United States Patent
`Ward et al.
`
`[19]
`
`[75]
`
`[54] MODIFIED POLYNUCLEOTIDES AND
`METHODS OF PREPARING SAME
`Inventors: David C. Ward, Guilford, Conn.;
`Pennina R. Langer, Monsey, N.Y.;
`Alexander A. Waldrop, m,
`Charlottesville, Va.
`[73] Assignee: Yale University, New Haven, Conn.
`[ *] Notice:
`The portion of the term of this patent
`subsequent to Dec. 8, 2004 has been
`disclaimed.
`[21] Appl. No.: 886,660
`[22] Filed:
`May 20, 1992
`
`[60]
`
`[51]
`[52]
`
`[58]
`
`[56]
`
`Related U.S. Application Data
`Continuation of Ser. No. 130,002, Dec. 8, 1987, aban(cid:173)
`doned, which is a division of Ser. No. 496,915, May 23,
`1983, Pat. No. 4,711,955, which is a continuation-in(cid:173)
`part of Ser. No. 255,223, Apr. 17, 1981, abandoned.
`Int. CJ.6 ............................................. C07H 21/00
`U.S. Cl ................................. 536/24.3; 536/25.32;
`.
`536/25.6; 536/26.6
`Field of Search ................... 536/24.3, 25.32, 25.6,
`536/26.6; 435/6
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,328,389 6/1967 Shimizu et al ................... 260/211.5
`(List continued on next page.)
`
`FOREIGN PATENT DOCUMENTS
`070685 1/1983 European Pat. Off ..
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`(List continued on next page.)
`
`OTHER PUBLICATIONS
`Trayer et al., "Preparation of Adenosine Nucleotide
`Derivatives Suitable for Affinity Chromatography",
`Biochem. J. (1974) 139:609-623.
`(List continued on next page.)
`
`Primary Examiner-Che S. Chereskin
`Attorney, Agent, or Firm-Ronald C. Fedus; John L.
`Santalone
`
`I IIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll llllll Ill lllll llll
`US005449767A
`5,449,767
`[11] Patent Number:
`[45] Date of Patent: * Sep. 12, 1995
`
`ABSTRACT
`[57]
`Compounds having the structure:
`
`x-v.O .~ ... A
`
`h'i--Yh
`
`y
`
`z
`
`wherein B represents 7-deazapurine, or pyrimidine moi(cid:173)
`ety covalently bonded to the Cl'-position of the sugar
`moiety, provided that when Bis 7-deazapurine, it is
`attached at the N9-position of the 7-deazapurine and
`when B is pyrimidine, it is attached at the NI.posi(cid:173)
`tion;
`wherein A represents a moiety consisting of at least
`three carbon atoms which is capable of forming a
`detectable complex with a polypeptide when the
`compound is incorporated into a double-stranded
`ribonucleic acid, deoxyribonucleic acid duplex, or
`DNA-RNA hybrid;
`wherein the dotted line represents a chemical linkage
`joining Band A, provided that if Bis 7-deazapurine,
`the linkage .is attached to the ?-position of the
`deazapurine, and if B is pyrimidine, the linkage is
`attached to the 5-position of the pyrimidine; and
`wherein each of x, y and z represents
`
`H-, HO-,
`
`0
`II
`HO-P-0-
`1
`OH
`
`'
`
`0
`0
`II
`II
`HO-P-O-P-0-,
`I
`I
`OH
`OH
`
`or
`
`0
`0
`O·
`II
`II
`II
`HO-P-0-P-O-P-O-,
`I
`I
`I
`OH
`OH
`OH
`
`either directly, or when incorporated into oligo- and
`polynucleotides, provide probes which are widely
`useful.
`
`71 Claims, No Drawings
`
`Page 1
`
`Illumina Ex. 1070
`IPR Petition - USP 10,435,742
`
`

`

`5,449,767
`
`Page 2
`
`U.S. PATENT DOCUMENTS
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`3,893,998 7/1975 Secrist, III et al .......... 260/211.5 R
`3,915,958 10/1975 Shuman et al ............... 260/211.5 R
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`3,968,101 7/1976 Christensen et al ............. 260/211.5
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`4,038,480 7/1977 Robins et al .......................... 536/27
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`4,086,417 4/1978 Ishida et al ........................... 536/29
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`4,171,432 10/1979 Carrico et al ......................... 536/26
`4,213,893 7/1980 Carrico et al .................... 260/112.5
`4,228,237 10/1980 Hevey et al ............................ 435/7
`4,230,698 10/1980 Bobek et al ......................... 424/180
`4,230,797 10/1980 Boguslaski et al ...................... 435/7
`4,247,544 1/l981 Bergstrom et al .................. 424/180
`4,261,893 4/!981 Boguslaski et al ............. 260/326 N
`4,267,171 5/1981 Bergstrom et al .................. 424/180
`4,302,204 11/1981 Wahl et al .......................... 23/230.3
`4,318,980 3/1982 Boguslaski et al ...................... 435/7
`4,318,981 3/1992 Burd et al ............................... 435/7
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`4,374,925 2/1983 Litman et al ........................... 435/7
`4,380,580 4/1983 Boguslaski et al ...................... 435/7
`4,383,031 5/1983 Boguslaski et al ...................... 435/7
`4,446,231 5/1984 Self .......................................... 435/7
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`
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`Page 2
`
`

`

`5,449,767
`
`Page 3
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`OTHER PUBLICATIONS
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`H. Hayashi et al. "Structure and Synthesis of Dihydrox(cid:173)
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`P.R. Langer et al., "Enzymatic Synthesis ofBiotin-La(cid:173)
`beled Polynucleotides: Novel Nucleic Acid Affinity
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`(1981) [Langer!].
`P.R. Langer and D. C. Ward, "A Rapid and Sensitive
`Immunological Method For In Situ Gene Mapping," in
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`II].
`P.R. Langer and D. C. Ward, Abstract 1153: "A Rapid
`and Sensitive Immunological Method For In Situ Gene
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`III].
`Manning et al., "A Method for Gene Enrichment Based
`on the Avdin-Biotin Interaction. Application to the
`Drosophila Ribosomal RNA Genes," Biochemistry, 16,
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`Method of Gene Enrichment to the Isolation of Long
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`J. L. Ruth and D. E. Bergstrom, "C-5 Substituted Py(cid:173)
`rimidine Nucleosides. 1. Synthesis of C-5 Allyl, Propyl,
`and Propenyl Uracil and Cytosine Nucleosides via Or(cid:173)
`ganopalladium Intermediates," J. Org. Chem., 43, pp.
`2870-2876 (1978).
`A. Sodja and N. Davidson, "Gene Mapping and Gene
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`Cytochrome-C as a Polyamine Bridge," Nucl Acids
`Res., 5, pp. 383-399 (1978).
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`Acid, A Polyuridylic Acid Analogue", J. Mo/. Biol, 47,
`pp. 57-67 (1970).
`J. G. J. Bauman et al., "A New Method for Fluores(cid:173)
`cence Microscopical Localization of Specific DNA
`Sequences by In Situ Hybridization of Fluoro(cid:173)
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`J. G. J. Bauman et al., "Rapid and High Resolution
`Detection of in situ Hybridisation to Polytene Chromo(cid:173)
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`Fluorochrome-Labeled
`RNA,"
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`
`D. Bergstrom and J. L. Ruth, "Preparation ofC-5 Mer(cid:173)
`curated Pyrimidine Nucleosides," J. Carbohydrates,
`Nucleotides and Nucleosides, 4, pp. 257-269 (1977)
`[Bergstrom II].
`R. M. K. Dale et al., "The Synthesis and Enzymatic
`Polymerization of Nucleotides Containing Mercury:
`Potential Tools for Nucleic Acid Sequencing and
`Structural Analysis", Proc. Natl. Acad. Sci. USA, 70, pp.
`2238-2242 (1973) [Dale II].
`W. S. Dallas and S. Falkow, "Molecular and Genetic
`Analysis of a DNA Sequence Encoding for Enterotoxin
`Snythesis in Escherichia coli, " Thirteenth Joint Confer(cid:173)
`ence on Cholera, The U.S.-Japan Cooperative Medical
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`ichia coli Plasmid Determinant That Encodes for the
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`and 5-Iododeoxyuridine: A New Reagent for Detec(cid:173)
`tion of DNA Replication," Science, 218, pp. 474-475
`(1982).
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`A Method for the Isolation of Cloned DNAs That Con(cid:173)
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`Their Application To Retrieval Of Streptanidin", Proc.
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`(1978).
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`
`Page 4
`
`

`

`1
`
`5,449,767
`
`MODIFIED POLYNUCLEOTIDES AND
`METHODS OF PREPARING SAME
`
`2
`biotin, similar reactions can be used for its detection.
`Moreover, the reversibility of the iminobiotin-avidin
`interaction, by decreasing solution pH, offers significant
`advantages in certain applications.
`The invention disclosed and/or claimed in this appli- 5
`The specificity and tenacity of the biotin-avidin com-
`cation was made in the course of work carried out
`plex has been used in recent years to develop methods
`under grants from the Public Health Service, National
`for visually localizing specific proteins, lipids, or carbo-
`lnstitute of General Medical Research Grant No. P50-
`hydrates on or within cells (reviewed by E. A. Bayer
`GM20124; Public Health Service, National Cancer In-
`and M. Wilchek in Methods of Biochemical Analysis,
`stitute Training Grant No. T32-CA09159; and Public 10 26, 1, 1980). Chromosomal location of RNA has been
`Health Service, National Institute of General Medical
`determined by electron microscopy using a biotinized
`Science Grant No. T32-GM07499. The Government
`protein, cytochrome C, chemically crosslinked to RNA
`has certain rights in the invention.
`as a hybridization probe. The site of hybridization was
`This
`is continuation of application Ser. No.
`visualized through the binding of avidin-ferritin or avi-
`07 /130,002, filed Dec. 8, 1987, now abandoned, which 15 din-methacrylate spheres mediated by the avidin-biotin
`is in tum a division of application Ser. No. 06/496,915,
`interaction. (J.E. Manning, N. D. Hershey, T. R. Bro-
`filed May 23, 1983, issued Dec. 8, 1987 as U.S. Pat. No.
`ker, M. Pellegrini, H. K. Mitchell, and N. Davidson,
`4,711,955. Application Ser. No. 06/496,915 is in tum a
`Chromosoma, 53, 107, 1975; J. E. Manning, M. Pelle-
`continuation of application Ser. No. 06/255,223, filed
`grini, and N. Davidson, Biochemistry, 61, 1364, 1977; T.
`Apr. 17, 1981, now abandoned.
`20 R. Broker, L. M. Angerer, P.H. Yen, N. D. Hersey, and
`N. Davidson, Nucleic Acid Res., 5, 363, 1978; A Sodja
`BACKGROUND OF THE INVENTION
`and N. Davidson, Nucleic Acid Res., 5, 383, 1978.) This
`Many procedures employed in biomedical research
`approach to the detection of polynucleotide sequences,
`and recombinant DNA technology rely heavily on the
`although successful in the specialized cases examined
`use of nucleotide or polynucleotide derivatives radioac- 25
`which were highly reitterated sequences, is not of gen(cid:173)
`tively labeled with isotopes of hydrogen (3H), phospho(cid:173)
`eral utility for analysis of polynucleotides present in
`rous (32p ), carbon (14C), or iodine (1251). Such radioac(cid:173)
`single or low copy number.
`tive compounds provide useful indicator probes that
`Moreover, methods for attaching chemical moieties
`permit the user to detect, monitor, localize, or isolate
`to pyrimidine and purine rings are known. Several years
`nucleic acids and other molecules of scientific or clini- 30
`ago a simple and rapid acetoxymercuration reaction
`cal interest, even when present in only extremely small
`was developed for introducing covalently bound mer(cid:173)
`amounts. To date, radioactive materials have provided
`cury atoms into the 5-position of the pyrimidine ring,
`the most sensitive, and in many cases the only, means to
`the C-8 position of the purine ring or the C-7 position of
`perform many important experimental or analytical
`a 7-deazapurine ring, both in nucleotides and polynu(cid:173)
`tests. There are, however, serious limitations and draw- 35
`cleotides. (R. M. K. Dale, D. C. Livingston and D. C.
`backs associated with the use of radioactive com(cid:173)
`Ward, Proc. Natl. Acad. Sci. U.S.A., 70, 2238, 1973; R.
`pounds. First, since personnel who handle radioactive
`M. K. Dale, E. Martin, D. C. Livingston and D. C.
`material can be exposed to potentially hazardous levels
`Ward, Biochemistry, 14, 2447, 1975.) It was also shown
`of radiation, elaborate safety precautions must be main(cid:173)
`several years ago that organomercurial compounds
`tained during the preparation, utilization, and disposal 40
`would react with olefinic compounds in the presence of
`of the radioisotopes. Secondly, radioactive nucleotides
`palladium catalysts to form carbon-carbon bonds (R. F.
`are extremely expensive to purchase and use, in large
`Heck, J. Am. Chem. Soc., 90, 5518, 1968; R. F. Heck,
`part due to the cost of equipment and manpower neces(cid:173)
`Ibid., 90, 5526, 1968; R. F. Heck, Ibid., 90, 5531, 1968;
`sary to provide the appropriate safeguards, producer(cid:173)
`R. F. Heck, Ibid., 90, 5535, 1968; and R. F. Heck, J. Am.
`/user health monitoring services, and waste-disposal 45
`Chem. Soc. 91, 6707, 1969.) Bergstrom and associates
`programs. Thirdly, radioactive materials are often very
`(J. L. Ruth and D. E. Berstrom, J. Org. Chem., 43, 2870,
`unstable and have a limited shelf-life, which further
`1978; and D. E. Bergstrom and M. K. Ogawa, J. Am.
`increases usage costs. This instability results from radio(cid:173)
`Chem. Soc., 100, 8106, 1978) and Bigge, et al. (C. F.
`lytic decomposition, due to the destructive effects asso(cid:173)
`Bigge, P. Kalaritis, J. R. Deck and M. P. Mertes, J. Am.
`ciated with the decay of the radioisotope itself, and 50
`Chem. Soc., 102, 2033, 1980) have recently applied this
`from the fact that many isotopes (e.g. 32p and 1251) have
`reaction scheme in the synthesis of C-5 substituted py(cid:173)
`half-lives of only a few days
`rimidine nucleotide compounds.
`It is known that haptens can combine with antibodies,
`Finally, it is known that antibodies specific for modi(cid:173)
`but can initiate an immune response only if bound to a
`fied nucleotides can be prepared and used for isolating
`carrier. This property can be exploited in detection and 55
`and characterizing specific constituents of the modified
`identification testing.
`nucleotides. (T. W. Munns and M. K. Liszewski,
`It is also known that biotin and iminobiotin strongly
`Progress in Nucleic Acid Research and Molecular Biol(cid:173)
`interact with avidin, a 68,000 dalton glycoprotein from
`ogy, 24, 109, 1980.) However, none of the antibodies
`egg white. This interaction exhibits one of the tightest,
`prepared to date against naturally occurring nucleotides
`non-covalent binding constants (K.iz:s= 10-15) seen in 60
`have been shown to react with their nucleotide determi(cid:173)
`nature. If avidin is coupled to potentially demonstrable
`nant when it exists in a double-stranded RNA or DNA
`indicator molecules, including fluorescent dyes, e.g.
`duplex or when in DNA-RNA hybrid molecules.
`fluorescein or rhodamine; electron-dense reagents, e.g.
`To circumvent the limitations of radioactively la(cid:173)
`ferritin, hemocyanin, or colloidal gold; or enzymes
`beled probes or previously utilized chemical and biolog(cid:173)
`capable of depositing insoluble reaction products, e.g. 65
`ical probes, a series of novel nucleotide derivatives that
`peroxidase or alkaline phosphatase, the presence, loca(cid:173)
`contain biotin, iminobiotin, lipoic acid, and other deter(cid:173)
`tion, or quantity of a biotin probe can be established.
`minants attached covalently to the pyrimidine or purine
`Although iminobiotin binds avidin less tightly than
`ring have been synthesized. These nucleotide deriva-
`
`Page 5
`
`

`

`5,449,767
`
`4
`-continued
`0
`0
`0
`II
`II
`II
`HO-P-O-P-0-P-O-,
`I
`I
`I
`OH
`OH
`OH
`
`3
`tives, as well as polynucleotides and coenzymes that
`contain them, will interact specifically and uniquely
`with proteins such as avidin or antibodies. The interac(cid:173)
`tion between modilied nucleotides and specific proteins
`can be utilized as an alternative to radioisotopes for the 5
`detection and localization of nucleic acid components in
`many of the procedures currently used in biomedical
`and recombinant-DNA technologies. Methods employ(cid:173)
`ing these modified nucleotide-protein interactions have 10
`detection capacities equal to or greater than procedures
`which utilize radioisotopes anc;i they often can be per(cid:173)
`formed more rapidly and with greater resolving power.
`These new nucleotide derivatives can be prepared
`relatively inexpensively by chemical procedures which 15
`have been developed and standarized as discussed more
`fully hereinafter. More significantly, since neither the
`nucleotide probes of this invention nor the protein rea(cid:173)
`gents employed with them are radioactive, the com- 20
`pounds can be prepared, utilized, and disposed of, with(cid:173)
`out the elaborate safety procedures required for radio(cid:173)
`isotopic protocols. Moreover, these nucleotide deriva(cid:173)
`tives are chemically stable and can be expected to have
`functional shelf-lives of several years or more. Finally, 25
`these compounds permit the development of safer, more
`economical, more rapid, and more reproducible re(cid:173)
`search and diagnostic procedures.
`
`SUMMARY OF THE INVENTION
`Compounds having the structure:
`
`x-v,O .~---A
`
`h1--Yh
`
`y
`
`z
`
`30
`
`35
`
`40
`
`wherein B represents a purine, deazapurine, or pyrimi(cid:173)
`dine moiety covalently bonded to the Cl-position of the
`sugar moiety, provided that when B is purine or 7-
`deazapurine, it is attached at the N9-position of the 45
`purine or deazapurine, and when B is pyrimidine, it is
`attached at the NI.position;
`wherein A represents a moiety consisting of at least
`three carbon atoms which is capable of forming a
`detectable complex with a polypeptide when the 50
`compound is incorporated into a double-stranded
`ribonucleic acid, deoxyribonucleic acid duplex, or
`DNA-RNA hybrid;
`wherein the dotted line represents a chemical linkage
`joining B and A, provided that if B is purine the 55
`linkage is attached to the 8-position of the purine, if
`B is 7-deazapurine, the linkage is attached to the
`7-position of the deazapurine, and if B is pyrimi(cid:173)
`dine, the linkage is attached to the 5-position of the 60
`pyrimidine; and
`wherein each of x, y, and z represents
`
`0
`0
`0
`II
`II
`II
`H-, HO-, HO-P-0-, HO-P-0-P-O-, or
`I
`I
`I
`OH
`OH
`OH
`
`65
`
`are widely useful as probes in biomedical research
`and recombinant DNA technology.
`Particularly useful are compounds encompassed
`within this structure which additionally have one or
`more of the following characteristics: A is non(cid:173)
`aromatic; A is at least CS; the chemical linkage joining
`B and A includes an a-olefinic bond; A is biotin or
`iminobiotin; and Bis a pyrimidine or 7-deazapurine.
`These compounds may be prepared by a process
`which involv~s:
`(a) reacting a compound having the structure:
`
`x-v.o.~;
`
`h1--Yh
`
`y
`
`z
`
`with a mercuric salt in a suitable solvent under
`suitable conditions so as to form a mercurated com(cid:173)
`pound having the structure:
`
`x-v,O .~-Hg+;
`h1--Yh
`
`y
`
`z
`
`(b) reacting said mercurated compound with a chemi(cid:173)
`cal moiety reactive with the -Hg+ portion of said
`mercurated compound and represented by the for(cid:173)
`mula ... N, said reaction :being carried out in an
`aqueous solvent and in the presence of K2PdC4
`under suitable conditions so as to form a compound
`having the structure:
`
`x -C~H2
`O
`B ... N
`
`H
`
`H
`
`H
`
`y
`
`z
`
`wherein N is a- reactive terminal functional group
`or is A; and
`(c) recovering said compound as said modified nucle(cid:173)
`otide when N is A, or when N is a reactive terminal
`group, reacting said compound with a compound
`having the structure M-A, wherein M represents a
`functional group reactive with N in an aqueous
`solvent under suitable conditions so as to form said
`modified nucleotide, which is then recovered.
`This invention also provides compounds having the
`structure:
`
`Page 6
`
`

`

`5
`
`5,449,767
`
`6
`
`0
`o
`II
`H0-1 --o-C~H2 B'
`
`H
`
`H H
`
`H
`0
`II
`z
`O--P
`I
`OH m
`
`o - c~H2
`B. .. A
`
`°
`
`H
`
`H
`
`H
`0
`II
`0 - -P
`I
`OH
`
`OH B''
`0 -C~H ,
`
`H
`
`H
`0
`I
`0--p-- H
`I
`OH n
`
`wherein each of B, B', and B" represents a purine, 25
`7-deazapurine, or pyrimidine moiety covalently
`bonded to the Cl'-position of the sugar moiety,
`provided that whenever B, B', or B" is purine or
`7-deazapurine, it is attached at the N9-position of
`the purine or 7-deazapurine, and whenever B, B', 30
`or B" is pyrimidine, it is attached at the N1-posi(cid:173)
`tion;
`wherein A represents a moiety consisting of at least
`three carbon atoms which is capable of forming a
`detectable complex with a polypeptide when the 35
`compound is incorporated into a double-stranded
`duplex formed with a complementary ribonucleic
`or deoxyribonucleic acid molecule.
`wherein the dotted line represents a chemical linkage
`joining B and A, provided that if B is purine the 40
`linkage is attached to the 8-position of the purine, if
`B is 7-deazapurine, the linkage is attached to the
`7-position of the deazapurine, and if B is pyrimi(cid:173)
`dine, the linkage is attached to the 5-position of the
`pyrimidine;
`wherein z represents H- or HO-; and
`wherein m and n represent integers from O up to
`about 100,000.
`These compounds can be prepared by enzymatic
`polymerization of a mixture of nucleotides which in- 50
`elude the modified nucleotides of this invention. Alter(cid:173)
`natively, nucleotides present in oligo- or polynucleo(cid:173)
`tides may be modified using chemical methods.
`Nucleotides modified in accordance with the prac(cid:173)
`tices of this invention and oligo- and polynucleotides 55
`into which the modified nucleotides have been incorpo(cid:173)
`rated may be used as probes in biomedical research,
`clinical diagnoJ;is, and recombinant DNA technology.
`These various utilities are based upon the ability of the
`molecules to form stable complexes with polypeptides 60
`which in turn can be detected, either by means of prop(cid:173)
`erties inherent in the polypeptide or by means of detect(cid:173)
`able moieties which are attached to, or which interact
`with, the polypeptide.
`Some uses include detecting and identifying nucleic 65
`acid-containing etiological agents, e.g. bacteria and
`viruses; screening bacteria for antibiotic resistance; di(cid:173)
`agnosing genetic disorders, e.g. thalassemia and sickle
`
`45
`
`cell anemia; chromosomal karyotyping; and identifying
`tumor cells.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`Several essential criteria must be satisfied in order for
`a modified nucleotide to be generally suitable as a sub(cid:173)
`stitute for a radioactively-labeled form of a naturally
`occurring nucleotide. First, the modified compound
`must contain a substituent or probe that is unique, i.e.,
`not normally found associated with nucleotides or
`polynucleotides. Second, the probe must react specifi(cid:173)
`cally with chemical or biological reagents to provide a
`sensitive detection system. Third, the analogs must be
`relatively efficient substrates for commonly studied
`nucleic acid enzymes, since numerous practical applica(cid:173)
`tions require that the analog be enzymatically metabo(cid:173)
`lized, e.g., the analogs must function as substrates for
`nucleic acid polymerases. For this purpose, probe moi(cid:173)
`eties should not be placed on ring positions that steri(cid:173)
`cally, or otherwise, interfere with the normal Watson(cid:173)
`Crick hydrogen bonding potential of the bases. Other(cid:173)
`wise, the substituents will yield compounds that are
`inactive as polymerase substrates. Substitution at ring
`positions that alter the normal "anti" nucleoside confor(cid:173)
`mation also must be avoided since such conformational
`changes usually render nucleotide derivatives unaccept(cid:173)
`able as polymerase substrates. Normally, such consider(cid:173)
`ations limit substitution positions to the 5-position of a
`pyrimidine and the 7-position of a purine or a 7-
`deazapurine.
`Fourth, the detection system should be capable of
`interacting with probe substituents incorporated into
`both single-stranded and double-stranded polynucleo(cid:173)
`tides in order to be compatible with nucleic acid hybrid(cid:173)
`ization methodologies. To satisfy this criterion, it is
`preferable that the probe moiety be attached to the
`purine or pyrimidine through a chemical linkage or
`"linker arm" so that it can readily interact with antibod(cid:173)
`ies, other detector proteins, or chemical reagents.
`Fifth, the physical and biochemical properties of
`polynucleotides containing small numbers of probe
`substituents should not be significantly altered so that
`current procedures using radioactive hybridization
`
`Page 7
`
`

`

`5,449,767
`
`8
`5-methylcytosine, and 5-hydroxymethylcytosine are
`not useful. Presently preferred bases are cytosine, ura(cid:173)
`cil, deazaadenine and deazaguanine.
`A may be any moiety which has at least three carbon
`atoms and is capable of forming a detectable complex
`with a polypeptide when the modified nucleotide is
`incorporated into a double-stranded duplex containing
`either deoxyribonucleic or ribonucleic acid.
`A therefore may be any ligand which possesses these
`properties, including haptens which are only immuno(cid:173)
`genic when attached to a suitable carrier, but are capa(cid:173)
`ble of interracting with appropriate antibodies to pro(cid:173)
`duce complexes. Examples of moieties which a