United States Patent [19J
`Hiatt et al.
`
`I 1111111111111111 11111 111111111111111 1111111111 1111111111 111111111111111111
`US005808045A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,808,045
`*Sep. 15, 1998
`
`[54]
`
`COMPOSITIONS FOR ENZYME
`CATALYZED TEMPLATE-INDEPENDENT
`CREATION OF PHOSPHODIESTER BONDS
`USING PROTECTED NUCLEOTIDES
`
`[75]
`
`Inventors: Andrew C. Hiatt, 660 Torrance St.,
`San Diego, Calif. 92103; Floyd Rose,
`Del Mar, Calif.
`
`[73]
`
`Assignees: Andrew C. Hiatt, San Diego; Floyd D.
`Rose, Del Mar, both of Calif.
`
`[ * l
`
`Notice:
`
`The term of this patent shall not extend
`beyond the expiration date of Pat. No.
`5,763,594.
`
`[21]
`
`Appl. No.: 486,897
`
`[22]
`
`Filed:
`
`Jun. 7, 1995
`
`[63]
`
`[51]
`[52]
`
`[58]
`
`[56]
`
`Related U.S. Application Data
`
`Continuation-in-part of Ser. No. 300,484, Sep. 2, 1994,
`abandoned.
`Int. Cl.6
`..................................................... C07H 19/04
`U.S. Cl. .................. 536/26.26; 536/26.7; 536/26.71;
`536/26.72; 536/26.74; 536/26.8
`Field of Search ................................ 536/26.26, 26.7,
`536/26.71, 26.72, 26.74, 26.8
`
`References Cited
`
`U.S. PATENT DOCUMENTS
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`6/1978 Kelly et al..
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`
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`
`9/1994 Reddy et al. .
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`11/1994 Ureda et al. .
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`
`FOREIGN PATENT DOCUMENTS
`
`55-38324
`
`3/1980
`
`Japan .
`
`OTHER PUBLICATIONS
`
`17,
`
`vol.
`
`Bollum, Fed Proc. Soc. Exp. Biol. Med., 17, 193 (1958).
`Deng and Wu, Meth. Enzymol., 100: 96-116 (1983).
`Kaufmann et al., Eur. J. Biochem, 24:4--11 (1971).
`Hinton and Gumport, Nucleic Acid Res., 7:453-464 (1979).
`Modak, Biochemistry, 17, 3116-3120 (1978).
`and Uhlenbeck, Biochemistry,
`England
`11:2069-2076 (1978).
`Chang and Bollum, Biochemistry, vol. 10, 3:536-542
`(1971).
`Bennett et al., Biochemistry, vol. 12, 20:3956-3960 (1973).
`Kassel and Roychoudrury, Eur J. Biochem., 22:271-276
`(1971).
`Flugel et al., Biochem. Biophys. Acta, 308:35-40 (1973).
`Sarfati et al., J. Biol Chem., 265(31), 18902-18906 (1990).
`Metzker et al., Nucleic Acids Res., 22(20), 4259-4267
`(1994).
`Canard et al.,Proc. Nat'l.Acad. Sci. USA, 92, 10859-10863
`(Nov. 1995).
`Canard et al., Gene, 148, 1-6 (1994).
`Kutateladze et al., FEES, 207(2), 205-212 (1986).
`
`Primary Examiner-James 0. Wilson
`Attorney, Agent, or Firm-Lerner, David, Littenberg,
`Krumholz & Mentlik
`
`[57]
`
`ABSTRACT
`
`A method for the stepwise creation of phosphodiester bonds
`between desired nucleosides resulting in the synthesis of
`polynucleotides having a predetermined nucleotide
`sequence by preparing an initiation substrate containing a
`free and unmodified 3'-hydroxyl group; attaching a mono(cid:173)
`nucleotide selected according to the order of the predeter(cid:173)
`mined nucleotide sequence to the 3'-hydroxyl of the initiat(cid:173)
`ing substrate in a solution containing a catalytic amount of
`an enzyme capable of catalyzing the 5' to 3' phosphodiester
`linkage of the 5'-phosphate of the mononucleotide to the
`3'-hydroxyl of the initiating substrate, wherein the mono(cid:173)
`nucleotide contains a protected 3'-hydroxyl group, whereby
`the protected mononucleotide is covalently linked to the
`initiating substrate and further additions are hindered by the
`3'-hydroxyl protecting group. Methods in which a mono(cid:173)
`nucleotide immobilized on a solid support is added to a free
`pol ynucleotide chain are also disclosed.
`
`7 Claims, No Drawings
`
`Page 1
`
`Columbia Ex. 2015
`Illumina, Inc. v. The Trustees
`of Columbia University
`in the City of New York
`IPR2020-01177
`
`

`

`5,808,045
`
`1
`COMPOSITIONS FOR ENZYME
`CATALYZED TEMPLATE-INDEPENDENT
`CREATION OF PHOSPHODIESTER BONDS
`USING PROTECTED NUCLEOTIDES
`
`RELATED APPLICATIONS
`
`This application is a continuation-in-part of U.S. patent
`application Ser. No. 08/300,484 filed Sep. 2, 1994.
`
`TECHNICAL FIELD
`
`This invention relates to the synthesis of oligonucleotides
`and other nucleic acid polymers using template independent
`enzymes.
`
`BACKGROUND OF THE INVENTION
`
`Oligonucleotides are presently synthesized in vitro using
`organic synthesis methods. These methods include the pho(cid:173)
`phoramidite method described in Adams et al., J. Amer.
`Chem. Soc., 105:661 (1983) and Froehler et al., Tetrahedron
`Lett., 24:3171 (1983) and the phosphotriester method
`described in German Offenlegungsshrift 264432. Other
`organic synthesis methods include those described by Froe(cid:173)
`hler et al., U.S. Pat. No. 5,264,566 in which H-phosphonates
`are used to produce oligonucleotides.
`The phosphoramidite method of phosphodiester bond
`formation and oligonucleotide synthesis represents the cur(cid:173)
`rent state of the art employed by most laboratories for the
`coupling of desired nucleotides without the use of a tem(cid:173)
`plate. In this method, the coupling reaction is initiated by a
`nucleoside attached to a solid support. The 5'-hydroxyl
`group of the immobilized nucleoside is free for coupling
`with the second nucleoside of the chain to be assembled.
`Since the growing oligonucleotide chain projects a
`5'-hydroxyl available for reaction with a mononucleotide,
`the direction of synthesis if referred to as 3' to 5'.
`Each successive mononucleotide to be added to the grow(cid:173)
`ing oligonucleotide chain contains a 3'-phosphoramidate
`moiety which reacts with the 5'-hydroxyl group of the
`support-bound nucleotide to form a 5' to 3' internucleotide
`phosphodiester bond. The 5'-hydroxyl group of the incom(cid:173)
`ing mononucleotide is protected, usually by a trityl group, in
`order to prevent the uncontrolled polymerization of the
`nucleosides. After each incoming nucleoside is added, the
`protected 5'-hydroxyl group is deprotected, so that it is
`available for reaction with the next incoming nucleoside
`having a 3'-phosphoramidite group and a protected
`5'-hydroxyl. This is followed by deprotection and addition
`of the next incoming nucleotide, and so forth.
`Between each nucleoside addition step, unreacted chains
`which fail to participate in phosphodiester bond formation
`with the desired nucleoside are chemically "capped" to
`prevent their further elongation. This usually involves
`chemical acetylation.
`This method and the other currently used organic methods
`while widely accepted require large amounts of costly
`monomers that require complex organic synthesis schemes
`to produce. In addition, these methods are complex in that
`the phosphoramidite method requires an oxidation step after
`each condensation reaction. The phosphotriester method
`requires that the subpopulation of oligonucleotides that have
`not had a monomer added in a particular cycle be capped in
`a separate reaction to prevent further chain elongation of
`these oligonucleotides.
`Other drawbacks of virtually all chemical methods of
`phosphodiester bond formation, is that the reaction must be
`
`5
`
`10
`
`2
`performed in organic solvents and in the absence of water.
`Many of these organic solvents are toxic or otherwise
`hazardous. Another drawback to chemical synthesis is that it
`is at best 98 percent efficient at each cycle. In other words,
`following each nucleotide addition, at least 2 percent of the
`growing oligonucleotide chains are capped, resulting in a
`yield loss. The total yield loss for the nucleotide chain being
`synthesized thus increases with each nucleotide added to the
`sequence.
`For example, assuming a yield of 98 percent per nucle-
`otide addition, the synthesis of a polynucleotide consisting
`of 70 mononucleotides would experience a yield loss of
`nearly 75 percent. Furthermore, the object nucleotide chain
`would require isolation from a reaction mixture of
`polynucleotides, nearly 75 percent of which consist of
`15 capped oligonucleotides ranging between 1 and 69 nucle(cid:173)
`otides in length.
`This inherent inefficiency in chemical synthesis of oligo(cid:173)
`nucleotides ultimately limits the length of oligonucleotide
`that can be efficiently produced to oligonucleotides having
`20 50 nucleic acid residues or less.
`A need exists for a method which improves the efficiency
`of phosphodiester bond formation and which could ulti(cid:173)
`mately be capable of producing shorter chain oligonucle-
`25 otides in higher yields and longer chain polynucleotides in
`acceptable yields. In addition, a need exists for a polynucle(cid:173)
`otide synthesis system which is compatible with pre-existing
`polynucleotides, such as vector DNAs, so that desired
`polynucleotide sequences can readily be added on to the
`30 pre-existing sequences. Chemical coupling by the phos(cid:173)
`phoramidite method is not compatible with "add-on" syn(cid:173)
`thesis to pre-existing polynucleotides. Enzyme catalyzed
`phosphodiester bond formation, however, can be performed
`in an aqueous environment utilizing either single or double
`35 stranded oligo- or polynucleotides to initiate the reaction.
`These reaction conditions also greatly minimize the use of
`toxic and hazardous materials.
`The 3' to 5' direction of synthesis inherent to the phos(cid:173)
`phoramidite method of phosphodiester bond formation can-
`40 not be enzyme catalyzed. All known enzymes capable of
`catalyzing the formation of phosphodiester bonds do so in
`the 5' to 3' direction since the growing polynucleotide strand
`always projects a 3'-hydroxyl available for attachment of the
`next nucleoside.
`There are many enzymes capable of catalyzing the for-
`mation of phosphodiester bonds. One class of such enzymes,
`the polymerases, are largely template dependent in that they
`add a complementary nucleotide to the 3' hydroxyl of the
`growing strand of a double stranded polynucleotide.
`50 However, some polymerases are template independent and
`primarily catalyze the formation of single stranded nucle(cid:173)
`otide polymers. Another class of enzyme, the ligases, are
`template independent and form a phosphodiester bond
`between two polynucleotides or between a polynucleotide
`55 and a mononucleotide.
`Addition of single nucleotides to DNA fragments, cata(cid:173)
`lyzed by deoxynucleotidyl terminal transferase (TdTase),
`has previously been described by Deng and Wu, Meth.
`Enzymol., 100:96-116, 1983. These reaction conditions did
`60 not involve transient protection of the 3'-hydroxyl nor were
`they intended to be used for the sequential creation of
`phosphodiester bonds to synthesize a predetermined nucle(cid:173)
`otide sequence. The presence of unprotected 3'-hydroxyls
`resulted in a highly heterogeneous population of reaction
`65 products.
`Similarly, prior attempts to catalyze synthesis of very
`short pieces of RNA or DNA using protected nucleotide
`
`45
`
`Page 2
`
`

`

`5,808,045
`
`3
`monophosphates or diphosphates resulted in unacceptably
`low levels of the desired phosphodiester bond formation or
`required excessive amounts of enzyme to achieve acceptable
`efficiencies. These problems were largely due to unavoid(cid:173)
`able heterogeneity of the mono nucleotide building blocks or 5
`to the very high turnover number of the enzyme, necessi(cid:173)
`tating extremely long incubation times (see, for example,
`Hinton and Gumport, Nucleic Acids Res. 7:453-464, 1979;
`Kaufman et al., Eur. J. Biochem., 24: 4-11, 1971). These
`experiments were limited to 5'-monophosphates and diphos- 10
`phates. No attempts have been made to catalyze controlled
`DNA synthesis using 5'-triphosphates protected at the 3'
`position.
`Enzyme catalyzed creation of a single phosphodiester
`bond between the free 3'-hydroxyl group of an oligonucle- 15
`otide chain and the 5'-phosphate of a mononucleotide
`requires protection of the 3'-hydroxyl of the mononucleotide
`in order to prevent multiple phosphodiester bond formations
`and hence repeated mononucleotide additions. Protection of
`the 3'-hydroxyl of the mononucleotide ideally involves a 20
`transient blocking group which can readily be removed in
`order to allow subsequent reactions. Flugel et al., Biochem.
`Biophys. Acta. 308:35-40, 1973, report that nucleoside
`triphosphates with blocked 3'-hydroxyl groups cannot be
`prepared directly. This lack of 3' blocked triphosphates 25
`necessitated previous processes to utilize lower energy and
`thus more inefficient 3' blocked monophosphates and dipos(cid:173)
`phates. Synthetic techniques to create 3' block triphosphates
`would be highly desirable, because this could enable step(cid:173)
`wise enzyme catalyzed phosphodiester bond formation lead- 30
`ing to polynucleotide synthesis.
`These prior attempts at synthesizing oligonucleotides
`using a template independent polymerase were extremely
`inefficient resulting in the production of very short oligo(cid:173)
`nucleotides. The inefficiency of these methods made these 35
`methods useless for practical synthesis of oligonucleotides.
`The present invention allows the creation of phosphodi(cid:173)
`ester bonds between nucleotides using a template indepen(cid:173)
`dent polymerase to create oligonucleotides having a prede(cid:173)
`termined sequence. This enzyme catalysis can vastly 40
`improve the efficiency of phosphodiester bond formation
`between desired nucleotides compared to current techniques
`of chemical coupling and can be carried out in the presence
`of other biological molecules such as pre-existing sequences
`of single or double stranded DNA as well as other types of 45
`enzymes. In addition, the very high specificity inherent to
`enzyme catalysis allows only coupling of a 5'-phosphate to
`a 3'-hydroxyl. The coupling of two mononucleosides, as
`well as various other side reactions inherent to chemical
`coupling techniques, simply do not occur.
`A further advantage of the present invention is realized by
`using 3' blocked triphosphates having high energy phosphate
`bonds which an enzyme can utilize to drive the reaction to
`greater completion level than when other monophosphates
`and diphosphates are used. In addition, triphosphates are less 55
`strongly hydrated than the diphosphate, which also tends to
`drive catalytic hydrolysis of the triphosphate to completion.
`Clearly, the availability of a homogeneous population of
`protected mononucleotide triphosphates and enzymes
`capable of efficiently joining protected nucleotides to initi- 60
`ating substrates will enable the creation of a highly uniform
`population of synthetic polynucleotides resulting from step(cid:173)
`wise phosphodiester bond formation.
`
`50
`
`4
`effectively protected and deprotected and wherein the pro(cid:173)
`tected nucleotide is utilized by a template independent
`polymerase to create a phosphodiester bond permitting the
`synthesis of oligonucleotides or polynucleotides having a
`desired predetermined sequence.
`Therefore, in accordance with the present invention, a
`method is provided for the synthesis of a polynucleotide of
`a predetermined sequence of which method includes the
`steps of:
`A providing an initiating substrate comprising a nucleo(cid:173)
`side having an unprotected 3'-hydroxyl group; and
`B. reacting under enzymatic conditions in the presence of
`a catalytic amount of an enzyme the 3'-hydroxyl group of the
`initiating substrate with a nucleoside 5'-triphosphate having
`a removable blocking moiety protecting the 3' position of the
`nucleoside 5'-triphosphate and selected according to the
`order of the predetermined sequence, so that enzyme cata(cid:173)
`lyzes the formation of a 5' to 3' phosphodiester linkage
`between the unprotected 3'-hydroxyl group of the initiating
`substrate and the 5'-phosphate of the nucleoside
`5'-triphosphate to produce the polynucleotide.
`In other embodiments of the present invention, the
`method further comprises the step:
`C. removing the blocking moiety protecting the 3' posi(cid:173)
`tion of said nucleotide 5'-triphosphate to produce an initi(cid:173)
`ating substrate having an unprotected 3'-hydroxyl group.
`In other embodiments, steps (b) and (c) are repeated at
`least once to add additional nucleotides to the initiating
`substrate by alternatively adding a nucleoside
`5'-triphosphate with a removable blocking moiety at its 3'
`position, deblocking the 3' position of the terminal nucleo(cid:173)
`side and then adding another nucleoside 5'-triphosphate with
`a removable blocking group at its 3' position. Repetition of
`steps (b) and (c) can also be carried out to produce an
`oligonucleotide or polynucleotide having a predetermined
`sequence.
`The present invention contemplates initiating substrates
`that are deoxynucleosides, nucleotides, single or double
`stranded oligonucleotides, single or double stranded
`polynucleotides, oligonucleotides attached to nonnucleoside
`molecules and the like.
`The present invention contemplates embodiments in
`which the substrate is immobilized on a solid support.
`Preferred solid supports include cellulose, Sepharose,
`controlled-pore glass, silica, Fractosil, polystyrene, styrene
`divinyl benzene, agarose, and crosslinked agarose and the
`like.
`The present invention contemplates the use of template
`independent polynucleotide polymerases such as terminal
`deoxynucleotidyl transferase from any number of sources
`including eukaryotes and protharyotes.
`The methods of the present invention utilize removable
`blocking moieties that block the 3' position of nucleoside
`5'-triphosphates used in the methods. Preferred removable
`blocking moieties can be removed in under 10 minutes to
`produce a hydroxyl group at the 3' position of the 3'
`nucleoside. Removable blocking groups contemplated
`include carbonitriles, phosphates, carbonates, carbamates,
`esters, ethers, borates, nitrates, sugars, phosphoramidates,
`phenylsulfenates, sulfates and sulfones.
`The methods of the present invention contemplate remov(cid:173)
`ing the removable blocking moiety using a deblocking
`65 solution that preferably contains divalent cations such as
`Co++ and a biological buffer such as comprises a buffer
`selected from the group consisting of dimethylarsinic acid,
`
`SUMMARY OF THE INVENTION
`A number of methods have been discovered by which the
`3'-hydroxyl group of a deoxynucleotide triphosphate can be
`
`Page 3
`
`

`

`5,808,045
`
`5
`tris[hydroxymethyl] amino methane, and 3-[ m-morpholine]
`propanosulphonic acid. Other embodiments of the present
`invention utilize an enzyme present in the deblocking solu(cid:173)
`tion to remove the removable blocking moiety.
`The present invention also contemplates methods in 5
`which the nucleoside 5'-triphosphate having the removable
`blocking moiety at its 3' position is immobilized in a solid
`support and reacted with free initiating substrates. The solid
`support is linked to the nucleoside 5'-triphosphate at the
`3'-hydroxyl group, thereby acting as a removable blocking 10
`moiety at the 3' position. Attachment of the nucleoside to the
`support is transient, thereby enabling the release of the
`newly synthesized product from the support and regenera(cid:173)
`tion of the free and unmodified 3'-hydroxyl to allow the next
`nucleotide addition to occur.
`Thus, in some embodiments of the present invention the 15
`deblocking solution would remove the removable blocking
`moiety at the position of the nucleoside and thus release the
`growing polynucleotide from the solid support.
`The present invention also includes polynucleotides hav(cid:173)
`ing a predetermined sequence provided according to the
`methods of this invention. Applications for using polynucle(cid:173)
`otides and oligonucleotides of the present invention in
`molecular cloning and/or expression of genes, peptides or
`proteins.
`Also contemplated by the present invention are compo- 25
`sitions of matter comprising a catalytic amount of a template
`independent enzyme and a nucleoside 5'-triphosphate hav(cid:173)
`ing a removable blocking moiety protecting the 3' position
`of said nucleoside 5'-triphosphate. Additional compositions
`of matter further comprising an initiating substrate are also
`contemplated.
`BRIEF DESCRIPTION OF IBE INVENTION
`A Definitions
`DNA: Deoxyribonucleic acid.
`RNA: Ribonucleic acid.
`Nucleotide: A subunit of a nucleic acid comprising a phos(cid:173)
`phate group, a 5-carbon sugar and nitrogen containing
`base. In RNA, the 5-carbon sugar is ribose. In DNA, it is
`a 2-deoxyribose. The term also includes analogs of such 40
`subunits.
`Nucleoside: Includes a nucleosidyl unit and is used inter(cid:173)
`changeably therewith, and refers to a subunit of a nucleic
`acid which comprises a 5-carbon sugar and a nitrogen
`containing base. The term includes not only those nude- 45
`osidyl units having A, G, C, T and U as their bases, but
`also analogs and modified forms of the naturally(cid:173)
`occurring bases, such as pseudoisocytosine and pseudou(cid:173)
`racil and other modified bases (such as 8-substituted
`purines). In RNA, the 5-carbon sugar is ribose; in DNA, 50
`it is 2'-deoxyribose. The term nucleoside also includes
`other analogs of such subunits, including those which
`have modified sugars such as 2'-O-alkyl ribose.
`Polynucleotide: A nucleotide multimer generally about 50
`nucleotides or more in length. These are usually of 55
`biological origin or are obtained by enzymatic means.
`Phosphodiester: The group
`
`6
`Hydrocarbyl: An organic radical composed of carbon and
`hydrogen which may be aliphatic (including alkyl,
`alkenyl, and alkynyl groups and groups which have a
`mixture of saturated and unsaturated bonds), alicyclic
`(carbocyclic), aryl (aromatic) or combination thereof; and
`may refer to straight-chained, branched-chain, or cyclic
`structures or to radicals having a combination thereof, as
`well as to radicals substituted with halogen atom(s) or
`heteroatoms, such as nitrogen, oxygen, and sulfur and
`their functional groups (such as amino, alkoxy, aryloxy,
`lactone groups and the like), which are commonly found
`in organic compounds and radicals.
`Non-nucleoside monomeric unit: A monomeric unit wherein
`the base, the sugar and/or the phosphorus backbone or
`other internuclosidyl linkage of a nucleoside has been
`replaced by other chemical moieties.
`Polypeptide and Peptide: A linear series of amino acid
`residues connected on to the other by peptide bonds
`between the alpha-amino and carboxyl groups of adjacent
`residues.
`20 Protein: A linear series of greater than about 50 amino acid
`residues connected one to the other as in a polypeptide.
`Gene: A segment of DNA coding for an RNA transcript that
`is itself a structural RNA, such as ribosomal RNA or
`codes for a polypeptide. The segment of DNA is also
`equipped with a suitable promoter, termination sequence
`and optionally other regulatory DNA sequences.
`Structural Gene: A gene coding for a structural RNA and
`being equipped with a suitable promoter, termination
`sequence and optionally other regulatory DNA sequences.
`30 Promoter: A recognition site on a DNA sequence or group of
`DNA sequences that provide an expression control ele(cid:173)
`ment for a gene and to which RNA polymerase specifi(cid:173)
`cally binds and initiates RNA synthesis (transcription) of
`that gene.
`35 Oligonucleotide: A chain of nucleosides which are linked by
`internucleoside linkages which is generally from about 2
`to about 50 nucleosides in length. They may be chemi(cid:173)
`cally synthesized from nucleoside monomers or produced
`by enzymatic means. The term oligonucleotide refers to a
`chain of nucleosides which have internucleosidyl linkages
`linking the nucleoside monomer and, thus, includes oli(cid:173)
`gonucleotide containing nucleoside analogs, oligonucle(cid:173)
`otide having internucleosidyl linkages such that one or
`more of the phosphorous group linkages between mono(cid:173)
`meric units has been replaced by a non-phosphorous
`linkage such as a formacetal linkage, a thioformacetal
`linkage, a sulfamate linkage, or a carbamate linkage. It
`also includes nucleoside/non-nucleoside polymers
`wherein both the sugar and the phosphorous moiety have
`been replaced or modified such as mopholino base
`analogs, or polyamide base analogs. It also includes
`nucleoside/non-nucleoside polymers wherein the vase,
`the sugar, and the phosphate backbone of a nucleoside are
`either replaced by a non-nucleoside moiety or wherein a
`non-nucleoside moiety is inserted into the nucleoside/
`non-nucleoside polymer. Thus an oligonucleotide may be
`partially or entirely phophonothioates, phosphorothioate
`phosphorodithioate phosphoramidate or neutral phos-
`phate ester such as phosphotriesters oligonucleotide ana(cid:173)
`logs.
`Removable Blocking Moiety: A removable blocking moiety
`is a moiety which is attached to the oxygen at the 3'
`position of a nucleoside or the equivalent position in a
`nucleoside analog. The removable blocking moiety pre(cid:173)
`vents reaction of the 3' oxygen when present and 1s
`removable under deblocking conditions so that the 3'
`oxygen can then participate in a chemical reaction.
`
`60
`
`O=P-0
`I
`0
`I
`
`wherein phosphodiester groups may be used as internucle- 65
`osidyl phosphorus group linkages (or links) to connect
`nucleosidyl units.
`
`Page 4
`
`

`

`5,808,045
`
`7
`
`A Methods
`Generally, the present invention provides methods for
`synthesizing oligonucleotides and polynucleotides having a
`predetermined sequence using a template independent poly(cid:173)
`merase and nucleoside having the 3' position blocked with
`a removable blocking moiety so that single nucleosides are
`added to the growing oligonucleotide. Single nucleosides
`are added to the growing chain by removing the blocking
`moiety at the 3' position of the terminal nucleoside of the
`growing oligonucleotide so that the next blocked nucleoside
`can be added to the oligonucleotide. This process is then
`repeated until the oligonucleotide having the predetermined
`sequence is produced.
`Thus, in accordance with this embodiment of the present
`invention, a method comprises the steps of:
`(a) providing an initiating substrate comprising a nucleo(cid:173)
`side having an unprotected 3'-hydroxyl group; and
`(b) reacting under enzymatic conditions in the presence of
`a catalytic amount of an enzyme said 3'-hydroxyl group
`of said initiating substrate with a nucleoside
`5'-triphosphate having a removable blocking moiety
`protecting the 3' position of said nucleoside
`5'-triphosphate and selected according to the order of
`said predetermined sequence, whereby said enzyme
`catalyzes the formation of a 5' to 3' phophodiester 25
`linkage between said unprotected 3'-hydroxyl group of
`said initiating substrate and the 5'-phosphate of said
`nucleoside 5'-triphosphate to produce said polynucle(cid:173)
`otide.
`In preferred embodiments, the methods of the present
`invention further comprises the step of:
`(c) removing the blocking moiety protecting the 3' posi(cid:173)
`tion of said nucleoside 5'-triphosphate to produce an
`initiating substrate having an unprotected 3'-hydroxyl
`group.
`This additional step regenerates a reactive atom at the 3'
`position of the terminal nucleoside so that this atom can be
`used to form a bond with the next nucleoside and thus extend
`the length of the oligonucleotide by one nucleoside.
`The methods of the present invention also include meth- 40
`ods in which the above steps (b) and ( c) are repeated at least
`once to produce an oligonucleotide. This process can be
`repeated many times to produce oligonucleotides of selected
`length. This process can also be repeated many times such
`that each particular nucleoside added to the oligonucleotide 45
`having a preselected sequence.
`1. Initiating Substrates
`An initiating substrate of the present invention is prepared
`containing a nucleoside with a free and unmodified
`3'-hydroxyl group. As is well understood by those of ordi- 50
`nary skill in the art, nucleotide derivatives of the nucleosides
`adenosine, cytidine, guanosine, uridine and thymidine can
`be assembled to form oligonucleotides and polynucleotides.
`According to the method of the present invention, the
`initiating substrate may contain a single nucleoside having a 55
`free and unmodified 3'-hydroxyl group, or a preassembled
`oligo- or polynucleotide may be provided as an initiating
`substrate, so long as the oligo- or polynucleotide has a free
`and unmodified 3'-hydroxyl group.
`One skilled in the art will understand that an initiating
`substrate could be provided in a form in which a nucleoside
`has a removable blocking moiety at its 3' position which is
`subsequently removed using a deblocking process so that the
`initiating substrate now has the free unprotected 3' hydroxyl
`group useful in the present invention.
`The initiating substrates of the present invention include
`the termini of polynucleotides frequently generated and used
`
`8
`in various cloning and molecular biology techniques.
`Examples of these initiating substrates include the termini of
`DNA or RNA vectors, single-stranded or double-stranded
`fragments, single-stranded or double-stranded RNA frag-
`5 ments and RNA or DNA oligonucleotides.
`In the preferred embodiments, initiating substrates will
`consist wholly or in part of an oligo- or polynucleotide. The
`initiating substrate can be any arrangement of nucleosides
`which enables the enzyme to create a phosphodiester bond
`10 between the 3'-hydroxyl of a nucleoside and the
`5'-phosphate of a mononucleotide. Initiating substrates may
`be based wholly or in part on ribonucleic acids (RNA) or
`deoxyribonucleic acids (DNA) and may be single stranded
`or multi-stranded. In addition, initiating substrates can
`15 include other types of naturally occurring or synthetic mol(cid:173)
`ecules (non-nucleosides) which may enable or enhance the
`ability of the enzyme to create a phosphodiester bond or
`which may facilitate the manipulation of reaction compo(cid:173)
`nents and by-products. An example of this would be a linker
`20 molecule (commonly used linkers consist of C, 0, N, and H
`e.g. Affi-Gel™ 10: R-OCH2 CONH(CH2) 2NHCO(CH2) 2
`COON(CH2 ) 2 which would serve to provide a convenient
`method for attaching an initiating substrate to a solid sup-
`port.
`The sequential creation of phosphodiester bonds and
`hence the addition of nucleotides to the initiating substrate
`may be performed entirely in solution, or the initiating
`substrate may be attached to an insoluble matrix. Attach(cid:173)
`ment to an insoluble matrix will permit the rapid separation
`30 of the substrate from unreacted reagents in order to prepare
`the substrate for the addition of the next nucleotide. For this
`reason, the substrate is preferably affixed to a solid support
`matrix during each reaction creating a phosphodiester bond.
`Insoluble matrices suitable for use as solid supports
`35 include cellulose, Sepharose™, controlled-pore glass
`(CPG), polystyrene, silica, agarose, and the like.
`Reagents, buffers and solvents suitable for use with the
`present invention are capable of flowing through the solid
`support matrix, by which means the initiating substrate is
`brought into contact with these materials. The growing
`nucleotide chain remains attached to the solid support as the
`various reagents, buffers and solvents sequentially flow
`therethrough. The solid support matrix is preferably con(cid:173)
`tained within a synthesis column, to which reagents, buffers
`and solvents are provided.
`Attachment of the initiating substrate to the solid support
`can be by covalent bonding. Numerous methods for the
`covalent attachment of molecules to insoluble matrices have
`been described and are well understood by those of ordinary
`skill in the art. In the preferred embodiment an oligonucle(cid:173)
`otide chain may be linked to alkylamine derivatized poly-
`styrene or CPG by way of a covalent phosphoramidate bond
`although numerous strategies for linking oligonucleotides to
`solid supports have been described. The choice of an appro(cid:173)
`priate linking strategy will depend on the specific require(cid:173)
`ments of stability, charge interactions, solubility and the like.
`Alternativ