[11]
`
`(45)
`
`4,362,867
`Dec. 7, 1982
`
`(57]
`ABSTRACT
`Compounds useful as complementary DNA (eDNA)
`include deoxyribonucleotides and at least one ribonu(cid:173)
`cleotide. They may be depicted by the general formula:
`
`3'(rNJb-(d
`I
`S'(dN)c3'
`
`1N
`
`Ja5'
`
`wherein (dN)a and (dN)c represent series of deox(cid:173)
`yribonucleotides and (rN)b[epresents a series of ribonu(cid:173)
`cleotides; wherein a, b, and c are the number of nucleo(cid:173)
`tides in the series, with the pro.,.iso that b is ~ 1, a is
`~35, and cis ~ 10; wherein the series of deoxyribonu(cid:173)
`cleotides (dN)a includes a series of deoxyribonucleo(cid:173)
`tides which is substantially complementary to the series
`of deoxyribonucleotides (dN)c and the dashed line rep(cid:173)
`resents noncovalent bonding between the complemen(cid:173)
`tary deoxyribonucleotide series; and wherein the solid
`line represents a coval!'!nt phosphodiester bond.
`These compounds may be prepared from messenger
`RNA (mRNA) containing the genetic information nec(cid:173)
`essary for cellular production of desired products such
`as polypeptides. After appropriate modification, they
`may be combined with DNA from a suitable cloning
`vehicle such as a plasmid and the resulting combined
`DNA used to transform bacterial cells. The trans(cid:173)
`formed bacterial cells may then be grown and har(cid:173)
`vested; and the desired product or products recovered.
`
`4 Claims, 1 Drawing Figure
`
`Sanofi/Regeneron Ex. 1037, pg 980
`
`United States Patent (19J
`Paddock
`
`(54) RECOMBINANT CDNA CONSTRUCTION
`METIIOD AND HYBRID NUCLEOTIDES
`USEFUL IN CLONING
`
`(75)
`
`Inventor: Gary V. Paddock, Mount Pleasant,
`S.C.
`
`[73] Assignee: Research Corporation, New York,
`N.Y.
`
`[21) Appl. No.: 214,648
`
`[22) Filed:
`
`Dec. 10, 1980
`
`(51)
`
`Int. CJ.l ...................... C07H 15/12; Cl2P 21/00;
`C12P 21/02; C12P 19/34; CI2P 15/00; CI2N
`1/00
`(52) U.S. Cl. ........................................ 536/27; 536/28;
`435/68; 435/70; 435/91; 435/172; 435/317
`(58) Field of Search ....................... 435/91; 536/27, 28
`
`(56)
`
`References Cited
`PUBLICATIONS
`Salser, in Genetic Engineering, Chakrabarty (ed.), CRC
`Press, West Palm Beach, Fla., pp. 53-81 (1978).
`Kavsan et al., Chern. Abstr. 92, 224 I23526h (1980).
`Romano eta!., Chern. Abstr. 91:170822r (1979).
`Hartman eta!., Chern. Abstr. 87:147246q (1977).
`Wagar eta!., Chern. Abstr. 84:100898b (1976).
`Ogawa eta!., Chern. Abstr. 87, 17513n (1977).
`
`Primary Examiner-Alvin E. Tanenholtz
`Assistant Examiner-James Martinell
`Attorney, Agent, or Firm-Cooper, Dunham, Clark,
`Griffin & Moran
`
`Merck Ex. 1037, pg 1006
`
`

`
`U.S. Patent
`
`Dec. 7, 1982
`
`4,362,867
`
`GLOBIIhn RNA rA
`5'
`~3'
`I '*JV REVERSE TRANSCRIPTASE
`dT i+dNTP.s; OLIGO dT
`~--;::-:;~VV\ 5 I
`C3' c DNA
`I DNA POLYMERASE (I<'LE"NCW
`y SUB FRACTION)+ rNTPs
`dT
`~~DNA POLYME~ASe(KLHJOW
`dT
`c::=:::
`,t,'ALINE dAl R~ASE H
`~~~IGEST OR
`I PHOSPHATASE
`t DIGEST
`
`fSUBfRACTIO~) tdijfP.s
`
`TERMINAL
`T~ANSFERAS E'
`4-dCTP
`
`!TE"RMINAL
`
`TRANSFt'RAS£
`+ctCTP
`dC N
`dT
`~r - -NV \
`
`...... -
`
`_.. _...
`
`Psr RESTRICTION
`E'NZYME
`
`l
`!TE'RMINAL
`
`·-------------
`
`TRANSFFRASE
`dG
`4 ~GTP
`~---- -------
`- - - - - - - - - - -- -tv"Y'w
`clG
`
`--Jv'VVVvV'.
`dA dC
`
`ANNEAL
`HETE ROGE.~E.OUS POPULATION OF HYBRID D~As
`IT~ANSFORM4T\ON OF E'.Coli Xl776 TO
`t TETRAC \"C Ll NE ~ESISTANCE
`1 SEltCTION OF CLONES SV CCLON'r' 'r\V6RID(cid:173)
`f 12ATION AND ABSENCE OF AMPICILLAN
`dC
`RE.SISTANCE
`_..- ------ ---- ----- -\1\1\N\AN----....
`
`((~---~~:::-~:::---~
`
`" ----------------- ---clG
`HOMOGENEOU.S POPULATION OF RECOMBINANT DNAs
`
`Sanofi/Regeneron Ex. 1037, pg 981
`
`Merck Ex. 1037, pg 1007
`
`

`
`1
`
`4,362,867
`
`RECOMBINANT CDNA CONSTRUCfiON
`METHOD AND HYBRID NUCLEOTIDES USEFUL
`IN CLONING
`
`The invention described herein was made in the
`course of work partly supported by Grant Number
`GM-24783 from the National Institute of Health, De(cid:173)
`partment of Health and Human Services.
`
`FIELD OF THE INVENTION
`This invention generally concerns the preparation of
`recombinant complementary DNAs (cDNAs) and
`eDNA analogs coding for cellular production of desir(cid:173)
`able products such as polypeptides. It also concerns
`novel compounds which include both deoxyribonucleo(cid:173)
`tides and ribonucleotides. Finally, it concerns the use of
`such compounds in bacterial cloning.
`
`2
`F. C., (1976), Cell, 8: 163-182; Rougeon, F., Kourilski,
`P., and Mach, B., (1975), Nucl. Acids Res. 2: 2365-2378;
`Efstratiadis, A., Kafatos, F. C., and Maniatis, T., (1977),
`Cell, 10: 571-585; Rabbits, T. H., (1976), Nature, 260:
`221-225; Rougeon, F. and Mach, B., (1976), Proc. Nat!.
`Acad. Sci. U.S.A., 73: 3418-3422; and Wood, K. 0. and
`Lee, J. C., (1976), Nucl. Acids Res., 3: 1961-1971.] have
`been successful in preserving these important terminal
`sequences. In fact, the most popular of these techniques
`10 is destined to destroy these sequences in part, because it
`relies upon use of Sl nuclease. [Higuchi, R., Paddock,
`G. V., Wall, R., and Salser, W., (1976), Proc. Nat!.
`Acad. Sci. U.S.A., 73: 3146-3150.]
`In order to preserve these important 5'-end signals,
`15 efforts have been undertaken to develop methodology
`which avoids the need for Sl nuclease. [Frankis, R.,
`Gaubatz, J., Lin, F. K., and Paddock, G. V., The
`Twelfth Miami Winter Symposium (ed. Whelan, W. J.,
`and Schultz, J., Academic Press, New York), val. 17, in
`BACKGROUND OF THE INVENTION
`20 press (1980); and Gaubatz, J, and Paddock, G. V.,
`(1980), Fed. Proc., 39: 1782.] These efforts have re-
`One of the major areas of research in molecular bioi-
`suited in the discovery of the floppy loop method de-
`ogy today concerns gene organization and expression in
`scribed herein. This method employs a ribosubstitution
`eukaryotic cells. Much effort has been spent on studies
`step so that cleavage of the hairpin loop can be carried
`of RNA transcription and its subsequent processing to
`mRNA. It is currently thought that the genome sequen- 25 out by alkali or ribonuclease. It avoids destruction of
`ces surrounding the cap site contain the signal for initia-
`nucleotide sequence information which is lost if the
`tion ofmRNA transcription or, alternatively, that very
`hairpin is opened in the conventional manner with Sl
`rapid processing cleaves a way the first few nucleotides
`nuclease. Thus, by elimination of the Sl nuclease step,
`followed by capping at the 5' end. [Konkel, D. A.,
`whole genes can be synthesized without loss of genetic
`Tilghman, S. M., and Leder, P., (1978), Cell, 15: 30 information. Moreover, the Sl nuclease technique is
`1125-1132; Konkel, D. A., Maize!, J. V., Jr., and Leder,
`known to introduce errors in the sequence [Richards, R.
`P., (1979), Cell, 18: 865-873; Gannon, F., O'Hare, K.,
`I., Shine, J., Ulbrich, A., Wells, J. R. E., and Goodman,
`H. M., (1979), Nucl. Acids Res. 7: 1137-1146] through
`Perrin. F., LePennec, J. P., Benoist, C., Cachet, M.,
`Breathnach, R., Royal, A., Garapin, A., Cami, B., and
`a mechanism which the present invention avoids. Fi-
`Chambon, P., {1979), Nature, 278: 428-434; Nishioka, 35 nally, although it has been demonstrated that hormones
`Y. and Leder, P., (1979), Cell, 18: 875-882; and Kin-
`(insulin) and interferon can be cloned via recombinant
`niburgh, A. J. and Ross, J., (1979), Cell, 17: 915-921.]
`eDNA, it may not be possible to clone some genes in
`In either case, the nucleotide sequences contained in the
`their entireties with the Sl nuclease technique because
`5' untranslated regions of mRNA, especially those near
`the hairpin loop may be extremely large and may even
`the cap site, are of ·prime importance to proper gene 40 include part of the structural gene (i.e., part of the
`mRNA coding for protein.)
`regulation, as illustrated by the extensive conservation
`of sequences found in this region for alpha and beta
`globin and other mRNA species. [Konkel, D. A., Tilgh(cid:173)
`man, S.M., and Leder, P., (1978), Cell, 15: 1125-1132;
`Konkel, D. A., Maize], J. V., Jr., and Leder, P., (1979), 45
`Cell, 18: 865-873; and Lockard, R. E. and RajBhand(cid:173)
`ary, U. L., (1976), Cell, 9: 747-760.] In addition to tran(cid:173)
`scription and processing of mRNA, these sequences
`undoubtedly play an important role in the translation of
`protein from mRNA. Indeed, the importance of the 50
`nucleotides contained in the 5' untranslated regions of
`mRNA is emphasized by the variety of methods de(cid:173)
`signed to sequence them. [Lockard, R. E. and RajB(cid:173)
`handary, U. L., (1976), Cell, 9: 747-760; Baralle, F. E.,
`(1977), Cell 10: 549 -558; Baralle, F. E., {1977), Nature, 55
`267: 279-281; Baralle, F. E., (1977), Cell, 12: 1085-1095;
`Legan, S., (1976). J. Mol. Bioi., 106: 37-53; Chang, J.
`C., Temple, G. F., Poon, R., Neumann, K. H. and Kan
`Y. W., (1977)., Proc. Nat!. Acad. Sci. U.S.A., 74:
`5145-5149; and Chang, J. C., Poon, R., Neumann, K. H. 60
`and Kan, Y. W., (1978), Nucl. Acids. Res., 5:
`3515-3522.] Yet none of these methods permits se(cid:173)
`quencing of the 5' end of an impure mRNA obtained in
`low yield as is the case for most mRNAs. Furthermore,
`none of the cloning techniques developed thus far, 65
`(Higuchi, R., Paddock, G. V., Wall, R., and Salser, W.,
`(1976), Proc. Nat!. Acad. Sci. U.S.A., 73: 3146-3150;
`Maniatis, T., Kee, S. G., Efstratiadis, A., and Kafatos,
`
`SUMMARY OF THE INVENTION
`This invention provides various compounds which
`include both deoxyribonucleotides and at least one ribo(cid:173)
`nucleotide. Certain of these compounds are useful in the
`preparation of eDNA and eDNA analogs. Others are
`useful in bacterial cloning. This invention also provides
`processes for preparing such molecules and using them
`in the production of desirable products such as polypep(cid:173)
`tides.
`Specifically, compounds useful in the preparation of
`cDNAs and eDNA analogs may be prepared. These
`compounds may be depicted by the formula:
`
`(dN)0 S'
`/ ' I
`(rN)b
`
`I "' (dN),3'
`
`wherein (dN)a and (dN)c rep,resent series of deox(cid:173)
`yribonucleotides and (rN)b represents a series of ribonu(cid:173)
`cleotides; wherein a, b, and c are numbers of nucleotides
`in the series provided that b is ~I, a is ~35, and c is
`~ 10; wherein the series of deoxyribonucleotides and
`( dN)a includes a series of deoxyribonucleotides which is
`substantially complementary to the series of deox-
`
`Sanofi/Regeneron Ex. 1037, pg 982
`
`Merck Ex. 1037, pg 1008
`
`

`
`3
`yribonucleotides (dN)c and the dashed line represents
`non-covalent bonding between the complementary de(cid:173)
`oxyribonucleotide series; and wherein the solid lines
`represent covalent phosphodiester bonds. Such com(cid:173)
`pounds may be prepared as follows. A first molecule
`having the formula 3'(dN)0 5' is prepared. At least one
`ribonucleotide is added to the 3'-end of this molecule to
`produce a molecule having the formula 3'(rN)b-(d(cid:173)
`N)a5', and additional deoxyribonucleotides are then
`added to the 3'-end of the latter to produce the com(cid:173)
`pounds.
`If these compounds are treated with a reagent capa(cid:173)
`ble of breaking or disrupting either 5'(rN)-(dN)3' or
`(rN)-(rN) bonds, or both, compounds useful as eDNA
`analogs or precursors may be prepared having the for- 15
`mula:
`
`4,362,867
`
`4
`cleotides (dN),; wherein the xxx-lines represent double(cid:173)
`stranded DNA derived from a cloning vehicle such as a
`plasmid, bacteriophage, or virus; wherein the dotted
`lines may be either no bond or covalent bonds; wherein
`the dashed lines represent non-covalent bonding be(cid:173)
`tween complementary deoxyribonucleotide series; and
`wherein the solid lines represent covalent phosphodi(cid:173)
`ester bonds.
`These molecules may be used to transform bacterial
`10 or eucaryotic cells, e.g., Escherichia coli cells, which
`may be grown in culture to produce desired products,
`including polypeptides.
`
`II
`
`20
`
`)a5'
`
`3'(rN)b-(d
`I
`5'(dN),3'
`
`1N
`
`wherein (dN)a and (dN)c represent series of deox(cid:173)
`yribonucleotides and (rN)brepresents a series of ribonu(cid:173)
`cleotides; wherein a, b, and care the numbers ofnucleo(cid:173)
`tides in the series; provided that b is ~ 1, a is ii; 35, and 25
`c is ii; 10; wherein the series of deoxyribonucleotides
`(dN)a includes a series of deoxyribonucleotides which is
`substantially complementary to the series of deox(cid:173)
`yribonucleotides (dN)c and the dashed line represents
`non-covalent bonding between the complementary de- 30
`oxyribonucleotide series; and wherein the solid line
`represents a covalent phosphodiester bond.
`Further compounds having the formula:
`
`3'(dN')a-(rN)b-{d['Ja5'
`I
`5'(dN),-(dN')e3'
`
`III 35
`
`may be prepared, wherein (dN)0 , (dN),, and (rN)bare as
`above and (dN')d and (dN')e represent series of identical 40
`deoxyribonucleotides; wherein a, b, and c are as above,
`and d and e are integral numbers of nucleotides in the
`series and are ~ 10; and wherein the solid lines represent
`covalent phosphodiester bonds. These compounds are
`prepared by adding deoxyribonucleotides dN' to the 45
`3'-ends of compounds II.
`Still other compounds may be prepared having the
`formula:
`
`BRIEF DESCRIPTION OF THE DRAWING
`FIG. 1 is a schematic illustration of the ribosubstitu(cid:173)
`tion floppy loop recombinant eDNA technique show(cid:173)
`ing alternative means for cleavage of the ribosubstituted
`hairpin double-stranded eDNA.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`Much publicity has been given to recent efforts to
`employ genetic engineering, particularly recombinant
`DNA technology, in the production of useful products
`such as insulin, interferon, growth hormone and the
`like. These attempts have often involved the insertion of
`a DNA molecule containing the genetic information
`necessary for cellular production of the desired product
`into host cells, especially bacterial cells. Bacterial cells
`into which such DNA molecules have been inserted can
`be grown in culture, resulting in the production of in(cid:173)
`creased quantities of recoverable products.
`One limitation upon such efforts is the availability of
`appropriate DNA molecules. Approaches which have
`been pursued to obtain these DNA molecules include
`synthesis of the molecules by conventional chemical
`methods and by reverse transcription of mRNA mole(cid:173)
`cules which have been recovered from cells which
`contain DNA sequences coding for the desired product.
`This invention is directed to improved methods of
`preparing these DNA molecules, called complementary
`DNA (eDNA). It is also directed to the preparation of
`novel hybrid molecules which include both deox(cid:173)
`yribonucleotides and at least one ribonucleotide. Cer(cid:173)
`tain of these molecules are useful in the preparation of
`eDNA and eDNA analogs. Others are useful in bacte(cid:173)
`rial cloning.
`One such compound may be represented by the gen(cid:173)
`eral formula:
`
`(dN)aS'
`/ I
`(rN)b I
`
`"' {dN),3'
`
`IV 50
`
`55
`
`XXX •.. (dN')a-(rN)b-=:l
`X
`',,
`XX(dN")f ... I
`X
`~-----~
`X
`I
`X
`xxxxxxxxxxxx {d
`
`XXXX ... (dN')e _j J
`
`(dN)c-(dN)a
`
`")g ..•
`
`1N
`
`wherein (dN)a and (dN), represent series of deox(cid:173)
`yribonucleotides, (dN')d, (dN')e. (dN")pnd (dN")grep(cid:173)
`resent series of identical deoxyribonucleotides, the se- 60
`ries (dN')dand (dN')e being complementary to the series
`(dN")Jand (dN")8 respectively, and (rN)b represents a
`series of ribonucleotides; wherein, a, b, c, d, e, f, and g
`are integral numbers of nucleotides in the series; pro(cid:173)
`vided that b is ii; I, a is ~ 35, and c, d, e, f, and g are 65
`~ 10; wherein the series of deoxyribonucleotides (dN)a
`includes a series of deoxyribonucleotides which is sub(cid:173)
`stantially complementary to the series of deoxyribonu-
`
`wherein (dN)a and (dN)c represent series of deox(cid:173)
`yribonucleotides and (rN)b represents a series of ribonu(cid:173)
`cleotides; wherein a, b, and care the numbers ofnucleo(cid:173)
`tides in the series; wherein b is ~I, a is ~ 35, and c is
`~ 10; wherein the serieS of deoxyribonucleotides (dN)a
`includes a ser'ies of deoxyribonucleotides which is sub(cid:173)
`stantially complementary to the series of deoxyribonu(cid:173)
`cleotides (dN),and the dashed line represents non-cova(cid:173)
`lent bonding, particularly hydrogen bonding, between
`complementary
`deoxyribonucleotide
`series;
`and
`wherein the solid lines represent covalent phosphodi(cid:173)
`ester bonds.
`
`Sanofi/Regeneron Ex. 1037, pg 983
`
`Merck Ex. 1037, pg 1009
`
`

`
`4,362,867
`
`5
`In such molecules the deoxyribonucleotide series
`(dN)a is an ordered polymer of deoxyribonucleotides
`which include the purines, adenine and guanine, and the
`pyrimidines, thymine and cytosine. The particular order
`of deoxyribonucleotides contains information necessary 5
`for cellular production of a desired product in accor(cid:173)
`dance with the established genetic code, whereby
`groups of three nucleotides correspond to single amino
`acids which are assembled by cells into polypeptides.
`The precise order of the nucleotides may vary widely, 10
`depending upon the product for which the series codes.
`However, in the aforementioned compound I, the num(cid:173)
`ber of nucleotides, a, must generally be E; 35 since fewer
`nucleotides do not permit sufficient formation of non(cid:173)
`covalent bonding between complementary nucleotides 15
`in the series (dN)a and (dN)c· More often, the number of
`nucleotides will be even greater, varying from as few as
`about 60 in the case of DNA sequences which code for
`oligopeptides, to about 103 nucleotides for an average
`protein containing about 300 amino acids, and to E; 105 20
`for particularly large polypeptides.
`The series (dN)cis also an ordered polymer, of at least
`10 deoxyribonucleotides. The order of nucleotides is
`such that the nucleotide series (dN)c is complementary
`to an equal number of nucleotides in the series (dN)0• In 25
`general, the length of the deoxyribonucleotide polymer
`(dN)c will be about 25 nucleotides shorter than the
`length of the series (dN)a. The approximately 25 nucle(cid:173)
`otide difference may be attributed to nucleotides in the
`series (dN)a which are not base-paired with nucleotides 30
`in the series (dN)c, but are involved in formation of a
`folded segment of nucleotides known to those skilled in
`the art as a hairpin structure. The minimum length of
`about ten nucleotides is necessary to permit sufficient
`non-covalent, hydrogen bonding between complemen- 35
`tary nucleotides in the (dN)c and (dN)a polymer strands
`to form a double-stranded helical structure.
`Situated between and covalently bonded to the series
`(dN)a and (dN), is a series of ribonucleotides (rN)b.
`wherein b is the integral number of nucleotides in the 40
`series and is E; I. It will be appreciated that, when b=O,
`the resulting molecule is a homopolymer of deox(cid:173)
`yribonucleotides. Although b may vary considerably, it
`will generally be less than about 50 and oftentimes less
`than about 20. Moreover, in practicing the invention, it 45
`may be preferable to first form a molecule having the
`formula 3'(rN)b-(dN)a5', wherein b is E;], then re(cid:173)
`move ribonucleotides until b= I, and finally add deox(cid:173)
`yribonucleotides (dN)c to form molecules I, wherein
`b =I. In all molecules I, the ribonucleotide series (rN)b 50
`is covalently joined to the series (dN)a and (dN)c by
`means of 5'~3' and 3'~5' phosphodiester bonds, re(cid:173)
`spectively.
`Although the experiments described hereinafter in(cid:173)
`volve globin polypeptides, it will be readily understood 55
`by those skilled in the art that the practices of this in(cid:173)
`vention are widely applicable to polypeptides generally
`and to other desirable products. Thus, the deoxyribonu(cid:173)
`cleotide series (dN)a may contain information in the
`form of its ordered nucleotide sequence for cellular 60
`production of any desired product, e.g., proinsulin, the
`polypeptide A chain of insulin, the polypeptide B chain
`of insulin, a growth hormone, an enzyme, a clotting
`factor, an antibody, or the polypeptide portion of one of
`the interferon glycoproteins. These examples are set 65
`forth to illustrate some of the better known commercial
`products which may be prepared in accordance with
`the present invention, but are not intended to be limit·
`
`6
`ing, since countless other products may also be pre(cid:173)
`pared.
`In general, the deoxyribonucleotide series (dN)a will
`be obtained by reverse transcription of a messenger
`RNA (mRNA) molecule which itself will have been
`obtained by standard methods from a natural source,
`such as a eukaryotlc cell known to contain a gene or
`genes coding for or otherwise associated with produc(cid:173)
`tion of the desired product. However, it is also contem(cid:173)
`plated that the series (dN)a might be directly synthe(cid:173)
`sized to create a polynucleotide having a sequence cod(cid:173)
`ing for a desired product.
`Compounds I may be prepared as follows. Initially, a
`first molecule having the formula 3'(dN)a5' is prepared
`either by conventional chemical synthesis, or preferably
`by reverse transcription of a mRNA molecule corre(cid:173)
`sponding thereto. One method of accomplishing this
`result is to treat the mRNA molecule with a suitable
`enzyme, such as AMY reverse transcriptase, and a mix(cid:173)
`ture of the deoxyribonucleotides, dATP, dCTP, dGTP,
`and dTTP, under appropriate conditions which permit
`formation of the (dN)a molecule. Suitable conditions are
`well known in the art and may include: a temperature of
`about 25'-45' C., e.g., 37' C.; a buffer having a pH of
`about 7.0 to 9.0, e.g., 8.3; a catalytic amount of enzyme;
`and a molar excess of the deoxyribonucleotide triphos(cid:173)
`phates.
`In one embodiment of the invention, the mRNA mol(cid:173)
`ecule will include a series of repeating adenylate ribonu(cid:173)
`cleotides at its 3' -end, the number of such being n, and
`formation of (dN)a involves addition of oligo dT, e.g.,
`oligo (dT)!2-18, to the reaction mixture in sufficient
`quantity to permit formation of a 3'(dN)a5' molecule
`having n repeating thymidylate deoxyribonucleotides at
`its 5' -end. In such molecules n is necessarily less than a.
`Next, at least one ribonucleotide is added to the 3' •
`end of the 3'(dN)a5' molecule to form a molecule hav(cid:173)
`ing the formula 3'(rN)b-(dN)a5', Although ribonucleo(cid:173)
`tide addition could be accomplished by conventional
`chemical methods, it is preferably effected by a ribosub(cid:173)
`stitution addition reaction utilizing a DNA polymerase,
`e.g., DNA polymerase I, and a mixture of the ribonucle(cid:173)
`otide triphosphates rATP, rGTP, rCTP, and rUTP
`under appropriate conditions to permit formation of the
`3'(rN)b-(dN)a5' molecule. Once again, suitable condi(cid:173)
`tions are known. They may include temperatures of
`about 25'-45' C., e.g., 37' C.; buffers having pH's of
`about 7.0, e.g., 7.4; molar excess of ribonucleotide tri(cid:173)
`phosphates; and catalytic amounts of enzyme.
`Finally, additional deoxyribonucleotides are added to
`the 3'-end of the 3'(rN)b-(dN)a5' molecule to form
`compound I. Here again, conventional chemical meth(cid:173)
`ods may be employed, or preferably enzymatic addition
`of the nucleotides may be utilized. In this regard, treat(cid:173)
`ment of the 3'(rN)b-(dN)a5' molecule with a DNA
`polymerase, e.g., DNA polymerase I, and a mixture of
`dATP, dCTP, dGTP, and dTTP under suitable reac(cid:173)
`tion conditions may be used. Suitable conditions may
`include temperatures from about 25"-45" C., e.g., 37"
`C.; buffers having pH's of about 7.0, e.g., 7.3; molar
`triphosphates; and catalytic
`excess of nucleotide
`amounts of polymerase.
`The resulting compound I may then be recovered and
`purified by conventional techniques. Thereafter, it may
`be converted to a double-stranded molecule useful in
`bacterial cloning as described more fully hereinafter.
`Also, it may have uses in other areas, e.g., in pharma(cid:173)
`ceutical preparations or diagnostic tests.
`
`Sanofi/Regeneron Ex. 1037, pg 984
`
`Merck Ex. 1037, pg 1010
`
`

`
`4,362,867
`
`7
`If compound I is treated with a reagent capable either
`of breaking 5'(rN)-(dN)3' bonds alone or of breaking
`both 5'(rN)-(dN)3' and (rN)-(rN) bonds under suit(cid:173)
`able conditions (suitability being determined by the
`nature of the reagent and the precise identity of com(cid:173)
`pound (I) a double-stranded compound can be prepared
`having the formula:
`
`3'(rN)b-(dN)a5'
`
`5'(d~)c3'
`
`n
`
`10
`
`20
`
`wherein (dN)a, (dN)c, (rN)b, a, b, c, and the dashed and
`solid lines are as indicated for compound I.
`Suitable reagents include alkali, alkaline phosphatase, 15
`and RNase, particularly RNase H. The appropriate
`conditions for employing each such reagent vary in
`terms of temperature, time, pH, and the like. Generally,
`the reagents will b~; employed at temperatures in the
`range 25"-45' C., e.g., 37' C.; for reaction times from
`about 10 minutes to about 5 hours; and in buffers having
`pH's of about 7.0.
`Compounds II can be converted to homogeneous
`~~t%~ ~~~~~~::~ ~!nal~:hr:;~~~::t~~o~~d~~r~~~a r~j 25
`wherein b = 0. Many such compounds are known. How(cid:173)
`ever, there may be some which are novel, particularly,
`in cases where the deoxyribonucleotide series (dN)a has
`been prepared by chemical synthesis and codes for an 30
`oligopeptide or polypeptide which does not occur in
`nature.
`This invention contemplates the use of compounds II
`as eDNA analogs in subsequent cloning. In this applica(cid:173)
`tion, it may be preferable to remove all ribonucieotides 35
`other than the first ribonucleotide joined to the (dN)a
`series of deoxyribonucleotides if this result has not al(cid:173)
`ready been effected by the reagent treatment.
`Compounds II may be stored for subsequent use in
`bacterial cloning. Alternatively, the compounds may be 40
`distributed or sold for such use. Finally, the compounds
`may be employed immediately in cloning.
`For cloning, it is generally desirable to add identical
`deoxyribonucleotides dN' to the 3'-ends of compounds
`II. This may be accomplished by known techniques, 45
`including treatment of compounds II with an enzyme,
`such as terminal transferase, and an excess of a single
`deoxyribonucleotide triphosphate dN'TP under suitable
`conditions. In this reaction N' may be any ofG, C, T, or
`A.
`This results in production of compounds which may
`be depicted by the formula:
`
`3'(dN')a-(rN)b-(dN)0 5'
`
`5'(d~),-(dN'),3'
`
`III
`
`wherein (rN)b. (dN)a, (dN)c, a, b, c, and the dashed and
`solid lines are as indicated hereinabove; (dN')d and
`(dN')e represent series of identical deoxyribonucleo(cid:173)
`tides; and wherein d and e are the number of nucleotides
`in the series, provided that d and e are ~ 10. It is gener(cid:173)
`ally necessary that d and e be ~ 10 in order for com(cid:173)
`pounds III to be used in subsequent cloning. Also, d and
`e will generally not exceed about 20-40, although this is 65
`not a critical upper limit, only one of convenience. The
`precise number of nucleotides may be varied by varying
`reaction conditions, including molar equivalents of nu-
`
`8
`cleotide triphosphates employed, as is true in the vari(cid:173)
`ous similar reactions discussed herein.
`Compounds III can be annealed with DNA mole(cid:173)
`cules which have been derived from a cloning vehicle,
`such as a plasmid, opened if circular, and tailed at both
`5'-ends with a series of deoxyribonucleotides dN"
`which is ~ 10 nucleotides in length. In order for anneal-
`ing to occur dN" must be complementary to dN'. An(cid:173)
`nealing is carried out under suitable conditions known
`to the art and results in formation of compounds which
`may be depicted by the formula:
`
`IV
`
`XXX ... (dN')a-(rN)b~
`X
`',,
`X
`XX(dN")J ... ---,
`X
`X
`I
`x-----x
`(dN)c-(dN)a
`X
`X
`I
`xxxxxxxxxxxx cdN"lc ...
`
`XXXX ... (dN'), _j J
`
`wherein (dN)a and (dN)c represent series of deox(cid:173)
`yribonucleotides, (dN')d, (dN')e, (dN").r. and (dN")g
`represent series of identical deoxyribonucieotides, the
`series (dN')d and (dN')e being complementary to the
`series (dN")Jand (dN")g respectively; and (rN)b repre-
`sents a series of ribonucleotides; wherein a, b, c, d, e, f,
`and g are the numbers of nucleotides in the series pro(cid:173)
`vided that b is ~I, a is ~35, and c, e, f, and g are ~ 10;
`wherein the series of deoxyribonucleotides (dN)a in(cid:173)
`cludes a series of deoxyribonucleotides which is sub-
`stantially complementary to the series of deoxyribonu(cid:173)
`cleotides (dN)c; wherein the xxx-lines represent double(cid:173)
`stranded DNA derived from a cloning vehicle; wherein
`the dotted lines may either be or not be covalent bonds;
`wherein the dashed lines represent non-covalent bond(cid:173)
`ing between complementary deoxyribonucleotide se(cid:173)
`ries; and wherein the solid lines represent covalent
`phosphodiester bonds.
`Generally, annealing alone will result in compounds
`IV wherein the dotted lines are not covalent bonds, but
`the presence of or subsequent treatment with a ligase or
`other suitable reagent can result in production of com(cid:173)
`pounds wherein the dotted lines represent covalent
`bonds. Compounds IV, both open and closed-loop ver-
`sions in which the dotted lines do and do not represent
`covalent bonds, are useful in cloning. Thus, for exam(cid:173)
`ple, when the cloning vehicle-derived DNA is plasmid
`50 DNA, e.g., pBR322 DNA, and the (dN)a deoxyribonu(cid:173)
`cleotide series includes the gene or genes coding for
`cellular production of a desired product or products,
`compounds IV can be used to transform bacterial cells,
`e.g., E. coli K-12 or )(1776. The transformed cells which
`55 then contain compounds IV may be cloned, grown in
`culture, harvested, disrupted, and the desired product
`recovered. By appropriate construction of compounds
`IV to insure that the series (dN)a includes the informa-
`60 tion necessary for cellular production of the desired
`product, and to insure that the cloning vehicle DNA is
`appropriate for use in transforming the contemplated
`host cells, it should be possible to successfully produce
`numerous, commercially valuable products.
`The following experiments are set forth to illustrate
`the practices of this invention, but should not be con(cid:173)
`strued as limiting the invention which is defined by the
`claims which follow.
`
`Sanofi/Regeneron Ex. 1037, pg 985
`
`Merck Ex. 1037, pg 1011
`
`

`
`4,362,867
`
`9
`EXPERIMENTAL DETAILS
`
`10
`was resuspended in 0.1 ml HzO, brought to 0.3 N in
`NaOH, and incubated at 90' C. for 30 min to hydrolyze
`the ribonucleotide link. The DNA was then precipi-
`MATERIALS AND METHODS
`tated with 0.1 val 3M NaAc, pH 5.5, 0.1 vol3 N HCl,
`Construction of recombinant cDNAs via the floppy
`40 /Lg/ml yeast tRNA, and 2.25 vol ethanol. The DNA
`loop technique was carried out as follows. Complemen-
`was then resuspended in 0.1 ml 0.1 M Tris-HCI, pH 8.0,
`tary DNA was synthesized in 0.1 ml to 0.4 ml 50 mM
`and treated with bacterial alkaline phosphatase (Wor-
`Tris-HCI, pH 8.3; 10 mM MgCh; 20 mM 2-mercaptoe-
`thanol; 30 /Lg/ml actinomycin D; 20 /Lg/ml oligo
`thington BAPF) as described by Shinagawa and Pad-
`manabhan. [Shinagawa, M. and padmanabhan, R.,
`(dT)tz-ts, obtained from Collaborative Research, Wal-
`tham, Mass.; 40-60 J.Lg/ml rabbit globin mRNA; I mM IO (1979), Anal. Biochem. 95: 458-464.] The reaction mix-
`each dATP, dGTP, dTTP, and dCTP [dCTP contain-
`ture was supplemented with 0.1% SDS and incubated
`ing 50-250 ILCii/Lmoi 3H or 32P]; and !50 U/ml AMV
`with 1.5-2.0 units of phosphatase which had been resus-
`pended in 0.1 M Tris-HCl (pH 8), 10 ILM ZnS04 for I hr
`reverse transcriptase, supplied by J. Beard through the
`Biological Carcinogenesis Branch, National Cancer
`at 37' C. [fhe removal of phosphate can be followed by
`Institute, NIH. Incubations in early experiments were 15 including 1 ~-tCi 32P-dNTP and at various times spotting
`carried out at 37' C. for 1 hr, but we have found incuba-
`1 ILl for cellulose thin-layer chromatography (me-
`thanol:HCI:H2o, 7:2:1). The reaction mixture can be
`lion at 45' C. for 15-20 min to be far superior. The
`reaction mixture was then extracted with an equal val-
`frozen until the degree of reaction is determined by
`ume of water-saturated phenol, and residual phenol was
`autoradiography of the chromatograms.] The reaction
`removed by extraction with an equal volume of ether. 20 mixture was then extracted four times with equal val(cid:173)
`The eDNA was precipitated with 40 /Lg/ml yeast
`umes of water-saturated phenol and one time with
`tRNA, 0.1 val 3 M NaAc (pH 5.5) and 2.25 val ethanol.
`The eDNA was resuspended in 0.2 ml H2o, brought to
`ether, and then chromatographed through Sephadex