I IIII IIIIIII II llll 111111111111111111 Ill lllll lllll 111111111111111111
`
`US005763594A
`Patent Number:
`Date of Patent:
`
`5,763,594
`Jun. 9, 1998
`
`United States Patent
`Hiatt et al.
`
`[19]
`
`[11]
`
`[45]
`
`[54] 3' PROTECTED NUCLEOTIDES FOR
`ENZYME CATALYZED
`TEMPLATE-INDEPENDENT CREATION OF
`PHOSPHODIESTER BONDS
`
`[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.
`
`[21] Appl. No.: 486,913
`Jun.7, 1995
`
`[22] Filed:
`
`Related U.S. Application Data
`
`[51]
`
`[63] Continuation-in-part of Ser. No. 300,484, Sep. 2, 1994.
`Int. CI.6
`.......................... C07H 21/00; C07H 21/02;
`C07H 19/04; Cl2Q 1/68
`[52) U.S. Cl . .................... 536/25.3; 536/25.1; 536/25.31;
`536/25.32; 536/25.33; 536/25.34; 536/26.1;
`435/6
`[58) Field of Search .................................. 536/25.1, 26.1.
`536/25.3. 25.31. 25.32. 25.33. 25.34; 435/6
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,096,324
`4,423,212
`4,605,735
`4,689,405
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`4,816,571
`4,820,812
`4,863,849
`4,876,335
`4,948,882
`4,950,745
`4,980,460
`5,003,059
`5,032,680
`5,039,796
`5,047,524
`5,091,519
`5,093,232
`5,112,963
`5,256,549
`5,258,506
`5,262,530
`5,262,536
`5,264,563
`5,264,566
`5,268,266
`5,268,464
`5,281,701
`5,302,509
`5,348,868
`5,362,866
`5,367,066
`5,380,833
`5,436,143
`
`6/1978 Kelly et al. ............................ 536/25.1
`12/1983 Shu1nick ................................ 536/25.1
`8/1986 Miyoshi et al.
`. ...................... 536/25.1
`8/1987 Frank et al.
`........................... 536/25.1
`1/1988 Klotz ...................................... 536/25.1
`3/1989 Andrus et al .......................... 536/25.1
`4/1989 Miyoshi et al ..•............•••••.... 536/25.1
`9/1989 Melamede .............................. 536/25.1
`10/1989 Yamane et al ......................... 536/25.1
`8/1990 Ruth ....................................... 536/25.1
`8/1990 Ishido et al ............................ 536/25.1
`12/1990 Molko et al ........................... 536/25.1
`3/1991 Brennan ................................. 536/25.1
`7/1991 Kawai et al ........................... 536/25.3
`8/1991 Engels et al ........................... 536/25.1
`9/1991 Andrus et al .......................... 536/25.l
`2/1992 Cruickshank .......................... 536/25.1
`3/1992 Urdea et al ............................ 536/25.1
`5/1992 Pieles et al ............................ 536/25.1
`10/1993 Urdea et al ............................ 536/25.1
`11/1993 Urdea et al .•..•••.•••..•.........•..•. 536/25.1
`11/1993 Andrus et al ........................ 536/25.31
`11/1993 Hobbs, Jr ............................. 536/25.32
`11/1993 Huse ...................................... 536/25.3
`11/1993 Froehler et al. . ...................... 536/25.3
`12/1993 Fritsch et al ........•.•••.•............ 536/25.1
`12/1993 Brill ....................................... 536/25.1
`1/1994 Vinayak ................................. 536/25.1
`4/1994 Cheeseman ................................. 435/6
`9/1994 Reddy et al ....•.•..•••.............•. 536/25.1
`11/1994 Arnold, Jr ............................ 536/25.34
`11/1994 Urdea et al ............................ 536/25.1
`1/1995 Urdea ..................................... 536/25.1
`7/1995 Hyman et al ........................ 536/25.33
`
`OTHER PUBLICATIONS
`
`Sarfati et al .• J. Biol. Chem., 265(31). 18902-18906 (1990)
`Month not available.
`Metzker et al., Nucleic Acids Res., 22(20). 4259-4267
`(1994) Month not available.
`Canard et al .• Proc. Nat'l. Acad. Sci. USA, 92. 10859-10863
`(Nov. 1995).
`Canard et al .. Gene, 148. 1-6 (1994) Month not availabe.
`Kutateladze et al .• FEBS, 207(2). 205-212 (1986) Month not
`available.
`Hyman. Edward David; 'The Hyman Method: Oligonucle(cid:173)
`otide Synthesis and Plasmid Preparation"; (1995) Month not
`available.
`Mukai et al (Abstract for JP 78-111456) 1978 Month not
`available.
`Bollum. Fed. Proc. Soc. E~. Biol. Med., 17. 193 (1958)
`Month not available.
`Deng and Wu. Meth. Enzymol., 100: 96-116 (1983) Month
`not available.
`Kaufmann et al .. Eur. J. Biochem., 24:4-11 (1971) Month
`not available.
`Hinton and Gurnport. Nucleic Acid Res., 7:453-464 (1979)
`Month not available.
`Modak. Biochemistry, 17. 3116-3120 (1978) Month not
`available.
`and Uhlenbeck, Biochemistry,
`England
`11:2069-2076 (1978) Month not available.
`Chang andBollum.Biochemistry vol. 10. 3:536-542 (1971)
`Month not available.
`Bennett et al .• Biochemistry vol. 12. 20:3956-3%0 (1973)
`Month not available.
`Kosse! and Roychourdrury. Eur. J. Biochem, 22:271-276
`(1971) Month not available.
`Augel et al .• Biochem. Biophys. Acta., 308:35-40 (1973)
`Month not available.
`
`vol.
`
`17.
`
`Primary Examiner-Louise N. Leary
`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
`polynucleotide chain are also disclosed.
`
`6 Claims, 4 Drawing Sheets
`
`Page 1
`
`Illumina Ex. 1043
`IPR Petition - USP 10,435,742
`
`

`

`U.S. Patent
`
`Jun. 9, 1998
`
`Sheet 1 of 4
`
`5,763,594
`
`catalyzing enzyme
`
`x. a blocking group
`
`N~
`
`FIGURE 1.
`
`1.
`
`N~
`
`of) N
`
`+
`
`DNA-0~
`
`OH H
`
`ONA·OyO~
`
`N~
`2. O~N)
`0 H N--'r
`O=~,oyO~
`OH H
`
`0
`
`H O~NJ
`
`X
`H
`INTERMEDIATE
`
`a{) N
`0 1--'r
`::I.......N J
`.AyN
`NH l .. J-)
`o=~,oyo~
`OH H
`
`DNA-0~
`remove blocking group,
`
`........ ep,.........
`
`blocked nucleotide
`
`INTERMEDIATE
`
`0
`
`N
`
`O H
`\
`
`0=~.-0~
`
`0
`
`OH
`
`0
`
`H
`
`2
`
`N
`
`N
`
`X H
`
`Page 2
`
`

`

`U.S. Patent
`
`Jun. 9, 1998
`
`Sheet 2 of 4
`
`5,763,594
`
`FIGURE 2.
`
`0
`
`cl~
`
`N
`
`-O(PO,lo~
`
`0 H
`I
`O=C
`
`B
`-O(PO,l,~
`
`OR H
`
`CH3
`3'-TOLUIC ACID ESTER OF'THYMIOINE 5'-TRIPHOSPHATE
`
`Page 3
`
`

`

`U.S. Patent
`
`Jun. 9, 1998
`
`Sheet 3 of 4
`
`5,763,594
`
`FIGURE 3.
`
`A
`21
`
`5 1
`
`C
`23
`
`53
`
`G
`25
`
`55
`
`T
`27
`
`57
`
`deblock wash
`28
`29
`
`release
`30
`
`58
`
`59
`
`60
`
`73
`........._RECOVERY
`
`69
`
`I
`
`DISCARD
`
`Page 4
`
`

`

`U.S. Patent
`
`Jun.9, 1998
`
`Sheet 4 of 4
`
`5,763,594
`
`FlGURE 4.
`
`A
`1 21
`
`151
`
`C
`123
`
`153
`
`G
`125
`
`155
`
`T
`127
`
`157
`
`deblock
`128
`
`water
`129
`
`158
`
`159
`
`131
`
`139
`
`1 41
`
`143
`
`145
`
`148
`
`149
`
`, a,
`
`183
`
`r
`DISCARD/RECOVERY
`
`to 151
`
`157
`
`Page 5
`
`

`

`5,763,594
`
`1
`3' PROTECTED NUCLEOTIDES FOR
`ENZYME CATALYZED TEMPLATE(cid:173)
`INDEPENDENT CREATION OF
`PBOSPHODIESTER BONDS
`
`RELATED APPLICATIONS
`
`This application is a Continuation-in-Part of U.S. patent
`application Ser. No. 08/300.484 filed Sep. 2, 1994. This
`application is also related to co-pending and commonly
`owned U.S. patent application Ser. Nos. 08/486,535; 08/486,
`536; 08/486,885 and 08/486.897; all of which, together with
`this application, have been filed on Jun. 7. 1995.
`
`TECHNICAL FJELD
`
`This invention relates to the synthesis of oligonucleotides
`and other nucleic acid polymers using template independent
`enzymes.
`
`BACKGROUND OF THE INVENTION
`
`2
`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 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
`10 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-
`15 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
`20 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
`25 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-
`30 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
`35 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
`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(cid:173)
`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(cid:173)
`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.
`However, some polymerases are template independent and
`primarily catalyze the formation of single stranded nucle-
`otide polymers. Another class of enzyme, the ligases, are
`template independent and form a phosphodiester bond
`between two polynucleotides or between a polynucleotide
`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.
`En:zymol., 100:96-116, 1983. These reaction conditions did
`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-
`
`40
`
`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. 5264.566 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 45
`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 50
`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 55
`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 60
`while widely accepted require latge 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 65
`requires that the subpopulation of oligonucleotides that have
`not had a monomer added in a particular cycle be capped in
`
`Page 6
`
`

`

`5,763,594
`
`3
`otide sequence. The presence of unprotected 3'-hydroxyls
`resulted in a highly heterogeneous population of reaction
`products.
`Similarly. prior attempts to catalyze synthesis of very
`short pieces of RNA or DNA using protected nucleotide 5
`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 mononucleotide building blocks or 10
`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-lL 1971). These
`experiments were limited to 5'-monophosphates and diphos- 15
`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- 20
`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 25
`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 30
`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- 35
`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-
`nucleotides. The inefficiency of these methods made these
`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- 45
`termined sequence. This enzyme catalysis can vastly
`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 50
`of single or double stranded DNA as well as other types of
`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 55
`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 60
`and diphosphates are used. In addition. triphosphates are less
`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 65
`capable of efficiently joining protected nucleotides to initi(cid:173)
`ating substrates will enable the creation of a highly uniform
`
`40
`
`4
`population of synthetic polynucleotides resulting from step(cid:173)
`wise phosphodiester bond formation.
`
`SUMMARY OF THE INVENTION
`A number of methods have been discovered by which the
`3'-hydroxyl group of a deoxynucleotide triphosphate can be
`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.
`
`Page 7
`
`

`

`5,763,594
`
`6
`analogs and modified forms of the naturally-occurring bases,
`such as pseudoisocytosine and pseudouracil and other modi(cid:173)
`fied bases (such as 8-substituted purines). In RNA. the
`5-carbon sugar is ribose; in DNA, it is 2'-deoxyribose. The
`term nucleoside also includes other analogs of such
`subunits. including those which have modified sugars such
`as 2'-0-alkyl ribose.
`Polynucleotide: A nucleotide multimer generally about 50
`nucleotides or more in length. These are usually of biologi-
`10 cal origin or are obtained by enzymatic means.
`
`5
`
`I
`Phosphodiester: Tuegroup O=P-0
`I
`0
`I
`
`5
`The methods of the present invention contemplate remov(cid:173)
`ing the removable blocking moiety using a deblocking
`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.
`tris[hydroxymethylj 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
`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 15
`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
`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(cid:173)
`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.
`
`wherein phosphodiester groups may be used as internucle(cid:173)
`osidyl phosphorus group linkages (or links) to connect
`20 nucleosidyl units.
`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 ),
`25 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
`30 (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
`35 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.
`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
`45 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.
`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 specifically
`binds and initiates RNA synthesis (transcription) of that
`gene.
`55 Oligonucleotide: A chain of nucleosides which are linked
`by intemucleoside linkages which is generally from about 2
`to about 50 nucleosides in length. They may be chemically
`synthesized from nucleoside monomers or produced by
`enzymatic means. The term oligonucleotide refers to a chain
`60 of nucleosides which have internucleosidyl linkages linking
`the nucleoside monomer and, thus, includes oligonucleotide
`containing nucleoside analogs, oligonucleotide having inter(cid:173)
`nucleosidyl linkages such that one or more of the phospho(cid:173)
`rous group linkages between monomeric units has been
`65 replaced by a non-phosphorous linkage such as a formacetal
`linkage, a thioforrnacetal linkage, a sulfarnate linkage. or a
`carbamate linkage. It also includes nucleoside/non-
`
`40
`
`50
`
`BRIEF DESCRIPTION OF TI1E DRAWINGS
`
`FIG. 1. A diagram showing enzymatic synthesis of an
`oligonucleotide using a template independent polymerase
`and a nucleoside 5' triphosphate having a removable block(cid:173)
`ing moiety at its 3' position is shown.
`FIG. 2. A diagram of a nucleotide having a removable
`blocking moiety at its 3' position is shown.
`FIG. 3. A diagram showing an apparatus for automating
`the enzymatic synthesis of polynucleotides is shown.
`FIG. 4. A diagram showing an apparatus for automating
`the enzymatic synthesis of polynucleotides is shown.
`
`BRIEF DESCRIPTION OF THE INVENTION
`
`A. Definitions
`DNA: Deoxyribonucleic acid.
`RNA: Ribonucleic acid.
`Nucleotide: A subunit of a nucleic acid comprising a
`phosphate 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
`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 nucleosi(cid:173)
`dyl units having A. G. C, T and U as their bases, but also
`
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`
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`5,763,594
`
`7
`nucleoside polymers wherein both the sugar and the phos(cid:173)
`phorous 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(cid:173)
`nucleoside polymer. Thus an oligonucleotide may be par(cid:173)
`tially or entirely phophonothioates. phosphorothioate phos(cid:173)
`phorodithioate phosphoramidate or neutral phosphate ester
`such as phosphotriesters oligonucleotide analogs.
`Removable Blocking Moiety: A removable blocking moi(cid:173)
`ety 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 prevents
`reaction of the 3' oxygen when present and is removable
`under deblocking conditions so that the 3' oxygen can then
`participate in a chemical reaction.
`A. Methods
`Generally. the present invention provides methods for 20
`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 25
`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 30
`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 nucleer
`side having an unprotected 3'-hydroxyl group; and
`(b) reacting under enzymatic conditions in the presence of
`a catalytic amount of an enzyme said3'-hydroxyl group
`of said initiating substrate with a nucleoside
`5'-triphosphate having a removable blocking moiety
`protecting the 3' position of said nucleoside 40
`5'-triphosphate and selected according to the order of
`said predetermined sequence. whereby said enzyme
`catalyzes the formation of a 5' to 3' phophodiester
`linkage between said unprotected 3'-hydroxyl group of
`said initiating substrate and the 5'-phosphate of said 45
`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- 50
`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 55
`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(cid:173)
`ods in which the above steps (b) and ( c) are repeated at least
`once to produce an oligonucleotide. This process can be 60
`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
`having a preselected sequence.
`1. Initiating Substrates
`An initiating substrate of the present invention is prepared
`containing a nucleoside with a free and unmodified
`
`8
`3'-hydroxyl group. As is well understood by those of ordi(cid:173)
`nary skill in the art, nucleotide derivatives of the nucleosides
`adenosine, cytidine. guanosine, uridine and thymidine can
`be assembled to form oligonucleotides and polynucleotides.
`s According to the method of the present invention, the
`initiating substrate may contain a single nucleoside having a
`free and unmodified 3'-hydroxyl group, or a preassembled
`oliger or polynucleotide may be provided as an initiating
`substrate. so long as the oligo- or polynucleotide has a free
`10 and unmodified 3'-hydroxyl group.
`One skilled in the art will understand that an initiating
`substrate could be prov