EP0100353A1 - Method for production of predetermined polyribonucleotides - Google Patents

Method for production of predetermined polyribonucleotides

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Publication number
EP0100353A1
EP0100353A1 EP19830900868 EP83900868A EP0100353A1 EP 0100353 A1 EP0100353 A1 EP 0100353A1 EP 19830900868 EP19830900868 EP 19830900868 EP 83900868 A EP83900868 A EP 83900868A EP 0100353 A1 EP0100353 A1 EP 0100353A1
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EP
European Patent Office
Prior art keywords
ribonucleotide
donor
recipient
phosphorylated
hydroxylated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP19830900868
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German (de)
French (fr)
Inventor
Hiroaki Shizuya
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Innovax Laboratories Ltd
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Innovax Laboratories Ltd
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Publication of EP0100353A1 publication Critical patent/EP0100353A1/en
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    • 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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Abstract

Les procédés de l'invention permettant la production de polyribonucléotides prédéterminés peuvent être utilisés pour synthétiser un codage ARN pour une protéine désirée. Les procédés décrits comprennent: a) l'addition d'un seul nucléotide sur l'extrémité 3' d'un ribonucléotide récepteur de composition prédéterminée en faisant réagir le composé de la forme AppNp avec le ribonucléotide récepteur en présence d'une ligase P4 ARN et l'isolation du ribonucléotide allongé où N est un ribonucléotide sélectionné parmi le groupe de l'adénine, la guanine, la cytosine, l'uracile et l'inosine; b) l'addition d'un ribonucléotide d'au moins deux bases par phosphorilation d'un ribonucléotide donneur d'au moins deux bases aux extrémités terminales 5' et 3', puis le mélange du ribonucléotide donneur phosphorilé avec un ribonucléotide récepteur en présence d'une ligase T4 ARN pour ajouter le donneur sur l'extrémité 3' du ribonucléotide récepteur, le nucléotide récepteur étant sélectionné parmi le groupe substitué d'un dinucléotide phosphorilé dans la position libre 5' et d'un nucléotide plus long que 2 paires de base, avec soit un groupe hydroxyle soit un groupe phosphate sur le terminus 5' et une extrémité 3' hydroxylée; c) la synthèse de l'acide ribonucléique d'une composition prédéterminée en construisant séquentiellement des polynucléotides à l'aide des procédés décrits ci-dessus.The methods of the invention for the production of predetermined polyribonucleotides can be used to synthesize RNA coding for a desired protein. The methods described include: a) adding a single nucleotide to the 3 'end of a receptor ribonucleotide of predetermined composition by reacting the compound of the AppNp form with the receptor ribonucleotide in the presence of a P4 RNA ligase and isolating the elongated ribonucleotide where N is a ribonucleotide selected from the group of adenine, guanine, cytosine, uracil and inosine; b) adding a ribonucleotide of at least two bases by phosphorilation of a donor ribonucleotide of at least two bases at the 5 ′ and 3 ′ terminal ends, then mixing the phosphorilated donor ribonucleotide with a receptor ribonucleotide in the presence a T4 RNA ligase to add the donor to the 3 'end of the receptor ribonucleotide, the receptor nucleotide being selected from the substituted group of a phosphorilated dinucleotide in the free position 5' and of a nucleotide longer than 2 pairs basic, with either a hydroxyl group or a phosphate group on the 5 'terminus and a 3' hydroxylated end; c) the synthesis of ribonucleic acid of a predetermined composition by sequentially constructing polynucleotides using the methods described above.

Description

METHOD FOR PRODUCTION OF PREDETERMINED POLYRIBONUCLEOTIDES
BACKGROUND AND PRIOR ART
There are three general steps involved in the production of a protein utilizing genetic engineering techniques. 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. Thus, synthesis of 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.
Enzymatic synthesis of RNA using polynucleotide phosphorylase and T4 RNA ligase have been reported. The polynucleotide phosphorylase method is limited to production of oligodeoxynucleo tides. Under controlled conditions, 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.
The existence, purification and mechanism of the enzyme 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. Additionally, 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. HOApApAOH + pCp + ATP >HOApApApCp + AMP + PPi
Abbreviations to be used in this application for convenience are provided in Table 1.
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.
SUMMARY OF THE INVENTION
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. If the recipient is a trinucl eotide or longer, the 5' terminal end may be either phosphorylated or hydroxylated to allow specific addition of the donor to the 3' end of the recipient. Referring to the method for adding a single base as a donor onto the end of a recipient, if the starting material for the recipient is a dinucl eotide either obtained commercially or synthesized by chemical techniques known in the art, 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. Sci. U.S.A. 54, 158 (1965) which specifically phosphorylates the 5' terminus of a nucleotide as depicted in equation 1. If the recipient is a trinucl eotide or longer, this phosphorylation step is not necessary.
(1) OHXpYOH + ATP pXpYOH + ADP
The second step is to mix the recipient dinucl eotide with the chemical compound p1-Adenosine, p2-(3' nucleotide monophosphate)-5' pyrophosphate of the form (AppNp), where N is a ribonucleotide selected from the group adenine, cytosine, guanine, uracil, and inosine. The compound AppNp and its synthesis are disclosed in the copending application
( ). The reaction of the known recipient ribonucleotide with the AppNp in the presence of RNA ligase resul ts in the production of a nucl eotide as depicted in equation 2. This reaction does not require ATP as an energy source.
(2) pXpYOH + AppNp > pXpYpNp + pA
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. Again for this method, if the recipient is a dinucl eotide it must be phosphorylated on the 5' end whereas if it is a trinucl eotide or longer, phosphorylation is unnecessary (equation 4).
(3 ) pXpYOH + pYpZp > pXpYpYpZp
ATP
(4) pXpY0H + Appnbzl pXpYpnbzl
To phosphorylate the 3 ' end of the donor mol ecul e, 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).
l ight
(5 ) pxpγpnbzl > pXpYp + nbzl 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).
(6)
The reactions described hereinabove are examples of a number of methods for production of nucleotides. These methods may be used effectively on longer oligonucleo tides as well as the dinucl eo tides described in this disclosure. In this way, entire RNA molecules coding for particular proteins may be synthesized.
For each of the reactions described below, it is necessary to determine the identity of the reaction products. Identification of reaction products throughout these examples is made by taking ratios of absorption at 250, 260 and 280 nm and comparing them to known standards. Identification and purity are also assayed by paper electrophoresis at pH 5.0 in sodium citrate buffer and at pH 2.8 in ammonium formate buffer at 800 volts for 1 hour.
Description of the Specific Embodiments
The present invention teaches the utilization of T4 RNA ligase in the preparation of nucleotide of a predetermined base sequence. As hereinbefore described in general, 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.
Monoribonucleotide Additions
In the synthesis of a trinucl eotide of predetermined base sequence from a known dinucleotide and an AppNp compound, it is first necessary to phosphorylate the free 5' end of the dinucl eotide. This is accomplished using the enzyme polynucl eotide kinase under conditions known in the art.
Following the phosphorylation step, it is necessary to isolate the phosphorylated dinucl eotide 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. The preferred reaction conditions are provided in the examples below.
Finally, the resultant nucleotide may be treated with 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.
Additions of More Than One Nucleotide
Addition of 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.
To phosphorylate the 3' end of the donor ribonucleotide, 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.
Following the addition of the donor ribonucleotide to the recipient, 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. Alternatively, the extended nucleotide may be treated with PNK to phosphorylate the 5' end and allow it to be used as a donor.
It is anticipated that all purification steps wherein the preferred method described is fractionation using a DEAE sephadex column may also be achieved by high pressure liquid chromatography, thin layer chromatography, gel and paper electrophoresis, and other known separation techniques without departing from the scope of this invention.
EXAMPLE I
Synthesis of ApGpA
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 H2O , 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 lyophilized.
To separate the ATP from the pApG the pool ed frac tion is treated with ATPase and fractionated on a column. 200 ul of the pApG/ATP mixture (A260=49.2) , 100 ul of 100mM DTT, 100 ul of 1M MOPS pH 7.9, 150 ul of 100 mM MgCl 2 , 800 ul of H2O and 250 ul of T4 ATPase (5000 units/ml ) are mixed and incubated for 1 hour at 37°C. 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.
The pApG from the above r eac tion was then used in the synthesis of pApGpAp. T4 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). 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 H2O 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.
EXAMPLE 2
Synthesis of pGUp from GAU
The trinucl eotide GAU (Collaborative Research) was assayed for purity by electrophoresis. A reaction mixture containing 225 ul 26 mM Appnbzl, 300 ul of 3.3 mM GpApU, 75 ul 1 M MOPS pH 7.9, 300 ul 0.1M MgCl2, 150 ul 100 mM DTT, 120 ul H2O, 180 ul RNA ligase and 150 ul 100% dimethyl sulfoxide DMSO were incubated at room temperature for 20 hours. The 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.
The 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. EXAMPLE
ApApA + pGpApnbzl >AAAGA
Th e r eac tion mix tur e consi s ting of 34 ul of 3.3 mM AAA (Boehringer) 70 ul of 1.0 mM pGpApnbzl (synthesized by the m ethod of EXAMPLE 2 ) , 40 ul of 1.0 M HEPES pH 7.83 , 5 ul of 1.0 M MgCl 2 , 25 ul of 20 mM ATP (SIGMA) , 2 ul of bovin e serum albumim (BSA) (1 mg/ml ) , 40 ul of RNA l igase (1500 units/ml ) , and 20 ul of 100% dimethyl sul foxide was incubated overnight at room temperature. The reaction mixture was fractionated on a DEAE Sephadex A-25 column. The sixth maj or peak with absorption ratios of 250/260 = 1.17 and 280/260 = .667 was identified as the product AAAGApnbzl . The ol igonucl eotide was then exposed to the mercury vapor lamp as describ ed in EXAMPLE 2 to remove the nbzl group.
EXAMPLE 4
ApCpCpU + pCpCpGpAp >ApCpCpUpCpCpGpAp
The reaction mixture containing 75 ul of (1.2 mM) ACCU (which may be synthesized using EXAMPLE 1), 90 ul of 1.0 mM pCCGAp (EXAMPLE 1), 30 ul of 1.0 M MOPS (pH 7.9), 120 ul of 100 mM MgCl2, 70 ul of 100 mM DTT, 60 ul of DMSO (100%), 60 ul of 20 mM ATP, 15 ul of BSA (10 mg/ml), 60 ul of RNA ligase (1500 units/ml) and 100 ul of H2O was incubated at room temperature (approximately 24°) for 16 hours. 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.

Claims

1. The method of synthesizing RNA of predetermined base sequence using the enzyme T4 RNA ligase which comprises adding a donor specifically to the hydroxylated 3' end of a recipient of at least two bases whereby if said donor consists of two or more nucleotide bases, adenosine triphosphate is provided as an energy source for the addition.
2. The method of adding a predetermined single ribonucleotide to a recipient ribonucleotide of at least two bases which comprises: mixing said recipient ribonucleotide with a compound of the form AppNp in the presence of T4 RNA ligase under conditions which favor transfer of the pNp moiety from the AppNp to said phosphorylated ribonucleotide at the 3' end; wherein the N of the AppNp is a ribonucleotide selected from the group consisting of adenine, guanine, cytosine, uracil and inosine.
3. The method of adding a predetermined ribonucleotide of at least 2 bases to a recipient ribonucleotide of at least two bases comprising the steps of: phosphorylating specifically the 3' end of the donor ribonucleotide; phosphorylating the 5' end of the donor ribonucleotide; incubating the recipient ribonucleotide with the phosphorylated donor ribonucleotide in the presence of T4 RNA ligase under conditions which permit covalent bonding between the recipient and donor ribonucleotide.
4. The method of Claim 2 wherein said recipient ribonucleotide is selected from the group consisting of a 5' phosphorylated 3' hydroxylated diribonucl eotide, an oligonucl eotide phosphorylated at the 5' end and hydroxylated at the 3' end, and an oligonucleotide hydroxylated at the 5' and 3' ends.
5. The method of Claim 3 wherein said recipient ribonucleotide is selected from the group consisting of a 5' phosphorylated 3' hydroxylated diribonucl eotide, an oligonucl eotide phosphorylated at the 5' end and hydroxylated at the 3' end, and an oligonucleotide hydroxylated at the 5' and 3' ends.
6. The method according to Claim 4 further comprising the step of synthesizing said 5' phosphorylated, 3' hydroxylated ribonucleotide by reacting a ribonucleotide hydroxylated at the free 3' and 5' termini with adenosine triphosphate (ATP) in the presence of polynucleotide kinase.
7. The method according to Claim 6 further comprising the steps of: reacting a ribonucleotide hydroxylated at the free 3' and 5' termini with ATP in the presence of polynucleotide kinase to form a phosphorylated ribonucleotide; fractionating the reaction mixture by an exchange column chromatography; treating the portion of the fractionated reaction mixture containing the phosphorylated ribonucleotide with an ATPase; fractionating the ATPase treated reaction mixture to isolate the phosphorylated ribonucleotide.
8. The method according to Claim 5 further comprising the step of phosphorylating specifically the 5' end of a recipient ribonucleotide by reacting said ribonucleotide with ATP in the presence of polynucl eotide kinase.
9. The method according to Claim 3 wherein the step of phosphorylating the 5' end of the donor ribonucleotide comprises the step of reacting said ribonucleotide with ATP in the presence of polynucleotide kinase.
10. The method according to Claim 3 wherein the step of phosphorylating the 3' end of the donor ribonucleotide comprises the step of reacting a ribonucleotide of at least two bases with the compound Appnbzl in the presence of T4 RNA Ligase under conditions which favor bonding of the pnbzl group to the 3' terminus of said ribonucleotide.
11. The method of synthesizing RNA of predetermined base sequence comprising the steps of: a) phosphorylating a ribonucleotide of known base sequence of at least 2 bases at the 5' terminus; b) reacting said RNA with AppNp in the presence of T4 RNA ligase to bond the pNp moiety of AppNp to the 3' terminus of said ribonucleotide; c) treat the resulting extended RNA with an enzyme which cleaves terminal phosphate groups; d) repeat steps b and c until the ribonucleic acid of predetermined sequence is produced; wherein N is a ribonucleotide selected from the group adenine, guanine, cytosine, uracil and inosine.
12. The method of Claim 2 wherein said conditions under which the transfer of said pNp moiety is favored consists of incubation of said recipient ribonucleotide with said AppNp compound in the presence of RNA ligase in a small volume of HEPES Buffer for 1 hour at 37°C.
EP19830900868 1982-02-01 1983-01-25 Method for production of predetermined polyribonucleotides Withdrawn EP0100353A1 (en)

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US6479262B1 (en) * 2000-05-16 2002-11-12 Hercules, Incorporated Solid phase enzymatic assembly of polynucleotides

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