is likely due to hydrolysis of the Schiff base under the HPLC conditions. Three additional peaks were observed following rewriting with aldehyde, which overlapped with those observed when this aldehyde was incubated with H.sub.2NOH.HCl (FIG. 7, line D). Analysis of the individual peaks was carried out by LC-MS which confirmed rewriting was successful and the nature of the chemical functionality on the oligo DNA. [0229] 5) Further Modification following Schiff-base cleavage. [0230] Fragments of DNA generated by PCR, containing 17 CpG sites, were site-selectively labelled with M.Mpel. Labelling was followed by incubation with H.sub.2NOH.HCl and reaction with a commercially available NHS-activated fluorophore Atto647N (further funtionalisation). The reaction was monitored via gel electrophoresis and shows specific conjugation of Atto647N to DNA modified with linker 21B. While no red-fluorescence was observed in the absence of Atto647N, this dye is positively charged and was able to non-specifically associate with the DNA in the control samples. Comparison of the intensities of the red and green (Sybr® Green) channels, to evaluate the degree of labelling with Atto647N per unit DNA showed a degree of labelling 4.8 times higher for the DNA that has undergone the write-erase than in the absence of the erase step, H.sub.2NOH.HCl treatment, and over 23 times higher than in the absence of the write step, MTase directed labelling. [0231] 6) Dual Modification [0232] Sequential modification with complementary fluorescent dyes was achieved by labelling short DNA fragments with two different fluorescent dyes (write-modify-erase-further modification). To this end, DNA fragments were first incubated with M.Mpel and dynamic linker 21A, to yield azide-functionalized DNA (FIG. 8, image A, Step 1). This modification resulted in a small shift in the migration time of the DNA on gel (FIG. 8, image B, Step 2) but, as expected, no fluorescence was observed (FIG. 8, image C, Step 1). A further shift in the migration time was observed when the azide-functionalized DNA was modified with TAMRA-DBCO 6 (FIG. 8, image B, Step 2) but, more importantly, emission from DNA-associated TAMRA fluorophore was clearly observed (FIG. 8, image, C, Step 2). Erasing the TAMRA modification was achieved by incubation with an excess of H.sub.2NOH.HCl. No fluorescence was observed from the resulting DNA fragments (FIG. 8, image C, Step 3) and a shift back to the original migration time was observed (FIG. 8, image B, Step 3), suggesting that this hydrazide linker had little impact on the physical properties of the DNA. Incubation of this hydrazide-functionalized DNA with NHS-activated Atto647N 7 resulted in a new shift in migration time (FIG. 8, image B, Step 4) and corresponding appearance of fluorescence, now visible under red illumination (FIG. 8, image C, Step 4). [0233] Modification was monitored using gel electrophoresis: DNA concentration; 7 ng/μL, release buffer; 10 mM Ammonium Acetate, pH 6.8, 1 M NaCl, 0.01% SDS. DNA stained with GelRed®. Gel was visualized using a Bio-Rad Pharos FX (GelRed®: excitation, trans-UV; emission filter, 590/110 nm; TAMRA: excitation, epi-green illumination; emission filter: 602/50 nm; Atto 647N 7: excitation, epi-red illumination; emission filter: 700/50 nm). TAMRA channel was colored yellow and Atto 647N 7was coloured red for visualization. (B) GelRed® channel and (C) Composite image of TAMRA and Atto647n channels.

Example of the Chemical Labelling of a Biomolecule: Cysteine Labelling of a Peptide

[0234] Referring now to FIG. 9, there is shown a synthetic route to a further linker molecule 31 using Precursor 1 as described in FIG. 3, wherein the first functional group LG is a bromine atom.

Synthesis of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-4-((2-(8-bromooct-6-ynoyl)hydrazoneylidene)methyl)benzamide 31

[0235] Activated linker (tert-butyl 2-(8-bromooct-6-ynoyl)hydrazone-1-carboxylate) 1 (20 mg, 0.06 mmol) was dissolved in 500 μL of TFA and stirred for 2 hours. TFA was evaporated to afford 14 mg of a yellow oil. This crude was then dissolved in 200 μL of PBS and Ald-Ph-PEG-Azide (22) (21 mg, 1 eq.) in solution in PBS was added (pH 7). Instantly, a white precipitate appeared. This white solid was filtered and dissolved in DCM for purification by HPLC preparative (0-60% ACN 40 min). MS: m/z [M+Na]=586.8/588.8 to afford linker molecule 31.

1) Chemical Labelling an Amino Acid (Step b of Method of the Invention)

[0236] A cysteine molecule 90 was labelled using linker molecule 31 in the following protocol.

[0237] Linker molecule 31 (1.2 mg) was dissolved in Ammonia 7M in Methanol. N-acetyl cysteine (Compound 90) (3 mg, 2.25 eq.) was added and mixture was stirred at room temperature for 2 hours. After reaction, compound 32 was formed, which was concentrated under pressure.

2) Schiff Base Reversal (Step c of Method of the Invention)

[0238] The Schiff base of compound 32 was hydrolysed in the following protocol. Compound 32 was dissolved in release buffer (release buffer: 231 mg NH.sub.2OH in 200 μl Ammonium buffer pH6) and heated at 50° C. for 1 hour. The products (compounds 33, 22) were purified by injection HPLC (analytical 0-60% ACN over 40 min and 100% ACN for 10 min).

[0239] The method of labelling an amino acid according to this Example of the invention shows that the method of the invention may be applied to label and release, and/or relabel, amino acids containing a thiol moiety other than cysteine. Moreover, this Example illustrates that peptides comprising amino acids containing a thiol moiety (e.g. cysteine) may be labelled and released, and relabeled, according to embodiments of the invention.

[0240] Advantageously, the method according to the invention is bio-orthogonal, that is, it may be performed in a biological system without interfering with the native biochemical processes. More advantageously, once reversed, the modification made to a biomolecule, e.g. a polynucleotide biomolecule, for example, DNA, is relatively small and hydrophilic meaning it will not affect the way that the biomolecule interacts in solution or with enzymes.

[0241] The linker molecule comprises a hydrolysable moiety, e.g. a Schiff base moiety, which is hydrolysable such that a label may be reversibly conjugated to the biomolecule. Advantageously, the conditions required to hydrolyse the Schiff base moiety are mild, which does not damage the structure of the biomolecule. Moreover, Schiff base chemistry is not commonly found in biomolecules, unlike prior art approaches such as the use of disulphide linkages. More advantageously, Schiff bases such as oximes and hydrazones are stable at physiological pH.

[0242] The covalent bonds formed using the linker molecule are reversible and rewritable. Advantageously, the label molecule may be used for repeated modifications of biomolecules, for example, labelling, capture, release, refunctionalisation for, e.g. fluorescent labelling, and/or imaging.

[0243] The analogue of S-adenosyl-1-methionine cofactor may be designed to comprise a linker molecule of any suitable structure.

[0244] Advantageously, the second functional group of the linker molecule is usable to further functionalise the biomolecule, e.g. polynucleotide, for example using click chemistry. This may be used for DNA capture, DNA complexation, drug attachment, and/or fluorescent labelling.

[0245] It will be appreciated by those skilled in the art that several variations to the aforementioned embodiments are envisaged without departing from the scope of the invention. For example, the biomolecule need not be a polynucleic acid. In embodiments, the first functional group of the linker molecule may be selected to be able to react with a moiety on a different biomolecule to form a covalent bond. For example, the biomolecule may comprise an azide moiety, and the first functional group may comprise an alkyne moiety, or vice versa, that is capable of forming a covalent bond via click chemistry.

[0246] It will also be appreciated by those skilled in the art that any number of combinations of the aforementioned features and/or those shown in the appended drawings provide clear advantages over the prior art and are therefore within the scope of the invention described herein.