MATE AND SEPARATE: A CONVENIENT AND GENERAL METHOD FOR THE SEPARATION AND PURIFICATION OF TARGET MOLECULES FROM BIOLOGICAL MEDIA BY PHASE TRANSITION OF PEGYLATED RECOGNITION AGENTS.
20220411463 · 2022-12-29
Inventors
- Khashayar KARIMIAN (Tehran, IR)
- Javad MOKHTARI (Tehran, IR)
- Amir Hassan ZARNANI (Tehran, IR)
- Aliakbar MOOSAVI-MOVAHEDI (Tehran, IR)
- Maryam NOURISEFAT (Bushehr, IR)
- Bita ZAMIRI (Tehran, IR)
- Leila FOTOUHI (Tehran, IR)
Cpc classification
A61K47/60
HUMAN NECESSITIES
C12N15/1006
CHEMISTRY; METALLURGY
International classification
Abstract
Biological small molecules, proteins or nucleic acids (target molecules, TM) are isolated in from biological media such as blood serum, cytoplasm, nucleoplasm etc. by a novel process (mate and separate) involving the use of PEGylated recognition molecule (PEG-RM) with high specificity and binding for TM, affording a macromolecular complex PEG-RM.TM, from which the target protein can be obtained in pure form.
Claims
1. A recognition macromolecule (PEG-RM) composed of a PEGylated recognition macromolecule (PEG-RM) and a target molecule (TM) in which the recognition molecule (RM) is covalently attached to a high molecular weight polyethylene glycol and TM is absorbed to PEG-RM by fundamental interactions including electrostatic (ionic and H-bonding, van der Waals (dipole-dipole), hydrophobic and p interactions, which afford high specificity and binding between PEG-RM and TM and where RM and TM can each be a protein, a nucleic acid or a small molecule and permutations thereof.
2. A product of claim 1 in which the recognition molecule (RM) is a small molecule.
3. A product of claim 1 in which the recognition molecule (RM) is a protein.
4. A product of claim 1 in which the recognition molecule (RM) is a nucleic acid.
5. A product of claim 1 in which the recognition molecule (TM) is a small molecule.
6. A product of claim 1 in which the recognition molecule (TM) is a protein.
7. A product of claim 1 in which the recognition molecule (TM) is a nucleic acid.
8. A process of formation of the macromolecular complex (PEG-RM. TM) in homogeneous media in which the recognition macromolecule (PEG-RM) is added to a biological medium such as blood serum, cell cytoplasm or nucleoplasm or mitochondrial matrix which contains the target molecule (TM).
9. A process of formation of a macromolecular complex (PEG-RM. TG) in which the recognition molecule (RM) is a small molecule, a protein or a nucleic acid and the target molecule (TM) can be a small molecule, a protein or a nucleic acid and the resulting nine permutations thereof.
10. A process of salt-assisted phase transition of a macromolecular complex (PEG-RM. TM) in which the water soluble macromolecular complex (PBG-RM.TM) separates as a solid or a semisolid from a biological medium such as blood serum, cell cytoplasm or nucleoplasm or mitochondrial matrix by the addition of a minimum quantity of salts, preferably ammonium sulfate.
11. A process of separating the solid or a semisolid macromolecular complex of claim 10 (PEG-RM.TM) by filtration.
12. A process of separating the solid or a semisolid macromolecule complex of claim 10 (PEG-RM.TM) by or centrifugation.
13. A process of convening the macromolecular complex (PEG-RM.TM) to its components (PBG-RM+TM) by firstly dissolving it in an appropriate buffer, preferably Tris buffer, and secondly reducing fee pH of the buffer.
14. A process of con veiling the macromolecular complex (PEG-RM.TM) to its components (PEG-RM+TM) by firstly dissolving it in an appropriate buffer, preferably Tris buffer, and secondly Increasing the ionic strength of the buffer by the addition of an appropriate quantity of salt.
15. A process of converting the recognition macromolecule (PEG-RM) from solution to a semisolid by the addition of a minimum quantity of salt, preferably ammonium sulfate.
16. A process of separating the semisolid recognition macromolecule (PEG-RM) by filtration and obtaining the pure or highly pure target (TM) molecule in solution.
17. A process of separating the semisolid recognition macromolecular (PEG-KM) by centrifugation and obtaining the pure or highly pure target (TM) molecule in solution
18. A homogenous process for separation of target molecules (TM) in which no leaching of the recognition molecule (RM) is observed.
19. A homogenous process for separation of proteins as target molecules (TM) in which no aggregate of the target molecule is observed
20. (canceled)
21. (canceled)
22. (canceled)
Description
BRIEF DESCRIPTION OF THE FIGURES
[0011]
[0012]
[0013]
[0014]
[0015]
[0016]
[0017]
DETAILED DESCRIPTION OF THE INVENTION
[0018] The separate and separate concept is based on 3 fundamental chemical principles:
[0019] First, biological target molecules (TM=protein, nucleic acid, small molecule with biological activity) are very likely to have a recognition molecule (RM) to which they are adsorbed with high specificity and binding.
[0020] Second, high specificity and binding of RM for TM is a result of electrostatic (ionic and H-bonding), van der Waals (dipole-dipole), hydrophobic and to a lesser extent π interaction between PEG-RM and TM. These are fundamental forces in binding (mating) of two molecules in chemistry and are therefore the mate and separate concept is applicable to most biological molecules.
[0021] Third, PEG has very unique physico-chemical properties and while it is very soluble in water, it undergoes phase transition with the addition of small quantity of a salt such as ammonium sulfate, separating as a solid or a semi-solid from aqueous solutions.
[0022] In light of the above, PEGylation of the recognition molecule (RM) affords a recognition macromolecule (PEG-RM) that will attach to its target molecule (TM) with high specificity and binding (mate) to form a macromolecular complex (PEG-RM.TM). Due to the presence of PEG, PEG-RM.TM can be easily separated from the aqueous biological medium by the addition of ammonium sulfate (separate). Separation occurs because the macromolecular complex (PEG-RM.TM) is endowed with the unique physico-chemical properties of PEG. The macromolecular complex is then isolated by filtration or centrifugation. It is then dissolved in a buffer and dissociated to its components (PEG-RM+TM) by decreasing the pH or increasing the ionic strength or the temperature of the solution which disrupts the fundamental parameters involved in absorption of two molecules to each other, namely electrostatic (ionic and H-bonding), van der Waals (dipole-dipole), hydrophobic and to a lesser extent π interaction between PEG-RM and TM. Being endowed with the unique physico-chemical properties of PEG, the recognition macromolecule (PEG-RM) can be separated from the solution as a semisolid by the addition of a salt such as ammonium sulfate. Separation of the recognition macromolecule leaves the target molecule in solution (Scheme 1).
[0023] Proof of principle for the mate and separate concept was obtained using proteins as RM and TM, small molecules as RM and TM and nucleic acids as RM and TM to afford 9 different permutations. To this end, RMs were attached to PEGs of various molecular weight using PEGylation reactions shown in Scheme 2-4.
##STR00002##
##STR00003##
##STR00004##
[0024] The mate and separate concept was then validated using proteins as RMs and as TMs as shown on Scheme 5.
##STR00005##
[0025] PEGylated Protein A was used for the isolation and purification of Igs (IgA, IgC, IgD, IgG, IgM) and sheep anti-human IgG was used for the isolation and purification of IgG. Results are provided in
[0026]
[0027]
##STR00006##
[0028] Results are shown in
[0029]
##STR00007##
[0030]
[0031] Proof of principle for the interchangeability of RM with TM and vice versa is summarized in Formulae 1 and 2. Where A and B can be either RM or TM and M.sub.n represents the innumerable molecules that are present in a biological solution such as blood serum, cell cytoplasm, nucleoplasm, etc.
##STR00008##
[0032] In Formulae 1 and 2 the steps are as follows: [0033] 1. Addition of PEGylated recognition molecule to biological medium containing the target molecule (A) as well as innumerable other molecules (M.sub.n) [0034] 2. Formation of macromolecular complex, its phase transition to a semisolid by the addition of salt and separation of the macromolecular complex by filtration or centrifugation [0035] 3. Dissociation of the isolated macromolecular complex to recognition macromolecule and target molecule [0036] 4. Phase transition of recognition macromolecule to a semisolid by the addition of salt to a semisolid and its separation by filtration or centrifugation, leaving the pure target molecule in solution
[0037] Interchangeability of A and B and vice versa is due to the commonality of the fundamental forces that result in high specificity and binding (i.e. electrostatic, van der Waals, hydrophobic and to a lesser extent π interaction). Perhaps the simplest example is the binding of an antibody (RM) to an antigen (TM) or the binding of an antigen (RM) to an antibody (TM).
[0038] Finally, the mate and separate concept was validated using 2 nucleic acid as RMs and as TMs as shown on Scheme 8.
##STR00009##
[0039] Results are shown in
[0040]
[0041] (see Experimental Section)
[0042] It should be noted that only m-RNA is capable of producing c-DNA by PCR. The formation of c-DNA shown in
[0043] The mate and separate processes for the isolation and purification of proteins, nucleic acids and small molecules has a number of other advantages. With regard to proteins, we used a very high molecular weight PEG for the PEGylation of the recognition molecule RM (especially when RM=protein) for the following reasons:
[0044] First, to prevent multi-PEGylation since this involves heteroatoms such as SH, NH and OH functional groups of the peptide which are also involved in the recognition of the TM by electrostatic (ionic and H-bonding), van der Waals (dipole-dipole), hydrophobic and to a lesser extent π interactions [50].
[0045] Second, to ensure dominance of the precipitative character of PEG over the physicochemical properties of the macromolecular complex (PEG-RM.RM), which guarantees phase transition of (PEG-RM.RM) to a semisolid at low salt concentration, concomitant with increased solubility of unbounded proteins.
[0046] Third, the use of a low salt concentration, in addition to increasing the solubility of proteins, is known to decrease nonspecific protein-protein interaction, thereby providing a better chance for high specificity and binding of PEG-RM to the target protein (TM) [51, 52] In light of the above, the salting-in of unbounded proteins and salting out of PEG-RM.TM as a function of salt concentration should follow the schematic representation in
[0047]
[0048] In fact, salting-in of unbounded proteins at low salt concentration concomitant with salting-out of high molecular weight PEG is a fortuitous coincidence that provides an ideal condition for salt-assisted phase transition of PEG-RM.TM.
[0049] Furthermore, it is important to note that in the absence of ammonium sulfate mating of PEG-RM with TM to afford PEG-RM.TM is an equilibrium phenomenon and according to the Le Chatelier Principle the addition of salt shifts the equilibrium to the formation of the PEG-RM.TM as it departs from the aqueous medium in the form of semisolid (salt-assisted phase transition) [53]. It is also important to note that protein isolation by the mate and separate concept does not cause leaching of recognition protein (e.g. protein A or sheep anti-human IgG). This can be seen in
[0050] Equally as important as the above-mentioned advantages is the fact that the proteins isolated and purified from the mate and separate process are devoid of aggregates. As indicated above, aggregate formation is a major challenge in mAb production, especially in view of the fact that such protein entities with immunogenic properties can form in both the upstream and the downstream production of mAbs as well as their formulation, transport and storage [54, 55]. Our initial results based on SDS PAGE (see
[0051]
[0052]
[0053] As regards the isolation and purification of nucleic acids using the mate and separate process, the use of Ammonium sulfate for the precipitation of PEG-Nucleic Acid. Nucleic acid has been reported to stabilize m-RNA [58] by inhibiting various RNases. Inhibition is caused by competition of sulfate moiety of ammonium sulfate with the phosphate moiety of RNA [59]. In light of the simplicity of the process, purity of the isolated product (proteins, nucleic acids or small molecules) as well as interchangeability of recognition molecule with the target molecules and vice versa, the actual and potential utility of the mate and separate process are summarized in Table 1.
TABLE-US-00001 TABLE 1 Various permutation of the mate and separate concept No (PEG-RM) (Target Molecule = TM) Examples of Utility 1 RM = Small Molecule Protein See Example 7 (Salicylic Acid) (Human Serum Albumin) 2 RM = Small Molecule TM = Nucleic Acid On-going work in our laboratories Bleomycin binding to a specific base sequence in nucleic acids 3 RM = Protein TM = Small Molecule See Example 2 (Human Serum Salicylic Acid Albumin) 4 RM = Protein TM = Protein See Examples 1 (Protein A or Sheep (Igs or IgG) Anti-human IgG) 5 RM = Protein TM = Nucleic Acid On-going work in our laboratories Isolation repressor or activator segments of genes 6 RM = Nucleic Acid TM = Small Molecule On-going work in our laboratories Daunomycin binding to a specific base sequence in nucleic acids 7 RM = Nucleic Acid TM = Protein On-going work in our laboratories Isolation repressor or activator segments of genes 8 RM = Nucleic Acid TM = Nucleic Acid Isolation of m-RNA using OdT.sub.40
Experimental
[0054] Material and Methods
[0055] All reagents including PEG-1,000,000 were obtained from commercially available sources such as Sigma-Aldrich, Fluka or Merck and were used without further purification. Human blood was obtained from the peripheral vein of healthy donor with sterilized disposable plastic syringe. The blood was preserved without anticoagulant in sterilized test tubes in vertical position until clot was formed. The clot was then removed from the test tube gently with a glass rod or a swab and the serum was centrifuged at 1000 g for 10 minutes.
[0056] 1. Isolation of Igs or IgG Using PEGylated-Protein A or PEGylated-Sheep Anti-Human IgG
[0057] 1.1. Monomethylation of PEG-1,000,000. To a solution of PEG-1,000,000 (50 g, 0.05 mmol) in 500 ml of dioxane was added freshly distilled triethylamine (7 μl, 0.05 mmol). Methyl iodide (3.1 μl, 0.05 mmol), dissolved in 20 ml of dry dioxane, was added dropwise over 1 h at 25° C. and the mixture was stirred for 24 h at room temperature. Diethyl ether (500 ml) was added slowly and the resulting white precipitate was filtered and washed with cooled dioxane and then diethyl ether. After drying to a constant weight, 49 g of product was obtained (97%).
[0058] 1.2. Activation of MeO-PEG-OH by N,N-Carbonyl Diimidazole. To the solution of monomethyl PEG-1,000,000 (20 g, 0.02 mmol) in 250 cc dioxane was added N,N-carbonyl diimidazole (0.032 g, 0.2 mmol). The reaction mixture was heated to 60° C. for 24 h, cooled to room temperature and 250 cc diethyl ether was slowly added. The resulting white precipitate was filtered and washed with cooled dioxane and diethyl ether. After drying to a constant weight, 19 g of activated PEG was obtained (95%).
[0059] 1.3. Coupling of Recognition Protein and activated PEG (RM=Sheep anti-human IgG or PEG-Protein A). To a solution of Sheep-Anti Human IgG (1 ml, 0.5 mg/ml, 3.3×10.sup.−6 mmol) or Protein A (1 ml, 0.5 mg/ml, 3.3×10.sup.−6 mmol) in 4 ml PBS buffer with pH=7.2 was added activated PEG (66 mg, 6.66×10.sup.−5 mmol). The resulting solution was gently shaken at 0-10° C. for 10 days.
[0060] Completion of the coupling was monitored by Native PAGE. A saturated solution of ammonium sulfate was then added to the reaction mixture. The semisolid material was separated by centrifugation. The semisolid was washed with saturated solution of ammonium sulfate (1 ml), and centrifuged again. The semisolid residue (PEG-Sheep anti-human IgG or PEG-Protein A) was used for the separation of IgG or Ig from the blood serum.
[0061] 1.4. solation and Purification of IgG from Blood Serum with PEG-RM (RM=Sheep-anti human IgG or PEG-Protein A). General procedure for IgG purification using the synthesized PEG-RM and human serum: Blood was withdrawn and diluted with 1 ml PBS buffer pH 7.2. Blood cells were separated from plasma by centrifugation (500×g during 5 min). PEG-RM was then added to the diluted sample of human serum and gently shaken for 45 min at room temperature. A saturated solution of ammonium sulfate (0.5 ml) was added to the mixture which resulted in salt assisted phase transformation of PEG-RM.IgG to a semisolid in about 2 minutes. The semisolid was separated by centrifugation at 3000 RPM for 5 minutes. PBS buffer (0.5 ml) was added to the semisolid and solution was gently mixed to wash away unbound serum components, followed by centrifugation and decantation. The wash procedure was repeated two more times by the addition of ammonium sulfate, separation of the semisolid and the addition of Phosphate-buffered saline (PBS, 1 ml). The macromolecular complex PEG-RM. IgG was then dissociated to its components (PEG-RM+IgG) by the addition of an acidic buffer (e.g., 0.15 M glycine-HCl, pH 2.5). PEG-RM was removed as a semisolid by the addition of ammonium sulfate and centrifugation as discussed above. The final solution contained pure IgG or Ig and ammonium sulfate, which was removed by dialysis in 0.15 M PBS buffer, pH 7.2, twice over a 2-h at a ratio of 1 volume of sample to 100 volume of buffer. PEG-RM was recovered in its purified form by washing 3 times with 0.15 M PBS buffer, pH 7.2 and stored in the buffer containing 0.01% NaN.sub.3.
[0062] 2. Isolation of Pure Serum Albumin Using PEGylated-Salicylic Acid
[0063] 2.1. Activation of OH-PEG-OH with Phosphorous Tribromide. The process of Bückmann et al. [60] was used with minor modification. PEG 1,000,000 (50 g, 0.05 mmol) was dissolved in 500 ml of toluene followed by distillation of 100 ml of the solvent to remove trace of moisture. After cooling to 35° C., freshly distilled anhydrous triethylamine (0.054 g, 0.54 mmol) was added. Phosphorus tribromide (0.146 g, 0.54 mmol), dissolved in 20 ml of dry toluene, and was then added dropwise over 1 h at 35° C. under a dry nitrogen atmosphere with continuous stirring. The mixture was refluxed for 1 h. Triethylammonium bromide byproduct was removed by passing the hot solution through a bed of Celite. The filtrate was stored at 4° C. overnight, affording activated PEG, which was filtered at 4° C. The solid product was further purified by dissolving in 2.5 l of absolute ethanol at 60° C. The ethanolic filtrate was stored overnight at 4° C. to recrystallize the product. The solid material was separated by filtration and washed with cold ethanol and then ether. After drying in a vacuum desiccator 49 g of a pale yellow product was obtained (98%).
[0064] 2.2. Coupling of salicylic acid and activated PEG. To a solution of salicylic acid (1.0 g, 7.24 mmol) in 100 ml DMF was added potassium carbonate (2.0 g, 14.48 mmol), followed by activated PEG (10 g, 0.01 mmol). The resulting solution was heated to 80-90° C. for 10 hours. Reaction completion was monitored by TLC. The mixture was then cooled to ambient temperature and diethyl ether (200 ml) was added drop-wise with stirring. The resulting precipitate was filtered and washed with diethyl ether and dried to a constant weight (9.8 g, 98% Yield).
[0065] 2.3. Isolation and Purification of Serum Albumin with PEGylated Salicylic Acid. Blood serum was obtained as above. PEG-salicylic acid was added to a diluted sample of human serum (10 μl) and gently shaken for 45 min at room temperature. A small quantity of a saturated solution of saturated ammonium sulfate (ca. 0.5 ml) was then added to the solution causing immediate formation of a PEG-Salicylic Acid.Serum Albumin as a semisolid, which was centrifuged at 4000 RMM for 5 minutes and decanted. The semisolid was washed twice with a saturated solution of ammonium sulfate (1 ml) to ensure removal of unbounded proteins and other potential impurity. The final semisolid was dissolved in 10% acetic acid to disrupt molecular interactions such as electrostatic, van der Waals, hydrophobic and to a lesser extent π interactions required for high affinity and specificity of RM for TM. The mixture was stirred for 10 minutes to cause dissociation of PEG-Salicylic Acid.Serum Albumin to PEG-Salicylic Acid+Serum Albumin. Addition of a small quantity of saturated solution of ammonium sulfate resulted in salt-assisted phase transition of PEG-Salicylic Acid to a semisolid, leaving pure serum albumin in solution. Ammonium sulfate was removed by dialysis in 0.15 M PBS buffer, pH 7.2, twice over a 2-h period at a ratio of 1 volume of sample to 100 volume of buffer. The recognition macromolecule PEG-RM was recovered in its purified form by washing 3 times with 0.15 M PBS buffer, pH 7.2 and stored in the buffer containing 0.01% NaN.sub.3. It can be used in the next cycle of isolation and purification of serum albumin.
[0066] 3. Isolation of Salicylic Acid from Mixture of Drugs Using PEGylated-Human Serum Albumin
[0067] 3.1.Coupling of Human Serum Albumin and Activated PEG
[0068] The process of experiment 3.1 (coupling of protein A and activated PEG) was used.
[0069] 3.2. Isolation and Purification of Salicylic Acid from an Equimolar Mixture of Drugs
[0070] Using PEGylated Human Serum Albumin. An equimolar mixture of salicylic acid (0.05 mg, 0.375 μmole), deferiprone (0.051 mg, 0.375 μmole), capecitabine (0.134 mg, 0.375 μmole) was prepared in 0.5 cc PBS buffer with pH 7.2. The PEGylated human serum albumin (375 mg, 0.375 μmole) was added to the drug mixture and the solution was gently shaken for 1 hour at room temperature. A small quantity of a saturated solution of saturated ammonium sulfate (ca. 0.5 ml) was then added to the solution causing immediate formation of a PEG-HSA. “Drug” as a semisolid, which was centrifuged at 4000 RMM for 5 minutes and decanted. The semisolid was washed twice with a saturated solution of ammonium sulfate (1 ml) to ensure removal of unbounded proteins and other potential impurity. The final semisolid was dissolved in 5% acetic acid to disrupt molecular interactions such as electrostatic, van der Waals, hydrophobic and to a lesser extent π interactions required for high affinity and specificity of RM for TM. The mixture was stirred for 10 minutes to cause dissociation of PEG-HSA. “Drug” to PEG-HAS+“Drug”. Addition of a small quantity of saturated solution of ammonium sulfate resulted in salt-assisted phase transition of PEG-HSA to a semisolid, leaving pure “Drug” in solution. The aqueous solution was evaporated under reduced pressure to dryness. To the solid mixture containing the “Drugs” was added ethyl acetate, filtered and the solvent was remove under reduced pressure to afford an oil which was subjected to HPLC chromatography which afford good separation of the drugs. Column: RP-C18, 300*4.0 mm, 10 micron: Mobile Phase: 550 ml 0.02 molar KH.sub.2PO.sub.4 buffer, pH 2.5 and 450 ml acetonitrile). The HPLC chromatogram showed 77% salicylic acid and 5% capecitabine and 3% deferiprone.
[0071] 4. Isolation of WBC m-RNA Using PEGylated-OdT.sub.40
[0072] 4.1. Synthesis of MeO-PEG-NH.sub.2. The process of Bückmann et al. [58] was used with minor modification. Monomethoxylated PEG 35,000 (20.0 g, 0.57 mmol) was dissolved in 500 ml dry toluene. After cooling to 35° C., freshly distilled anhydrous triethylamine (0.06 g, 0.6 mmol) was added. Phosphorus tribromide (0.162 g, 0.6 mmol), dissolved in 20 ml of dry toluene, and was then added dropwise over 1 h at 30° C. under a dry nitrogen atmosphere with continuous stirring. The mixture was refluxed for 1 h. Triethylammonium bromide byproduct was removed by passing the hot solution through a bed of Celite. The filtrate was stored at 4° C. overnight, affording activated PEG, which was filtered at 4° C. The solid product was further purified by dissolving in 100 ml of absolute ethanol at 60° C., treated with decolorizing charcoal, filtered and the ethanolic filtrate was stored overnight at 4° C. to recrystallize the product. The solid material was separated by filtration and washed with cold ethanol and then ether. After drying in a vacuum desiccator 18 g of a pale yellow product was obtained (90%). The halogenated PEG was added slowly to a 100 ml 15% ethanolic solution of ammonia (large excess) and refluxed for 24 hours. The solvent was removed under reduced pressure and residual ammonia was removed by the addition of 50 ml ethanol followed by its removal under reduced pressure. The resulting solid was uses in the next step.
[0073] 4.2. Coupling of OdT.sub.40 and activated PEG. A solution of 0.1 molar imidazole was prepared by the addition of 340 mg imidazole to 50 ml PBS buffer, containing 10 mM EDTA, pH 7.2. MeO-PEG-NH.sub.2 (PEG 35,000, 8.75 g, 0.25 mmol) was added to the imidazole buffer solution, resulting in a 0.25 molar solution of MeO-PEG-NH.sub.2. A 100 μl aliquot portion of this solution was added to a microtube (Solution A). In a separate microtube, 1.25 mg (8 μmole) EDCE (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide was prepared in 20 μl of the PBS buffer followed by the addition of 252 μl of a 1000 μmolar solution of OdT.sub.40 corresponding to 60 μg OdT.sub.40(Solution B). Solution A (20 μl) was then added to Solution B and mixed well, followed by the addition of a second portion of solution A (80 μl). The mixture was gently shaken for 24 hour at 37° C. Agarose gel electrophoresis was used to ascertain reaction completion, at which point the mixture was passed through a small anion exchange resin (1.0 g) using the PBS buffer.
[0074] 4.3 Separation and Isolation of pure m-RNA from White Blood Cells. To 1 ml human blood was added RBC Lysis buffer (10 ml) and the mixture was gently shaken for 10 minutes. The mixture was then centrifuged (4° C., 4000 rpm, 5 minutes). Red blood cells were removed by repeating the process. Total RNA was extracted using RNX-Plus from CinnaClone (1 ml) and gently vortexed for 5-10 seconds and incubated the mixture for 5 minutes at room temperature. To the resulting suspension was added chloroform (200 μl) and the mixture was gently shaken for 15 seconds and incubated on ice for 5 minutes, followed by centrifugation (4° C., 12,000 rpm, 15 minutes). The top layer containing total RNA was carefully transferred to a 1.5 ml microtube. An equivalent volume of isopropanol was added and the mixture gently shaken and incubated on ice for 15 minutes, followed by centrifugation (4° C., 12,000 rpm, 15 minutes). The process was repeated using 75% ethanol and the mixture was centrifuged (4° C., 7500 rpm, 15 minutes). The top layer was carefully removed and to the pellet was added DEPC water (50 μl) and the solution was examined for total RNA content by measuring OD ratio at 260/280 nm using a Nanodrop instrument (Thermo-Fischer) and agarose gel electrophoresis (
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