Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

• the first step includes laboratory production of or isolation and purification of the desired gene which codes for a particular protein.
• the second step is the recombination of the gene with a proper transfer vector such as a plasmid.
• the third step includes transferring the recombined vector into a particular microorganism and inducing the microorganism to produce the particular gene product.
• the present invention is directed towards a method of accomplishing the first step.
• Current methodology for in vitro production of genes by sequential addition of nucl eo tides consists of using chemical techniques (Itakura, K. and Riggs, A., Science 109:1401 (1980), (Khorana, H.G., Science 203:614 (1979), enzymatic procedures (S. Gillam, P. Jahnke, C. Astell, S. Phillips, CA. Hutchinson, M. Smith, Nucelic Acids Res. 6,2973 (1979) and solid phase techniques to produce the desired DNA or RNA molecules.
• the chemical procedures of DNA synthesis are tedious because the reactions involved are non-specific such that extensive purification of the desired product is necessary after each operative step.
• RNA and DNA by chemical techniques is significantly less desirable than enzymatic systems because greater amounts of starting material are required to produce comparable yields thereby increasing costs, the purification techniques are both time consuming and wasteful where some product is lost at each step, and the large number of steps involved in blocking and unblocking reactive sites provide many opportunities for error and risk of contamination by degradative enzymes which destroy the synthesized product.
• RNA using polynucleotide phosphorylase and T4 RNA ligase have been reported.
• the polynucleotide phosphorylase method is limited to production of oligodeoxynucleo tides.
• poly nucleotide phosphorylase adds predominantly a single nucleotide to a short oligodeoxynucl eotide which is then isolated by chromatogrpahic techniques.
• Solid phase techniques are performed by binding the nucleotide chain to a solid support material and using the above chemical methodology to add nucleotides stepwise.
• the present invention teaches the composition and method of production of a compound useful in the process of synthesizing a predetermined sequence of RNA using the enzyme T4 RNA ligase.
• Processes for transcribing the synthesized RNA into DNA so that the synthesized gene may be incorporated into a plasmid, inserted into a microorganism and expressed, are known in the art.
• T4 RNA ligase which is isolated from Escherichia coli infected with bacteriophage T4 have been described (Silber, R., Malathi, V.G. & Hurwitz, J., Proc. Natl. Acad. Sci. (1972) 69:3009). This enzyme catalyzes the formation of a phosphodiester bond between the phosphate group on the 5' end of a donor nucleotide and the hydroxyl group on the 3' end of the recipient oligonucl eotide as shown below.
• Japanese Patent Application 1980-19003 (published February 9, 1980 teaches the utilization of T4 RNA ligase to extend a polynucleotide by adding a single mononucleoside diphosphates (pNp) onto the 3' end of a nucleotide sequence with no terminal phosphate groups.
• This methodology requires as the starting substrate a trinucl eotide, which must either be obtained commercially or synthesized by chemical means, and requires adenosine triphosphate (ATP) as an energy source for the reaction.
• ATP adenosine triphosphate
• the invention teaches only the addition of a single base and is not capable of producing oligo or polynucleotides for which sequential addition is required.
• HO A p A p A OH + pCp + ATP >HO A p A p A p C p + AMP + PPi
• This invention teaches the synthesis of biologically active RNA and DNA molecules. All nucleotide sequences are indicated by their abbreviations according to Table 1. All phosphodiester linkages between nucleotides are 3'-5' unless otherwise indicated.
• the invention disclosed herein teaches methods of synthesizing single stranded RNA molecules of predetermined base sequence using the enzyme T4 RNA ligase to specifically add a mono-, di-, oligo- or polynucl eotide (the donor) onto the 3' end of a ribonucleotide sequence two bases or longer (the recipient) in a 5'--3' linkage.
• the shortest recipient which the T4 RNA ligase will act upon in the catalysis is a dinucl eotide phosphorylated in the 5' position.
• the 5' terminal end may be either phosphorylated or hydroxylated to allow specific addition of the donor to the 3' end of the recipient.
• the first step is to phosphorylate the free 5' end. This step is performed by mixing the dinucl eotide with ATP in the presence of the enzyme polynucl eotide kinase (PNK) (Richardson, C.C., Proc. Natl. Acad.
• PNK polynucl eotide kinase
• the second step is to mix the recipient dinucl eotide with the chemical compound p 1 -Adenosine, p 2 -(3' nucleotide monophosphate)-5' pyrophosphate of the form (AppNp), where N is a ribonucleotide selected from the group adenine, cytosine, guanine, uracil, and inosine.
• AppNp a ribonucleotide selected from the group adenine, cytosine, guanine, uracil, and inosine.
• This invention also discloses a method of adding a donor RNA molecule of 2 bases or longer to a recipient ribonucleotide such that 5' phosphate on the donor binds specifically with the 3' hydroxyl group of the recipient in the presence of ATP and RNA ligase to form an extended ribonucleotide (equation 3).
• the phosphate group on the 3' end of the donor molecule makes that end unreactive to RNA ligase so as to prevent further addition of the donor molecules.
• the 5' phosphoryl ated dinucl eotide is reacted with the compound Appnbzl in the presence of RNA ligase (equation 4).
• the nbzl moiety may be removed from the resul ting nucl eotide by exposure to strong light (equation 5).
• the resulting extended ribonucleotide can then be treated with bacterial alkaline phosphatase (BAP) which cleaves terminal phosphate groups preparing it to act as a recipient or it can be treated with polynucl eotide kinase (PNK) phosphorylating the 5' end thereby converting it to a donor (equation 6).
• BAP bacterial alkaline phosphatase
• PNK polynucl eotide kinase
• RNA molecules coding for particular proteins may be synthesized.
• the present invention teaches the utilization of T4 RNA ligase in the preparation of nucleotide of a predetermined base sequence.
• two methods are disclosed.
• One method teaches the addition of donor mononucl eotide to a recipient ribonucleotide of at least two bases.
• the other method teaches the addition of donor nucleotides having two baes or longer. The decision as to which of these methods should be used is dependent on the composition of the nucleotide base sequence desired and the availability of commercially prepared nucleotides to be used as starting material.
• the phosphorylated dinucl eotide is isolated from the other components of the reaction mixture. This is achieved by fractionating the reaction mixture and treating the pooled fractions containing the phosphorylated dinucl eotide with ATPase which cleaves a pyrophosphate from ATP contaminating the phosphorylated dinucl eotide pool. Isolation of the phosphorylated dinucl eotide by fractionation is then possible.
• the ATPase used in Example 1 below was obtained and purified as a side product of the T4 RNA ligase purification however, any ATPase should be effective.
• the phosphorylated dinucl eotide or other form of recipient nucleotide is then incubated with an AppNp compound where the specific compound to be used is one for which N is the nucleotide to be added to the 3' end of the dinucl eotide.
• AppNp compound where the specific compound to be used is one for which N is the nucleotide to be added to the 3' end of the dinucl eotide.
• the resultant nucleotide may be treated with a phosphatase which removes temrinal phosphates leaving hydroxyl groups on the 5' and 3' termini.
• a phosphatase which removes temrinal phosphates leaving hydroxyl groups on the 5' and 3' termini.
• the conditions for the phosphatase reactions, dependent on which phosphatase is used, are well-known in the art.
• the phosphatase treated nucleotide is then prepared for subsequent additions as described below.
• Donor ribonucleo tides of two bases or more can be made to the 3' end of a recipient ribonucleotide by phosphorylating the 5' and 3' ends of said donor and incubating said phosphorylated donor with said recipient in the presence of RNA ligase and ATP.
• the recipient ribonucleotide for this reaction must be a 5' phosphorylated dinucl eotide or an oligonucl eotide with a phosphorylated or free 5' terminus.
• the ribonucleotide is incubated with Appnbzl in the presence of RNA ligase, thereby bonding the pnbzl moiety to the 3' end.
• the reaction mixture is boiled to inactivate the RNA ligase and then mixed with ATP in the presence of polynucl eotide kinase to phosphorylate the 5' end of said donor molecule.
• the donor is isolated and may then be exposed to strong light such as 300 watt mercury vapor lamp to remove the nbzl moiety leaving the donor phosphorylate at the 5' and 3' ends.
• the donor may be added to the recipient with or without removing the nbzl moiety.
• the resultant extended nucleotide may be treated with a phosphatase to remove all terminal phosphate groups thereby converting it to a form wherein it may act as a recipient.
• the extended nucleotide may be treated with PNK to phosphorylate the 5' end and allow it to be used as a donor.
• Adenylyl (3 ' - 5') guanosine (ApG) is first mixed with ATP in the presence of polynucl eotide kinase to form pApG.
• the reac tion mixture containing 70 ul of 15 mM AG (S IGMA) , 60 ul of 1M Tris-HCl (pH 8.1 ), 10 ul 2-mercaptoethanol , 100 ul of 100 mM MgCl 2 , 150 ul of 20 mM ATP , 50 ul H 2 O , and 100 ul PNK (1500 units/ml ) was incubated at 37° for 1 hour then fractionated by a DEAE Sephadex A-25 column on a linear gradien t of 0.2 to 0.8 M TEAB (pH 7.6 ).
• the fourth peak containing ATP and pApG as identified by UV absorption is pooled, brought to dryness with methanol under a vacuum, and then
• the pool ed frac tion is treated with ATPase and fractionated on a column.
• the resul ting product was fractionated on a DEAE Sephadex A-25 column on a 0.2 - 0.8 M TEAB (pH 7.6) linear gradient.
• the second major peak at 260 nm, the pApG fraction was pool ed.
• RNA l igase was obtained using the procedure of Sil b er et. al . 112 ul pApG ( .87 mM ) , 58 ul AppAp (3.4 mM ) , 15 ul 1 M HEPES pH 7.5 and 50 ul RNA l igase (1500 units/ml ) were mixed and incubated for 1 hour at room temperature. The resultant mixture was fractionated on DEAE Sephadex A-25, and the pApGpAp peak was identified by paper electrophoresis at pH 5.0 in 50 mM sodium citrate buffer. The pooled pApGpAp fraction was brought to dryness in the presence of methanol under vacuum and lyophilized.
• the sample was then treated with bacterial alkaline phosphatase (BAP).
• BAP bacterial alkaline phosphatase
• 40 ul pApGpAp, (approximately 1.0 unit at 260 nm.), 5ul 1M Tris-Hcl pH 8.1, 5 ul BAP C (200 units/ml, Worthington Biochemical), and 50 ul H 2 O was incubated for 30 min at 65°C and fractioned on a DEAE Sephadex A-25 column with a 0.1 to 1.0 M TEAB pH 7.6 linear gradient. The ApGpA containing fractions were pooled.
• the trinucl eotide GAU (Collaborative Research) was assayed for purity by electrophoresis.
• reaction mixture was then boiled for two minutes and 1400 ul of the boiled mixture was then mixed with 300 ul of 20 mM ATP, 200 ul of 100 mM DTT, and 100 ul PNK (1500 units/ml), and incubated for 90 minutes at 37°C.
• reaction mixture was then fractionated on a DEAE Sephadex A-25 column with linear gradient of 0.1 M to 1.0 M TEAB pH 7.6.
• the fourth peak containing the pGpApUpnbzl was pooled and then exposed to a 300 watt mercury vapor lamp at a distance of 5 cm for 15 minutes in a water jacketed cooling chamber.
• SIGMA bovin e serum albumim
• the reaction mixture was fractionated on a DEAE Sephadex A-25 column.
• the ol igonucl eotide was then exposed to the mercury vapor lamp as describ ed in EXAMPLE 2 to remove the nbzl group.
• the mixture was fractionated on a DEAE Sephadex A-24 column with a 0.1 to 2.0 M TEAB pH 7.5 linear gradient and the sixth peak identified by UV absorption as being the polynucl eotide ApCpCpUpCpCpGpAp.

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• 1983
  • 1983-01-25 WO PCT/US1983/000115 patent/WO1983002626A1/en not_active Ceased
  • 1983-01-25 JP JP83500949A patent/JPS59500081A/ja active Pending
  • 1983-01-25 AU AU13349/83A patent/AU1334983A/en not_active Abandoned
  • 1983-01-25 EP EP19830900868 patent/EP0100353A1/de not_active Withdrawn
  • 1983-01-25 GB GB08324625A patent/GB2123428B/en not_active Expired
  • 1983-01-25 DE DE19833328937 patent/DE3328937T1/de not_active Withdrawn
  • 1983-01-31 FR FR8301430A patent/FR2522682A1/fr not_active Withdrawn

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