GB2542427A

GB2542427A - Assay for methyltrasferase

Info

Publication number: GB2542427A
Application number: GB1516680.4A
Authority: GB
Inventors: Aharoni Amir; Levy Dan
Original assignee: National Institute for Biotechnology in the Negev Ltd
Current assignee: National Institute for Biotechnology in the Negev Ltd
Priority date: 2015-09-21
Filing date: 2015-09-21
Publication date: 2017-03-22
Also published as: GB201516680D0

Abstract

Methods for assaying methyl transfer reactions e.g., methyltransferase (MT) activity and in particular S-adenosylmethionine (SAM) dependent MT activity by spectrophotometric analysis at a wavelength of 300nm or above. The methyltransferases require S-adenosylmethionine (SAM) as the source of the methyl group. On transferring the methyl group SAM generates S-adenosylhomocysteine (SAH) which is cleaved by a nucleosidease to give S-ribosylhomocysteine and adenine. The adenine is deaminated to provide ammonia which may be analysed by the addition of α-ketoglutarate, NADPH and glutamate dehydrogenase, wherein the consumption of NADPH is followed at 340nm. Additional enzymatic coupled reactions to the generation of ammonia may provide pyruvate or hydrogen peroxide.

Description

ASSAY FOR METHYLTRASFERASE

FIELD OF THE INVENTION

The present invention, in some embodiments thereof, relates to a rapid enzyme coupled UV absorption assay for the characterization of enzymes that catalyze methyl transfer reactions.

BACKGROUND OF THE INVENTION

Methyltransferases (MTs) catalyze the S-adenosylmethionine (SAM)-dependent methylation of a wide variety of protein and DNA substrates. Methylation of lysine, arginine or cytosine regulates a variety of biological processes including transcriptional activation and gene silencing. Despite extensive studies of the cellular roles of MTs, their quantitative kinetic characterization remains challenging. In the past decade several assays have been developed to monitor methyl transfer activity utilizing different approaches including radiolabeling, antibodies or mass-spectrometry analysis. However, each approach suffers from different limitation and no easy one step continuous assay for detection of MT activity exists.

To establish a continuous assay for MTs activity that monitors Λ'-adenosyl-L-homocysteine (SAH) formation, the transfer of a methyl group to peptide, protein or DNA must be coupled to additional reactions that lead to measurable change in absorbance or fluorescence. Previous work has utilized SAH nucleosidase (SAHN), and adenine deaminase (ADE) for the conversion of SAH to hypoxanthin and ammonia (Luo, M. ACS Chem. Biol. 7, 443 (2012)). These two coupled reactions were shown to be non-rate limiting enabling the efficient coupling of methyl transfer reaction to hypoxanthine formation that is monitored at 265 nm. However, measuring MTs activity at 265 nm is highly problematic due to the high absorbance of proteins at 280 nm and the inability to utilize standard cuvetes or multi-well plates due to their high absorbance at this wavelength.

SUMMARY OF THE INVENTION

In a search for novel methodologies for a rapid, one step, characterization of enzymes that catalyze methyl transfer reactions, the inventors have developed, inter aha, a versatile continuous enzyme coupled UV absorption assay for monitoring the activity of various methyl transferase proteins.

According to an aspect of some embodiments of the present invention, there is provided a method for assaying a methyl transfer reaction in a reaction mixture, the reaction mixture comprising a substrate, a methyl donor and a catalyst, the method comprising the steps of coupling: (i) one or more reactions to the methyl transfer reaction, thereby forming one or more intermediate products, and (ii) one or more subsequent enzymatic or chemical reactions coupled thereto, thereby forming one or more final products characterized by an absorption of above 300 nm; and further comprising the step of spectrophotometric analyzing or monitoring the reaction mixture or an aliquant thereof, wherein the spectrophotometric analyzing or monitoring is performed at a wavelength of above 300 nm, thereby assessing the amount of the one or more final products.

In some embodiments, the catalyst is an enzyme selected from methyltransferase and a derivative thereof.

In some embodiments, the methyl donor is adenosylmethionine (SAM).

In some embodiments, the methyl transfer reaction is a rate-limiting reaction with respect to any reaction coupled and/or subsequent thereto.

In some embodiments, the intermediate products are selected from ammonia, pyruvate, hydrogen peroxide, hypoxanthine, and any respective derivative thereof.

In some embodiments, the final product is NAD(P)+, or a respective derivative thereof.

According to an aspect of some embodiments of the present invention, there is provided a method for assaying methyltransferase activity on a substrate, comprising the following steps: preparing a reaction mixture comprising a methyltransferase, a substrate, and adenosylmethionine (SAM) or respective derivative thereof, thereby transmethylating the substrate and forming adenosylhomocysteine (AdoHcy); contacting the reaction mixture to AdoHcy nucleosidase, thereby forming ribosylhomocysteine and adenine or respective derivative thereof; contacting the reaction mixture to adenine deaminase (ADE), thereby forming or respective derivative thereof and ammonia; and determining the amount of the ammonia in the reaction mixture by an enzymatic conversion of the ammonia to one or more products characterized by a defined absorbance of above 300 nm, thereby determining the methyltransferase activity.

In some embodiments, the enzymatic conversion of the ammonia to the product characterized by a defined absorbance of above 300 nm is a process selected from: a one-step process, a two-step process, a three-step process, or four-step process.

In some embodiments, the one step-process comprises a step of reacting the ammonia with NADPH and α-ketoglutarate in the presence of glutamate dehydrogenase and wherein the product is NAD(P)+.

In some embodiments, the three step-process comprises the steps of: reacting the ammonia with Glutamate and ATP in the presence of glutamine synthetase, thereby forming ADP; enzymatic converting ADP to pyruvate; and enzymatic converting pyruvate and NADPH to lactate and NAD(P)+.

In some embodiments, the four step-process comprises the steps of: reacting the ammonia with Glutamate and ATP in the presence of glutamate synthetase, thereby forming ADP; enzymatic converting ADP to pyruvate; enzymatic converting pyruvate to acetyl phosphate, CO2, H2O2; and contacting a phenol and/or a dye molecule characterized by a defined absorbance of above 300 nm to H2O2.

In some embodiments, the absorbance of above 300 nm is determined by a spectrophotometric monitoring being performed at a wavelength in the range of 320 nm to 600 nm.

In some embodiments, the substrate is selected from DNA, protein, peptide and any combination thereof.

In some embodiments, the step of transmethylating the substrate is rate-limiting with respect to the other steps.

In some embodiments, the spectrophotometric monitoring is performed continuously.

In some embodiments, at least two steps are performed in one-pot.

In some embodiments, the steps (i) to (v) are performed sequentially.

In some embodiments, the methyltransferase is nucleic acid methyltransferase.

In some embodiments, the nucleic acid methyltransferase is DNA methyltransferase.

In some embodiments, the reaction mixture further comprises at least one solution selected from the group consisting of: aqueous buffer, water, organic solvent and oil.

According to an aspect of some embodiments of the present invention, there is provided kit for quantitatively assaying methyltransferase activity comprising effective amounts of: (a) Glutamate dehydrogenase; (b) α-ketoglutarate; and (c) NADPH.

In some embodiments, the kit further comprises in a buffer.

In some embodiments, the kit further comprises AdoHcy nucleosidase.

In some embodiments, the kit further comprises ADE.

In some embodiments, the kit further comprises each of a, b, and c are packaged in separate compartments.

According to an aspect of some embodiments of the present invention, there is provided a composition comprising: (a) a compound selected from SAM, methyltransferase, AdoHcy, adenine, ADE, hypoxanthine, and ammonia; and (b) a compound selected from: α-ketoglutarate, NAD(P)H, glutamate dehydrogenase, and NAD(P)+; or (c) a compound selected from pyruvate, glutamate, glutamate synthetase, ATP, ADP, glutamine, pyrophosphate, phosphoenolpyruvate, pyruvate, acetyl phosphate, CO2, and H2O2.

In some embodiments, the compound of (b) is selected from a-ketoglutarate, NAD(P)H, glutamate dehydrogenase and the compound of (c) is selected from glutamate synthetase and ATP.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

Figures 1A-B present a general concept underlying the assays of some embodiments of the invention: a general diagram for the continuous coupled assay for Methyltransferases (MTs) activity in which MTs activity is coupled to three enzymes: SAH nucleosidase (SAHN), adenine deaminase (ADE) and glutamate dehydrogenase to couple methyl transfer activity to NADPH oxidation; as a result methyl transfer activity is directly monitored by a decrease in absorbance at 340 nm (Fig. 1A); and a diagram describing the chemical structure of SAM, SAH and all other products derived from the activity of the three coupled enzymes (Fig. IB).

Figures 2A-B present graphs demonstrating the establishment of the continuous coupled assay for MTs using short peptide as a substrate, showing that the detection of MTs activity is dependent on all assay components: the reaction cannot be monitored in the absence of SAM, SAHN, ADE or Glutamate dehydrogenase. In addition, activity cannot be monitored in the presence of SET7/9 inactive mutant. Activity is measured only when all reaction components are present including the peptide substrate (TAT peptide as an example, blue) (Fig. 2A); the SET7/9 activity is the rate limiting step in the three enzyme coupled assay (Fig. 2B). Each enzyme was monitored separately to ensure the determination of SET7/9 activity. Absorbance values at 340 nm in all reactions are normalized to 1 and the change in absorbance over time is presented as 1 -the absorbance value obtains for each measurement.

Figure 3 presents a graph showing that the detection of MTs activity is dependent on all assay components; the reaction cannot be monitored in the absence of Glutamate dehydrogenase or adenine deaminase. Activity is measured only when all reaction components are present including the peptide substrate (TAT peptide as an example).

Figures 4A-B present graphs showing raw measurement of MT activity coupled with NADPH oxidation leads: the rate of absorbance decrease at 340 nm reflects the rate of SET7/9 activity with the TAT peptide (Fig. 4A); and absolute absorbance values transformed to change in absorbance at 340 nm and the values of 1-absorbance change shown to highlight the rate of methylation (Fig. 4B).

Figures 5A-B present graphs demonstrating the steady state kinetics of SET7/9 methyl transfer activity to peptide derived from Fox03 substrate: kinetic traces of SET7/9 at increased Fox03 peptide concentrations (0 μΜ to 700 μΜ) (initial rates at each Fox03 substrate concentration were calculated by fitting the raw data to a linear equation; absorbance values at 340 nm in all reactions are normalized to 1 and the change in absorbance over time is presented as 1-the absorbance value obtains for each measurement) (Fig. 5A), and Michaelis-Menten (MM) plots for SET7/9 activity with Fox03 peptide (the kinetic parameters derived from the fit to the MM equations are kcat of 32±0.023 min-1 and KM of 165.4±20.2 μΜ (Fig. 5B).

Figures 6A-C present a steady state kinetic analysis of SETD6 activity with RelA protein (residues 1-431): kinetic traces of SETD6 at increased RelA concentrations of 0 to 2.5 μΜ. Initial rate at each RelA concentration were calculated by fitting the raw data to a linear equation (absorbance values at 340 nm in all reactions are normalized to 1 and the change in absorbance over time is presented as 1 -the absorbance value obtains for each measurement) (Fig. 6A); fitting the initial rate data to linear equation allowing the determination of kcat/KM to be 57.1 min"1 μΜ"1 (Fig. 6B); and radioactive gel analysis of SETD6 activity with SAHN, ADE and RelA protein (1-431) showing that methylation do not take place on SAHN and ADE and only RelA is recognized as a substrate for SETD6 (Fig. 6C).

Figures 7A-C present monitoring DNA methyl transferase activity using the coupled kinetic assay - a steady state kinetics of Haelll methyl transfer activity with DNA fragment containing 6 methylation sites: a scheme of DNA substrate for Haelll containing six methylation sites including one methylation site that is located at Notl recognition sequence; Notl cleavage at this site leads to two fragments of 550 bp and 1050 bp (Fig. 7A); kinetic traces of Haelll at increased DNA concentrations (1.5 μΜ, 3 μΜ and 6 μΜ) showing the increase in methylation rate (Fig. 7B); and DNA methylation by Haelll preventing cleavage by Notl; gel electrophoresis analysis of the DNA substrate cleavage by Notl prior and following methylation by Haelll; methylation of the DNA substrate by Haelll performed in the Haelll optimal reaction buffer and in the coupled assay buffer to ensure that the later does not interfere with DNA methylation (Fig. 7C).

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in some embodiments thereof, relates to a characterization of enzymes that catalyze methyl transfer reactions, using enzyme coupled UV absorption assay.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

In some embodiments, there is provided a method for assaying methyltransferase activity on a substrate, comprising the steps of preparing a reaction mixture comprising a methyltransferase, a substrate, and a methyl donor e.g., adenosylmethionine (SAM) or respective derivative thereof, thereby transmethylating the substrate and forming adenosylhomocysteine ("AdoHcy"); contacting the reaction mixture to AdoHcy nucleosidase, thereby forming ribosylhomocysteine and adenine or respective derivative thereof; contacting the reaction mixture to adenine deaminase (ADE), thereby forming or respective derivative thereof and ammonia, and determining the amount of the ammonia in the reaction mixture by an enzymatic conversion of the ammonia to one or more products characterized by a defined absorbance of above 280 nm, and/or by a defined fluorescence, thereby determining the methyltransferase activity.

In some embodiments, the absorption assay is performed at UV wavelength i.e. in the range from about 280-400 nm.

In some embodiments, the enzymatic conversion of the ammonia may be performed by any NH3 binding protein e.g., gulatmate synthetase, as noted hereinbelow.

As used herein, the term “methyltransferase" (also referred to hereinthroughout as "MT or "MTase") refers to a family of enzymes that has an activity described as EC 2.11, according to the IUMBM enzyme nomenclature. In certain cases, methyltransferase catalyzes the transfer of a methyl group from a donor to an acceptor.

In some embodiments, the methyltransferase is protein methyltransferases.

In some embodiments, the methyltransferase is nucleic acid the methyltransferase. The methylation conditions of the method will include provision of a suitable methyl donor source for use by the nucleic acid methyltransferase such as S-adenosylmethionine, as described hereinabove, for example, where the nucleic acid methyltransferase is a Dam or Dnmt enzyme.

In some embodiments, the nucleic acid methyltransferase is a DNA methyltransferase. For example, in some embodiments of the invention the DNA methyltransferase is a prokaryotic DNA adenine N-6 methyltransferase for which the binding and methylation site is GATC, and the restriction enzyme is DpnI. In some embodiments the DNA methyltransferase is a DNA adenine N-6 methyltransferase from Yersinia species, or from Y. Pestis. In some embodiments the DNA adenine N-6 methyltransferase is from E. coli.

The acceptor may be a nucleotide base in a DNA. When methylation occurs on a DNA, the methyltransferase is referred to as a DNA methyltransferase. DNA methyltransferases can use a cofactor such as s-adenosyl methionine (SAM) as the methyl donor in the methyltransferase reaction. In addition to methyl groups, methyltransferase may also transfer other functional groups to an acceptor from a cofactor, e.g. amino group, if used with an appropriate cofactor. RNA methyltransferase may also be used in some embodiments.

The DNA MTase may be characterized by the acceptor site to which the functional group is transferred: C5 carbon of cytosine, N4 nitrogen of cytosine, or N6 nitrogen of adenine. In certain embodiments, the MTase is a site-specific MTase that recognizes a specific nucleotide sequence in the genome. In some cases, the recognition sequence may comprise 2, 3, 4, 5, 6, 8, 10 or more nucleotides or nucleotide pairs. Under suitable conditions, the site-specific DNA MTase specifically transfers a functional group from the cofactor to a nucleotide within or close to the recognition sequence. As such, in certain cases, the recognition sequence comprises an acceptor site for the functional group from the cofactor. As a result, the genome is modified by a covalent linkage to the functional group in a sequence- and base-specific manner. If the recognition sequence of the site-specific MTase does not exist in the genome, no functional group may be transferred from the cofactor to the genome.

In some embodiments, the MTase may be a variant that exists in nature or a recombinant variant. Variants of MTase that may be used in the subject method include MTase protein variants or derivatives that are still enabled to transfer the functional group from a donor to an acceptor. It would be apparent to one of skilled in the art the variants of MTase that can be employed in the subject method since the structure and function relationships of MTase are known in the art, as illustrated in Chen X et al. 2008 “Mammalian DNA methyltransferases: A structural perspective” Cell 16:341-50.

The MTase may be of a bacterial restriction modification system or of a mammalian origin. In certain embodiments, bacterial MTases include but are not limited to M. Taql, M. Hhal, M.BcnlB, M.BseCl, M. Rsrl, M2.Bfil, M2.Eco311. In certain cases, mammalian DNA MTases include but are not limited to DNMT1, DNMT2, DNMT3 A and DNMT3B. Nucleotide and protein sequences of these exemplary bacterial or mammalian MTases are known and deposited in databases such as the NCBI's GenBank database.

In some embodiments, the DNA MTases modifies the genome in a sequence- and base-specific manner. In certain cases, the recognition sequence may comprise e.g., 2, 3, 4, 5, 6, 8, 10 or more nucleotides or nucleotide pairs. The acceptor nucleotide onto which the functional group is transferred may be within or close to the recognition site. For example, the bacterial Haelll MTase recognizes 4 consecutive nucleotide bases of the sequence GGCC and transfers the functional group onto the internal cytosines (C5) of the recognition sequence. As an another example, the bacterial Alul MTase recognizes the panlindromic sequence of AGCT and transfers the functional group onto the internal cytosines (C5) of the recognition sequence. Many other MTases and the information relating to their recognition and acceptor sites are known in the art and commercially available.

Additional experiments conducted during the course of development of the present invention utilized the methods of the present invention to study Human SET7/9, a protein lysine methyltransferase (PKMT).

Additional experiments conducted during the course of development of the present invention utilized the methods of the present invention to study N-lysine methyltransferase SETD6, which is a SET domain protein that, inter alia, specifically monomethylates 'Lys-310' of the RELA subunit of NF-kappa-B complex, leading to down-regulation of NF-kappa-B transcription factor activity

In some embodiments, the methyl donor is adenosylmethionine.

In some embodiments, the adenosylmethionine is S-Adenosyl methionine.

As known in the art, S-Adenosyl methionine (also referred to as: "SAM-e", "SAMe", "SAM", "S-Adenosyl-L-methionine", "AdoMet", "ademetionine") is a common co-substrate involved in methyl group transfers.

The term "reaction" is used to denote one or more reactions or interactions carried out at once or in sequence, to attach the acceptor, directly or indirectly, to the recognition agent.

In some embodiments, the reactions described hereinthroughout refers to conditions suitable for an enzyme to be active. For example, “methyltransferase reaction conditions” refers to conditions suitable for a methyltransferase to be active in transferring a functional group from a cofactor/donor to a substrate/acceptor.

In some embodiments, the substrate is a methyl-acceptor.

Hereinthroughout, the substrate may be, without being limited thereto, a nucleic acid (e.g., RNA or DNA), a protein, a peptide, a polypeptide, an antigen, a lipid, carbohydrate, or proteoglycan DNA, and any combination thereof.

It is to be understood that any enzyme in any embodiments hereinthroughout may be purified or recombinant. Furthermore, the enzymes may additionally include functional variants, including, without limitation, conservative amino acid sequence variants, fragments, muteins, derivatives and fusion proteins thereof.

The terms "determination", "determining" or "detection" are used to refer collectively to both qualitative and quantitative assay of the analyte in the assayed sample. The terms may also refer to the act of detecting, perceiving, uncovering, exposing, visualizing or identifying a compound.

The term "assay" encompasses all the conditions, substances or actions necessary or useful for the appropriate reaction to take place, including sequences of varying conditions or actions.

The term "enzymatic conversion" in general refers to the modification of a substrate by an enzyme action.

The term "transmethylation", or any grammatical derivative thereof refers to organic chemical reaction in which a methyl group is transferred from one compound to another.

In some embodiments of the present invention, suitable wavelengths for measurement are 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, 790 nm, 800, nm 810 nm, 820 nm, 830 nm, 840, 850 nm, 860 nm, 870 nm, 880 nm, 890 nm, 900 nm, 910 nm, 920 nm, 930 nm, 940 nm, 950 nm, 960 nm, 970 nm, 980 nm, 990 nm, or 1000 nm, including any value therebetween, as wavelengths at which the final product shows a satisfactory absorption, but it will be evident to one skilled in the art that also other individual wavelengths in the range in question could be used for the measurement.

The calculation of the concentration of each of the products or the parameters derived from the concentration of the final products may be performed using any suitable calculation means, such as in a separate calculator, an analog computer connected to the spectrophotometric equipment or, preferably, in a digital microprocessor after an analog/digital conversion of the spectrophotometrically measured absorbance.

In some embodiments, one or more of the final products can be detected by means designed for fluorescence detection. "Methyl donor substrate" as used herein refers to a transmethylation substrate (methyl donor) for a methyltransferease enzymatic activity, including but not limited to the methyl donor substrate S-adenosyl-L-metbionine (AdoMet/SAM). "Methyltransferase activity" as used herein refers to a methyltransferease enzymatic activity that catalyzes transfer of a methyl group from a methyl donor substrate (e.g., from S-adenosyl-L-methionine (AdoMet/SAM)) to a methyl group recipient molecule (e.g., peptide, protein nucleic acid, etc.) and converting the methyl donor substrate into a transmethylation product. Such methyltransferase activities include, but are not limited to S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activities. "Transmethylation product" as used herein refers to the conversion product of the methyltransferase activity on the methyl donor substrate, includes, but is not limited to S-adenosylhomocysteine (AdoHcy), 5'-methylthioadenosine (MTA), and structural analogs of AdoHcy or MTA with hydrophobic residues at the C5 position. "Adensosine" or derivative thereof as used herein refers to adenosine or adenosine derivates that are suitable substrates for an adenosine nucleosidase activity. "Adenosine nucleosidase" is, inter alia, capable of releasing adenine or a derivative thereof from at substrate adensosine or adenosine derivative moiety.

Adenine deaminase is, inter alia, capable of releasing ammonia from adenine or from an adenine derivative, and includes but is not limited to adenine deaminase and may convert adenine to hypoxanthine and ammonia.

The term "coupling reactions", and the like, as used herein is understood to mean, inter aha, reactions and/or conditions that may be used to couple a molecule of one reaction e.g., enzymatic conversion, to another molecule or enzyme.

In some embodiments, one or more enzymes referred to hereinthroughout exhibit Michaelis-Menten (MM) kinetics. The person skilled in the art will readily be able to determine, if an enzyme exhibits MM kinetics. Such an enzyme will e.g., have to obey or substantially obey the kinetics described herein.

In some embodiments, the disclosed assay can be used for the determination of steady-state kinetic enzymatic parameters, e.g., Kcat and Km.

Without being bound by any particular theory or mechanism, in some embodiments, in order to yield valid kinetic parameters in the coupled assay, the coupled reactions (enzymes) used should not be rate limiting, so that the measured rate is determined solely by the methyltransferase activity.

In some embodiments, the reaction or the corresponding enzyme may also exhibit kinetics selected from the group consisting of zero order kinetics, first order kinetics, second order kinetics and combinations hereof. Likewise, the person skilled in the art will readily be able to recognize such zero order kinetics, first order kinetics, and second order kinetics based on the results of a suitable enzymatic assay. In some embodiments the enzyme exhibits MM first order kinetics.

In exemplary embodiments, the intermediate products are one or more products selected from ammonia, hypoxanthine, and any respective derivative thereof (see Figures 1A-B).

In other exemplary embodiments, the intermediate products are one or more products selected from pyruvate, hydrogen peroxide and any respective derivative thereof.

In exemplary embodiments, the final product is NAD(P)+, or a respective derivative thereof (see Figure IB).

The person skilled in the art, numerous methods are known with which enzymatically active polypeptides can be over-expressed in suitable cells and purified or isolated. Thus, all those skilled in the available expression systems can be used for expression of the polypeptides.

As used herein, by NAD(P)+ is meant to refer to either or both of NAD+ (nicotinamide adenine dinucleotide, oxidized form) and NADP+ (nicotinamide adenine dinucleotide phosphate, oxidized form). NAD(P)H is used herein to mean either or both of NADH (nicotinamide adenine dinucleotide, reduced form) and NADPH (nicotinamide adenine dinucleotide phosphate, reduced form).

In some embodiments, there is provided a method for assaying methyltransferase activity on a substrate, comprising the following steps: preparing a reaction mixture comprising a methyltransferase, a substrate, and adenosylmethionine (SAM) or respective derivative thereof, thereby transmethylating the substrate and forming adenosylhomocysteine (AdoHcy); contacting the reaction mixture to AdoHcy nucleosidase, thereby forming ribosylhomocysteine and adenine or respective derivative thereof; contacting the reaction mixture to adenine deaminase (ADE), thereby forming or respective derivative thereof and ammonia; and determining the amount of the ammonia in the reaction mixture by an enzymatic conversion of the ammonia to one or more products characterized by a defined absorbance of above 300 nm, thereby determining the methyltransferase activity.

Typically, but not exclusively, the enzyme-catalyzed reactions are performed in a mixture, solvent or solvent mixture with a high water content, in the presence of a suitable buffer system for setting a compatible with enzymatic activity pH. In the case of hydrophobic reactants, in particular in alcohols having a carbon chain comprising more than three carbon atoms, however, the additional presence of an organic co-solvent is advantageous, which can provide the contact of the enzyme with the substrate. The one or more than one co-solvent is in an overall proportion of the solvent mixture of or less than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50 45, 40, 35, 30, 25, 20, 15, 10 or 5 volume percent.

Hereinthroughout, the reaction mixture may further comprise at least one solution selected from the group consisting of: aqueous buffer, water, organic solvent and oil.

As used herein, "contacting" means that the compound, composition, dye or agent affecting a reaction, is introduced into a sample comprising at least one reagent in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and for a time sufficient to permit the desired effect or reaction.

Methods for contacting the samples with the compounds, compositions, dyes or agents are known to those skilled in the art and may be selected depending on the type of assay protocol to be run. Protocol may also standard and are known to those skilled in the art.

The term "forming", as used herein, refers interchangeably to providing, obtaining, procuring, or supplying a sample.

The term "sample" as referred to herein encompasses, but is not limited to, a reaction vessel, biological sample, for example any tissue, cell-comprising tissue, cell line, cell culture, a primary cell culture, arising from or derived from an organism and processes to form a specimen suitable for cell staining.

In some embodiments, the enzymatic conversion of the ammonia to the product is a one-step process. In some embodiments, the enzymatic conversion of the ammonia to the product is a two-step process (also referred to as "two-enzyme coupled assay"). In some embodiments, the enzymatic conversion of the ammonia to the product is a three-step process (also referred to as "three-enzyme coupled assay"). In some embodiments, the enzymatic conversion of the ammonia to the product is a four-step process. In some embodiments, the enzymatic conversion of the ammonia to the product is an n-step process. "n" may represents a number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20, including any value therebetween.

The term "one step process", as used herein, means that the ammonia, optionally with an enzyme, is involved in a single step process in the production of the detectable final product as disclosed hereinthroghout. This term is to be distinguished from two, three or n-step processes in which the ammonia, optionally with an enzyme, is involved in a two, three or n-step process in the production of the final product as disclosed hereinthroghout.

An exemplary and non-limiting one-step process comprises a step of reacting the ammonia with NADPH and α-ketoglutarate in the presence of glutamate dehydrogenase and wherein the product is NAD(P)+.

An exemplary and non-limiting three-step process comprises the steps of: (i) reacting the ammonia with Glutamate and ATP in the presence of Glutamine synthetase, thereby forming ADP; (ii) enzymatic converting ADP to pyruvate; and (iii) enzymatic converting pyruvate and NADPH to lactate and NAD(P)+.

An exemplary and non-limiting four-step process comprises: (i) reacting the ammonia with Glutamate and ATP in the presence of glutamate synthetase, thereby forming ADP; (ii) enzymatic converting ADP to pyruvate; (iii) enzymatic converting pyruvate to acetyl phosphate, CO2, H2O2; and (iv) contacting a phenol and/or a dye molecule characterized by a defined absorbance of above 300 nm to H2O2. In some embodiments, the dye molecule is characterized by a defined fluorescence of above 300 nm.

It is to be understood that there are a person skilled in the art will recognize that there are many ways to detect H2O2.

As used herein, the term "dye" refers to any substance that imparts color to a biological tissue, animal, plant, or otherwise, and may be also useful for cytological or histological visualization of tissue, cells, or the parts that comprise them. The dyes which have been found to be of immediate utility for the purpose of implementing the present invention are disclosed herein, although other dyes not herein described may be useful as well for this purpose. Thus, although the description of the present invention provides dyes which manifest color in the UV or visible range of the spectrum, it may easily be envisioned that, through the use of appropriate filters, illuminations, configurations, or means for detection, dyes which are invisible to direct observation may prove equivalent to the visible dyes described herein for implementing the present invention .

In some embodiments, the dye is an acidic dye. In some embodiments, the dye is a basic dye. In some embodiments, the dye is a triamnotriphenylmethane derivative. In some embodiments, the dye is a diazo derivative. In some embodiments, the dye is any combination of an acidic dye, a basic dye, a triaminotriphenylmethane derivative a diazo derivative .

Non-limiting examples of dyes are selected from Hematoxylin Gill, Azure, A, Eosin Yellow, Phloxine, Light Green, New Fuchsin, Dahlia, Basic Fuchsin, Methyl Violet, Gentian Violet, Methyl Violet 6b, Crystal Violet, Pararosanilin, Rosanilin, Magenta I, Isorubin, Fuchsin NB, FIAT-764, Spider's Purple, Bismark Brown R, Bismark Brown Y, Bismark Brown Eosine Conjugate, Bismark Brown Phloxine Conugate, Phoenix Brown A, or any combination thereof.

In some embodiments, the dye is a red dye. As used herein, the term "red" refers not only to the color red but as well to related shades and hues such as violet, pink or pinkish, purple or purplish, or magenta. In one embodiment, the red dye is New Fuchsin.

In some embodiments, the dye is a green dye. As used herein, the term "green" refers not only to the color green but as well to related shades and hues such as light green, dark green, emerald, olive and lime.

In some embodiments, the dye is aminophenazone (detected at 500 nm) or 4-aminoantipyrine (detected at 505 nm). In some embodiments, the dye is N-ethyl-N-2-hydroxyethyl-m-toluidine (detect at 550 nm).

The concentration of the dye will be determined empirically based on the need of the assay. A suitable concentration of a dye is one that enables the detection of the staining of the cell with the dye, using any of the detection methods as described herein.

As used herein, the term “spectrophotometer” or any grammatical derivative thereof, refers to a device configured for, and effective to, measure optical intensity within a particular range of wavelengths, or at a particular wavelength, or at multiple particular wavelengths. A spectrophotometer may be effective to measure absorbance in a sample. A spectrophotometer may be effective to measure light emitted from a sample (e.g., fluorescence and/or luminescence). A spectrophotometer may be effective to measure absorbance in a sample and to measure light emitted from a sample.

The term “spectrophotometry” may refer to the making of measurements using a spectrophotometer; e.g., to the making of optical measurements within a particular range of wavelengths, or at a particular wavelength, or at multiple particular wavelengths. "Spectrophotometric monitoring" as used herein refers to any spectroscopic methodology (e.g., UV-vis, Fluorescence, Vibrational, Mass) known in the art used to monitor the concentration of a chemical species present within the reaction mixture either through direct analysis of the reaction mixture or analysis of a quenched aliquot.

As used herein, the term “optical measurement” and means for measuring an optical property (e.g., of a colored solution) refer to any suitable optical means, optical device, photodetector, and optical device element for detecting electromagnetic radiation (e.g., light of any wavelength). For example, an optical detector, and optical detection means, may be used to detect absorbance, transmittance, turbidity, luminescence (including chemiluminescence), fluorescence and/or other optical signal. Optical detectors include, but are not limited to, imaging devices. Optical means, optical devices, photodetectors, and optical device elements include, but are not limited to, electronic detectors such as digital cameras, charge coupled devices (CCDs, including super-cooled CCDs), photodiodes (including, e.g., pin diodes and avalanche photodiodes), photomultipliers, phototubes, photon counting detector, arrays of photodiodes (including, e.g., pin diode arrays and avalanche photodiode arrays), arrays of charge coupled devices (including super-cooled CCD arrays), arrays of photodiodes, arrays of photomultipliers, arrays of phototubes, arrays of photon counting detectors, and other detection devices and detection elements. In some embodiments a pin diode or other element may be coupled to an amplifier.

In some embodiments, an optical detector may include a camera (e.g., a digital camera). A camera may include a lens, or may operate without a lens. In some instances, cameras may include CCDs, may use complementary metal-oxide semiconductor (CMOS) elements, may be lensless cameras, microlens-array cameras, open-source cameras and may use any visual detection technology known or later developed in the art. Cameras may acquire conventional and/or non-conventional images, e.g., holographic images, tomographic images, interferometric images, Fourier-transformed spectra, any or all of which may be interpreted with or without the aid of computational methods. Cameras may include one or more features that may focus the camera during use, or may capture images that can be later focused. In some embodiments, imaging devices may employ two-dimensional (2-D) imaging, three-dimensional (3-D) imaging, and/or four-dimensional (4-D) imaging (incorporating changes over time). Imaging devices may capture static images.

Optical schemes used to achieve 3-D and 4-D imaging may be one or more of the several known to those skilled in the art, e.g. structured illumination microscopy (SLM), digital holographic microscopy (DHM), confocal microscopy, light field microscopy etc. Static images may be captured at one or more point in time. Imaging devices may capture video and/or dynamic images. Video images may be captured continuously over a single period, or may be captured over one or more periods of time. An imaging device may collect signal from an optical system which scans a target (e.g., a sample used for an assay) in arbitrary scan patterns (e.g., in a raster scan).

An optical detector, and optical detection means, include without limitation, a microscope, and means for optical detection may include microscopy, visual inspection, via photographic film, or may include the use of electronic detectors such as digital cameras, charge coupled devices (CCDs), super-cooled CCD arrays, phototubes, photodetectors, and other detection devices known in the art, and as disclosed herein. An optical detector, and optical detection means, may include an optical fiber or a plurality of optical fibers (e.g., fiber optic cables) which may, for example, be functionally connected to a CCD detector or to a PMT array. A fiber optic bundle may comprise discrete fibers and/or many small fibers fused together to form a solid bundle.

An optical detector may include a light source, such as an electric bulb, incandescent bulb, electroluminescent lamp, laser, laser diode, light emitting diode (LED), gas discharge lamp, high-intensity discharge lamp, a chemiluminescent light source, a bioluminescent light source, a phosphorescent light source, a fluorescent light source, and natural sunlight. In embodiments, e.g., where chemiluminesence is to be detected, light may be produced by the assay chemistry. In some embodiments, a light source can illuminate a component in order to assist with detecting the results. For example, a light source can illuminate a solution in assay in order to detect the results of the assay. For example, an assay can be a fluorescence assay or an absorbance assay, as are commonly used with nucleic acid assays. A detector may comprise optical elements effective to deliver light from a light source to an assay or assay chamber. Such an optical element may include, without limitation, for example, a lens, a mirror (e.g., a scanning or galvano-mirror), a prism, a fiber optic fiber or bundle of fibers, a light guide (e.g., a liquid light guide), and/or other optical element. An optical detector may include such optical elements, where such optical elements are disposed effective to deliver light to a detector. For example, an optical detector may be configured to detect selected wavelengths or ranges of wavelengths of electromagnetic radiation. An optical detector may be configured to move over, or to view portions of, a sample. An optical detector may include a mirror, a motor, a piezoelectric element, or other element effective to allow detection of light from different portions of a target location at different times, e.g., to scan a sample.

An optical detector may be used to detect one or more optical signal. For example, a detector may be used to detect the presence of, or progress of a reaction providing luminescence. A detector may be used to detect a reaction providing one or more of fluorescence, chemiluminscence, photoluminescence, electroluminescence, sonoluminescence, absorbance, turbidity, optical-rotary-dispersion (ORD), circular dichroism (CD), or polarization. An optical detector may be able to detect optical signals relating to color intensity and phase or spatial or temporal gradients thereof.

In some embodiments, the device (e.g., spectrophotometer) comprises a means for communicating information from the device to a computer or other external device, and/or a channel for communicating information to a computer or other external device.

As used herein, a means for communicating information from the device to a computer or other external device, and a channel for communicating information to a computer or other external device, without limitation, refers to a computer network, a telephone, a telephone network, and a device configured to display information communicated from the device. In embodiments, a means for communicating information, and a channel for communicating information include direct links using wires (including twisted pair, coaxial, ribbon, and other cables), wireless means and wireless technology (e.g., Bluetooth technology or RTM (retransmission mode) technology). Communicating means and channels for communication include any suitable communication method, including a dial-up wired connection with a modem, a direct link using a wire, a wireless connection including infrared, cellular, wimax, wifi, satellite, pager, general packet radio service (GPRS), local data transport system (such as, e.g., ethernet or token ring over a local area network (LAN) or other network).

An external device to be communicated with may be any device capable of receiving such a communication. For example, an external device may be a networked device, including a server, a personal computer, a laptop computer, a tablet, a mobile device, a “dumb” cell phone, a satellite phone, a smart phone, a pager or any other device. In embodiments, an external device may be a diagnostic device. In some embodiments where an external device comprises a diagnostic device, the relationship between devices and systems disclosed herein and an external device may comprise a master-slave relationship, a peer-to-peer relationship, or a distributed relationship.

In some embodiments, the spectroscopic monitoring is performed at 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, 790 nm, 800, nm 810 nm, 820 nm, 830 nm, 840, 850 nm, 860 nm, 870 nm, 880 nm, 890 nm, 900 nm, 910 nm, 920 nm, 930 nm, 940 nm, 950 nm, 960 nm, 970 nm, 980 nm, 990 nm, or 1000 nm, including any value therebetween.

In some embodiments, the spectrophometric monitoring is performed continuously. In some embodiments, the spectrophometric monitoring is performed continuously with predefined intervals. In some embodiments, the spectrophometric monitoring is performed periodically. In some embodiments, the spectrophometric monitoring is performed using multi-well plate.

In some embodiments, at least two of steps (i) to (iv), in any embodiment described hereinabove may be performed in one pot. In some embodiments, at least two of steps (i) to (iv) are performed sequentially.

By “one pot” it is meant to refer to a synthesis that may be carried out in a single reaction vessel without removing any intermediate product.

In some embodiments, the hereinthroughout disclosed method is devoid of using and/or forming radioactive materials.

Additional embodiments provide a kit for assaying of methyltransferase activity.

In some embodiments, the kit comprises effective amounts of: glutamate dehydrogenase, α-ketoglutarate, and NADPH.

The kits of the present disclosure may, if desired, be presented in a pack which may contain one or more units of the disclosed kit. The pack may be accompanied by instructions for using the kit. The pack may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of laboratory supplements, which notice is reflective of approval by the agency of the form of the compositions.

The kits may be useful in a variety of detection assays. Detection may be performed in spectrophotometer as described herein above e.g., at wavelength higher than 300 nm.

The kits of this aspect of the present invention may also include a chemiluminescence enhancer. Generally, the enhancer used herein comprises an organic compound which is soluble in an organic solvent or in a buffer and which enhances the luminescent reaction between the chemiluminescent organic compound, the oxidant and the enzyme or other biological molecule. Suitable enhancers include, for example, halogenated phenols, such as p-iodophenol, p-bromophenol, p-chlorophenol, 4-bromo-2-chlorophenol, 3,4-dichlorophenol, alkylated phenols, such as 4-methylphenol and, 4-tert-butylphenol, 3-(4-hydroxyphenyl) propionate and the like, 4-benzylphenol, 4-(2',4'-dinitrostyryl) phenol, 2,4-dichlorophenol, p-hydroxycinnamic acid, p-fluorocinnamic acid, p-nitroicinnamic acid, p-aminocinnamic acid, m-hydroxycinnamic acid, o-hydroxycinnamic acid, 4-phenoxyphenol, 4-(4-hydroxyphenoxy) phenol, p-phenylphenol, 2-chloro-4-phenylphenol, 4'-(4'-hydroxyphenyl) benzophenone, A-(phenylazo) phenol, 4-(2'-carboxyphenylaza) phenol, l,6-dibromonaphtho-2-ol, 1-bromonaphtho-2-ol, 2-naphthol, 6-bromonaphth-2-ol, 6-hydroxybenzothiazole, 2-amino-6-hydroxybenzothiazol-e, 2,6-dihydroxybenzothiazole, 2-cyano-6-hydroxybenzothiazole, dehydroluciferin, firefly luciferin, phenolindophenol, 2,6-dichlorophenolindophenol, 2,6-dichlorophenol-o-cresol, phenolindoaniline, N-alkylphenoxazine or substituted N-alkylphenoxazine, N-alkylphenothiazine or substituted N-aucylphenothiazine,N-alkylpyrimidyl- phenoxazine or substituted N-alkylpyrimidylphenoxazine, N-alkylpyridylphenoxazine, 2-hydroxy-9-fluorenone or substituted 2-hydroxy-9-fluorenone, 6-hydroxybenzoxazole or substituted 6-hydroxybenzoxazole. Still other useful compounds include a protected enhancer that can be cleaved by the enzyme such as p-phenylphenol phosphate or p-iodophenol phosphate or other phenolic phosphates having other enzyme cleavable groups, as well as p-phenylene diamine and tetramethyl benzidine. Other useful enhancers include fluorescein, such as 5-(n-tetradecanyl) amino fluorescein and the like.

The term "effective amount" means a level (e.g., concentration or unit) of a compound sufficient to effect the purpose of the compound.

In some embodiments, the concentration of α-ketoglutarate is 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, including any value therebetween.

In exemplary embodiments, the concentration of α-ketoglutarate is 3.3 mM.

In some embodiments, the concentration of NADPH is 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, including any value therebetween.

In exemplary embodiments, the concentration of NADPH is 0.2 mM.

In some embodiments, the kit comprises 1, 2, 3, 4, or 5 units, including any value therebetween, of glutamate dehydrogenase.

In exemplary embodiments, the kit comprises 3 units of glutamate dehydrogenase. As used herein and in the art, the terms “enzyme unit” or "unit" refer to the amount of enzyme that produces 1 micromole of product per minute under the specified conditions of the assay, for example reducing 1.0 mmol of a-ketoglutarate by glutamate dehydrogenase to glutamate per minute.

As noted hereinabove, the kit may further comprise a buffer. Buffer may allow to control a desired pH condition (e.g., 6, 6.5, 7, 7.5, 8, 8.5 or 9, including any value therebetween) of a medium.

Exemplary buffer comprises sodium phosphate or potassium phosphate.

Buffer may be in a concentration of e.g., 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM, including any value therebetween.

In some embodiments, the kit further comprises any methyltransferase enzyme described hereinthroughout.

The concentration of methyltransferase enzyme may be e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, or 1.5 μΜ, including any value therebetween.

In some embodiments, the kit further comprises a substrate as described hereinthroughout (e.g., a peptide or protein substrate).

The concentration of methyltransferase enzyme may be in the range of e.g., 0-700 μΜ.

In exemplary embodiments, the kit comprises 20 Mm of buffer so as to keep the pH at a desired range of e.g., 7.5-7.8.

In some embodiments, the kit further comprises AdoHcy nucleosidase, as described hereinabove.

AdoHcy nucleosidase may be in a concentration of 10 μΜ, 50 μΜ, 100 μΜ, 150 μΜ, 200 μΜ, 250 μΜ, 300 μΜ, 350 μΜ, 400 μΜ, 450 μΜ, or 500 μΜ, including any value therebetween.

In some embodiments, the kit further comprises ADE.

In some embodiments, ADE is in concentration of 0.2 μΜ, 0.4 μΜ, 0.6 μΜ, 0.8 μΜ, 1 μΜ, 1.2 μΜ, 1.4 μΜ, 1.6 μΜ, 1.8 μΜ, 2 μΜ, 2.2 μΜ, 2.4 μΜ, 2.6 μΜ, 2.8 μΜ, 3 μΜ, 3.2 μΜ, 3.4 μΜ, 3.6 μΜ, 3.8 μΜ, 4 μΜ, 4.2 μΜ, 4.4 μΜ, 4.6 μΜ, 4.8 μΜ, 5 μΜ, 5.2 μΜ, 5.4 μΜ, 5.6 μΜ, 5.8 μΜ, or 6 μΜ, including any value therebetween.

In some embodiments, each the compounds (e.g., glutamate dehydrogenase, a-ketoglutarate, and NAD(P)H of the kit are packaged in separate compartments.

In some embodiments, there is provided a composition comprising one or more compounds selected from SAM, methyltransferase, AdoHcy, adenine, ADE, hypoxanthine, and ammonia; and one or more compounds selected from: a-ketoglutarate, NAD(P)H, glutamate dehydrogenase, and NAD(P)+.

In some embodiments, there is provided a composition comprising one or more compounds selected from SAM, methyltransferase, AdoHcy, adenine, ADE, hypoxanthine, and ammonia; and one or more compounds selected from pyruvate, glutamate, glutamate synthetase, ATP, ADP, glutamine, pyrophosphate, phosphoenolpyruvate, pyruvate, acetyl phosphate, CO2, and H2O2.

In some embodiments, there is provided one or more compounds selected from a-ketoglutarate, NAD(P)H, glutamate dehydrogenase and one or more compounds of selected from glutamate synthetase and ATP.

In some embodiments, the kit may be used for clinical diagnosis.

General

As used herein the term “about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of means “including and limited to”. The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

The word “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

The word “optionally” is used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to "at least one of A, B, or C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion. EXAMPLE 1

Materials

Molecular biology: In exemplary procedures, SET7/9 gene was cloned into pET-Duet plasmid for E. coli expression and purification. The SET7/9 gene was PCR amplified from pGex-6pl plasmid containing the gene as a template. The amplified DNA fragment was then cleaved by Spel and Xhol restriction enzymes and ligated into a pET-Duet plasmid containing a Maltose Binding Domain (MBP) tag. The Adenine deaminase (ADE, E.C. 3.5.4.4) and -S'-adenosyl-L-homocysteine nucleosidase (SAHN, E.C. 3.2.2.9) genes were amplified from the genomic DNA of an XL1 blue E. coli strain using PCR. The amplified DNA fragments were then cleaved using Nhel and Hindlll restriction enzymes and ligated into a pET28a plasmid containing a Hisx6 tag at the N-terminal of the protein.

Protein Expression and purification: In exemplary procedures, SET7/9 enzyme was expressed from a pET-Duet plasmid containing the SET7/9 gene fused to a MBP tag in E. coli BL21 (DE3). Expression was induced using 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) for 6h at 30 °C. Following inductions, cells were centrifuged and resuspended at a buffer containing 25 mM Tris-Hcl (pH 7.5), 200 mM NaCl and 1 mM DTT. The cells were lysed using a French press and the resulting cell extract was centrifuged at 13000g for lh. The SET7/9 protein was then purified from the clear lysate using amylose beads (Amersham) according to standard procedures. The MBP tag was then cleaved using TEV protease for 4h at 4°C and purified using gel-filtration chromatography to obtain a monomeric SET7/9 protein. GST-SETD6 was expressed and purified as previously described (Levy, D. etal., Nat Immunol 12, 29-36).

In additional exemplary procedures, the RelA protein was expressed using a pGEX-6pl plasmid containing the RelA gene fused to a GST tag in E. coli BL21 (DE3). Expression was induced with 0.5 mM IPTG for 16h at 20 °C. Following inductions, cells were centrifuged and resuspended in buffer containing 25 mM Tris-Hcl (pH 7.5), 200 mM NaCl and 1 mM DTT. The cells were lysed using a French press and the cell extract was centrifuged at 13000g for lh. The RelA protein was then purified using glutathione beads (Amersham) according to standard procedures.

In additional exemplary procedures, the ADE enzyme was expressed from pET28a plasmid containing the ADE gene fused to a 6xHis tag at the N-terminal in E.coli BL21 (DE3). Expression was induced with 0.5 mM IPTG for 16h at 20°C. In order to replace the

Fe2+ metal at the ADE active site with Mn2+, 50 μΜ of 2,2’-dipyridyl and 1.0 mM MnCh were added at time of the induction. Following induction, cell were centrifuged and resuspended at a buffer containing 25 mM Tris-Hcl (pH 7.5), 200 mM NaCl and 1 mM DTT. The cells were lysed using French press and the cell extract was centrifuged at 13000g for lh. The ADE protein was then purified using nickel-NTA beads using standard procedures. Following purification, a dialysism against the original buffer was performed to remove the imidazole. The SAHN protein was purified using the same purification protocol without the metal replacement procedure performed for the ADE purification. For DNA methylation assays commercial Haelll enzyme was purchased from NEB. EXAMPLE 2 ASSAYS Methods

Methylation assays: In exemplary procedures, the continuous coupled methylation assay was carried out with clear flat bottom 96 well plates, containing 4.5 μΜ SAHN, 3 μΜ ADE, 5 μΐ of glutamate dehydrogenase (Ammonia detection kit, Sigma), 300 μΜ SAM and a varying concentration of methyltransferase enzyme and methyl-acceptor. A concentration of 300 μΜ SAM was used to ensure saturation of the methyl donor. A final volume of 250 μΐ was reached in the well using the ammonia assay kit buffer (Sigma). The assay was performed at 30 °C, and the reaction was monitored at 340 nm using Tecan Infinite M200 plate reader. Kinetic parameters were derived by fitting to Michaelis-Menten Vo= ME]o[S]o/([S]o+Km) model. The TAT and Fox03 peptide substrate sequences are GISYGRKKRRQRRRP (residues 44-58) and RGRAAKKKAALQTA (residues 264-277), respectively (methylated lysine is in bold).

Radioactive in-vitro methylation assay: In exemplary procedures, assays were performed as described (Levy, D. et al., Nat Immunol 12, 29-36). Briefly, recombinant proteins were incubated with recombinant SETD6, and 2 mCi 3H-SAM (Amersham Pharmacia Biotech Inc, Piscataway, NJ, USA) in methylation buffer (50 mM Tris-HCl (pH 8.0), 10% glycerol, 20 mM KC1, 5 mM MgC'h and 1 mM PMSF) at 30 °C overnight. The reaction mixture was resolved by SDS-PAGE, followed by either autoradiography or Coomassie blue stain.

Results

Development of a coupled continuous assay for MTs

To overcome these limitations, we have coupled the activity of SAHN and ADE with glutamate dehydrogenase that utilizes ammonia and alpha-ketoglutarate to generate glutamate while oxidizing NADPH. The coupling of this reaction to MTs activity allows monitoring the continuous change in absorbance at 340 nm due to NADPH oxidation (as shown in Figure 1A) which linearly correlates with the reduction of the SAM concentration.

To establish the continuous coupled assay for MTs activity, we initially utilized the PKMTs SET7/9 as a model enzyme. Previous works have shown that SET7/9 exhibits broad substrate specificity catalyzing methyl transfer to a variety of histone and nonhistone proteins including H3, TAF10, TAT, RelA, p53 and Fox03. Methylation of these proteins by SET7/9 was shown to regulate protein-protein and protein-DNA interactions thus modulating the target protein activity.

The coupled assay was initially utilized to monitor SET7/9 activity with a peptide substrate derived from the HIV trans-activator TAT protein. SET7/9 was previously shown to monomethylate TAT protein at K51, thus, a peptide was designed to include the K51 acceptor residue (see peptide sequence in the Methods section). It was found that when all components of the coupled reaction are present, a gradual change in absorbance at 340 nm was observed indicating the successful coupling of SET7/9 activity with NADPH oxidation (as shown in Figure 2A). To verify that the coupled assay specifically measures MT activity, several control reactions were performed in which one of the reaction components was omitted. It was observed that when each of the coupled enzymes was absent from the reaction mix no activity was measured (as shown in Figure 2A and Figures 3 and 4A-B). In addition, in the absence of SAM or in the presence of inactive SET7/9 containing the E254A mutation, no activity was observed (as shown in Figure 2A). To further verify that SET7/9 is the rate determining step in the kinetic measurements, the catalytic rate of each step was examined in the coupled reaction independently by monitoring NADPH oxidation. It was found that under the reaction mixture as detailed hereinabove the three coupling enzymes were significantly faster than SET7/9 activity (as shown in Figure 2B).

Overall, these controls ensure that the change in absorbance at 340 nm reflects the true measurement of SET7/9 methylation activity and provides a wide dynamic range for activity measurements at different conditions (e.g., substrate concentrations see below).

Measurements of SET7/9 and SETD6 activity with peptide and protein substrates

To examine whether the coupled kinetic assay can be utilized for the MM kinetic analysis of SET7/9 activity, a peptide derived from the Fox03 protein was utilized. Previous work has shown that SET7/9 methylates Fox03 at K271 modulating its transcriptional activity and stability. Thus, a 15 amino-acid peptide substrate derived from Fox03 containing K271 was designed to quantitatively measure SET7/9 catalytic activity as described hereinabove. Using the coupled assay, the initial rates of SET7/9 activity at different peptide substrate concentrations ranging from 0 to 700 μΜ was measured (as shown in Figure 5A). A gradual increase in initial rates that is correlated with the increase in Fox03 peptide concentration was observed. Fitting the initial rate data to the MM equation allowed deriving kinetic parameters for the activity of SET7/9 with the Fox03 peptide (as shown in Figure 5B). It was found that the Km and kcat parameters for this activity werel65 μΜ and 32 min"1, respectively. The values are in excellent correlation with previous analysis of SET7/9 activity with H3K4 and DNMT1K142 utilizing radioactive MT assay. The previously measured Km and Kcat for H3K4 and DNMTK142 were 143 μΜ and 134 μΜ and 48 min''and 42 min"1, respectively (Esteve, V.O. et al,. Proc Natl Acad Sci U SA 106, 5076). This correlation provides strong support for our continuous assay and confirms that the Fox03 peptide is a substrate for SET7/9.

To further examine whether the coupled assay can monitor MTs activity with full length protein substrates the activity of SETD6 with RelA protein (residues 1-431) was examined. Previously, SETD6 was shown to methylate RelA on K310 leading to a dramatic modulation of NFkB transcriptional activity. To measure SETD6 activity with RelA protein, RelA concentrations of up to 3 μΜ was utilized (as shown in Figure 6A). Analysis of SETD6 activity at these RelA concentrations showed a linear increase in reaction rate with a slope of kcat/KM of 57.1 min"1 μΜ"1 (as shown in Figure 6B). These results demonstrate that the disclosed assay can be utilized to examine MTs activity with full length proteins as substrates, paving the way for additional studies to examine MTs activity with natural protein substrates.

To further validate that the coupling enzymes utilized in the MTs kinetic assay do not act as substrates for methylation, radioactive [3H]-SAM as part of the SETD6 RelA coupled assay was utilized. The utilization of [3H]-SAM in the assay allows monitoring methylation of each protein in the reaction mixture using SDS-PAGE followed by autoradiography. It was found that while RelA serves as a substrate for SETD6, the SAH and ADE coupling enzymes do not and thus no significant background is observed in the absence of substrate (as shown in Figure 6C and Figure IB).

Measurements of Haelll methyl transfer activity with DNA substrate

To further examine the versatility of the assay for the general monitoring of MTs activity, the methylation activity of Haelll from Haemophilus aegypticus with DNA substrate was examined. Haelll belongs to a large family of bacterial DNA MTs that catalyses cytosine C5 DNA methylation. Haelll methylates the internal cytosine of the canonical sequence GGCC and is utilized in the restriction-modification bacterial defense system against phage infection.

To examine Haelll activity using the disclosed coupled assay, first DNA substrate was prepared by PCR amplification of 1.6 kb DNA fragment from pGex plasmid containing 6 predicted methylation sites for Haelll (as shown in Figure 7A). The coupled assay was utilized to monitor Haelll activity with different DNA concentrations and measured the initial reaction rates. An increase in the initial rate of Haelll catalysed DNA methylation was observed at increased DNA substrate concentrations, demonstrating the ability to measure the kinetics of MTs with DNA substrates (as shown in Figure 7B).

To verify that Haelll methylates DNA under the coupled assay conditions, Notl digestion analysis was performed. This DNA substrate contains one Notl cleavage site (GCGGC*CGC) that is located 1 kb from the 5’ of the DNA. Methylation of this site will prevent Noil cleavage leading to intact 1.6 kb substrate even in the presence of Notl.

It was found that methylation of the DNA substrate by Haelll in the presence of all coupled assay components prevents Notl cleavage leading to the presence of undigested 1.6 kb fragment (as shown in Figure 7C).

These results highlight the utility of the coupled assay to efficiently monitor MTs activity with DNA substrates paving the way for quantitative analysis of many DNA methylation enzymes and sites.

Claims

WHAT IS CLAIMED IS:

1. A method for assaying a methyl transfer reaction in a reaction mixture, said reaction mixture comprising a substrate, a methyl donor and a catalyst, the method comprising the steps of: (a) coupling: (i) one or more reactions to said methyl transfer reaction, thereby forming one or more intermediate products, and (11) one or more subsequent enzymatic or chemical reactions coupled thereto, thereby forming one or more final products characterized by an absorption of above 300 nm; and (b) spectrophotometric analyzing or monitoring the reaction mixture or an aliquant thereof, wherein said spectrophotometric analyzing or monitoring is performed at a wavelength of above 300 nm, thereby assessing the amount of said one or more final products.

2. The method of claim 1, wherein said catalyst is an enzyme selected from methyltransferase and a derivative thereof.

3. The method of claim 1, wherein said methyl donor is adenosylmethiomne (SAM).

4. The method of claim 1, wherein said methyl transfer reaction is a rate-limiting reaction with respect to any reaction coupled and/or subsequent thereto.

5. The method of claim 1, wherein said one or more intermediate products are selected from ammonia, pyruvate, hydrogen peroxide, hypoxanthine, and any respective derivative thereof.

6. The method of claim 1, wherein said one or more final products is NAD(P)+, or a respective derivative thereof.

7. A method for assaying methyltransferase activity on a substrate, comprising the following steps: i) preparing a reaction mixture comprising a methyltransferase, a substrate, and adenosylmethionine (SAM) or respective derivative thereof, thereby transmethylating said substrate and forming adenosylhomocysteine (AdoHcy); ii) contacting the reaction mixture to AdoHcy nucleosidase, thereby forming ribosylhomocysteine and adenine or respective derivative thereof; iii) contacting the reaction mixture to adenine deaminase (ADE), thereby forming or respective derivative thereof and ammonia; and iv) determining the amount of the ammonia in the reaction mixture by an enzymatic conversion of said ammonia to one or more products characterized by a defined absorbance of above 300 nm, thereby determining the methyltransferase activity.

8. The method of claim 7, wherein said enzymatic conversion of said ammonia to said product characterized by a defined absorbance of above 300 nm is a process selected from: a one-step process, a two-step process, a three-step process, or four-step process.

9. The method of claim 8, wherein said one step-process comprises a step of reacting the ammonia withNADPH and α-ketoglutarate in the presence of glutamate dehydrogenase and wherein said product is NAD(P)+.

10. The method of claim 8, wherein said three step-process comprises the steps of: (a) reacting the ammonia with Glutamate and ATP in the presence of Glutamine synthetase, thereby forming ADP; (b) enzymatic converting ADP to pyruvate; and (c) enzymatic converting pyruvate and NADPH to lactate and NAD(P)+.

11. The method of claim 8, wherein said four step-process comprises the steps of: (a) reacting the ammonia with Glutamate and ATP in the presence of glutamate synthetase, thereby forming ADP; (b) enzymatic converting ADP to pyruvate; (c) enzymatic converting pyruvate to acetyl phosphate, CO2, H2O2; and (d) contacting a phenol and/or a dye molecule characterized by a defined absorbance of above 300 nm to H2O2.

12. The method of claim 7, wherein step (iv) is determined by a spectrophotometric monitoring being performed at a wavelength in the range of 320 nm to 600 nm.

13. The method of any one of claims 1 and 7, wherein said substrate is selected from DNA, protein, peptide and any combination thereof.

14. The method of claim 7, wherein the step of transmethylating the substrate is rate-limiting with respect to the steps (ii) and (iii).

15. The method of claim 12, wherein said spectrophotometric monitoring is performed continuously.

16. The method of claim 7, wherein at least two steps of steps (i) to (iv) are performed in one-pot.

17. The method of claim 7, wherein said steps (i) to (v) are performed sequentially.

18. The method of any one of claims 1 and 7, wherein said methyltransferase is nucleic acid methyltransferase.

19. The method of claim 18, wherein said nucleic acid methyltransferase is DNA methyltransferase.

20. The methods of claims 1 and 7, wherein the reaction mixture further comprises at least one solution selected from the group consisting of: aqueous buffer, water, organic solvent and oil.

21. A kit for quantitatively assaying methyltransferase activity comprising effective amounts of: (a) Glutamate dehydrogenase; (b) α-ketoglutarate; and (c) NADPH.

22. The kit of claim 21, further comprising in a buffer.

23. The kit of claim 21, further comprising AdoHcy nucleosidase.

24. The kit of claim 21, further comprising ADE.

25. The kit of claim 21, wherein each of a, b, and c are packaged in separate compartments.

26. A composition comprising: (a) one or more compounds selected from SAM, methyltransferase, AdoHcy, adenine, ADE, hypoxanthine, and ammonia; and (b) one or more compounds selected from: α-ketoglutarate, NAD(P)H, glutamate dehydrogenase, and NAD(P)+; or (c) one or more compounds selected from pyruvate, glutamate, glutamate synthetase, ATP, ADP, glutamine, pyrophosphate, phosphoenolpyruvate, pyruvate, acetyl phosphate, CO2, and H2O2.

27. The composition of claim 26, wherein said one or more compounds of (b) are selected from α-ketoglutarate, NAD(P)H, glutamate dehydrogenase and said one or more compounds of (c) are selected from glutamate synthetase and ATP.