ANION POLYMER POLY (CYTIDINE DIPHOSPHATE RIBOSE) AND ITS METHOD OF PREPARATION

Abstract

An anionic polymer comprising Poly (cytidine diphosphate ribose) of Formula (I), (I) wherein n is 1 to 50 units. The present disclosure also relates to method of preparation thereof.

Specification

Description:FIELD OF THE INVENTION:
[0001] The present disclosure relates to an anionic polymer. More particularly, the disclosure relates to the anionic biopolymer comprising poly (cytidine diphosphate ribose) and its method of preparation.

BACKGROUND OF THE INVENTION
[0002] Biopolymers have numerous biomedical, agricultural, and environmental applications due to their biocompatibility, bioavailability, and capability to be tailored for particular purposes. There are a number of anionic biopolymers like hyaluronic acid, alginate, poly acrylic acid, poly styrene sulfonate, etc. which serves different purposes due to their unique properties but also hold certain limitations. For example, for oral and injectable delivery of drugs, alginate is considered as a good option as it can easily form gels in presence of calcium. But it relies on calcium ions for gel formation and variations in ionic strength can affect the gel stability. Also, it can degrade quickly in physiological conditions causing premature release of encapsulated drugs. It is highly used anionic biopolymer for making creams and gels and thus used in cosmetics and regenerative medicine is hyaluronic acid. Being a polymer that is found naturally in humans, hyaluronic acid is naturally broken down by the body's enzymes, which may restrict its potency and duration of action in medicinal uses.
[0003] Another example of a widely used anionic biopolymer is polyacrylates. It is often used to improve soil moisture retention and products like diapers due to their ability to absorb large amounts of liquid. A major drawback of polyacrylates is that it is predominantly chemically synthesized using petrochemicals.
[0004] Poly styrene sulfonates is another anionic polymer which is synthesized using chemical methods. It is ubiquitously used in medical devices, drug delivery and wastewater treatment and purification because of its negative charge and water solubility property. But the compromised mechanical strength and sensitivity to high temperature and extreme pH limits use.
[0005] All of the above-mentioned biopolymers have their advantages as well as certain disadvantages which limit their usage. A lot of research is going on to develop biocompatible and sustainable biopolymers and recently several patents has been granted to further promote and support the relevant inventions. To mention a few, Patent No. 295062 was given to a process for preparing chitosan derivatives for its application in drug delivery and antimicrobial properties. Another very relevant patent was approved for the microbial production method of polyhyroxyalkanoate (PHA) (Patent No. 330170). PHA can be attractive alternative to conventional petroleum-based plastics providing an eco-friendly option and have also have potential in biomedical field.
[0006] Thus, there is a need for developing the anionic biopolymer that addresses the limitations of the above-described polymers.

OBJECTS OF THE INVENTION
[0007] Some of the objectives of the present disclosure, with at least one embodiment herein satisfied, are listed herein below:
[0008] It is the primary objective of the present disclosure to provide an anionic biopolymer
[0009] It is yet another objective of the present disclosure to provide a simple and environment-friendly method for the preparation of the anionic biopolymer.

SUMMARY OF INVENTION
[0010] The present disclosure relates to an anionic polymer comprising
Poly (cytidine diphosphate ribose) of Formula (I),
(I)
wherein n is 1 to 50 units.

[0011] The present disclosure also relates to a method of preparing an anionic polymer comprising Poly (cytidine diphosphate ribose), comprising the steps of
a) incubating a poly(ADP-ribose) polymerase 1 (PARP-1) enzyme with 18 base pairs double strand DNA in reaction buffer to obtain reaction mixture containing PARP1 bound DNA;
b) adding an aqueous solution of Nicotinamide cytosine dinucleotide (NCD) to the reaction mixture;
c) adding ice cold trichloroacetic acid (TCA) for a time period of 10 mins to 20 minutes to precipitate out pellet of Poly (cytidine diphosphate ribose) covalently attached to PARP1;
a) cleaving Poly (cytidine diphosphate ribose) from the PARP1 and separating it from the reaction mixture.


BRIEF DESCRIPTION OF DRAWINGS
[0012] The present disclosure contains the following drawings that simply illustrates certain selected embodiments of the anionic polymer and processes that are consistent with the subject matter as claimed herein, wherein:
[0013] Figure 1: Schematic representation of steps used for recombinant expression and purification of human PARP1 protein from Escherichia coli to make Poly(cytidine diphosphate ribose).
[0014] Figure 2: (A) The chromatogram of size exclusion chromatography (SEC) on HiLoad 16/ 600 Superdex 200 pg of PARP1. (B) The SDS-PAGE gel of eluted fractions of PARP1 from SEC, which were used to make Poly(cytidine diphosphate ribose) The last lane shows the protein marker for molecular weight estimation. All the molecular weights are in kilodalton (kDa).
[0015] Figure 3: (A) SDS-PAGE gel of in vitro PCRylation reaction showing the formation of desired product (Poly(cytidine diphosphate ribose)) on PARP1, which can be seen as smear over PARP1 protein band. (B) Quantification of the activity of PARP1 in presence of NAD+ (natural substrate) and NCD (non-natural substrate).
[0016] Figure 4: Native PAGE gel showing purified Poly(cytidine diphosphate ribose) as smear.
[0017] Figure 5: Mass spectrometry of Poly(cytidine diphosphate ribose)

DESCRIPTION OF THE INVENTION:
[0018] A detailed description of various exemplary embodiments of the disclosure is described herein. It should be noted that the embodiments are described herein in such detail as to communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.
[0019] The terminology used herein is to describe particular embodiments only and is not intended to be limiting to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising", or "includes" and/or "including" or "has" and/or "having" when used in this specification specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.
[0020] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0021] In an embodiment, the present disclosure relates to an anionic polymer comprising
Poly (cytidine diphosphate ribose) of Formula (I),
(I)
wherein n is 1 to 50 units.

[0022] In an embodiment of present disclosure, the molecular weight of the anionic polymer is in the range of 534- 26,700 Da (Daltons).
[0023] In another embodiment of present disclosure, Poly(cytidine diphosphate ribose) is a highly negatively charged (or anionic) polymer, which has not been reported to be present in any cellular system in any organism.
[0024] In an embodiment, the present disclosure relates to the synthesis of a novel non-natural polymer called Poly(cytidine diphosphate ribose), using an enzyme Poly(ADP-ribose) polymerase 1 (PARP1). The method of preparing an anionic polymer comprising Poly (cytidine diphosphate ribose), comprising the steps of
a) incubating a poly(ADP-ribose) polymerase 1 (PARP-1) enzyme with 18 base pairs double strand DNA in reaction buffer to obtain reaction mixture containing PARP1 bound DNA;
b) adding an aqueous solution of Nicotinamide cytosine dinucleotide (NCD) in the reaction mixture;
c) adding ice cold trichloroacetic acid (TCA) for a time period of 10 mins to 20 minutes to precipitate out the pellet of Poly (cytidine diphosphate ribose) covalently attached to PARP1;
d) cleaving the Poly (cytidine diphosphate ribose) from PARP1 and separating it from the reaction mixture.
[0025] In another embodiment of the present disclosure, the reaction mixture is incubated for a period of 2 to 4 hours after adding NCD to the reaction mixture.
[0026] In an embodiment of present disclosure, the PARP1 uptake a non-natural co-substrate called 'Nicotinamide cytosine dinucleotide' (NCD+) and catalyse the synthesis of Poly(cytidine diphosphate ribose).
[0027] In an embodiment of present disclosure, the process for the synthesis of Poly(cytidine diphosphate ribose) is classified as green synthesis as it uses a biocatalyst or enzyme.
[0028] In yet another embodiment of the present disclosure, concentration of PARP1 enzyme is in range of 1 µM- 20 µM
[0029] In another embodiment of the present the concentration of NCD is in range of 2- 5 mM
[0030] In yet another embodiment of the present disclosure, the poly (cytidine diphosphate ribose) is extracted from Poly (cytidine diphosphate ribose) covalently attached to PARP1 by the method comprising the steps of:
a) suspending the pellet of Poly (cytidine diphosphate ribose) covalently attached to PARP1 in Tris Ethylenediamine tetraacetic acid (EDTA) buffer;
b) adding DNasel to digest the DNA followed by adding Proteinase K to degrade the PARP1; and
c) cleaving the Poly (cytidine diphosphate ribose) from PARP1; and
d) isolating Poly (cytidine diphosphate ribose) from the the reaction mixture.
[0031] In yet another embodiment of the present disclosure, the solution in step b) is incubated for a period of 30 minutes to 2 hours at a temperature of 35 to 40 °C.
[0032] In another embodiment of the present disclosure, the sodium dodecyl sulfate (SDS) and proteinase K is incubated for 2 to 3 hours at a temperature of 45 to 50 °C.
[0033] In another embodiment of the present disclosure, the Poly (cytidine diphosphate ribose) is cleaved from the PARP1 using potassium hydroxide (KOH) and ethylene diamine tetra acetic acid (EDTA) by incubating at a temperature of 60 to 65 °C for 2 to 3 hours.
[0034] In an embodiment of present disclosure, PARP proteins are present across all domains of life and some of them catalyze the formation of a biopolymer called Poly(Adenine diphosphate ribose) using the nicotinamide adenine dinucleotide (NAD+) as a co-factor to transfer ADP-ribose moiety onto the target molecule.
[0035] In an embodiment of present disclosure, NCD+ is a chemical analogue of NAD+ in which the adenine base is replaced with cytosine.
[0036] In another embodiment of the present disclosure, recombinant DNA technology is used to express and purify the human PARP1 from Escherichia coli Rossetta2(DE3) strain to synthesize Poly(cytidine diphosphate ribose).
[0037] In an embodiment, Poly(cytidine diphosphate ribose), can be tested for various biomedical applications, such as drug delivery cargo, that can encapsulate drugs for better delivery and bioavailability. Due to the high biocompatibility, biopolymers can serve as scaffold for tissue engineering and cell growth. Most importantly, dressings made of anionic polymers are especially beneficial for chronic wounds and burns. Some anionic polymers have antibacterial properties, making them suitable for medical devices and coatings. The anionic biopolymer hydrogels can be used in skin care and cosmetics products and can have environmental usages for controlled release of fertilizers in agricultural fields or can be used for adsorption of pollutants for wastewater treatment. Another unrelated yet very important use of Poly(cytidine diphosphate ribose) could be to investigate if the Poly(Adenine diphosphate ribose) binding proteins like poly(adenine dinucleotide phosphate) glycohydrolase (PARG), X-ray cross complementing protein 1 (XRCC1), etc. can recognize Poly(cytidine diphosphate ribose) or not. These proteins are targets for designing anti-cancer drugs as they are either directly involved or indirectly regulate DNA damage repair. In future, Poly(cytidine diphosphate ribose) can be used as parent molecule for generation of Poly(cytidine diphosphate ribose) based drugs to treat chronic diseases like cancer.
ADVANTAGES OF THE PRESENT INVENTION
[0038] In accordance with the present disclosure anionic polymer comprising Poly (cytidine diphosphate ribose) and its method of preparation has the following advantages:
• simpler process without any additional step of its modification is involved.
• the Poly(cytidine diphosphate ribose) formed is of different lengths which can be successfully fractionated, can come handy depending on the application.
• green or environment friendly method with very less use of chemicals
• No hazardous waste is generated while producing Poly(cytidine diphosphate ribose).
[0039] The present disclosure will be explained using the following examples:

EXAMPLE
Materials:
[0040] Gene encoding full length human PARP1 with N-terminal hexa-Histidine (6x-His) tag cloned in pRSFDuet-1 expression vector was purchased from GenScript <Please
Example 1
Expression of human PARP1 protein in E. coli
[0041] In pRSFDuet-1 vector, PARP1 gene was cloned between NdeI and XhoI restriction site and was under the control of Isopropyl β- d-1-thiogalactopyranoside (IPTG) inducible promoter. The construct was transformed in E. coli expression strain Rosetta 2 (DE3) and plated on Luria Bertani (LB) Agar plate containing the antibiotics Kanamycin (50 g/mL) and Chloramphenicol (35 g/mL) for selection (Figure 1). A single transformed colony was inoculated into the 100 mL LB Broth with same antibiotics and incubated overnight at 37°C while shaking at 200 rpm (rotations per minute) to prepare a starter culture. 10 mL of this starter culture was inoculated per litre of LB broth. A total of 4-6 Litres of LB broth were inoculated (Figure 1) and incubated at 37 °C while shaking at 200 rpm. When the larger cultures were grown to log phase, i.e., the optical density measured at 600 nm reached 0.6- 0.8, the cultures were cooled down to 16 °C. Since, PARP1 contains three domains which bind Zn2+ ions for its stability and functionality, 100 M ZnSO4 was added to the cultures at this stage and again incubated at 16 °C for another hour. To induce the expression of PARP1 in the bacterial cells 0.2 mM IPTG was added to the cultures. The cultures were then again incubated for overnight at 16°C. After 14-16 hours, the cells were pelleted down by centrifugation at 3,500 rpm using Sorval Lynx centrifuge. The supernatant was discarded and the cell pellets of PARP1were stored.

Example 2:
Purification of PARP1 protein
[0042] The pellets were resuspended in the 70-100 mL lysis buffer containing 500 mM NaCl, 50 mM (2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid) (HEPES) pH 7.5, 20 mM Imidazole and 3mM -mercaptoethanol. The resuspended cells were lysed using sonication (i.e., using ultrasonic sound waves to lyse the cells). The sonicated cell lysate was centrifuged for 1h at 10,000 rpm at 4 °C to separate the lysate debris from the protein solution. PARP1 is a cytosolic protein (that comes out into solution or buffer upon cell lysis). Thus, the supernatant was kept and pellete was discarded. PARP1 was then purified from the protein-pool in the supernatant using a the three-step purification protocol using chromatography techniques, to obtain pure PARP1 protein to be used to make Poly(cytidine diphosphate ribose).
[0043] The first step is the affinity purification which utilizes a nickel (Ni2+)-charged column to separate the 6xHis-tagged PARP1 protein from the clarified supernatant. First, the Ni2+-charged (Ni2+-Nitrilotriacetic acid aka Ni-NTA) column (HisTrap HP 5 mL column from Cytiva) was washed with 100 mL of double distilled deionized water and equilibrated with 30 mL of the lysis buffer. Then, the lysate supernatant was passed through the HisTrap column. The column was, then, washed with 50 mL lysis buffer and 30 mL high-salt buffer (1 M NaCl, 50 mM HEPES pH 7.5, 20 mM Imidazole and 3mM -mercaptoethanol) to reduce the protein contaminants. Finally, the column-bound proteins were eluted using the elution buffer (500 mM NaCl, 50 mM HEPES pH 7.5, 500 mM Imidazole and 3mM -mercaptoethanol) (Figure 1) and collected in 5 mL fractions. These elution fractions were analyzed on 12% sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) for the presence of expressed PARP1.
[0044] In the second step, the fractions containing PARP1 was further passed through anion exchange chromatography column to remove contaminant DNA since PARP1 binds to DNA with very high affinity. The heparin column (HiTrap Heparin HP, 5 mL column from Cytiva) was washed with 50 mL of double distilled deionized water and equilibrated with 30 mL of the low salt buffer (250 mM NaCl, 50 mM HEPES pH 7.5 and 3mM -mercaptoethanol). The elution fractions from nickel column containing PARP1 were diluted two folds using no-salt buffer (50 mM HEPES pH 7.5 and 3mM -mercaptoethanol) to bring the salt concentration to 250 mM, to match the salt concentration in the low salt buffer and loaded on the column. The proteins were eluted in 3 mL volume fractions on a salt concentration gradient of 250 mM- 1M NaCl over 200 ml. The elution fractions were analyzed on 12% SDS-PAGE for the purity of PARP1.
[0045] Finally, in the third step, homogeneity of the purified PARP1 was assessed by size exclusion chromatography (SEC) using Hiload 16/600 Superdex 200 pg column from Cytiva. The SEC column was washed with 150 mL double distilled deionized water and equilibrated with 150 mL of SEC buffer containing 150 mM NaCl, 10 mM HEPES pH 7.5, 2% glycerol and 2mM -mercaptoethanol. The ion-exchange chromatography fractions containing pure PARP1 protein was passed through the SEC column and the elution fractions were collected in 500 L volume (Figure 1 and 2A). The elution fractions were again analysed on 12% SDS-PAGE (Figure 2B). The fractions containing PARP1 were pooled together and concentrated to 30 M using a 50 kDa spin concentrator from Merck Millipore. The entire purification step was performed at 4 °C. This protein was used to set up the in vitro reactions to make Poly(cytidine diphosphate ribose).

Example 3:
In vitro reaction to generate Poly(cytidine diphosphate ribose) and quantification of PARP1 activity
[0046] DNA-bound PARP1 uses NAD+ as native cofactor to synthesize Poly(Adenine diphosphate ribose). Poly(Adenine diphosphate ribose) is a natural anionic polymer which is short lived in the cell before getting degraded by PAR-hydrolyzing enzymes. NCD is a non-natural analogue of NAD+. NCD was used as a co-substrate for PARP1 to synthesize Poly(cytidine diphosphate ribose) under in vitro conditions. NCD was dissolved in water to a concentration of 100 mM. A reaction buffer containing 20 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl2 and 0.1 mM -mercaptoethanol was prepared. To set up the reaction, 1 µM PARP1 was incubated with 1 µM 18 base-pairs double strand DNA in the reaction buffer at room temperature for 10 min, so that PARP1 can bind to DNA. To this reaction mixture, NCD (5 mM) was added, and the reaction was again incubated for 2 hours at room temperature. Lamilli buffer containing SDS detergent was added to the reaction mixture to stop the reaction by denaturing PARP1.
[0047] The mixture was resolved on 12% SDS-PAGE and then stained using Coomassie Brilliant Blue (a protein staining dye). The presence of high molecular weight bands/ smear above PARP1 band indicates the formation of polymer Poly(cytidine diphosphate ribose) covalently attached to PARP1. As a positive control, PARP1-DNA complex was also incubated with NAD+ to generate Poly(Adenine diphosphate ribose). Relative shorter polymer chains of Poly(cytidine diphosphate ribose) are formed compared to the long Poly(Adenine diphosphate ribose) chains by PARP1 (Figure 3A).
Example 4:
The enzymatic activity of PARP1 by PNC1-OPT assay.
[0048] The enzymatic activity of PARP1 to synthesize Poly(cytidine diphosphate ribose) was quantified using a well standardised PNC1-OPT assay. The assay estimates the amount of nicotinamide released during the PARylation reaction. The amount of nicotinamide produced is equal to the NCD consumption by PARP1 and/or CDPr units added. The assay uses an enzyme pyrazinamidase/nicotinamidase 1 (PNC1) found in budding yeast classified as a nicotinamidase. PNC1 converts the PCRylation by-product nicotinamide to nicotinic acid with the release of ammonia. Furthermore, ammonia reacts with ortho-pthalaldehyde (OPT) to give fluorescence proportional to the catalytic activity of the PARP1. To measure activity, the reaction was set up in similar manner with 1 μM PARP1, 1 μM DNA, and 5 mM NCD in a 96-well plate and incubated on an orbital shaker for 2 hrs. The fluorescence was measured at 420 nm (excitation wavelength) and 450 nm (emission wavelength). The fluorescence in the reaction sample with NAD was more as compared to the reaction sample with NCD. This indicates PARP1 utilized more NAD than NCD which points towards formation of more Poly(Adenine diphosphate ribose) over Poly(cytidine diphosphate ribose). When quantified PARP1 showed 30% activity for Poly(cytidine diphosphate ribose) synthesis compared to Poly(Adenine diphosphate ribose) synthesis (Figure 3B).
Example 5:
Extraction and purification of Poly(cytidine diphosphate ribose)
[0049] To extract the Poly(cytidine diphosphate ribose), the same in vitro reaction as described above was set up at larger scale (20 mL reaction). The reaction was stopped by precipitation with ice-cold trichloroacetic acid (20%, v/v) on ice for 15 minutes. The precipitates were collected by centrifugation at 14,000 rpm for 15 minutes at 4 °C in a microcentrifuge. The obtained pellets were washed twice with ice-cold 20% trichloroacetic acid, followed by three times washing with 100% ethanol. The residual ethanol was removed by air drying the pellet. The pellets were dissolved in 100 μL of Tris-EDTA buffer. The DNaseI (10 units/μL) was added in the sample and incubated for an hour at 37 °C to digest the DNA, followed by adding SDS to a final concentration of 0.1%. To this, proteinase K was added to 0.2 mg/mL from a 20 mg/mL stock and incubated at 50°C for 2 to 3 hours to degrade the PARP1. Equal volume of 1 M KOH/50 mM EDTA was added and incubated for 2 hours at 60 °C to detach the Poly(cytidine diphosphate ribose) polymers from the digested proteins. Following the alkaline treatment, the pH was adjusted to 8.0 using HCl. Equal volume of PCI (phenol/chloroform/isoamyl alcohol) solution was added to the pH adjusted mixture and vortex for 30 seconds and then centrifuged at 14,000 rpm for 5 minutes at room temperature in a microcentrifuge to separate the aqueous and organic phases. The upper aqueous phase contains the Poly(cytidine diphosphate ribose) which was collected. Equal volume of chloroform was added to it and vortexed for 30 seconds and centrifuged again at 14,000 rpm. Aqueous upper phase was collected, and the chloroform treatment was repeated to remove any organic contaminants. To the finally obtained aqueous phase solution, 1/10 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold ethanol was added to precipitate Poly(cytidine diphosphate ribose). The solution was mixed well and incubated overnight at −20 °C. Following day, the solution was centrifuged at 14,000 rpm for 30 minutes at 4°C in a microcentrifuge. The supernatant was carefully discarded, and pellet was washed with 1 mL of 70% ethanol and re-centrifuge at 14,000 rpm for 5 minutes at room temperature in a microcentrifuge. The obtained pellets were air dried and dissolved in 20-30 l of water. The sample was analyzed on 12% native PAGE and silver stained to confirm the formation of Poly(cytidine diphosphate ribose). Since PARP1 synthesized Poly(cytidine diphosphate ribose) of variable length, Poly(cytidine diphosphate ribose) appeared as a smear on the native PAGE gel (Figure 4). The samples were aliquoted and stored at −20°C until further needed.

Example 6:
Mass spectrometry of Poly(cytidine diphosphate ribose)
[0050] Sample of Poly(cytidine diphosphate ribose) dissolved in water was submitted for MALDI-TOF mass spectrometry analysis at the Centre for Cellular and Molecular Biology (CCMB), Hyderabad. There were two major peaks at 3922.4 and 3671.8 m/z which roughly correspond to the 7 and 8 mer Poly(cytidine diphosphate ribose), respectively.
Comparative Example
The poly (cytidine diphosphate ribose) has more negative charge per repeating unit compared to before mentioned highly used anionic biopolymers like hyaluronic acid, polyacrylates, alginate, etc.
Anionic Polymer Negative Charge per unit
Poly (cytidine diphosphate ribose) -2
Hyaluronic acid -1
Polyacrylates -1
Alginate -1
Poly styrene sulfonate -1


SPECIFIC EMBODIMENTS OF THE PRESENT INVENTION
[0051] The present disclosure relates to an anionic polymer comprising Poly (cytidine diphosphate ribose) of Formula (I),
(I)
wherein n is 1 to 50 units.

[0052] Such an anionic polymer wherein the molecular weight of the anionic polymer is in the range of 534- 26,700 Da (Daltons).
[0053] The present disclosure also relates to a method of preparing an anionic polymer comprising Poly (cytidine diphosphate ribose), comprising the steps of
b) incubating a poly(ADP-ribose) polymerase 1 (PARP-1) enzyme with 18 base pairs double strand DNA in reaction buffer to obtain reaction mixture containing PARP1 bound DNA;
c) adding an aqueous solution of Nicotinamide cytosine dinucleotide (NCD) in the reaction mixture;
d) adding ice cold trichloroacetic acid (TCA) for a time period of 10 mins to 20 minutes to precipitate out the pellet of Poly (cytidine diphosphate ribose) covalently attached to PARP1;
e) cleaving Poly (cytidine diphosphate ribose) from the Poly (cytidine diphosphate ribose) covalently attached to PARP1 and separating it from the reaction mixture.

[0054] Such a method, wherein in step b) the reaction mixture is incubated for a period of 2 to 4 hours after adding NCD
[0055] Such a method, wherein concentration of PARP1 enzyme is in range of 1 µM- 20 µM
[0056] Such a method, wherein the concentration of NCD is in range of 2- 5 mM
[0057] Such a method, wherein the Poly (cytidine diphosphate ribose) is extracted from Poly (cytidine diphosphate ribose) covalently attached to PARP1 by the method comprising the steps of:
a) resuspending the pellet in Tris EDTA buffer;
b) adding DNasel to digest the DNA followed by adding Proteinase K to degrade the kiPARP1; and
c) cleaving Poly (cytidine diphosphate ribose) from the PARP 1.

[0058] Such a method, wherein in step b) the solution is incubated for a period of 30 minutes to 2 hours at a temperature of 35 to 40 °C.
[0059] Such a method, wherein the sodium dodecyl sulfate (SDS) and proteinase K is incubated for 2 to 3 hours at a temperature of 45 to 50 °C.
[0060] Such a method, wherein the Poly (cytidine diphosphate ribose) is cleaved from the PARP1 using potassium hydroxide (KOH) and ethylene diamine tetra acetic acid (EDTA) by incubating at a temperature of 60 to 65 °C for 2 to 3 hours.
, Claims:WE CLAIM
1. An anionic polymer comprising
Poly (cytidine diphosphate ribose) of Formula (I),
(I)
wherein n is 1 to 50 units.

2. The anionic polymer as claimed in claim 1, wherein the molecular weight of the anionic polymer is in the range of 534- 26,700 Da (Daltons).

3. A method of preparing an anionic polymer comprising Poly (cytidine diphosphate ribose), comprising the steps of
a) incubating a poly(ADP-ribose) polymerase 1 (PARP-1) enzyme with 18 base pairs double strand DNA in reaction buffer to obtain reaction mixture containing PARP1 bound DNA;
b) adding an aqueous solution of Nicotinamide cytosine dinucleotide (NCD) in the reaction mixture;
c) adding ice cold trichloroacetic acid (TCA) for a time period of 10 mins to 20 minutes to precipitate out the pellet of Poly (cytidine diphosphate ribose) covalently attached to PARP1;
f) cleaving Poly (cytidine diphosphate ribose) from the Poly (cytidine diphosphate ribose) covalently attached to PARP1 and separating it from the reaction mixture.


4. The method as claimed in claim 3, wherein in step b) the reaction mixture is incubated for a period of 2 to 4 hours after adding NCD

5. The method as claimed in claim 3, wherein concentration of PARP1 enzyme is in range of 1 µM- 20 µM

6. The method as claimed in claim 3, wherein the concentration of NCD is in range of 2- 5 mM

7. The method as claimed in claim 3, wherein the Poly (cytidine diphosphate ribose) is extracted from Poly (cytidine diphosphate ribose) covalently attached to PARP1 by the method comprising the steps of:
a) suspending the pellet in Tris EDTA buffer;
b) adding DNasel to digest the DNA followed by adding Proteinase K to degrade the PARP1;and
c) cleaving Poly (cytidine diphosphate ribose) from the PARP1

8. The method as claimed in claim 7, wherein in step b) the solution is incubated for a period of 30 minutes to 2 hours at a temperature of 35 to 40 °C.

9. The method as claimed in claim 7, wherein the sodium dodecyl sulfate (SDS) and proteinase K is incubated for 2 to 3 hours at a temperature of 45 to 50 °C.

10. The method as claimed in claim 7, wherein the Poly (cytidine diphosphate ribose) is cleaved from the PARP1 using potassium hydroxide (KOH) and ethylene diamine tetra acetic acid (EDTA) by incubating at a temperature of 60 to 65 °C for 2 to 3 hours.

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