United States Patent
`
`[19]
`
`Paddock
`
`[54] RECOMBINANT CDNA CONSTRUCI‘ION
`METHOD 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. Cl.’ .................... .. C07H 15/12; C12P 21/00;
`Cl2P 21/O2; C12P 19/34; C12P 15/00; C12N
`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., Chem. Abstr. 92, 224 l23526h (1980).
`Romano et al., Chem. Abstr. 91:170822r (1979).
`Hartman et al., Chem. Abstr. 87:147246q (1977).
`Waqar et al., Chem. Abstr. 84:100898b (1976).
`Ogawa et al., Chem. Abstr. 87, 17513n (1977).
`
`Primary Examiner—Alvin E. Tanenholtz
`Assistant Examiner-James Martinell
`Attorney, Agent, or Firm—Cooper, Dunham, Clark,
`Griffin & Moran
`
`[11]
`
`[45]
`
`- 4,362,867
`
`Dec. 7, 1982
`
`[57]
`
`ABSTRACI‘
`
`Compounds useful as complementary DNA (cDNA)
`include deoxyribonucleotides and at least one ribonu-
`cleotide. They may be depicted by the general formula:
`
`3'(!N)b"(dIN)a5'
`s'(di~I),3'
`
`wherein (dN),, and (dN)c represent series of deox-
`yribonucleotides and (rN)1, represents a series of ribonu-
`cleotides; wherein a, b, and c are the number of nucleo-
`tides in the series, with the proviso that b is 21, a is
`:35, and c is E 10; wherein the series of deoxyribonu-
`cleotides (dN),, includes a‘ series of deoxyribonucleo-
`tides which is substantially complementary to the series
`of deoxyribonucleotides (dN)c and the dashed line rep-
`resents noncovalent bonding between the complemen-
`tary deoxyribonucleotide series; and wherein the solid
`line represents a covalent phosphodiester bond.
`These compounds may be prepared from messenger
`RNA (mRNA) containing the genetic information nec-
`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-
`formed bacterial cells may then be grown and har-
`vested; and the desired product or products recovered.
`
`4 Claims, 1 Drawing Figure
`
`Genzyme Ex. 1037, pg 983
`
`Genzyme Ex. 1037, pg 983
`
`

`
`U.S. Patent
`
`Dec. 7, 1982
`
`
`
`4,362,867
`
`EGLOBIN M RNA #1 3,
`lAMV REVERSE TRANSC'R$PTv°«SE
`
`+dNTPs -+ OLIGO dT
`
`dT
`
`s‘°DNA
`
`5|
`
`DN/-\ POLYMERASE (KLENOW
`iSUBFRACTlON)+»-NTP5
`
`x\\\
`
`\\
`
`I
`\
`
`2
`;
`
`‘.
`,
`
`(XL,
`W
`
`PBR§??f”A
`/
`// /z
`«’
`
`DNA POLYMER/-\SE~(KLENOW
`dTlSUBFRACT|OH)+dNTF.s
`V51“ Resrmcnow CN::M,w.
`ENZYME
`V ‘V MW
`''''''"£9-,§,"4fN°,;L
`ipnosmmse
`ITRANSFERASE
`DIGEST
`/civ§A______ id eff
`-
`_____ _ _'__:__ _W_
`TERMINAL
`as
`JTRANSFERASE
`
`ALKALINE
`DIGEST
`
`RNA“ H
`'
`
`OR
`
`TERMINAL
`TRANSFERASE
`+°‘C'”’
`
`+acrp
`
`dT
`
`
`
`
`A d
`
`dc:
`./W\ArN
`
`z; dC
`
`ANNEAL
`
`Hsmoeemeous POPULATION or H‘/BRID DMAS
`
`lTRANsroRMzmoN or E.Coli mm TO
`TETRACYCLINE Res\s“"TANcE
`
`sascnou or CLONES av COLONV mauv-
`
`lmmow AND ABSENCE on AMPICILLAN
`dc
`RESISTANCE
`/, _.__...._-..- ...__.~vv\AAN
`// I,” ______________ __ de
`l\\ \\
`. _ _ — - _ _-dC
`
`\ ‘"""""""" "116
`HOMOGENEOUS POPULATION OF RECOMBINANT DNAS
`
`Genzyme Ex. 1037, pg 984
`
`Genzyme Ex. 1037, pg 984
`
`

`
`1
`
`4,362,867
`
`RECOMBINANT CDNA CONSTRUCTION
`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-
`partment of Health and Human Services.
`FIELD OF THE INVENTION
`
`This invention generally concerns the preparation of
`recombinant complementary DNAs
`(cDNAs) and
`CDNA analogs coding for cellular production of desir-
`able products such as polypeptides. It also concerns
`novel compounds which include both deoxyribonucleo-
`tides and ribonucleotides. Finally, it concerns the use of
`such compounds in bacterial cloning.
`BACKGROUND OF THE INVENTION
`
`One of the major areas of research in molecular biol-
`ogy today concerns gene organization and expression in
`eukaryotic cells. Much effort has been spent on studies,
`of RNA transcription and its subsequent processing to
`mRNA. It is currently thought that the genome sequen-
`ces surrounding the cap site contain the signal for initia-
`tion of mRNA transcription or, alternatively, that very
`rapid processing cleaves away the first few nucleotides
`followed by capping at the 5' end. [Konkel, D. A.,
`Tilghman, S. M., and Leder, P.,
`(1978), Cell,
`15:
`1125-1132; Konkel, D. A., Maizel, J. V., Jr., and Leder,
`P., (1979), Cell, 18: 865-873; Gannon, F., O’Hare, K.,
`Perrin. F., LePennec, J. P., Benoist, C., Cochet, M.,
`Breathnach, R., Royal, A., Garapin, A., Cami, B., and
`Chambon, P., (1979), Nature, 278: 428-434; Nishioka,
`Y. and Leder, P., (1979), Cell, 18: 875-882; and Kin-
`niburgh, A. J. and Ross, J., (1979), Cell, 17: 915-921. ]
`In either case, the nucleotide sequences contained in the
`5’ untranslated regions of mRNA, especially those near
`the cap site, are of -prime importance to proper gene
`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-
`man, S. M., and Leder, P., (1978), Cell, 15: 1125-1132;
`Konkel, D. A., Maizel, J. V., Jr., and Leder, P., (1979),
`Cell, 18: 865-873; and Lockard, R. E. and RajBhand-
`ary, U. L., (1976), Cell, 9: 747-760.] In addition to tran-
`scription and processing of mRNA, these sequences
`undoubtedly play an important role in the translation of
`protein from mRNA. Indeed, the importance of the
`nucleotides contained in the 5’ untranslated regions of
`mRNA is emphasized by the variety of methods de-
`signed to sequence them. [Lockard, R. E. and RajB-
`handary, U. L., (1976), Cell, 9: 747-760; Baralle, F. E.,
`(1977), Cell 10: 549 -558; Baralle, F. B., (1977), Nature,
`267: 279-281; Baralle, F. B., (1977), Cell, 12: 1085-1095;
`Legon, S., (1976). J. Mol. Biol., 106: 37-53; Chang, 1.
`C., Temple, G. F., Poon, R., Neumann, K. H. and Kan
`Y. W.,
`(1977)., Proc. Natl. Acad. Sci. U.S.A., 74:
`5145-5149; and Chang, J. C., Poon, R., Neumann, K. H.
`and Kan, Y. W.,
`(1978), Nucl. Acids. Res.,
`5:
`3515-3522.] Yet none of these methods permits se-
`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,
`(Higuchi, R., Paddock, G. V., .Wall,vR., and Salser, W.,
`(1976), Proc. Natl. Acad. Sci. U.S.A., 73: 3146-3150;
`Maniatis, T., Kee, S. G., Efstratiadis, A., and Kafatos,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`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. Natl.
`Acad. Sci. U.S.A., 73: 3418-3422; and Wood, K. O. 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
`is destined to destroy these sequences in part, because it
`relies upon use of S1 nuclease. [Higuchi, R., Paddock,
`G. V., Wall, R., and Salser, W., (1976), Proc. Natl.
`Acad. Sci. U.S.A., 73: 3146-3150.]
`In order to preserve these important 5’-end signals,
`efforts have been undertaken to develop methodology
`which avoids the need for S1 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), vol. 17, in
`press (1980); and Gaubat_z, I. and Paddock, G. V.,
`(1980), Fed. Proc., 39: 1782.] These efforts have re-
`sulted in the discovery of the floppy loop method de-
`scribed herein. This method employs a ribosubstitution
`step so that cleavage of the ‘hairpin loop can be carried
`out by alkali or ribonuclease. It avoids destruction of
`nucleotide sequence information which is lost if the
`hairpin is opened in the conventional manner with S1
`nuclease. Thus, by elimination of the S1 nuclease step,
`whole genes can be synthesized without loss of genetic
`information. Moreover,
`the S1 nuclease technique is
`known to introduce errors in the sequence [Richards, R.
`I., Shine, J., Ulbrich, A., Wells, J. R. E., and Goodman,
`H. M., (1979), Nucl. Acids Res. 7: 1137-1146] through
`a mechanism which the present invention avoids. Fi-
`nally, although it has been demonstrated that hormones
`(insulin) and interferon can be cloned via recombinant
`cDNA, it may not be possible to clone some genes in
`their entireties with the S1 nuclease technique because
`the hairpin loop may be extremely large and may even
`include part of the structural gene (i.e., part of the
`mRNA coding for protein.)
`
`SUMMARY OF THEINVENTION
`
`This invention provides various compounds which
`include both deoxyribonucleotides and at least one ribo-
`nucleotide. Certain of these compounds are useful in the
`preparation of cDNA and cDNA 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-
`tides.
`
`Specifically, compounds useful in the preparation of
`cDNAs and cDNA analogs may be prepared. These
`compounds may be depicted by the formula:
`
`/(d:N),,5’
`
`(1'N)b }\ I
`(dN)c3'
`
`I
`
`-65
`
`wherein (dN)a and (dN)c represent series of deox-
`yribonucleotides and (rN)1, represents a series of ribonu-
`cleotides; wherein a, b, and c are numbers of nucleotides
`in the series provided that b is 51, a is 235, and c is
`_>—_l0; wherein the series of deoxyribonucleotides and
`(dN),, includes a series of deoxyribonucleotides which is
`substantially complementary to the series of deox-
`
`Genzyme Ex. 1037, pg 985
`
`Genzyme Ex. 1037, pg 985
`
`

`
`4,362,867
`
`3
`yribonucleotides (dN)c and the dashed line represents
`non-covalent bonding between the complementary de-
`oxyribonucleotide series; and wherein the solid lines
`represent covalent phosphodiester bonds. Such com-
`pounds may be prepared as follows. A first molecule
`having the formula 3’(dN),,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-
`N),,5’, and additional deoxyribonucleotides are then
`added to the 3’-end of the latter to produce the com-
`pounds.
`If these compounds are treated with a reagent capa-
`ble of breaking or disrupting either 5’(rN)——(dN)3’ or
`(rN)—-(rN) bonds, or both, compounds useful as cDNA
`analogs or precursors may be prepared having the for-
`mula:
`
`5
`
`10
`
`15
`
`3'(rN)b‘(dlN)a5'
`5'(dlN),_-3’
`
`"
`
`20
`
`wherein (dN),, and (dN)c represent series of deox-
`yribonucleotides and (rN)1, represents a series of ribonu-
`cleotides; wherein a, b, and c are the numbers of nucleo-
`tides in the series; provided that bis :1, a is :35, and
`c is 210; wherein the series of deoxyribonucleotides
`(dN),, includes a series of deoxyribonucleotides which is
`substantially complementary to the series of deox-
`yribonucleotides (dN)c and the dashed line represents
`non-covalent bonding between the complementary de-
`oxyribonucleotide series; and wherein the solid line
`represents a covalent phosphodiester bond.
`Further compounds having the formula:
`
`25
`
`30
`
`3'(dN')d"(1'N)b"“(d'N)a5'
`5'(dIN)c"(dN')e3'
`
`35
`
`"1
`
`may be prepared, wherein (dN)a, (dN)c, and (rN)1, are as
`above and (dN'),1 and (dN’)e represent series of identical
`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
`3'-ends of compounds II.
`Still other compounds may be prepared having the
`formula:
`
`45
`
`4
`cleotides (dN)c; wherein the xxx- lines represent double-
`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-
`tween complementary deoxyribonucleotide series; and
`wherein the solid lines represent covalent phosphodi-
`ester bonds.
`These molecules may be used to transform bacterial
`or eucaryotic cells, e.g., Escherichia cali cells, which
`may be grown in culture to produce desired products,
`including polypeptides.
`BRIEF DESCRIPTION OF THE DRAWING
`
`FIG. 1 is a schematic illustration of the ribosubstitu-
`tion floppy loop recombinant cDNA technique show-
`ing alternative means for cleavage of the ribosubstituted
`hairpin double-stranded cDNA.
`
`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-
`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-
`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 (cDNA). It is also directed to the preparation of
`novel hybrid molecules which include both deox-
`yribonucleotides and at least one ribonucleotide. Cer-
`tain of these molecules are useful in the preparation of
`cDNA and cDNA analogs. Others are useful in bacte-
`rial cloning.
`One such compound may be represented by the gen-
`eral formula:
`
`(|'N)b
`
`(dN),5'
`I
`
`:
`(dl\1)¢3'
`
`I
`
`wherein (dN)a and (dN)c represent series of deox-
`yribonucleotides and (rN)1, represents a series of ribonu-
`cleotides; wherein a, b, and c are the numbers of nucleo-
`tides in the series; wherein b is _Z.l, a is £35, and c is
`Z 10; wherein the series of deoxyribonucleotides (dN)_,,
`includes a series of deoxyribonucleotides which is sub-
`stantially complementary to the series of deoxyribonu-
`cleotides (dN)c and the dashed line represents non-cova-
`lent bonding, particularly hydrogen bonding, between
`complementary
`deoxyribonucleotide
`series;
`and
`wherein the solid lines represent covalent phosphodi-
`ester bonds.
`«
`
`Genzyme Ex. 1037, pg 986
`
`XX(dN”)f. .. ——|
`
`50
`
`' 1V
`
`0
`
`i
`
`d —(dN)
`( N)c
`
`55
`
`65
`
`.
`
`x X
`
`wherein (dN),, and (dN)c represent series of deox-
`yribonucleotides, (dN’),1, (dN‘)e, (dN”)fand (dN”)g rep-
`resent series of identical deoxyribonucleotides, the se-
`ries (dN’),1 and (dN’)e being complementary to the series
`(dN")fand (dN")g respectively, and (rN)1, represents a
`series of ribonucleotides; wherein, a, b, c, d, e, f, and g
`are integral numbers of nucleotides in the series; pro-
`vided that b is 5 l, a is £35, and c, d, e, f, and g are
`:10; wherein the series of deoxyribonucleotides (dN),,
`includes a series of deoxyribonucleotides which is sub-
`stantially complementary to the series of deoxyribonu-
`
`xxx. . .(dN’),1-(rN);,
`X
`\
`
`X
`
`X-----—"
`X
`X
`. (dl\l')e
`X
`xxxx. .
`XXXXXXXXXXX(dlN")g. .
`
`Genzyme Ex. 1037, pg 986
`
`

`
`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
`for cellular production of a desired product in accor-
`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,
`depending upon the product for which the series codes.
`However, in the aforementioned compound I, the num-
`ber of nucleotides, a, must generally be 2 35 since fewer
`nucleotides do not permit sufficient formation of non-
`covalent bonding between complementary nucleotides
`in the series (dN),, 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 2105
`for particularly large polypeptides.
`The series (dN)¢ is 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),,. In
`general, the length of the deoxyribonucleotide polymer
`(dN),,- will be about 25 nucleotides shorter than the
`length of the series (dN),,. The approximately 25 nucle-
`otide difference may be attributed to nucleotides in the
`series (dN)a which are not base-paired with nucleotides
`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-
`tary nucleotides in the (dN)c and (dN)g polymer strands
`to form a double-stranded helical structure.
`Situated between and covalently bonded to the series
`(dN)a and (dN)c is a series of ribonucleotides (rN)1,,
`wherein b is the integral number of nucleotides in the
`series and is E 1. It will be appreciated that, when b=O,
`the resulting molecule is a homopolymer of deox-
`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
`may be preferable to first form a molecule having the
`formula 3’(rN):,—(dN),,5’, wherein b is :1, then re-
`move ribonucleotides until b=1, and finally add deox-
`yribonucleotides (dN)., to form molecules 1, wherein
`b= 1. In all molecules 1, the ribonucleotide series (rN)1,
`is covalently joined to the series (dN)a and (dN)c by
`means of 5'—>3’ and 3’——>5’ phosphodiester bonds, re-
`spectively.
`Although the experiments described hereinafter in-
`volve globin polypeptides, it will be readily understood
`by those skilled in the art that the practices of this in-
`vention are widely applicable to polypeptides generally
`and to other desirable products. Thus, the deoxyribonu-
`cleotide series (dN),, may contain information in the
`form of its ordered nucleotide sequence for cellular
`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
`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-
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Genzyme Ex. 1037, pg 987
`
`4,362,867
`
`10
`
`15
`
`20
`
`6
`ing, since countless other products may also be pre-
`pared.
`In general, the deoxyribonucleotide series (dN),, 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 eukaryotic cell known to contain a gene or
`genes coding for or otherwise associated with produc-
`tion of the desired product. However, it is also contem-
`plated that the series (dN),, might be directly synthe-
`sized to create a polynucleotide having a sequence cod-
`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-
`sponding thereto. One method of accomplishing this
`result is to treat the mRNA molecule with a suitable
`enzyme, such as AMV reverse transcriptase, and a mix-
`-ture of the deoxyribonucleotides, dATP, dCTP, dGTP,
`and dTTP, under appropriate conditions which permit
`formation of the (dN),, 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-
`phates.
`'
`In one embodiment of the invention, the mRNA mol-
`ecule will include a series of repeating adenylate ribonu-
`cleotides at its 3’-end, the number of such being 11, and
`formation of (dN)a involves addition of oligo dT, e.g.,
`oligo (dT)12.1g,
`to the reaction mixture in sufficient
`quantity to permit formation of a 3’(dN),,5’ 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),,5’ molecule to form a molecule hav-
`ing the formula 3'(rN)b—(dN),,5’. Although ribonucleo-
`tide addition could be accomplished by conventional
`chemical methods, it is preferably effected by a ribosub-
`' stitution addition reaction utilizing a DNA polymerase,
`e.g., DNA polymerase I, and a mixture of theribonucle-
`otide triphosphates rATP, rGTP, rCTP, and rUTP
`under appropriate conditions to permit formation of the
`3'(rN)[,—(dN)a5’ molecule. Once again, suitable condi-
`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-
`phosphates; and catalytic amounts of enzyme.
`7
`Finally, additional deoxyribonucleotides are added to
`the 3’-end of the 3’(rN)g,—(dN)g5' molecule to form
`compound I. Here again, conventional chemical meth-
`ods may be employed, or preferably enzymatic addition
`of the nucleotides may be utilized. In this regard, treat-
`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-
`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
`excess of nucleotide triphosphates;
`and catalytic
`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-
`ceutical preparations or diagnostic tests.
`
`Genzyme Ex. 1037, pg 987
`
`

`
`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-
`able conditions (suitability being determined by the
`nature of the reagent and the precise identity of com-
`pound (I) a double-stranded compound can be prepared
`having the formula:
`
`I
`3'(rN)b“‘ (d'N)a5'
`5’(dN),_-3’
`
`wherein (dN),,, (dN)c, (rN)b, a, b, c, and the dashed and
`solid lines are as indicated for compound 1.
`Suitable reagents include alkali, alkaline phosphatase,
`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 be 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
`cDNA by removal of all the ribonucleotides. The re-
`sulting compounds can be represented by formula 11
`wherein b= 0. Many such compounds are known. How-
`ever, there may be some which are novel, particularly,
`in cases where the deoxyribonucleotide series (dN),, has
`been prepared by chemical synthesis and codes for an
`oligopeptide or polypeptide which does not occur in
`nature.
`
`This invention contemplates the use of compounds II
`as CDNA analogs in subsequent cloning. In this applica-
`tion, it may be preferable to remove all ribonucleotides
`other than the first ribonucleotide joined to the (dN)a
`series of deoxyribonucleotides if this result has not al-
`ready been effected by the reagent treatment.
`Compounds II may be stored for subsequent use in
`bacterial cloning. Alternatively, the compounds may be
`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,
`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 of G, C, T, or
`A.
`
`This results in production of compounds which may
`be depicted by the formula:
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`3'(dN')a'-(rN)b"(d|N)a5'
`5'(d'N)c—(dN')e3’
`
`"1
`
`55
`
`wherein (rN)g,, (dN)g, (dN)c, a, b, c, and the dashed and
`solid lines are as indicated hereinabove;
`(dN'),1 and
`(dN’)e represent series of identical deoxyribonucleo-
`tides; and wherein d and e are the number of nucleotides
`in the series, provided that d and e are 210. It is gener-
`ally necessary that d and e be ->_-10 in order for com-
`pounds III to be used in subsequent cloning. Also, d and
`e will generally not exceed about 20-40, although this is
`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-
`
`60
`
`65
`
`8
`cleotide triphosphates employed, as is true in the vari-
`ous similar reactions discussed herein.
`Compounds III can be annealed with DNA mole-
`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 E 10 nucleotides in length. In order for anneal-
`ing to occur dN” must be complementary to dN’. An-
`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
`
`.
`
`. (dN’),1--(rN)b
`xxx .
`\‘
`X
`XX(dN")f. .
`X
`’,§_____§§
`. (dN’),.
`X
`xxxx. .
`:
`x
`xxxxxxxxxxxx (dN")g. .
`
`. --—|
`(dN)r(dN)a
`
`.
`
`wherein (dN),, and (dN)c represent series of deox-
`yribonucleotides,
`(dN‘),1,
`(dN’)e,
`(dN")f, and (dN")g
`represent series of identical deoxyribonucleotides, the
`series (dN’),1 and (dN’), being complementary to the
`series (dN")fand (dN”)g respectively; and (rN)1, repre-
`sents a series of ribonucleotides; wherein a, b, c, d, e, f,
`and g are the numbers of nucleotides in the series pro-
`vided that b is :1, a is :35, and c, e, f, and g are Z10;
`wherein the series of deoxyribonucleotides (dN),, in-
`cludes a series of deoxyribonucleotides which is sub-
`stantially complementary to the series of deoxyribonu-
`cleotides (dN)¢; wherein the xxx-lines represent double-
`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-
`ing between complementary deoxyribonucleotide se-
`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-
`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-
`ple, when the cloning vehicle-derived DNA is plasmid
`DNA, e.g., pBR322 DNA, and the (dN),, deoxyribonu-
`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 X1776. The transformed cells which
`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),, includes the informa-
`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-
`strued as limiting the invention which is defined by the
`claims which follow.
`
`Genzyme Ex. 1037, pg 988
`
`Genzyme Ex. 1037, pg 988
`
`

`
`9
`
`EXPERIMENTAL DETAILS
`MATERIALS AND METHODS
`
`4,362,867
`
`Construction of recombinant cDNAs via the floppy
`loop technique was carried out as follows. Complemen-
`tary DNA was synthesized in 0.1 ml to 0.4 ml 50 mM
`Tris-HC1, pH 8.3; 10 mM MgCl2; 20 mM 2-mercaptoe-
`thanol; 30 p.g/ml actinomycin D; 20 pg/ml oligo
`(dT)12.13, obtained from Collaborative Research, Wal-
`th-am, Mass.; 40-60 p.g/ml rabbit globin mRNA; 1 mM
`each dATP, dGTP, dTTP, and dCTP [dCTP contain-
`ing 50-250 p.Ci/p.mol 3H or 32P]; and 150 U/ml AMV
`reverse transcriptase, supplied by J. Beard through the
`Biological Carcinogenesis Branch, National Cancer
`Institute, NIH. Incubations in early‘ experiments were
`carried out at 37° C. for 1 hr, but we have found incuba-
`tion at 45° C. for 15-20 min to be far superior. The
`reaction mixture was then extracted with an equal vol-
`ume of water-saturated phenol, and residual phenol was
`removed by extraction with an equal volume of ether.
`The cDNA was precipitated with 40 pg/ml yeast
`tRNA, 0.1 vol 3 M NaAc (pH 5.5) and 2.25 vol ethanol.
`The cDNA was resuspended in 0.2 ml H20, brought to
`0.3 M in NaOH, and incubated at 90° C. for 30 min to
`hydrolyze the mRNA. The cDNA was then precipi-
`tated with 0.1 vol 3 M NaAc (pH 5.5), 0.1 vol 3 N HC1,
`40 pg/ml yeast tRNA, and 2.5 vol ethanol. The cDNA
`was then chromatographed through a 0.6X15 cm col-
`umn of Sephadex G-100 in 0.01 M triethylammonium
`bicarbonate, pH 8.5. The columns were prepared in
`silanized glass or plastic pipettes. One-ml
`tuberculin
`syringes have also been used, but in this case the oligo
`(dT) may not be removed. Yeast tRNA (40 pg) was
`chromatographed on a column prior to cDNA samples
`to fill nonspecific binding sites. The peak fractions were
`pooled and precipitated with 0.1 vol 3 M NaAc (pH
`5.5), 40 pg/ml tRNA, and 2.25 vol EtOH.
`The ribosubstitution step, modified from Whitcome,
`et al., [Whitcome, P., Fry, K. and Salser, W., (1974),
`Methods Enzymol. 29: 295-321], was carried out in 0.1
`ml 67 mM Tris-HCl (pH 7.4), 0.67 mM MnCl2; 1.0 mM
`2-mercaptoethanol; 330 p.M each rATP, rGTP, rUTP,
`and rCTP; 100 U/ml DNA polymerase I (Klenow large
`fragment
`from Boehringer-Mannheim); and 10-40
`pg/ml cDNA for 10 min at 37° C. Recently, 5’-rAMP
`has also been added at 0.3 mM to prevent any possible
`degradation by the 3'-exonuclease activity contained in
`DNA polymerase I [Byrnes, J. J., Downey, K. M., Que,
`B. G., Lee, M. Y. W., Black, V. L. and S0. A. G.,
`(1977), Biochemistry 16: 3740-3746] during the slow
`ribosubstitution reaction. The cDNA reaction mixture
`was then extracted with water-saturated phenol and the
`residual phenol removed by ether extraction. Following
`the extraction, cDNA was chromatographed through
`Sephadex G-100, and the excluded material was precipi-
`tated with EtOH as above.
`Second-strand synthesis was carried out as described
`by Higuchi, et al. [Higuchi, R., Paddock, G. V., Wall,
`R., and Salser, W., (1976), Proc. Natl. Acad. Sci. U.S.A.
`73: 3146-3150.] The cDNA was resuspended in 0.12 ml
`67 mM potassium phosphate, pH 7.3; 6.7 mM MgC12; 1
`mM 2-mercaptoethanol; 33 p.M each of dTTP, dCTP,
`dATP, and dGTP (dCTP containing 10 to 20 mCi/p.-
`mo] 3H or 32P) and incubated with 40 U/ml DNA poly-
`merase I (Klenow large fragment) for 30 min at 37° C.
`The double-stranded hairpin cDNA reaction mixture
`was extracted with water-saturated phenol and ether,
`and precipitated with ethanol as above. The precipitate
`
`15
`
`20
`
`50
`
`10
`was resuspended in 0.1 ml H20, 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-
`tated with 0.1 vol 3 M NaAc, pH 5.5, 0.1 vol 3 N HC1,
`5